Compositions and methods of treatment using nicotinamide mononucleotide

Abstract

To provide various methods and compositions for treating age-associated conditions and other medical conditions, such as muscle diseases, type 2 diabetes and / or obesity.SOLUTION: A method comprises administering to a subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one additional compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, esters of any thereof, pharmaceutically acceptable salts of any thereof, and combinations of any thereof.SELECTED DRAWING: None

Description

Government Aid Declaration

[0001] This invention was made with Government support under awards AG047902 and AG037457 from the National Institutes of Health. The Government has certain rights in the invention.

[0002] [Reference to array list] The sequence listing that is part of this disclosure includes a text file that contains the primers, nucleotide and / or amino acid sequences of the present invention. The contents of the sequence listing are incorporated herein by reference in their entirety. The information recorded in computer readable format is identical to the written sequence listing. [Technical field]

[0003] The present disclosure relates to various methods and compositions for the treatment of age-related and other medical conditions, such as muscle disease, type 2 diabetes, and / or obesity. Methods for enhancing cellular uptake of NMN and stimulating production of NAD+ are also described. Various mammalian cells and mammalian cell lines are described, including those that contain cDNA encoding Slc12a8 protein. Gene therapy vectors containing nucleic acids encoding Slc12a8 and non-human animals containing inactivating mutations in the Slc12a8 gene are also disclosed. Additionally, methods for screening candidate compounds to identify compounds that enhance transport of NMN are also described. [Background technology]

[0004] NAD+ is a universal and essential coenzyme involved in many cellular redox reactions. NAD+ is also required for the activity of NAD+-consuming enzymes, including the NAD+-dependent protein deacetylases of the sirtuin family, poly-ADP-ribose polymerases (PARPs), and CD38 / 157 NAD+ hydrolase / cyclic ADP-ribose synthase (Canto, C., et al., Cell Metab., 22, 304, 31-53, 2015; Imai, S. and Guarente, L., Trends in Cell Biology, 24, 306, 464-471, 2014). To produce NAD+, mammals utilize a variety of precursors, such as tryptophan, nicotinamide and nicotinic acid (also known as the two forms of vitamin B3), and intermediates including nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). The salvage pathway starting from nicotinamide is the major NAD+ biosynthetic pathway in mammals (Garten, A., et al., Nat. Rev. Endocrinol., 11, 535-546, 2015; Imai, S., Curr. Pharm. Des., 15, 20-28, 2009). In this pathway, nicotinamide phosphoribosyltransferase (NAMPT) produces NMN from nicotinamide and 5'-phosphoribose pyrophosphate. NMN is then converted to NAD+ by NMN adenylyltransferases, NMNAT1-3, together with ATP. The product of the NAMPT reaction is NMN, a key NAD+ intermediate. In mammals, NAMPT is the rate-limiting enzyme in NAD+ biosynthesis. Without being bound by any particular theory, NMN is believed to be useful in preventing and / or treating various disease symptoms and in relation to mitigating age-related physiological decline. However, the mechanism of NMN transport remains controversial.

[0005] Slc12a8 was first identified by Gagnon,KB and Delpire,E. (Am. J. Physiol. Cell Physiol., 304, C693-714, 2013). However, Gagnon et al. did not identify the function of Slc12a8. Kubo,Y. et al. identified Slc12a8 as a spermine transporter (Exp. Eye Res., 124, 17-23, 2014), but did not disclose that it is involved in NMN transport. Summary of the Invention

[0006] Various methods are provided for treating age-related symptoms of a subject in need thereof.Typically, the methods include administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one additional compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, hexanicotinic acid inositol, any ester thereof, any pharma-ceutically acceptable salt thereof, and any combination thereof, wherein age-related symptoms include at least one symptom selected from the group consisting of loss of insulin sensitivity, loss of insulin secretion, loss of insulin action and secretion, impaired memory function, decreased eye function, retinal degeneration, decreased function, and any combination thereof.

[0007] Other methods relate to treating a medical condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharma- ceutically acceptable salt thereof, and any combination thereof, wherein the medical condition comprises at least one selected from the group consisting of muscle disease, type 2 diabetes, obesity, and any combination thereof.

[0008] Also provided are methods for enhancing the cellular uptake of NMN in a subject in need thereof. These methods include administering to the subject a therapeutically effective amount of a compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharma- ceutically acceptable salt thereof, and any combination thereof.

[0009] Methods are provided for stimulating NAD+ production and / or enhancing NMN uptake into cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding Slc12a8.

[0010] A gene therapy vector is provided, the gene therapy vector comprising a nucleic acid encoding Slc12a8.

[0011] A non-human animal is provided, the non-human animal comprising an inactivating mutation in the Slc12a8 gene.

[0012] A mammalian cell or a mammalian cell line is provided. The mammalian cell or the mammalian cell line comprises a cDNA encoding Slc12a8 protein. The cDNA comprises SEQ ID NO:12 (mouse Slc12a8 protein), SEQ ID NO:13 (mouse Slc12a8 mutant A protein), SEQ ID NO:14 (mouse Slc12a8 mutant B protein), or SEQ ID NO:15 (human Slc12a8 protein).

[0013] Further mammalian cells or mammalian cell lines are provided. The mammalian cells or mammalian cell lines contain a cDNA encoding a Slc12a8 protein. The cDNA includes a cDNA encoding a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0014] Further, another mammalian cell or mammalian cell line is provided. The mammalian cell or mammalian cell line comprises a cDNA encoding Slc12a8 protein. The cDNA comprises a cDNA sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence homology with GenBank reference sequence: NM_134251 (SEQ ID NO:1) or Slc12a8 human full-length cDNA (SEQ ID NO:11).

[0015] Another mammalian cell or mammalian cell line is provided. The cell or cell line comprises a cDNA encoding the Slc12a8 protein. The cell or cell line does not include a placenta-derived cell.

[0016] A method for screening candidate compounds to identify compounds that regulate the transport of NMN is provided. The method includes (a) contacting a candidate compound with a cell expressing an NMN transporter protein or a proteoliposome containing an NMN transporter protein, and (b) detecting a change in the expression or activity of the NMN transporter protein in the cell, or a change in the activity of the NMN transporter protein in the proteoliposome. A change in the expression or activity of the NMN transporter protein in the cell, or a change in the activity of the NMN transporter protein in the proteoliposome following contact with the candidate compound, suggests that the candidate compound regulates the transport of NMN.

[0017] A pharmaceutical composition is provided. The pharmaceutical composition comprises NMN and an agonist of the NMN transporter.

[0018] Further pharmaceutical compositions are provided, the pharmaceutical compositions comprising NMN and an inducer of gene expression of Slc12a8 or a homolog thereof. [Brief description of the drawings]

[0019] FIG. 1 is a Venn diagram of genes commonly upregulated in primary hepatocytes, pancreatic islets and hippocampal spheroids treated with FK866.

[0020] FIG. 2 shows the expression of Slc12a8 mRNA in different tissues from 3-month-old B6 male mice.

[0021] FIG. 3 shows the changes in expression of Slc12a8 mRNA in primary mouse hepatocytes, NIH3T3 fibroblasts, and ex vivo explants of jejunum and ileum treated with 0.1% DMSO, FK866 alone, or FK866 plus NMN.

[0022] FIG. 4 shows intracellular NAD+ content in primary mouse hepatocytes and NIH3T3 fibroblasts treated with DMSO, FK866 alone, and FK866 plus NMN.

[0023] FIG. 5 shows flow cytometry data of surface and intracellular Slc12a8 protein expression levels measured in NIH3T3 cells treated with FK866 and FK866 plus NMN for 48 hours.

[0024] FIG. 6 shows the time course of exogenous NMN uptake in primary mouse hepatocytes treated with the NMN pathway inhibitors described below.

[0025] FIG. 7 shows the knockdown efficiency for Slc12a8 and Nrk1 mRNA silencing in primary mouse hepatocytes.

[0026] FIG. 8 shows intracellular NMN content measured by HPLC in cells subjected to Slc12a8 and Nrk1 mRNA silencing.

[0027] FIG. 9 shows Slc12a8 protein expression revealed by SDS-PAGE of plasma membrane fractions of control and Slc12a8-OE NIH3T3 cells.

[0028] FIG. 10 shows Slc12a8 protein levels normalized to caveolin-1 protein levels in control and Slc12a8-OE NIH3T3 cells.

[0029] FIG. 11 shows the intracellular uptake of 3H-NMN in Slc12a8-overexpressing and control NIH3T3 cells.

[0030] Figure 12 shows the calculation of Michaelis-Menten kinetics by plotting intracellular NMN against the cellular uptake of 3H-NMN.

[0031] FIG. 13 shows the uptake of 3H-NMN in proteoliposomes derived from Slc12a8-OE cells or control cells.

[0032] FIG. 14 shows the displacement of 3H-NMN against unlabeled NAD+ related compounds to determine substrate specificity of Slc12a8.

[0033] FIG. 15 shows the determination of IC50 of NMN and NR using the proteoliposome system.

[0034] FIG. 16 shows the effect of replacing Na+ with Li+ on the uptake of 3H-NMN by proteoliposomes.

[0035] FIG. 17 shows representative Western blots of jejunum and ileum tissue samples from mice infected with firefly luciferase (fLuc) shRNA and Slc12a8 shRNA.

[0036] FIG. 18 shows a bar graph of expression levels normalized to GAPDH from jejunum and ileum tissue samples from mice infected with firefly luciferase (fLuc) shRNA and Slc12a8 shRNA.

[0037] FIG. 19 shows in vivo mouse plasma NMN levels measured by HPLC over a 60 minute period following oral gavage of NMN in control and Slc12a8 knockdown mice.

[0038] FIG. 20 shows in vivo mouse plasma nicotinamide levels measured by HPLC over a 60 minute period following oral gavage of NMN in control and Slc12a8 knockdown mice.

[0039] FIG. 21 shows NAD concentrations in jejunal tissue samples collected over 60 minutes following oral gavage of NMN in control and Slc12a8 knockdown mice.

[0040] FIG. 22 shows the expression of Slc12a8 mRNA in various tissues of control and Slc12a8 knockout mice.

[0041] FIG. 23 shows the absence of Slc12a8 protein in the duodenum, jejunum, ileum, and pancreas of Slc12a8 knockout mice by Western blot.

[0042] FIG. 24 shows the absence of Slc12a8 protein in the hypothalamus of Slc12a8 knockout mice by Western blot.

[0043] 25A-B show NAD+ levels during the light and dark periods in various tissue samples taken from Slc12a8 knockout mice.

[0044] FIG. 26 shows the uptake of 18O-D-NMN following gavage in Slc12a8 knockout mice and their wild-type littermates.

[0045] FIG. 27 shows food intake of Slc12a8 knockout mice compared to wild-type littermates.

[0046] FIG. 28 shows the percentage of body fat and lean mass in Slc12a8 knockout mice compared to wild-type littermates.

[0047] FIG. 29 shows the fecal lipid content of Slc12a8 knockout mice compared to wild-type littermates.

[0048] FIG. 30 shows oxygen consumption in Slc12a8 knockout mice compared to wild-type littermates.

[0049] FIG. 31 shows energy expenditure in Slc12a8 knockout mice compared to wild-type littermates.

[0050] FIG. 32 shows the ambulation rate of Slc12a8 knockout mice compared to wild-type littermates.

[0051] FIG. 33 shows the lower airway respiratory exchange ratio of Slc12a8 knockout mice compared to wild-type littermates.

[0052] FIG. 34 shows glucose levels in Slc12a8 knockout mice compared to wild-type littermates.

[0053] FIG. 35 shows insulin levels in Slc12a8 knockout mice compared to wild-type littermates.

[0054] FIG. 36 shows triglyceride levels in Slc12a8 knockout mice compared to wild-type littermates.

[0055] FIG. 37 shows plasma GLP-2 levels in fasted and refed Slc12a8 knockout mice compared to wild-type littermates.

[0056] FIG. 38 shows the relative mRNA expression of Chop and Socs3 in the arcuate nucleus of Slc12a8 knockout mice compared to wild-type littermates.

[0057] FIG. 39 shows the expression of Pepck and G6pc in the liver of ad libitum fed Slc12a8 knockout mice compared to wild-type littermates.

[0058] FIG. 40 shows expression of PMCH mRNA in Slc12a8 knockout mice compared to wild-type littermates.

[0059] FIG. 41 shows the effect of NAD+ biosynthesis inhibitors in NIH3T3 cells with or without the Slc12a8 overexpression construct.

[0060] FIG. 42 shows the enhancement of 3H-labeled NMN (3H-NMN) uptake into proteoliposomes containing Slc12a8 by nicotinic acid (NA).

[0061] FIG. 43 shows reduced NAD+ levels in the jejunum and ileum of 24 month old mice versus 2 month old mice.

[0062] FIG. 44 shows the expression of Slc12a8 in the jejunum and ileum of 2-month-old and 24-month-old mice.

[0063] FIG. 45 shows food intake in 2-month-old and 24-month-old intestinal Slc12a8 knockdown mice over a 4-day period.

[0064] FIG. 46 shows fasting glucose levels in 2-month-old and 24-month-old mice with or without Slc12a8 knockdown in the gut.

[0065] FIG. 47 shows the effect of refeeding after a 24-hour fast on GLP-1 levels in Slc12a8 and control mice.

[0066] FIG. 48 shows Western blots of Slc12a8 and GAPDH in ileal lysates from 2-month-old intestinal Slc12a8 knockdown and control mice.

[0067] FIG. 49 shows Western blots of Slc12a8 and GAPDH in ileal lysates of 24-month-old intestinal Slc12a8 knockdown and control mice.

[0068] FIG. 50 shows NAD+ levels in the ileum of intestinal Slc12a8 knockdown and control mice at 2 and 24 months of age.

[0069] FIG. 51 shows ELISA of GLP-1 levels in supernatants from ex vivo explants of 24-month-old intestinal Slc12a8 knockdown and control mice.

[0070] FIG. 52 shows GLP-1 levels in explants (left) and NAD+ levels in samples (right) from 12-month-old Slc12a8 knockout and control mice.

[0071] FIG. 53 shows GLP-1 levels in explants treated with SIRT family inhibitors or controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Disclosed herein is a method for enhancing the uptake of NMN into cells. Also disclosed is a cell line useful for studying Slc12a8 and the transport of NMN for this purpose. The present inventor also identified the transporter Slc12a8, which enhances the uptake of NMN into cells.

[0073] The methods disclosed herein can be used to treat, ameliorate, alleviate, or reverse any disease or condition in which NMN metabolism is implicated, including, but not limited to, type 2 diabetes, obesity, age-related obesity, age-related elevated blood lipid levels, age-related decreased insulin sensitivity, age-related loss or decline in memory function, age-related loss or decline in eye function, age-related decline in physiological function, impaired glucose-stimulated insulin secretion, diabetes, improving mitochondrial function, neuronal death, and / or cognitive function in Alzheimer's disease, protecting the heart from ischemia / reperfusion injury, preserving neural stem / progenitor cell populations, restoring skeletal muscle mitochondrial function and arterial function after injury, and age-related functional decline.

[0074] The present teachings include, but are not limited to, 1. A full-length cDNA of a mammalian Slc12a8 mRNA, such as mouse Slc12a8 mRNA or human Slc12a8 mRNA; 2. A cDNA encoding a mouse Slc12a8 polypeptide or a human Slc12a8 polypeptide; 3. A mammalian expression vector comprising a Slc12a8 cDNA, such as a full-length Slc12a8 cDNA; 4. Cell lines, such as the NIH3T3 cell line, that harbor Slc12a8 cDNA, including cell lines that overexpress Slc12a8 cDNA, such as the full-length mouse Slc12a8 cDNA (Slc12a8-OE cells) or the full-length human Slc12a8 cDNA; 5. A lentivirus containing an shRNA that reduces or silences the expression of Slc12a8 mRNA. 6. Polyclonal antibodies, including rabbit polyclonal antibodies, against the N-terminal and internal domains of Slc12a8 proteins, such as mouse Slc12a8 protein or human Slc12a8 protein; 7. Whole-organ Slc12a8 knockout mice Includes.

[0075] The present teachings include proteoliposome systems comprising liposomes having a transport protein, such as Slc12a8 protein, encapsulated in a membrane bilayer, such that such systems can be used to analyze Slc12a8 protein and test candidate drugs for their affinity to Slc12a8 or for their activity as agonists or antagonists of Slc12a8 activity.

[0076] The present teachings include methods for promoting NMN uptake in cells, tissues, and organs, such as the digestive tract. These methods can include administering cDNA of Slc12a8 to the digestive tract. These methods can include administering lentiviruses containing cDNA encoding Slc12a8 to the digestive tract.

[0077] Methods for enhancing NMN uptake can include administering NMN in combination with a sodium salt. For example, administration can be oral.

[0078] The present teachings include administering nicotinic acid (NA) or NA derivative compounds (i.e., structural analogs of NA) to a subject in need thereof to enhance the uptake of NMN. The present teachings include administering nicotinic acid (NA) or NA derivative compounds to enhance the uptake function of the Slc12a8 NMN transporter in a subject in need thereof. Nicotinic acid (NA) or NA derivative compounds (i.e., structural analogs of NA) can be administered to a subject in need thereof at a dose range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day of NA or NA derivative compounds. Administration can be oral, parenteral, or any combination thereof.

[0079] The present teachings include administering NA or NA derivative compounds in combination with NMN to promote or accelerate uptake of NMN in a subject in need thereof by enhancing the NMN uptake function of the Slc12a8 NMN transporter. Such combinations can be administered to a subject in need thereof at a dosage range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day of NMN and NA or NA derivative compounds. Administration can be oral, parenteral, or any combination thereof.

[0080] The present teachings include co-administration of NMN and a salt (e.g., Na+) to enhance uptake of NMN in a subject in need thereof. The co-administration can be an oral combination. The co-administration can be a parenteral combination. NMN and an NMN uptake enhancing salt, such as a sodium salt, can each be administered in a dosage range, including but not limited to, 10-500 mg / day or 50-500 mg / day.

[0081] The present teachings can include the combined administration of NMN, sodium salt, and nicotinic acid (NA) or NA derivative compounds to promote uptake of NMN in a subject in need thereof. One or any combination of NMN, sodium salt, and nicotinic acid (NA) or NA derivative compounds can be orally administered. The administration can include the oral administration of any or all of NMN, sodium salt, and nicotinic acid (NA) or NA derivative compounds. NMN, sodium salt, and nicotinic acid (NA) or NA derivative compounds can be administered to a subject in need thereof in a dosage range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day, respectively.

[0082] The present teachings also include administration of nicotinamide riboside (NR) or NR derivative compounds to inhibit the NMN uptake function of the Slc12a8 NMN transporter in a subject in need thereof. The nicotinamide riboside (NR) or NR derivative compounds can be administered orally. The NR or NR derivative compounds can be administered to a subject in need thereof in a dosage range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day.

[0083] The present teachings also include administration of a non-sodium salt, such as a lithium salt, to inhibit the NMN uptake function of the Slc12a8 NMN transporter in a subject in need thereof. The non-sodium salt can be administered orally. The non-sodium salt can be administered to a subject in need thereof in a dosage range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day.

[0084] The present teachings include administration of nicotinamide riboside (NR) or an NR derivative compound, e.g., in combination with a non-sodium salt, e.g., a lithium salt, to inhibit the NMN uptake function of the Slc12a8 NMN transporter in a subject in need thereof. The NR or NR derivative compound, and / or the non-sodium salt, can be administered orally. The NR or NR derivative compound and the non-sodium salt can be administered to a subject in need thereof in a dosage range, such as, but not limited to, 10-500 mg / day or 50-500 mg / day.

[0085] The present teachings disclose a method for stimulating NAD+ production in a subject. The method can include administering to a subject in need thereof a nucleic acid comprising a gene DNA or cDNA encoding Slc12a8. The nucleic acid can be or include a lentivirus comprising a gene DNA or cDNA encoding Slc12a8, such as a cDNA having a sequence designated, but not limited to, NM_134251 (SEQ ID NO:1), Slc12a8 mouse mutant A (SEQ ID NO:9), Slc12a8 mouse mutant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). The subject can be a mammal (e.g., a mouse, a rat, or a human). The cDNA encoding Slc12a8 can be a sequence having at least 70% sequence identity to GenBank reference sequence: NM_134251 (SEQ ID NO:1), at least 70% sequence identity to Slc12a8 mouse variant A (SEQ ID NO:9), at least 70% sequence identity to Slc12a8 mouse variant B (SEQ ID NO:10), or at least 70% sequence identity to Slc12a8 human full-length cDNA (SEQ ID NO:11). The cDNA encoding Slc12a8 can be the sequence of GenBank Reference Sequence: NM_134251 or can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to GenBank Reference Sequence: NM_134251 (SEQ ID NO:1).

[0086] The cDNA encoding Slc12a8 can be the sequence of SEQ ID NO:9 or can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.

[0087] The cDNA encoding Slc12a8 can be the sequence of Slc12a8 mouse mutant B designated SEQ ID NO:10, or can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 mouse mutant B (SEQ ID NO:10).

[0088] The cDNA encoding Slc12a8 can be the sequence of the full-length human Slc12a8 sequence designated SEQ ID NO:11, or can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the full-length human Slc12a8 sequence (SEQ ID NO:11).

[0089] The present teachings include a method for promoting or enhancing NMN uptake into cells in a subject. The method can include administering to a subject in need thereof a cDNA encoding Slc12a8 or a nucleic acid comprising a sequence having at least 70% sequence identity with GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). A sequence having at least 70% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1). A sequence having at least 70% sequence identity to Slc12a8 mouse mutant A (SEQ ID NO:9) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 mouse mutant A (SEQ ID NO:9). A sequence having at least 70% sequence identity to Slc12a8 mouse mutant B (SEQ ID NO:10) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 mouse mutant B (SEQ ID NO:10).The sequence having at least 70% sequence homology with Slc12a8 human full-length cDNA (SEQ ID NO:11) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with Slc12a8 human full-length cDNA (SEQ ID NO:11). The cDNA can be administered to the gastrointestinal tract. This administration can include administration by gavage. This administration can include an oral formulation designed to dissolve in the small intestine.

[0090] The present teachings include knockout mice comprising a defect in the Slc12a8 gene. The defect can be a complete defect in the Slc12a8 gene, or a partial defect in the Slc12a8 gene, such as, but not limited to, a defect in exon 4 of the Slc12a8 gene, or a defect within exon 4 of the Slc12a8 gene.

[0091] The present teachings include mammalian cell lines that contain an introduced sequence of Slc12a8 gene or cDNA, such as the sequence designated GenBank Reference Sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). The introduced Slc12a8 gene or cDNA can be of exogenous origin or homologous to the host mammalian cell line. The mammalian cell line can express Slc12a8 polypeptide at a higher level than a parent cell line into which the Slc12a8 gene or cDNA was not introduced.

[0092] The present teachings include mammalian cell lines that are non-native, i.e., introduced into a cell by transformation or transfection, and contain a sequence that has at least 70% sequence identity to the sequence of GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). A sequence having at least 70% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1). A sequence having at least 70% sequence identity to Slc12a8 mouse mutant A (SEQ ID NO:9) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 mouse mutant A (SEQ ID NO:9). A sequence having at least 70% sequence identity to Slc12a8 mouse mutant B (SEQ ID NO:10) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 mouse mutant B (SEQ ID NO:10).A sequence having at least 70% sequence identity to Slc12a8 human full-length cDNA (SEQ ID NO:11) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Slc12a8 human full-length cDNA (SEQ ID NO:11).

[0093] The present teachings include mammalian cell lines that contain a Slc12a8 sequence, such as GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). The Slc12a8 sequence of the cell lines of the present teachings can also be heterologous to the host cell.

[0094] The cell line of the present teachings can include a Slc12a8 sequence, which can be a sequence having at least 70% sequence homology with GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). The sequence can be a sequence of a gene or cDNA encoding Slc12a8. The gene or cDNA encoding Slc12a8 of the cell line of the present teachings can be heterologous to the host cell. The cell line comprising a sequence encoding Slc12a8 can further include a promoter operably linked to the sequence encoding Slc12a8. The cell line can be a mammalian cell line. The mammalian cell line can also lose CD73 activity, lose CD38 activity, or lose both CD73 activity and CD38 activity. The mammalian cell line can be an NIH3T3 cell line. The mammalian cell line can be a stably transformed cell line comprising a sequence having at least 70% sequence identity to GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11). A sequence having at least 70% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1). Mammalian cell lines can be transiently transfected with a sequence having at least 70% sequence identity to the GenBank reference sequence: NM_134251 (SEQ ID NO:1).A sequence having at least 70% sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO:1) can have at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO:1). The mammalian cell line can be a mouse cell line, a rat cell line, or a human cell line. The mammalian cell line can be selected from the group consisting of a mouse cell line and a rat cell line. The mammalian cell line can be a human cell line. A sequence having at least 70% sequence identity with GenBank reference sequence: NM_134251 (SEQ ID NO:1), Slc12a8 mouse variant A (SEQ ID NO:9), Slc12a8 mouse variant B (SEQ ID NO:10), or Slc12a8 human full-length cDNA (SEQ ID NO:11) may be a full-length Slc12a8 cDNA.

[0095] The methods of the present teachings include methods of enhancing the intracellular uptake of NMN in a subject in need thereof. These methods can include administering to a subject in need thereof a therapeutically effective amount of nicotinic acid or an ester thereof or a pharma- ceutically acceptable salt thereof. The ester of nicotinic acid or a pharma-ceutically acceptable salt thereof can be

[0096] [ka] or a pharma- ceutically acceptable salt thereof, where R can be selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, and p-nitrophenyl. R can be H, 2-dimethylaminoethyl, p-chlorophenyl, or p-nitrophenyl. R can be selected from the group consisting of H, 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl. R can be selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl. R can be H, C 1 ~C 6 Straight chain alkyl, C 3 ~C 6 Branched chain alkyl, C 3 ~C 6 Cycloalkyl, C 1 ~C 6 Linear alkoxy, C 3 ~C 6 Branched alkoxy and C 3 ~C 6 R can be selected from the group consisting of H, C 1 ~C 6 Linear alkoxy, C 3 ~C 6 Branched Alkoxy, C 3 ~C 6 Cycloalkoxy, C 1 ~C 6 Linear haloalkoxy, C 3 ~C 6 Branched Chain Haloalkoxy, C 3 ~C 6 Cyclohaloalkoxy, C 1 ~C 6 Straight chain hydroxyalkoxy, C 3 ~C 6 Branched hydroxyalkoxy, C 3 ~C 6It may be selected from the group consisting of cyclohydroxyalkoxy, alkoxymethyl, alkoxyethyl, alkoxyalkoxymethyl, alkoxyalkoxyethyl, benzyl, alkoxybenzyl, naphthyl and alkoxynaphthyl.

[0097] R can be selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, and p-nitrophenyl. R can be 2-dimethylaminoethyl, p-chlorophenyl, or p-nitrophenyl. R can be selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl. R can be selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl. R can be selected from the group consisting of C 1 ~C 6 Straight chain alkyl, C 3 ~C 6 Branched chain alkyl, C 3 ~C 6 Cycloalkyl, C 1 ~C 6 Linear alkoxy, C 3 ~C 6 Branched alkoxy and C 3 ~C 6 R can be selected from the group consisting of cycloalkoxy. 1 ~C 6 Linear alkoxy, C 3 ~C 6 Branched Alkoxy, C 3 ~C 6 Cycloalkoxy, C 1 ~C 6 Linear haloalkoxy, C 3 ~C 6 Branched Chain Haloalkoxy, C 3 ~C 6Cyclohaloalkoxy, C 1 ~C 6 Straight chain hydroxyalkoxy, C 3 ~C 6 Branched hydroxyalkoxy, C 3 ~C 6 It may be selected from the group consisting of cyclohydroxyalkoxy, alkoxymethyl, alkoxyethyl, alkoxyalkoxymethyl, alkoxyalkoxyethyl, benzyl, alkoxybenzyl, naphthyl and alkoxynaphthyl.

[0098] The present teachings include methods of enhancing the cellular uptake of NMN in a subject in need thereof. These methods can include administering to the subject a compound or a pharma- ceutically acceptable salt thereof, the compound being nicotinic acid.

[0099] [ka] The compound may be niceritrol, tocopherol nicotinate, β-pyridyl carbinol, or inositol hexanicotinate. The compound may be selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridyl carbinol, and inositol hexanicotinate. The compound or a pharmaceutically acceptable salt thereof may be nicotinic acid or a pharmaceutically acceptable salt thereof. The administration may include administering 50-500 mg of nicotinic acid or a pharmaceutically acceptable salt thereof per day. Dosages were 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 400, 450, 460, 470, 480, 495, 500, 510, 520, 530, 540, 550, 560, 570, 580, 595, 600, 610, 620, 630, 640, 650, 660, 670, 680, 695, 700, 710, 720, 730, 740, 750, 760, 770, 780, 795, 800, 810, 820, 830, 840, 850, 850, 860, 870, 880, 895, 900, 910, 920, 930, 940, , 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500 mg of nicotinic acid or a pharma- ceutically acceptable salt thereof.Dosage is 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 105-110, 110-115, 115-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-170, 175-175 ... 5, 175-180, 180-185, 185-190, 190-195, 195-200, 200-205, 205-210, 210-215, 215-220, 220-225, 225-230, 230-235, 235-240, 240-245, 245-250, 250-255, 255-260, 260-265, 265-270, 270-275, 275-280, 280-285, 285-290, 29 0-295, 295-300, 300-305, 305-310, 310-315, 315-320, 320-325, 325-330, 330-335, 335-340, 340-345, 345-350, 350-355, 355-360, 360-365, 365-370, 370-375, 375-380, 380-385, 385-390, 390-395, 395-400, 400-405, 405-41 The dose may include administering 0, 410-415, 415-420, 420-425, 425-430, 430-435, 435-440, 440-445, 445-450, 450-455, 455-460, 460-465, 465-470, 470-475, 475-480, 480-485, 485-490, 490-495, or 495-500 mg of nicotinic acid or a pharma- ceutically acceptable salt thereof.Dosage is 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300 per day. , 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500 mg of nicotinic acid or a pharma- ceutically acceptable salt thereof. Administering the compound or a pharma-ceutically acceptable salt thereof can include administering the compound or a pharma-ceutically acceptable salt thereof with a pharma-ceutically acceptable excipient.

[0100] The present teachings include methods for treating, improving, reducing, preventing or reversing age-related loss of insulin sensitivity and / or insulin secretion in a subject in need thereof. A method for treating, improving, reducing, preventing or reversing age-related loss of insulin sensitivity in a subject can include administering an effective amount of NMN or a pharma- ceutical acceptable salt thereof, and an effective amount of at least one of nicotinic acid or a pharma-ceutical acceptable salt thereof, niceritrol or a pharma-ceutical acceptable salt thereof, tocopherol nicotinate or a pharma-ceutical acceptable salt thereof, β-pyridylcarbinol or a pharma-ceutical acceptable salt thereof, or inositol hexanicotinate or a pharma-ceutical acceptable salt thereof. A method for treating, ameliorating, reducing, preventing or reversing age-related loss of insulin secretion in a subject in need thereof can include administering an effective amount of NMN or a pharma- ceutically acceptable salt thereof and at least one effective amount of nicotinic acid or a pharma- ceutically acceptable salt thereof, niceritrol or a pharma- ceutically acceptable salt thereof, tocopherol nicotinate or a pharma- ceutically acceptable salt thereof, β-pyridylcarbinol or a pharma- ceutically acceptable salt thereof, or inositol hexanicotinate or a pharma- ceutically acceptable salt thereof.

[0101] The present teachings include a method for treating, improving, alleviating, preventing or reversing age-related memory impairment in a subject in need thereof. The method for treating, improving, alleviating, preventing or reversing age-related memory impairment in a subject in need thereof can include administering an effective amount of NMN or a pharma- ceutically acceptable salt thereof, and an effective amount of at least one of nicotinic acid or a pharma-ceutically acceptable salt thereof, niceritrol or a pharma-ceutically acceptable salt thereof, tocopherol nicotinate or a pharma-ceutically acceptable salt thereof, β-pyridylcarbinol or a pharma-ceutically acceptable salt thereof, or inositol hexanicotinate or a pharma-ceutically acceptable salt thereof.

[0102] The present teachings include a method for treating, improving, reducing, preventing or reversing age-related decline in ocular function in a subject in need thereof. The method for treating, improving, reducing, preventing or reversing age-related decline in ocular function in a subject in need thereof can include administering an effective amount of NMN or a pharma- ceutically acceptable salt thereof, and an effective amount of at least one of nicotinic acid or a pharma-ceutically acceptable salt thereof, niceritrol or a pharma-ceutically acceptable salt thereof, tocopherol nicotinate or a pharma-ceutically acceptable salt thereof, β-pyridylcarbinol or a pharma-ceutically acceptable salt thereof, or inositol hexanicotinate or a pharma-ceutically acceptable salt thereof.

[0103] Provided is a method for treating the age-related retinal degeneration of a subject who needs it.The method comprises administering NMN or its pharmaceutically acceptable salt, and at least one of nicotinic acid or its pharmaceutically acceptable salt, niceritrol or its pharmaceutically acceptable salt, tocopherol nicotinate or its pharmaceutically acceptable salt, β-pyridylcarbinol or its pharmaceutically acceptable salt, or inositol hexanicotinate or its pharmaceutically acceptable salt.

[0104] Provided is a method for treating age-related retinal degeneration, age-related ocular function decline, age-related memory function impairment, or age-related insulin sensitivity loss.The method comprises administering NMN or its pharmaceutically acceptable salt, and at least one of nicotinic acid or its pharmaceutically acceptable salt, niceritrol or its pharmaceutically acceptable salt, tocopherol nicotinate or its pharmaceutically acceptable salt, β-pyridylcarbinol or its pharmaceutically acceptable salt, or inositol hexanicotinate or its pharmaceutically acceptable salt.

[0105] Provided is a method for treating, improving, preventing or restoring the decline in function associated with aging.The method can include administering NMN or its pharmaceutically acceptable salt and at least one of nicotinic acid or its pharmaceutically acceptable salt, niceritrol or its pharmaceutically acceptable salt, tocopherol nicotinate or its pharmaceutically acceptable salt, β-pyridylcarbinol or its pharmaceutically acceptable salt, or inositol hexanicotinate or its pharmaceutically acceptable salt.The decline in function associated with aging can include anorexia, low glucose level, muscle weakness, sarcopenia, or a combination thereof.

[0106] Provided is a method for treating, improving, preventing or reversing diabetes.The method can include administering NMN or its pharmaceutically acceptable salt and at least one of nicotinic acid or its pharmaceutically acceptable salt, niceritrol or its pharmaceutically acceptable salt, tocopherol nicotinate or its pharmaceutically acceptable salt, β-pyridylcarbinol or its pharmaceutically acceptable salt, or inositol hexanicotinate or its pharmaceutically acceptable salt.Diabetes can be type 1 diabetes or type 2 diabetes.

[0107] Provided is a method for treating, improving, preventing or reversing obesity.The method comprises administering NMN or its pharmaceutically acceptable salt and at least one of nicotinic acid or its pharmaceutically acceptable salt, niceritrol or its pharmaceutically acceptable salt, tocopherol nicotinate or its pharmaceutically acceptable salt, β-pyridylcarbinol or its pharmaceutically acceptable salt, or inositol hexanicotinate or its pharmaceutically acceptable salt.

[0108] A pharmaceutical composition is provided. The pharmaceutical composition can include NMN or a pharmaceutically acceptable salt thereof, and an agonist of NMN transporter. The agonist of NMN transporter can be nicotinic acid or a pharmaceutically acceptable salt thereof, niceritrol or a pharmaceutically acceptable salt thereof, tocopherol nicotinate or a pharmaceutically acceptable salt thereof, β-pyridyl carbinol or a pharmaceutically acceptable salt thereof, or inositol hexanicotinate or a pharmaceutically acceptable salt thereof. The agonist of NMN transporter can be selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate or a pharmaceutically acceptable salt thereof, β-pyridyl carbinol or a pharmaceutically acceptable salt thereof, or inositol hexanicotinate or a pharmaceutically acceptable salt thereof. The NMN transporter can be Slc12a8 polypeptide or a homolog thereof.

[0109] Further pharmaceutical compositions are provided. The pharmaceutical compositions can include NMN and an inducer of gene expression of Slc12a8 or its homologue. Slc12a8 can have the amino acid sequence identified as accession number NM_134251 (SEQ ID NO: 12), Slc12a8 mouse variant A (SEQ ID NO: 13), Slc12a8 mouse variant B (SEQ ID NO: 14) or Slc12a8 human (SEQ ID NO: 15).

[0110] The present teachings include a method of stimulating NAD+ production in a subject and a method of enhancing NMN uptake into cells in a subject. The subject may be a subject in need of treatment for a disease involving NMN metabolism, such as age-related obesity. These methods include administering a nucleic acid comprising a cDNA encoding Slc12a8 to a subject in need thereof. The cDNA encoding Slc12a8 may be Slc12a8 cDNA of a Slc12a8 mRNA transcript from a mammal, such as a human or a rodent, such as a mouse or a rat. The nucleic acid may comprise a lentivirus comprising a cDNA encoding Slc12a8. The cDNA encoding Slc12a8 may comprise a sequence set forth in GenBank Reference Sequence: NM_134251 (SEQ ID NO:1) or may have 70% or more sequence homology to the sequence set forth in GenBank Reference Sequence: NM_134251 (SEQ ID NO:1). The lentivirus of the present teachings may be administered to the small intestine of a subject in need of treatment.

[0111] The present teachings can also include a knockout mouse comprising a defect in the Slc12a8 gene. The defect can be, but is not limited to, a defect in exon 4 of the Slc12a8 gene.

[0112] The present teachings further include a mammalian cell line comprising a cDNA encoding a Slc12a8 polypeptide or a cDNA sequence having at least 70% or more sequence homology with the GenBank reference sequence: NM_134251 (SEQ ID NO: 1). The cell line can be stably transformed with the cDNA or can be transiently transformed with the cDNA. The cDNA can be operably linked to a control sequence, such as, but not limited to, a promoter or enhancer operably linked to the cDNA.

[0113] Mammalian cell lines can lose CD73 and / or CD38 activity.

[0114] The mammalian cell line can be a mouse cell line, a rat cell line, or a human cell line. The mammalian cell line can be an NIH3T3 cell line transformed with a cDNA sequence having at least 70% or more sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO: 1). The transformation can be stable transformation or transient transformation.

[0115] The present teachings also include methods of enhancing the cellular uptake of NMN in a subject in need thereof. These methods include administering a therapeutically effective amount of nicotinic acid or an ester thereof, or a pharma- ceutically acceptable salt thereof. The ester of nicotinic acid is

[0116] [ka] where R can be, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, or p-nitrophenyl. R can be H, 2-dimethylaminoethyl, p-chlorophenyl, or p-nitrophenyl. R can be 2-dimethylaminoethyl, p-chlorophenyl, or p-nitrophenyl.

[0117] The present teachings also include methods of enhancing the intracellular uptake of NMN in a subject in need thereof. These methods can include administering a compound or a pharmaceutically acceptable salt thereof, where the compound is nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate. Administration can include administering 50-500 mg of nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate, or a pharmaceutically acceptable salt of any of these compounds per day. The compound or a pharmaceutically acceptable salt thereof can be administered with a pharmaceutically acceptable excipient.

[0118] The present teachings also include a method for treating age-related loss of insulin sensitivity in a subject in need thereof, a method for treating age-related loss of insulin secretion in a subject in need thereof, a method for treating age-related loss of insulin action in a subject in need thereof, a method for treating age-related loss of insulin action and secretion in a subject in need thereof, a method for treating age-related memory function impairment in a subject in need thereof, a method for treating age-related ocular function decline in a subject in need thereof, and a method for treating age-related retinal degeneration in a subject in need thereof. These methods can include administering a pharmacologic amount of NMN and nicotinic acid, nicotinic acid ester, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or hexanicotinate inositol, or a pharmacologic acceptable salt thereof.

[0119] The present teachings also include a method for treating a muscle disease in a subject in need thereof. The muscle diseases that can be treated according to the present teachings include, but are not limited to, muscle weakness, muscle atrophy, muscle wasting, and muscle weakness. The muscle diseases that can be treated according to the present teachings include, but are not limited to, sarcopenia, dynapenia, cachexia, muscular dystrophy, muscle tone disorder, spinal muscular atrophy, and myopathy. The muscular dystrophy can be, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, or oculopharyngeal muscular dystrophy. The muscle tone disorder can be myotonic dystrophy, congenital myotonia, or congenital paramyotonia. The myopathy may be Bethlem myopathy, congenital fiber type disproportion, fibrodysplasia ossificans, hyperthyroid myopathy, hypothyroid myopathy, minicore myopathy, multicore myopathy, myotubular myopathy, nemaline myopathy, periodic paralysis, hypokalemic myopathy, or hyperkalemic myopathy. The muscle disease may be acid maltase deficiency, carnitine deficiency, carnitine palmityltransferase deficiency, debranching enzyme deficiency, lactate dehydrogenase deficiency, mitochondrial myopathy, myoadenylate deaminase deficiency, phosphorylase deficiency, phosphofructokinase deficiency, or phosphoglycerate kinase deficiency. The muscle disease may be sarcopenia, dynapenia, or cachexia. The muscle disease may be sarcopenia. These methods can include administering a pharma- ceutical effective amount of NMN and a compound such as nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate, or a pharma- ceutical acceptable salt thereof.

[0120] The present teachings also include pharmaceutical compositions comprising NMN and an agonist of the NMN transporter. The agonist of the NMN transporter can be, but is not limited to, nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate.

[0121] The NMN transporter can be a Slc12a8 polypeptide or a homolog thereof.

[0122] The present teachings also include a method for preventing age-related functional decline in a subject in need thereof.Age-related functional decline can be caused by or associated with, but is not limited to, anorexia, low glucose level, muscle weakness, malnutrition, or anorexia due to aging.These methods can include administering a pharmacologic effective amount of NMN and a compound such as nicotinic acid, nicotinic acid ester, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or hexanicotinic acid inositol, or a pharmacologic acceptable salt thereof.

[0123] The present teachings also include a method for preventing age-related functional decline in a subject in need thereof.Age-related functional decline can be caused by or associated with, but is not limited to, anorexia, low glucose level, muscle weakness, malnutrition, or anorexia due to aging.These methods can include administering a pharmacologic effective amount of NMN and a compound such as nicotinic acid, nicotinic acid ester, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or hexanicotinic acid inositol, or a pharmacologic acceptable salt thereof.

[0124] The present teachings also include methods for treating type 2 diabetes in a subject in need thereof. These methods can include administering a pharmacologic amount of NMN and a compound such as nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate, or a pharmacologic acceptable salt thereof.

[0125] The present teachings also include methods of treating obesity in a subject in need thereof. These methods can include administering a pharma- ceutical effective amount of NMN and a compound such as nicotinic acid, an ester of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, or inositol hexanicotinate, or a pharma- ceutical acceptable salt thereof.

[0126] The work presented herein demonstrates that the Slc12a8 gene encodes a novel NMN transporter in mammals. Slc12a8 gene mRNA expression is upregulated in response to low NAD+, which, without being limited by theory, allows cells to respond to the urgent demand for NAD+ biosynthesis. The Slc12a8 NMN transporter may be specific for NMN under physiological conditions. Knocking down this gene in cell culture and small intestine completely abolishes fast uptake of NMN, whereas overexpression of its full-length cDNA enhances NMN transport into cells that otherwise show minimal NMN transport.

[0127] Various aspects of the invention are described in additional detail in the following sections. Treatment methods

[0128] Various methods are provided for treating age-related symptoms in a subject in need thereof.Typically, the method comprises administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharma-ceutically acceptable salt thereof, and any combination thereof, and the age-related symptoms comprise at least one symptom selected from the group consisting of loss of insulin sensitivity, loss of insulin secretion, loss of insulin action and secretion, impaired memory function, decreased ocular function, retinal degeneration, decreased function, and any combination thereof.

[0129] Age-related symptoms can include loss of insulin sensitivity.

[0130] Age-related conditions can include loss of insulin secretion.

[0131] Age-related conditions can include loss of insulin action and secretion.

[0132] Age-related symptoms can include impaired memory function.

[0133] Age-related symptoms can include a decline in eye function.

[0134] Age-related conditions can include degeneration of the retina.

[0135] Age-related symptoms can include decline in function (eg, loss of appetite, low glucose levels, muscle weakness, malnutrition, age-related anorexia, or any combination thereof).

[0136] The age-related symptoms can include any combination of the age-related symptoms described herein.

[0137] Other methods relate to treating a medical condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharma- ceutically acceptable salt thereof, and any combination thereof, wherein the medical condition comprises at least one selected from the group consisting of muscle disease, type 2 diabetes, obesity, and any combination thereof.

[0138] The medical condition can include a muscle disorder, and if the subject is a subject in need of treatment for a muscle disorder, the muscle disorder can include muscle weakness, muscle atrophy, muscle wasting, muscle weakness, and any combination thereof.

[0139] Muscle diseases include sarcopenia, dynapenia, cachexia, muscular dystrophies (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, or any combination thereof), muscle tone disorders (e.g., myotonic dystrophy, myotonia congenita, paramyotonia congenita, or any combination thereof), and muscle tone disorders (e.g., myotonic dystrophy, myotonia congenita, paramyotonia congenita, or any combination thereof). or any combination thereof), spinal muscular atrophy, and myopathy (e.g., Bethlem myopathy, congenital fiber type disproportion, fibrous dysplasia ossificans progressiva, hyperthyroid myopathy, hypothyroid myopathy, minicore myopathy, multicore myopathy, myotubular myopathy, nemaline myopathy, periodic paralysis, hypokalemic or hyperkalemic myopathy, or any combination thereof), or any combination thereof.

[0140] The muscle disease can include acid maltase deficiency, carnitine deficiency, carnitine palmityltransferase deficiency, debranching enzyme deficiency, lactate dehydrogenase deficiency, mitochondrial myopathy, myoadenylate deaminase deficiency, phosphorylase deficiency, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, or any combination thereof.

[0141] The muscle disorder may be selected from sarcopenia, dynapenia, cachexia, and any combination thereof.

[0142] The muscle disease can include sarcopenia.

[0143] The medical condition can include type 2 diabetes.

[0144] The medical condition can include obesity.

[0145] A muscular disorder can include any combination of the medical conditions described herein.

[0146] In the various methods described herein, the at least one additional compound can include nicotinic acid, any ester thereof, or any pharma- ceutically acceptable salt thereof.

[0147] The ester or pharma- ceutically acceptable salt of nicotinic acid has the structural formula (I):

[0148] [ka] wherein R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, and p-nitrophenyl (e.g., R can be selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl).

[0149] In various methods described herein, about 50 mg to about 500 mg of at least one additional compound is administered to the subject per day. Also, about 10 to about 500 mg of NMN can be administered to the subject per day.

[0150] Additionally, a pharmaceutical composition comprising NMN and at least one additional compound can be administered to the subject.

[0151] Also provided are methods for enhancing the cellular uptake of NMN in a subject in need thereof. These methods include administering to the subject a therapeutically effective amount of a compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharma- ceutically acceptable salt thereof, and any combination thereof.

[0152] The compound can include nicotinic acid, an ester thereof, or a pharma- ceutically acceptable salt thereof. The ester or pharma- ceutically acceptable salt of nicotinic acid is represented by the structural formula (I):

[0153] [ka] wherein R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, and p-nitrophenyl (e.g., R is selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl).

[0154] From about 50 mg to about 500 mg of the compound per day can be administered to a subject.

[0155] Methods are provided for stimulating NAD+ production and / or enhancing cellular uptake of NMN in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding Slc12a8.

[0156] The nucleic acid encoding Slc12a8 can include a cDNA encoding Slc12a8.

[0157] In any method of stimulating NAD+ production and / or enhancing cellular uptake of NMN in a subject, administering a nucleic acid encoding Slc12a8 can include administering a gene therapy vector encoding Slc12a8.

[0158] Gene therapy vectors can include retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, vaccinia viruses, or any combination thereof.

[0159] For example, the gene therapy vector can include a retrovirus.

[0160] When the gene therapy vector comprises a retrovirus, the retrovirus preferably comprises a lentivirus.

[0161] In any method involving administration of a cDNA encoding Slc12a8 to stimulate NAD+ production and / or enhance cellular uptake of NMN in a subject, the cDNA encoding Slc12a8 can include a sequence having at least 70% sequence identity to GenBank reference sequence: NM_134251 (SEQ ID NO:1).

[0162] A cDNA encoding Slc12a8 can comprise a sequence having at least 75% sequence identity with SEQ ID NO:1.

[0163] A cDNA encoding Slc12a8 can comprise a sequence having at least 80% sequence identity with SEQ ID NO:1.

[0164] A cDNA encoding Slc12a8 can comprise a sequence having at least 85% sequence identity with SEQ ID NO:1.

[0165] A cDNA encoding Slc12a8 can comprise a sequence having at least 90% sequence identity with SEQ ID NO:1.

[0166] A cDNA encoding Slc12a8 can comprise a sequence having at least 95% sequence identity with SEQ ID NO:1.

[0167] A cDNA encoding Slc12a8 can comprise a sequence having at least 99% sequence identity to SEQ ID NO:1.

[0168] A cDNA encoding Slc12a8 can comprise a sequence having 100% sequence identity with SEQ ID NO:1.

[0169] Alternatively or additionally, the cDNA encoding Slc12a8 can include any Slc12a8 disclosed herein.

[0170] In any method comprising administering a therapeutically effective amount of a nucleic acid encoding Slc12a8 to a subject to stimulate NAD+ production and / or enhance cellular uptake of NMN in the subject, the method can include administering a nucleic acid encoding Slc12a8 to the digestive tract of the subject. For example, the method can include administering a nucleic acid encoding Slc12a8 to the small intestine of the subject.

[0171] In any of the methods involving administration of a therapeutically effective amount of a nucleic acid encoding Slc12a8 to a subject to stimulate NAD+ production and / or enhance cellular uptake of NMN in the subject, the subject may be one in need of treatment for a muscle disease, type 2 diabetes, obesity, an age-related condition, or any combination thereof.

[0172] The age-related condition may be selected from the group consisting of loss of insulin sensitivity, loss of insulin secretion, loss of insulin action and secretion, impaired memory function, decreased eye function, retinal degeneration, decreased function, obesity, and any combination thereof.

[0173] Age-related symptoms can include loss of insulin sensitivity.

[0174] Age-related conditions can include loss of insulin secretion.

[0175] Age-related conditions can include loss of insulin action and secretion.

[0176] Age-related symptoms can include impairment of memory function.

[0177] Age-related symptoms can include a decline in eye function.

[0178] Age-related conditions can include degeneration of the retina.

[0179] Age-related symptoms can include a decline in function (eg, loss of appetite, low glucose levels, muscle weakness, malnutrition, age-related anorexia, or any combination thereof).

[0180] Age-related conditions can include age-related obesity.

[0181] When the subject is one in need of treatment for a muscle disorder, the muscle disorder can include muscle weakness, muscle atrophy, muscle wasting, muscle weakness, and any combination thereof.

[0182] Muscle diseases include sarcopenia, dynapenia, cachexia, muscular dystrophies (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, or any combination thereof), muscle tone disorders (e.g., myotonic dystrophy, myotonia congenita, paramyotonia congenita, or any combination thereof), and muscle tone disorders (e.g., myotonic dystrophy, myotonia congenita, paramyotonia congenita, or any combination thereof). or any combination thereof), spinal muscular atrophy, and myopathy (e.g., Bethlem myopathy, congenital fiber type disproportion, fibrous dysplasia ossificans progressiva, hyperthyroid myopathy, hypothyroid myopathy, minicore myopathy, multicore myopathy, myotubular myopathy, nemaline myopathy, periodic paralysis, hypokalemic or hyperkalemic myopathy, or any combination thereof), or any combination thereof.

[0183] The muscle disease can include acid maltase deficiency, carnitine deficiency, carnitine palmityltransferase deficiency, debranching enzyme deficiency, lactate dehydrogenase deficiency, mitochondrial myopathy, myoadenylate deaminase deficiency, phosphorylase deficiency, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, or any combination thereof.

[0184] The muscle disorder may be selected from sarcopenia, dynapenia, cachexia, and any combination thereof.

[0185] The muscle disease can include sarcopenia.

[0186] In any of the methods involving administration to a subject of a therapeutically effective amount of a nucleic acid encoding Slc12a8 to stimulate NAD+ production and / or enhance cellular uptake of NMN in the subject, the subject may be one in need of treatment for type 2 diabetes.

[0187] In any of the methods involving administration of a therapeutically effective amount of a nucleic acid encoding Slc12a8 to a subject to stimulate NAD+ production and / or enhance cellular uptake of NMN in the subject, the subject may be one in need of treatment for obesity.

[0188] In any of the methods described herein, the subject can be a mammal.

[0189] For example, the subject can be a mouse or a rat.

[0190] The subject can be a human. When the subject is a human, the human can be at least 30 years old, at least 40 years old, at least 50 years old, at least 60 years old, or at least 70 years old. Gene Therapy Vectors

[0191] A gene therapy vector is provided, the vector comprising a nucleic acid encoding Slc12a8.

[0192] The nucleic acid encoding Slc12a8 can include a cDNA encoding Slc12a8.

[0193] The vector can comprise a retrovirus, adenovirus, adeno-associated virus, alphavirus, herpes virus, vaccinia virus, or any combination thereof.

[0194] For example, the gene therapy vector can include a retrovirus.

[0195] The retrovirus can include a lentivirus.

[0196] A cDNA encoding Slc12a8 can comprise a sequence having at least 70% sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO:1).

[0197] A cDNA encoding Slc12a8 can comprise a sequence having at least 75% sequence identity with SEQ ID NO:1.

[0198] The cDNA encoding Slc12a8 can comprise a sequence having at least 80% sequence identity to SEQ ID NO: 1. The cDNA encoding Slc12a8 can comprise a sequence having at least 70% sequence identity to SEQ ID NO: 1.

[0199] A cDNA encoding Slc12a8 can comprise a sequence having at least 85% sequence identity with SEQ ID NO:1.

[0200] A cDNA encoding Slc12a8 can comprise a sequence having at least 90% sequence identity with SEQ ID NO:1.

[0201] A cDNA encoding Slc12a8 can comprise a sequence having at least 95% sequence identity with SEQ ID NO:1.

[0202] A cDNA encoding Slc12a8 can comprise a sequence having at least 99% or more sequence identity with SEQ ID NO:1.

[0203] A cDNA encoding Slc12a8 can comprise a sequence having 100% sequence identity with SEQ ID NO:1.

[0204] Alternatively, or in addition, the cDNA encoding Slc12a8 can include any Slc12a8 disclosed herein. Non-human animals containing mutations in Slc12a8

[0205] A non-human animal is provided, the non-human animal comprising an inactivating mutation in the Slc12a8 gene.

[0206] The non-human animal preferably comprises a mouse or a rat.

[0207] An inactivating mutation in the Slc12a8 gene can include a deletion in the Slc12a8 gene, an insertion in the Slc12a8 gene, or a combination thereof.

[0208] For example, an inactivating mutation in the Slc12a8 gene can include a deletion in the Slc12a8 gene. The deletion can include a partial deletion of the Slc12a8 gene, or a complete deletion of the lc12a8 gene.

[0209] The defect can include a deletion of exon 4 of the Slc12a8 gene.

[0210] The defect can involve the deletion of part of exon 4 of the Slc12a8 gene. Mammalian Cells and Mammalian Cell Lines

[0211] Mammalian cells and mammalian cell lines are also provided.

[0212] A mammalian cell or a mammalian cell line is provided. The mammalian cell or the mammalian cell line comprises a cDNA encoding a Slc12a8 protein. The cDNA comprises a cDNA encoding SEQ ID NO:12 (mouse Slc12a8 protein), SEQ ID NO:13 (mouse Slc12a8 mutant A protein), SEQ ID NO:14 (mouse Slc12a8 mutant B protein), or SEQ ID NO:15 (human Slc12a8 protein).

[0213] Further provided are mammalian cells or mammalian cell lines. The mammalian cells or mammalian cell lines contain a cDNA encoding a Slc12a8 protein. The cDNA includes a cDNA encoding a protein having at least 70% sequence identity with SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0214] The cDNA can include a cDNA encoding a protein having at least 75% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0215] The cDNA can include a cDNA encoding a protein having at least 80% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0216] The cDNA can include a cDNA encoding a protein having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0217] The cDNA can include a cDNA encoding a protein having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0218] The cDNA can include a cDNA encoding a protein having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0219] The cDNA can include a cDNA encoding a protein having at least 99% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0220] Further, a mammalian cell or a mammalian cell line is provided. The mammalian cell or the mammalian cell line comprises a cDNA encoding Slc12a8 protein. The cDNA comprises a cDNA sequence having at least 70% sequence identity with GenBank reference sequence: NM_134251 (SEQ ID NO: 1) or Slc12a8 human full-length cDNA (SEQ ID NO: 11).

[0221] The cDNA can include a cDNA sequence having at least 75% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0222] The cDNA can include a cDNA sequence having at least 80% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0223] The cDNA can include a cDNA sequence having at least 85% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0224] The cDNA can include a cDNA sequence having at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0225] The cDNA can include a cDNA sequence having at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0226] The cDNA can include a cDNA sequence having at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0227] The cDNA can include a cDNA sequence having 100% sequence identity to SEQ ID NO:1 or SEQ ID NO:11.

[0228] For any mammalian cells or mammalian cell lines, the mammalian cells or mammalian cell lines preferably do not include cells of placental origin.

[0229] Further mammalian cells or mammalian cell lines are provided. The cells or cell lines comprise a cDNA encoding the Slc12a8 protein. The cells or cell lines do not include cells derived from the placenta.

[0230] The cDNA can encode the mouse Slc12a8 protein or a variant thereof.

[0231] The cDNA can encode the human Slc12a8 protein or a variant thereof.

[0232] In any mammalian cell or mammalian cell line, the cDNA can comprise a cDNA encoding SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0233] In any mammalian cell or mammalian cell line, the cell or cell line can further comprise a promoter operably linked to the cDNA.

[0234] In any mammalian cell or mammalian cell line, expression of Slc12a8 protein is preferably enhanced compared to expression of Slc12a8 protein in the same mammalian cell or mammalian cell line that does not contain the cDNA.

[0235] In any mammalian cell or mammalian cell line, the mammalian cell or mammalian cell line can lack detectable CD73 activity, can lack detectable CD38 activity, or can lack both detectable CD73 activity and detectable CD38 activity.

[0236] In any mammalian cell or mammalian cell line, the mammalian cell or mammalian cell line preferably exhibits enhanced NMN uptake.

[0237] Mammalian cells can include fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells.

[0238] Mammalian cell lines can include fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells.

[0239] The mammalian cell can be an NIH3T3 cell.

[0240] The mammalian cell line may be an NIH3T3 cell line.

[0241] In any mammalian cell or mammalian cell line, the mammalian cell or mammalian cell line may be stably transformed with the cDNA sequence.

[0242] In any mammalian cell or mammalian cell line, the mammalian cell or mammalian cell line can be transiently transfected with the cDNA sequence.

[0243] For any mammalian cell or mammalian cell line, the cDNA sequence can have at least 70% sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO:1).

[0244] The cDNA sequence can have at least 75% sequence identity with SEQ ID NO:1.

[0245] The cDNA sequence can have at least 80% sequence identity with SEQ ID NO:1.

[0246] The cDNA sequence can have at least 85% sequence identity with SEQ ID NO:1.

[0247] The cDNA sequence can have at least 90% sequence identity with SEQ ID NO:1.

[0248] The cDNA sequence can have at least 95% sequence identity with SEQ ID NO:1.

[0249] The cDNA sequence can have at least 99% sequence identity with SEQ ID NO:1.

[0250] The cDNA sequence can have 100% sequence identity with SEQ ID NO:1.

[0251] Any mammalian cell can be a mouse or rat cell.

[0252] Any mammalian cell line may be a mouse or rat cell line.

[0253] Any mammalian cell can be a human cell.

[0254] Any mammalian cell line may be a human cell line. Drug Screening Methods

[0255] A method for screening candidate compounds is provided to identify compounds that regulate (e.g., promote) NMN transport.Such methods can be used to identify novel therapeutic compounds that can be used for preventing or treating diseases or conditions that are improved by promoting the uptake of NMN into cells.

[0256] A method for screening candidate compounds to identify compounds that regulate NMN transport is provided. The method includes (a) contacting a candidate compound with a cell expressing an NMN transporter protein or a proteoliposome containing an NMN transporter protein, and (b) detecting a change in the expression or activity of the NMN transporter protein in the cell, or a change in the activity of the NMN transporter protein in the proteoliposome. A change in the expression or activity of the NMN transporter protein in the cell after contact with the candidate compound, or a change in the activity of the NMN transporter protein in the proteoliposome, indicates that the candidate compound regulates the transport of NMN.

[0257] The NMN transporter protein can include the Slc12a8 protein.

[0258] The Slc12a8 protein can include an amino acid sequence having at least 70% sequence identity to SEQ ID NO:12 (mouse Slc12a8 protein), SEQ ID NO:13 (mouse Slc12a8 mutant A protein), SEQ ID NO:14 (mouse Slc12a8 mutant B protein), or SEQ ID NO:15 (human Slc12a8 protein).

[0259] The Slc12a8 protein can comprise an amino acid sequence having at least 75% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0260] The Slc12a8 protein can comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0261] The Slc12a8 protein can comprise an amino acid sequence having at least 85% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0262] The Slc12a8 protein can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0263] The Slc12a8 protein can comprise an amino acid sequence having at least 95% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0264] The Slc12a8 protein can comprise an amino acid sequence having at least 99% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0265] The Slc12a8 protein can include an amino acid sequence having 100% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0266] The method can further include comparing (i) the expression or activity of the NMN transporter protein in a cell that expresses the NMN transporter protein, after contact with the candidate compound, to (ii) the expression or activity of the NMN transporter protein in a cell that does not express the NMN transporter protein or in a cell in which NMN protein expression or activity has been inhibited, after contact with the candidate compound.

[0267] Cells that do not express the NMN transporter protein can include cells from a Slc12a8 knockout animal.

[0268] The method can further include comparing (i) the activity of the NMN transporter protein in proteoliposomes containing the NMN transporter protein, after contact with the candidate compound, with (ii) the activity of the NMN transporter protein in proteoliposomes that do not contain the NMN transporter protein or in proteoliposomes in which NMN protein activity has been inhibited, after contact with the candidate compound.

[0269] Proteoliposomes that do not contain the NMN transporter protein can include proteoliposomes derived from cells of an Slc12a8 knockout animal.

[0270] In either method, the cells can include mammalian fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells.

[0271] In either method, the proteoliposomes can include proteoliposomes derived from mammalian fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells.

[0272] In either method, the cell can include any mammalian cell described herein.

[0273] In either method, the proteoliposomes can include proteoliposomes derived from any of the mammalian cells or mammalian cell lines described herein.

[0274] In either method, the proteoliposomes can comprise proteoliposomes derived from mammalian cells or mammalian cell lines that contain Slc12a8 cDNA. Pharmaceutical Compositions

[0275] A pharmaceutical composition is provided. The pharmaceutical composition comprises NMN and an agonist of the NMN transporter.

[0276] Agonists of the NMN transporter can include compounds selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharmaceutically acceptable salt thereof, and any combination thereof. NMN transporters can include nicotinic acid, any ester thereof, any pharmaceutically acceptable salt thereof.

[0277] The ester or pharma- ceutically acceptable salt of nicotinic acid has the structural formula (I):

[0278] [ka] wherein R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, n-octyl, 2-chloroethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-methoxyethyl, 2-butoxyethyl, carbamoylmethyl, 1-carbamoylethyl, 2-dimethylaminoethyl, 3-aminopropyl, tetrahydrofurfuryl, benzyl, phenoxyethyl, p-chlorophenyl, and p-nitrophenyl (e.g., R can be selected from the group consisting of 2-dimethylaminoethyl, p-chlorophenyl, and p-nitrophenyl).

[0279] Furthermore, the NMN transporter can include a Slc12a8 polypeptide or a homolog thereof.

[0280] Further pharmaceutical compositions are provided, which include NMN and an inducer of gene expression of Slc12a8 polypeptide or a homolog thereof.

[0281] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. EXAMPLES

[0282] [method] The methods and compositions described herein employ laboratory techniques familiar to those of skill in the art, which may be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Spector, DL, et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, NY, 2003; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. Methods and regimens for administering pharmaceutical agents can be determined according to standard principles of pharmacology well known to those of skill in the art using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, JG, et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, RC, et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003.As used in this description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0283] Microarray analysis. Total RNA was isolated from primary hepatocytes, pancreatic islets, and hippocampal spheroids treated with 0.1% DMSO (control) or FK866 (200 nM for primary hepatocytes, 10 nM for islets and hippocampal spheroids). To evaluate transcriptional changes induced by FK866 treatment, microarray analysis was performed using Illumina Mouse Ref8 Whole Genome Microarrays (version 2). Background-subtracted raw microarray data were subjected to Z-score transformation to calculate Z-ratios as previously reported (Yoshino, J., et al., Cell Metab., 14, 528-536, 2011). All data were analyzed with the R statistical software package.

[0284] Cell culture and ex vivo small intestinal explant culture. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. For Slc12a8 mRNA expression analysis, 2.5 × 105 cells per well were cultured in DMEM with 1% FBS containing 0.1% DMSO or 100 nM FK866 or 100 nM FK866 plus 100 μM NMN for 24 h in 6-well plates. Small intestines from 3-month-old B6 male mice were cut into three segments with a duodenum / jejunum / ileum length ratio of 1:3:2 (Wang, HH, et al., Hepatology, 45, 998-1006, 2007). One-centimeter segments were opened longitudinally, washed once with cold PBS, and cultured for 4 h at 37°C with 0.1% DMSO or 100 nM FK866 or 100 nM FK866 plus 500 μM NMN in a 1:1 mixture of DMEM and Ham's F-12 medium (Sigma) containing 5% FBS and the following additives: 5 μg / mL insulin (Sigma), 20 ng / mL epidermal growth factor (Sigma), 1× B27 supplement (GIBCO), 1 mM sodium pyruvate (Corning), 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM GlutaMAX™ (GIBCO). Cell and tissue RNA samples were analyzed by quantitative RT-PCR as previously described (Stein, LR & Imai, S., EMBO J., 33, 1321-1340, 2014).

[0285] Measurement of NAD+ and NMN by HPLC. NAD+ and NMN were extracted from cells and tissues and quantified by HPLC as previously reported (Yoshino, J., et al., Cell Metab., 14, 528-536, 2011; Yoshino, J. & Imai, S., Methods Mol. Biol., 1077, 203-215, 2013).

[0286] Flow cytometry analysis. 2 × 106 NIH3T3 cells were cultured with DMEM with 1% FBS containing 0.1% DMSO or 100 nM FK866 or 100 nM FK866 plus 100 μM NMN in 10 cm culture dishes at 37 °C and 5% CO2 for 48 h. Cells were then washed once with cold PBS, treated with 0.02% EDTA in PBS, and stained for flow cytometry at 4°C for 25 min using a commercial polyclonal rabbit anti-mouse Slc12a8 antibody (ARP44039, Aviva, CA) at 1:200, a secondary goat anti-rabbit IgG (H+L) antibody conjugated with ALEXA FLUOR® 488 (ThermoFisher Scientific, Waltham, MA, USA) at 1:2000 (Invitrogen), and the viability marker ZOMBIE AQUA™ Dye (Biolegend, San Diego, CA) at 1:400. Cells were then washed and analyzed on a GALLOIOS™ flow cytometer (BeckmanCoulter, Indianapolis, IN). For intracellular staining, cells were first fixed in 2% PFA for 10 min at room temperature, then permeabilized in saponin-containing buffer for an additional 10 min at room temperature. Slc12a8 staining was performed in permeabilization buffer for 25 min at 4° C. Samples were analyzed on a GALLOIOS™ flow cytometer (Beckman Coulter, Indianapolis, IN) and data were analyzed with KALUZA™ 1.3 (Beckman Coulter, Indianapolis, IN). Dead cells were excluded using a modified Fixable Viability kit from ZOMBIE AQUA™ (Biolegend, San Diego, CA).

[0287] Isolation of hepatocytes, assay of 5'-nucleotidase activity, measurement of NMN uptake, and silencing of Slc12a8 and Nrk1 expression. Primary hepatocytes were isolated from 3-month-old C57BL / 6J (B6) male mice as previously reported (Grimm, AA, et al., Aging Cell, 10, 305-317, 2011). Cells were cultured overnight in poly-L-lysine-coated 6-well plates at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin (pen / strep) before performing any experiments. Slc12a8 mRNA expression and NAD+ content were assessed by incubating hepatocytes with 500 nM FK866 or 500 nM FK866 plus 500 μM NMN in DMEM containing 1% FBS for 24 h. To test whether adenosine-5'-[α,β-methylene]diphosphate (AOPCP) inhibits 5'-nucleotidase activity, 1.5 × 105 cells per well were grown in DMEM containing 1% FBS with 500 nM FK866 for 24 h in 12-well plates and then incubated in the presence of 100 μM adenosine monophosphate (AMP) or 100 μM AMP plus 500 μM AOPCP in 1.4 mL of Hank's buffered saline (HBSS, GIBCO) containing Ca2+ and Mg2+ at pH 7.5. At different time points (0, 1, 5, 15, and 30 min), 200 μL of each culture supernatant was taken and extracted by adding 28 μL of 70% perchloric acid. The amount of adenosine produced was measured by HPLC. The elution times of AMP and adenosine were 4.7 and 17.4 min, respectively. To examine cell viability, the absorbance was measured at λ=490 using CELLTITER96(R) AQueous One Solution Cell Proliferation Solution (Promega, Madison, WI) after 4 h of incubation.For NMN uptake measurements, 1.5x105 cells per well were grown in DMEM containing 1% FBS with 500nM FK866 for 24 hours in 12-well plates and then cultured in 1mL HBSS in the presence of 500μM AOPCP, 20μM dipyridamole, and 500nM FK866 or these inhibitors plus 100μM NMN. At different time points (0, 0.25, 1, 5, 15, and 30 min), cells were washed once with cold HBSS and lysed in cold 10% perchloric acid. Intracellular NMN levels were measured by HPLC as previously reported (Yoshino, J., et al., Cell Metab., 14, 528-536, 2011). For gene silencing experiments, 10 μg of ON-TARGETPLUSTM (Dharmacon, Inc, Lafeyette, CO) mouse siRNA (Thermo Scientific) specific for Slc12a8 (J-042450-12-0020) or Nrk1 (J051839-11-0010) or negative control siRNA (NON-targeting siRNA#1, D-001810-01-20) were electroporated into 1 million cells per condition and mixed with 100 μL of AMAXA® Mouse Hepatocyte NUCLEOFECTOR® solution (Lonza, Basel, Switzerland) using the NUCLEOFECTOR® program H-26 (Lonza, Basel, Switzerland) according to the manufacturer's instructions. Electroporated cells were incubated in the cuvette for 15 min before adding medium. After electroporation, 2.5x105 cells per well were seeded in poly-L-lysine-coated 6-well plates in DMEM containing 10% FBS and penicillin-streptomycin for 48 hours at 37°C and 5% CO2. The cells were incubated with 500nM FK866 in DMEM containing 1% FBS for 24 hours. NMN uptake was measured by HPLC after incubating cells with 500μM AOPCP, 20μM dipyridamole and 500nM FK866, or these inhibitors plus 100μM NMN in HBSS for 1 minute at room temperature. Silencing efficiency was assessed by quantitative RT-PCR.

[0288] Generation of NIH3T3 cells stably overexpressing full-length mouse Slc12a8 cDNA.

[0289] GenBank Reference Sequence: NM_134251 was used as the sequence. The sequence of GenBank Reference Sequence: NM_134251 is presented herein as SEQ ID NO:1.

[0290] The coding region of full-length mouse Slc12a8 cDNA (GenBank reference sequence: NM_134251) was amplified from mouse liver by PCR using PFUULTRATMII fusion HS DNA polymerase (Agilent, Santa Clara, CA) with the following forward and reverse primers containing an XhoI site (Table 1).

[0291] [Table 1]

[0292] The resulting 2118-bp full-length Slc12a8 DNA was digested with XhoI and cloned into pBlueScript SK vector. The Slc12a8 cDNA fragment was then subcloned into the mammalian expression vector pCXN2 (Revollo, JR, et al., J. Biol. Chem., 279, 50754-50763, 2004). The sequence of Slc12a8 cDNA in the final vector was confirmed by sequence analysis. NIH3T3 cells were transfected with 5 μg of pCXN2 carrying full-length Slc12a8 cDNA (Slc12a8-OE) or empty pCXN2 vector (control) using SUPERFECT® transfection reagent (QIAGEN, Fredrick, MD) and cultured for 2 weeks in DMEM supplemented with 10% FBS, antibiotics, and 300 μg / mL G418 (Invitrogen). Resistant cells were pooled, split, and frozen for further experiments. To confirm the expression of Slc12a8 protein in Slc12a8-OE cells, plasma membrane (PM) fractions were prepared from control and Slc12a8-OE NIH3T3 cells as previously reported (Bruzzone, S., et al., FASEB J., 26, 1251-1260, 2012). Briefly, 7.5 × 107 cells cultured in five 10 cm dishes were used. After washing twice with ice-cold HES buffer (20 mM HEPES, 1 mM EDTA, 255 mM sucrose, pH 7.4), cells were scraped in HES buffer (3 mL / dish) containing protease inhibitor cocktail (Roche) and homogenized by passing five times through a 22-gauge needle. All subsequent steps were performed at 4 °C. The homogenate was centrifuged (Avanti JE) at 16,000 g for 30 min in a JA25.5 rotor (Beckman-Coulter). The pellet was resuspended in 10 mL HES buffer and centrifuged again at 16,000 g for 30 min. The resulting pellet was resuspended in 10 mL HES buffer, layered on top of a 10 mL sucrose cushion (38.5% sucrose, 20 mM HEPES, and 1 mM EDTA, pH 7), and centrifuged at 53,000 g for 120 min.The interface containing the PM fraction was removed and resuspended in 10 mL HES buffer and centrifuged at 40,000 × g for 30 min to generate the PM fraction in the pellet. PM fractions from control or Slc12a8OE cells were lysed in RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5, 2 mM EDTA, 1 mM PMSF, 0.5 mM DTT, and protease inhibitor cocktail) and boiled for 5 min in 1 × Laemmli buffer. Western blots were performed using polyclonal rabbit anti-mouse Slc12a8 (1:500, ARP44039, Aviva, CA) and anti-caveolin-1 (1:2000, #3238, Cell Signaling, MA). Band intensity was quantified on AMERSHAM HYPERFILM™ ECL (GE Healthcare, Marlborough, MA) using ADOBE® PHOTOSHOP® (Adobe systems Inc, San Jones, CA).

[0293] NMN uptake assay using radiolabeled NMN. Control and Slc12a8-OE NIH3T3 cells were harvested by centrifugation (400×g, 5 min), washed once with HBSS, and cultured at 37°C in HBSS [pH 7.5] (5×106 cells / mL) containing 100 nM 3H-β-nicotinamide mononucleotide (9 Ci / mmol, Moravek Biochemicals, CA) and unlabeled NMN to a final concentration of 25 μM. At the indicated time points (1, 3, 5, and 10 min), aliquots of cells (100 μL) were harvested and placed in 1.5 mL microcentrifuge tubes containing silicone-mineral oil (density, 1.015, Sigma-Aldrich) on top of 2 M potassium hydroxide solution and centrifuged at 16,000×g for 30 s. Cells were separated from the buffer and pelleted through the silicone-mineral oil layer. The radioactivity of these cell pellets was measured by liquid scintillation counter. The same conditions as above were used to calculate Km and Vmax, but the total concentration of NMN varied from 1 μM to 100 μM. At 4 min, 100 μL of cell suspension was collected and pelleted in the same manner as above. The radioactivity of the cell pellets was measured by liquid scintillation counter. The amount of Slc12a8-specific NMN uptake in Slc12a8-OE NIH3T3 cells was calculated by subtracting the radioactivity measured in control NIH3T3 cells.

[0294] Proteoliposome experiments. Proteoliposome preparation was performed as previously described (Bruzzone, S., et al., FASEB J., 15, 10-12, 2001). Briefly, 2 × 107 control or Slc12a8-OE NIH3T3 cells were resuspended in 1 mL of lysis buffer (10 mM Tris-HCl, pH 8.3, 150 mM NaCl, 0.3 M sucrose, protease inhibitor cocktail) and disrupted using a homogenizer. The lysates were centrifuged at 3,000 × g for 10 min, and the supernatants were collected and centrifuged at 100,000 × g for 15 min. Membrane proteins were solubilized in buffer A (10 mM Tris-HCl, pH 8.3, containing 150 mM NaCl and 0.5% n-octyl β-glucopyranoside). Separately, total lipids were extracted from hemoglobin-free erythrocyte membranes (ghosts) as previously reported (Franco, L., et al., FASEB J., 12, 1507-1520, 1998). Total lipids from human erythrocyte membranes (3 mg) were dried and resuspended with 600 μL of solubilized membrane protein (protein concentration, approximately 0.7 mg / mL). The resulting emulsion was sonicated for 1 min in ice and dialyzed against 5 liters of buffer A (dialysis buffer) without n-octyl-β-glucopyranoside for 24 h at 4°C. Proteoliposomes were collected, centrifuged at 100,000×g for 15 min, resuspended in 900 μL dialysis buffer, and passed five times through a 30-gauge needle.

[0295] To investigate the Na+ dependence of Slc12a8, NaCl was replaced with 150 mM LiCl. All steps were performed at 4°C. Proteoliposomes (30 μL in triplicate for each condition) were incubated in the presence or absence of unlabeled nucleotides (NMN, NR, NAD, AMP, NAM, or NaMN) at 105 cpm / mL 3H-β-NMN (specific activity, 9.1 Ci / mmol) for 2, 5, or 10 min at 25°C. At the end of the incubation, samples were filtered through glass fiber paper. Filters were washed with 3 mL of dialysis buffer, dried, and radioactivity was counted.

[0296] In vivo Slc12a8 knockdown experiments. To generate shRNA-expressing lentivirus constructs, 56-bp double-stranded oligonucleotides containing a sense target sequence, a microRNA-based loop sequence (CTTCCTGTCA, SEQ ID NO:4), an antisense sequence, a terminal sequence of four thymidines, and appropriate restriction enzymes at both ends were generated for mouse Slc12a8 and firefly luciferase (fLuc), respectively, and cloned into the U6-PGK-GFP vector provided by the Washington University School of Medicine Viral Vector Core. The sense Slc12a8 sequence is 5'-GCCTAGAGTGAACAGAGAAGA-3' (SEQ ID NO:5). Lentiviruses were generated as previously reported (Satoh, A. et al., Cell Metab., 18, 416-430, 2013). Knockdown efficiency was tested using primary intestinal cultures (Satoh, T. & Clevers, H., Methods Mol. Biol., 945, 319-328, 2013). Large-scale lentivirus production was performed at the Viral Vector Core of the Hope Center for Neurological Disorders, University of Washington. After two consecutive overnight fasts, 3-week-old C57BL / 6J male mice (Jackson Laboratories) were orally administered (gavage) fLuc or Slc12a8shRNA lentivirus with a titer of 5×106 transforming units. Six days later, mice administered fLuc or Slc12a8shRNA lentivirus were fasted overnight and gavaged with NMN (500mg / kg) or PBS. Blood was collected from the tail vein at 0, 5, and 60 minutes following gavage, and plasma was separated by centrifugation. Tissue samples were then collected 60 minutes following gavage. Plasma NMN and tissue NAD+ levels were measured as previously reported (Yoshino, J., et al., Cell Metab., 14, 528-536, 2011; Grimm, AA, et al., Aging Cell, 10, 305-317, 2011).

[0297] Generation of antibodies against mouse Slc12a8. Two different polyclonal rabbit antisera were generated against a synthetic N-terminal peptide of mouse Slc12a8 (AQRSPQELFHEAAQQGC; SEQ ID NO:6) (Convance, Denver, PA). Slc12a8-specific antibodies were affinity purified from one of these antisera. Antisera or affinity-purified antibodies against the N-terminal peptide of Slc12a8 were used for Western blotting at a dilution of 1:500.

[0298] Generation of whole-body Slc12a8 knockout mice. Whole-body Slc12a8 knockout (Slc12a8KO) mice were generated by the Transgenic Vector Core at the University of Washington using CRISPR-CAS9 technology. CRISPR gRNAs were designed to flank exon 4 of the Slc12a8 gene. The sequences of the gRNAs were 5'gRNA 5'-AGTGCATGTATAGACGTATG-3' (SEQ ID NO:7) and 3'gRNA 5'-CCTCACAAATATTTACAGGC-3' (SEQ ID NO:8). gRNAs were obtained as gBlocks (IDT). Cleavage activity was assessed by transfecting N2A cells with gBlock and Cas9 plasmids (addgene #42230) using XTREMEGENETMHP (Roche, Basel, Switzerland). Cleavage activity was determined by T7E1 assay using standard methods. gRNA was transcribed in vitro using the T7 MEGASHORTSCRIPT™ kit (Ambicon, Waltham, MA, USA). Cas9RNA was transcribed in vitro using the MMESSAGE MMACHINE® T7 Ultra kit (Ambicon, Waltham, MA). All RNA was purified using MEGACLEAR® columns (Ambicon, Waltham, MA). RNA was microinjected into C57BL / 6J x CBA hybrid zygotes at the University of Washington Mouse Genetics Core Facility at concentrations of 50ng / μL Cas9, 25ng / μL gRNA, and 100ng / μL ssODN. Whole-body knockout alleles were detected by PCR of the cleavage site and confirmed by sequence analysis. One heterozygous founder was established and backcrossed to wild-type C57BL / 6J mice (Jackson Laboratories) for five generations prior to analysis. Heterozygous Slc12a8-deficient mice were crossed to generate homozygous Slc12a8KO mice, and wild-type littermates served as controls.

[0299] Preparation of 18O-D-NMN. 18O-nicotinamide was prepared by hydrolysis of cyanopyridine in 18O water (Kolodziejska-Huben, M., et al., J. Label. Compd. Radiopharm., 45, 1005-1010, 2002). 1,2-2H,3,5-tetraacetate was synthesized from D-[2-2H]-ribose (purchased from Omicron Biochemicals) (Chatterjee, A., et al., Angew. Chem. Int. Ed. Engl., 49, 8653-8656, 2010). 18O-2H-labeled nicotinamide riboside was synthesized from 18O-nicotinamide and D-ribofuranose 1,2-2H,3,5-tetraacetate (Fouquerel, E., et al., Cell Rep., 8, 1819-1831, 2014). 18O-2H-nicotinamide mononucleotide (18O-D-NMN) was synthesized from 18O-2H-nicotinamide riboside as previously reported (Mills, KF, et al., Cell Metab., 24, 795-806, 2016).

[0300] Isotope tracing experiments. Seven to eight month old Slc12a8KO mice and their wild type littermates were orally administered 500 mg / kg 18O-D-NMN or PBS after an overnight fast. Ten minutes after gavage, the jejunum and ileum were harvested. A 1:1 mixture of reagent grade methanol and water (4°C) was added to the frozen tissue (60 μL / mg tissue). After sonication, the extract was centrifuged at 12,000×g for 15 minutes at 4°C. Chloroform was added to the extract in a 1:1 (v / v) ratio, vortexed well for 30 seconds, and centrifuged at 12,000×g for 10 minutes at 4°C. The upper layer (methanol and water) was separated from the lower layer (organic layer), lyophilized at room temperature by speed vacuum, reconstituted in 5 mM ammonium formate, and centrifuged at 12,000×g for 10 minutes. NMN and 18O-D-NMN serially diluted to concentrations ranging from 128 to 1000 nmol / L in 5 mM ammonium formate were used for calibration. Liquid chromatography was performed using 5 mM ammonium formate in mobile phase A and 100% methanol in mobile phase B at a flow rate of 0.15 mL / min using an HPLC (1290, Agilent) equipped with an ATLANTIS® T3 (LC2.1 × 150 mm, 3 mm, Waters, Millford, MA). Metabolites were eluted with a gradient of 0–70% B for 0–10 min, 70% B for 10–15 min, and 0% B for 16–20 min. Metabolites were analyzed using a triple quadrupole mass spectrometer (6470, Agilent) under positive ESI multiple reaction monitoring (MRM) using parameters for NMN (335>123) and 18O-D-NMN (338>125). The fragmentation, collision, and post-acceleration voltages for NMN were 135, 8, and 7. NMN and 18O-D-NMN peaks were identified using the MassHunter quantitative analysis tool (Agilent).

[0301] Animal experiments. All mice were group-housed in an isolation facility with a 12-h light / 12-h dark cycle. Mice were maintained on a standard diet ad libitum (LabDiet5053, LabDiet, St. Louis, MO). Measurements of food intake, fed and fasting glucose, triglyceride, free fatty acid, and insulin levels were performed as previously reported (Yoshino, J., et al., Cell Metab., 14, 528-536, 2011; Mills, KF, et al., Cell Metab., 24, 795-806, 2016; Yoon, MJ, et al., Cell Metab., 21, 706-717, 2015). Feces were collected from Slc12a8KO mice and their wild-type littermates between 9:00 and 10:00 a.m. and dried overnight at 55°C before being weighed. Total fecal fat was extracted with a chloroform / methanol mixture (2:1, vol / vol) (Sigma, St. Louis, MO) as previously reported (Folch, J., et al., J. Biol. Chem., 226, 497-509, 1957). One milliliter of the organic layer was transferred to a pre-weighed tube, vacuum dried, and weighed again to measure fat mass. Fat content percentages were normalized to the initial mass (g) of extracted feces. Tissues were collected at 9 am or 9 pm from 8-10 month-old Slc12a8KO mice and their wild-type littermates to measure NAD+ levels by HPLC. Body composition was measured using a whole-body NMR facility (EchoMRI®, Echo Medical Systems LLC, Waco, TX). Indirect calorimetry and locomotor activity measurements were performed by a PhenoMaster system (TSE Systems, MO). All animal studies were approved by the University of Washington Animal Care and Use Committee and conformed to NIH guidelines.

[0302] Plasma GLP-2 measurements. Blood was collected from the tail vein of 7-9 month old Slc12a8KO mice and their control wild-type littermates after 24 h fasting (9 am to 9 pm) and 6 h re-feeding (10 am to 4 pm) using EDTA-coated microvette tubes. Blood was immediately chilled on ice and centrifuged at 5,000 × g at 4 °C, and plasma samples were stored at -80 °C. GLP2-levels in plasma were measured using a Mouse GLP-2 ELISA kit (Alpco, Salem, NH) according to the manufacturer's instructions.

[0303] Measurement of GLP-1 in plasma and ex vivo small intestinal explant culture supernatant. Blood was collected from the tail vein of 2-month-old or 24-month-old female Slc12a8 knockdown mice and their controls after 24-hour fasting (9:00 AM to 9:00 AM) followed by 6-hour refeeding (10:00 AM to 4:00 PM) using EDTA-coated tubes. Blood was immediately cooled on ice and centrifuged at 5,000×g at 4°C, and plasma samples were stored at −80°C. Small intestines of gut-specific female Slc12a8 knockdown mice or 12-month-old female Slc12a8KO mice were cut into three segments of duodenum, jejunum, and ileum with a length ratio of 1:3:2 (Wang, HH, et al., Hepatology, 2007, 45, 998-1006). One centimeter of ileum or colon was opened longitudinally, washed once with cold PBS, and incubated in 500 μL DMEM without nicotinamide and glucose (GIBCO, secretion medium) with 0.25% fatty acid-free bovine serum albumin (Sigma, St. Louis, MO) for 2 h at 37°C. For experiments with Sirt1 and Sirt6 inhibitors, ileum from 24-month-old female B6 mice was preincubated in 500 μL secretion medium containing 0.2% DMSO (vehicle), 20 μM EX527 (Cayman chemical, MI) or 20 μM STK665401 (ChemDiv, cat#8018-9378) for 2 h at 37°C (Sociali, G., et al., Eur. J. Med. Chem., 2015, 102, 530-539). After pre-incubation, the medium was replaced and ex vivo ileal explants were pre-incubated with the same compounds for 2 h at 37°C. At the end of the incubation in a humidified incubator (95% O2, 5% v / v CO2), the medium was collected, centrifuged at 800g for 5 min at 4°C to remove any floating debris, and frozen at -80°C for subsequent GLP-1 measurement. GLP-1 levels in plasma and medium were measured by GLP-1 Total ELISA kit (EMD Millipore, MO) according to the manufacturer's instructions. GLP-1 values ​​were normalized by total protein amount analyzed by Bradford assay.

[0304] Laser microdissection. Each hypothalamic nucleus was microdissected, and total RNA was extracted and analyzed by quantitative RT-PCR as previously reported (Satoh, A., et al., Cell Metab., 18, 416-430, 2013).

[0305] Statistical analysis. Unless otherwise indicated, differences between two groups were evaluated using Student's t-test. Comparisons between some groups were performed using one-way ANOVA with various post-hoc tests shown in the examples. Wilcoxon signed rank test was used to compare differences in oxygen consumption, energy expenditure, respiratory exchange ratio, and total locomotor activity. In comparisons, a p-value <0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism (version 7). All values ​​are presented as mean ± SEM. Unless otherwise indicated, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0306] The present teachings include statements that are provided in examples and are not intended to limit the scope of claims or aspects. Unless expressly presented in the past tense, examples may be expected or actual examples. The following non-limiting examples are provided to further illustrate the present teachings. Those skilled in the art will understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present teachings.

[0307] [Example 1] This example demonstrates the identification of the NMN transporter gene.

[0308] When NAMPT-mediated NAD+ biosynthesis was inhibited by FK866, a potent NAMPT inhibitor, in various types of primary cells, co-administration of NMN consistently led to a higher increase in NAD+ than that induced by NMN in the absence of FK866 (Revollo, JR, et al., Cell Metab., 6, 363-375, 2007; Stein, LR & Imai, S., EMBO J., 33, 1321-1340, 2014; Yoshino, J., et al., Cell Metab., 14, 528-536, 2011). We performed gene expression profiling in FK866-treated mouse primary hepatocytes, pancreatic islets, and hippocampal neural stem cell clusters to explore genes commonly upregulated in these three types of primary cultures. We focused our search on genes encoding transporters or transmembrane proteins and found only one gene of unknown function that met these criteria. This gene, Slc12a8, showed Z-ratios of 2.06, 1.69, and 4.91 in primary hepatocytes, pancreatic islets, and hippocampal neural stem cell clusters, respectively (Figure 1). Slc12a8 was identified in the section indicated by the arrow in the Venn diagram in Figure 1. The Z-ratios and p-values ​​of Slc12a8 in each cell type are shown in Figure 1. The Slc12a8 gene belongs to the SLC12 gene family of electroneutral cation-chloride binding cotransporters, and the function of the protein encoded by this gene is still unknown (Hebert, SC, et al., Pflugers Arch., 447, 580-593, 2004).

[0309] [Example 2] This example shows the differential expression of Slc12a8 in B6 mice.

[0310] Figure 2 shows that Slc12a8 is highly expressed in the small intestine and pancreas and moderately expressed in the liver and white adipose tissue of 3-month-old B6 male mice (n=3 mice). Further changes in Slc12a8 mRNA expression were observed in primary mouse hepatocytes and are shown in Figure 3 (n=4 mice). NIH3T3 fibroblasts (n=4) and ex vivo explants of jejunum and ileum (n=4 mice for DMSO and FK866 only, n=3 mice for FK866 plus NMN) treated with 0.1% DMSO, FK866 alone or FK866 plus NMN (24 hours for cells, 4 hours for explants, analyzed using ANOVA with Tukey's test). The expression of Slc12a8 was significantly induced in mouse primary hepatocytes, mouse NIH3T3 fibroblasts, and ex vivo explants of jejunum and ileum when treated with FK866, but this induction was suppressed by the addition of NMN (Figures 3-5). Figure 4 shows the intracellular NAD+ content of primary mouse hepatocytes and NIH3T3 fibroblasts treated with DMSO, FK866 alone, and FK866 plus NMN. Cells were treated with these compounds for 24 h (n=4 mice for hepatocytes treated with DMSO and FK866, n=3 for hepatocytes treated with FK866 and NMN, n=6 for NIH3T3 cells, analyzed using ANOVA with Tukey's test). Figure 5 shows the expression levels of surface and intracellular Slc12a8 protein, measured by flow cytometry analysis, in NIH3T3 cells treated with FK866 or FK866 plus NMN for 48 h. The percentage of NIH3T3 cells positive for Slc12a8 staining (Slc12a8+) was calculated among cells negatively selected for markers of apoptosis (Zombi-) (n=8 for surface staining, n=5 for intracellular staining, analyzed by unpaired t-test).

[0311] [Example 3] This example shows the effect of Slc12a8 on the kinetics of NMN uptake.

[0312] The kinetics of NMN uptake in mouse primary hepatocytes was determined. Extracellular degradation of NMN to NR by CD73 was inhibited by AOPCP (adenosine-5'-[α,β-methylene]diphosphate). Uptake of NR into cells via nucleoside transporters was inhibited by dipyridamole, and intracellular NMN synthesis by NAMPT was inhibited by FK866. Untreated primary hepatocytes were cultured with AMP or AMP plus AOPCP. The production of extracellular adenosine was measured by HPLC at various time points (0, 1, 5, 15, and 30 min). AOPCP inhibits 5'-nucleotidase activity by 97%. The cocktail of AOPCP, dipyridamole, and FK866 did not affect cell viability up to 30 min (data not shown).

[0313] Primary mouse hepatocytes were pretreated with 500 nM FK866 for 24 h and then incubated with a cocktail of 20 μM dipyridamole, 500 μM AOPCP, and 500 nM FK866 in the presence and absence of 100 μM NMN. NMN was measured by HPLC (n=4 mice, except for three data sets at 15 and 30 min for inhibitor only, analyzed using ANOVA with Sidak's test). This treatment significantly increased intracellular NMN levels at 1 min in primary mouse hepatocytes compared to controls (Figure 6).

[0314] [Example 4] This example shows the effect of reducing Slc12a8 and Nrk1 mRNA on NMN uptake.

[0315] To measure knockdown efficiency of Slc12a8 and Nrk1 mRNA in primary mouse hepatocytes, cells were treated with scrambled and Slc12a8 or Nrk1 siRNA for 72 hours (n=5 mice for Slc12a8 silencing, n=3 for Nrk1 silencing, analyzed by independent 700t test). Without being limited by theory, these conditions knocked down the expression of Slc12a8 and Nrk1, the major NR kinases that convert NR to NMN in cells (Belenky, P., et al., Cell, 129, 473-484, 2007). Primary hepatocytes treated with scrambled, Slc12a8, and Nrk1 siRNA were assayed by HPLC 1 min after addition of 100 μM NMN. Culture conditions were the same as those described in Example 3 (n=5 mice for Slc12a8 silencing, n=3 for Nrk1 silencing, analyzed by ANOVA with Tukey's test). The knockdown efficiency of both genes is about 80% (Figure 7). Fast NMN uptake was completely abolished in Slc12a8 knockdown hepatocytes, whereas no significant decrease in NMN uptake was observed in Nrk1 knockdown hepatocytes (Figure 8). These data indicate that Slc12a8 is required for fast NMN uptake in primary hepatocytes and that the observed increase in intracellular NMN is not due to the conversion of NR or nicotinamide to NMN.

[0316] [Example 5] This example demonstrates overexpression of full-length mouse Slc12a8 cDNA in mouse NIH3T3 cells.

[0317] The NIH3T3 cell line was chosen because it does not have any detectable extracellular activity of CD73 (which converts NMN to NR) and CD38 (which degrades NMN to nicotinamide and phosphoribose), and has very weak NMN uptake activity. Full-length mouse Slc12a8 cDNA was transfected into cells and Slc12a8 protein expression was assayed as described in Methods. Figure 9 shows Slc12a8 protein expression in plasma membrane fractions from control and Slc12a8-OE NIH3T3 cells. Figure 10 shows Slc12a8 protein levels normalized to caveolin-1 protein levels for each cell line (right panel, n=3, analyzed by unpaired t-test). Slc12a8 protein levels were significantly increased by about 2.2-fold in Slc12a8-overexpressing NIH3T3 (Slc12a8-OE) cells.

[0318] [Example 6] This example shows the kinetics of NMN uptake in Slc12a8-OE and control cells using 3H-labeled NMN.

[0319] As described in the method "NMN uptake analysis using radiolabeled NMN", NMN uptake was measured for more than 10 min at 37 °C in control and Slc12a8-OE NIH3T3 cells using 25 μM 3H-NMN in Hank's buffer [pH 7.5] containing Mg2+ and Ca2+ (n = 12, analyzed by ANOVA with Sidak's test). 3H-NMN uptake was significantly enhanced at 3 and 5 min in Slc12a8-OE cells compared to control cells (Figure 11). To calculate the Michaelis-Menten constant of Slc12a8 protein, control and Slc12a8-OE NIH3T3 cells were incubated with 100 nM 3H-NMN and increasing concentrations of non-radioactive NMN in Hank's buffer [pH 7.5] containing Mg2+ and Ca2+ for 4 min at 37 °C. Km and Vmax values ​​were determined by nonlinear regression analysis by subtracting the background of control cells (n=5 at 1 and 10 μM, n=4 at 25 and 100 μM). For NMN, Km was 34.1±8.3 μM and Vmax was 11.5±1.2 pmol / min / mg (FIG. 12). This Km qualitatively matches the NMN concentration range detected in mouse plasma and erythrocytes (Revollo, JR, et al., Cell Metab., 6, 363-375, 2007; Ramsey, KM, et al., Science, 324, 651-654, 2009; Yamada, K., et al., Anal. Biochem., 352, 282-285, 2006).

[0320] [Example 7] This example demonstrates the specificity of the Slc12a8 protein.

[0321] Proteoliposomes were prepared from plasma membrane fractions of Slc12a8-OE and control NIH3T3 cells in transport buffer by combining membrane fractions of Slc12a8-OE or control NIH3T3 cells with phospholipid bilayers from deproteinized erythrocyte plasma membranes at 25°C as described above (n=3, analyzed by one-way ANOVA with Sidak's multiple comparison test). Slc12a8-OE-derived proteoliposomes incorporated significantly higher levels of H-NMN than control-derived proteoliposomes within 2 min (Figure 13).

[0322] In some experiments, it was found that nicotinic acid (NA) could enhance the uptake of radiolabeled 3H-NMN into proteoliposomes overexpressing Slc12a8 (Figure 42). The enhancement of 3H-NMN uptake by NA was reproduced under several different conditions (Figure 42).

[0323] To determine the substrate specificity of Slc12a8, these Slc12a8-OE proteoliposomes were used in displacement experiments with 3H-NMN and various non-radioactive NAD+ related compounds. The uptake of 3H-NMN (150 nM, 25°C) was measured in Slc12a8-OE proteoliposomes at 2 min in the presence of 150 μM of competing non-radioactive compounds (NMN, NR, NAD, AMP, NaMN, and nicotinamide) in transport buffer (n=3, analyzed by ANOVA using Dunnett's test). 150 μM non-radioactive NMN showed complete displacement with 3H-NMN, whereas NAD+, AMP, nicotinic acid mononucleotide (NaMN), and nicotinamide showed very low or negligible displacement at the same concentrations (Figure 14). Since 150 μM NR showed approximately 70% displacement, the IC50 concentrations of NMN and NR were determined using the proteoliposome system. The IC50 of NMN was 22.8 ± 3.6 μM and that of NR was 77.4 ± 10.8 μM (Figure 15). Data are presented as percentage of 3H-NMN uptake (n=3, IC50 calculated by nonlinear regression analysis). Without being limited by theory, this result suggests that Slc12a8 protein is primarily specific for NMN under physiological conditions, since NR levels have not been shown to reach such high concentrations under pathophysiological conditions, such as in blood (Frederick, DW, et al., Cell Metab., 24, 269-282, 2016) or ascites exudate (Sociali, G., et al., Oncotarget, 7, 2968-2984, 2016).

[0324] These findings suggest that NA and some NA derivative compounds can be used to promote the function of the Slc12a8 NMN transporter.

[0325] [Example 8] This example shows the sodium ion dependence of Slc12a8 on NMN transport.

[0326] In these experiments, we used a proteoliposome system to evaluate the sodium ion dependency of Slc12a8 on NMN transport. When sodium (Na+) was replaced with equimolar lithium (Li+) during the preparation of proteoliposomes and during the measurement of NMN influx, the uptake of 3H-NMN was dramatically reduced by about 80% (Figure 16, n=3, analyzed by unpaired t-test), indicating that NMN transport by Slc12a8 is sodium ion dependent.

[0327] These results indicate that sodium salts can enhance Slc12a8-mediated NMN transport and that other cations, such as lithium, can inhibit Slc12a8-mediated NMN transport.

[0328] [Example 9] This example shows the measurement of the effect of overexpression of Slc12a8 on NAD+ biosynthesis in NIH3T3 cells.

[0329] Pretreatment of control NIH3T3 and Slc12a8-OE cells with a cocktail of 100 nM FK866, 2 μM dipyridamole, and 500 μM AOPCP for 1 h significantly reduced intracellular NAD+ levels in both control and Slc12a8-OE cells (Figure 41). However, an additional 1 h incubation with 100 μM NMN was able to restore NAD+ levels to their original levels only in Slc12a8-OE cells, but not in control NIH3T3 cells (Figure 41, n=9, analyzed by ANOVA using Tukey's test).

[0330] [Example 10] This example shows the in vivo validation of NMN transport.

[0331] Lentiviruses carrying control firefly luciferase (fLuc) shRNA and Slc12a8 shRNA were generated as described above. We administered each virus directly into the intestine of mice by force. The expression levels of Slc12a8 protein in samples of jejunum and ileum infected with control shfLuc lentivirus and shSlc12a8 lentivirus were measured by Western blot (Figure 17). Figure 18 shows a bar graph of Slc12a8 protein levels in jejunum and ileum normalized to GAPDH protein levels (n=6, 3-4 month old B6 males, analyzed by unpaired t-test). Compared to mice administered fLuc shRNA-expressing lentivirus, Slc12a8 protein levels were reduced by approximately 60% in the jejunum and approximately 30% in the ileum of mice administered Slc12a8 shRNA-expressing lentivirus in the intestine (Figures 17-18). When NMN (500 mg / kg body weight) was administered by oral gavage to these mice, plasma NMN levels were significantly increased at 5 min in control mice, but not at all in Slc12a8 knockdown mice (Figure 19, NMN was measured by HPLC, n=6, 3-4 month old B6 males, analyzed by ANOVA using Sidak's test). Instead, plasma nicotinamide levels tended to be higher in Slc12a8-knockdown mice compared to control mice (Figure 20), possibly because higher levels of NMN in Slc12a8-knockdown mice underwent degradation to nicotinamide. In addition to plasma samples, tissue samples were collected from the jejunum at 60 min after oral gavage of phosphate-buffered saline (PBS) or NMN (500 mg / kg body weight) in control shfLuc lentivirus- and shSlc12a8 lentivirus-infected mice (n=5 mice for PBS, n=8 for NMN, 3-4 month old B6 males). The NAD+ levels of these samples were then measured by HPLC (analyzed by ANOVA with Tukey's test). NAD+ levels were significantly reduced in the jejunum of Slc12a8 knockdown mice compared to control mice (Figure 21).These results strongly suggest that intestinal Slc12a8 is important for the absorption of NMN from the gut into the blood circulation, affecting intestinal NAD+ levels and systemic supply of NMN in vivo.

[0332] [Example 11] The Examples provide the characterization of Slc12a8 knockout mice.

[0333] Exon 4 of the Slc12a8 gene was excised from mice using the CRISPR-CAS9 system described above to generate whole-body Slc12a8 knockout (Slc12a8KO) mice. The birth rate of these knockout mice was lower than the expected Mendelian ratio, implying that there was some premature death during the embryonic stage. However, the mice born became adults and no gross abnormalities were observed. Tissue samples of the duodenum, jejunum, ileum, and pancreas from Slc12a8KO mice and control wild-type littermates (WT) were collected during the light phase (9:00 am to 10:00 pm) or dark phase (9:00 pm to 10:00 pm). NAD+ levels in these samples were measured by HPLC (except for three data points in the jejunum of Slc12a8KO mice, n=5 mice in the light phase and n=4 mice in the dark phase, 8–10 month old females, analyzed by unpaired t-test). In adult Slc12a8KO mice, expression of full-length Slc12a8 mRNA was completely abolished in tissues (n=3, 2-3 month old males, Figure 22). Western blotting showed that Slc12a8 protein expression was also abolished in total tissue lysates of the jejunum and ileum (Figure 23), pancreas (Figure 23) and hypothalamus (Figure 24) of Slc12a8KO mice. Antiserum against the first 17 amino acids of the N-terminal domain of Slc12a8 was used. Despite expressing high levels of Slc12a8 mRNA in wild-type mice (Figure 2), the duodenum of knockout mice does not express full-length Slc12a8 protein (Figure 23). Consistent with the protein expression profile in the small intestine, Slc12a8KO mice showed a significant reduction in NAD+ levels in the jejunum and ileum, but not in the duodenum, especially during the dark phase when NAD+198 levels are normally elevated (Figure 25A-B). Pancreatic NAD+ was also decreased during both the light and dark periods (Figure 25A-B).To confirm whether NMN transport was impaired in the small intestine of Slc12a8KO mice, we administered doubly labeled 3-D heavier isotope NMN (18O-D-NMN) by gavage and measured the direct uptake of 18O-D-NMN into the jejunum and ileum by mass spectrometry.Ten minutes after oral gavage of 500 mg / kg 18O-D-NMN to Slc12a8KO mice and wild-type (WT) littermates, this isotopic NMN was clearly detected in the wild-type jejunum and ileum, whereas 18O-D-NMN uptake was reduced by 46% and 36%, respectively, in the jejunum and ileum of Slc12a8KO mice (Figure 26, n=6 mice, 3 males and 3 females aged 7-8 months, excluding 2 males and 2 females in wild-type ileum, analyzed by unpaired t-test). The area under the peak for 18O-D-NMN was calculated by subtracting the background value of the PBS control. Values ​​are expressed relative to the 18O-D-NMN levels detected in the WT. In this example, all values ​​are presented as mean ± SEM. *p<0.05, **p<0.01.

[0334] [Example 12] This example shows that Slc12a8KO mice exhibit hyperphagia without affecting fat and lean mass.

[0335] At 6-7 months of age, female Slc12a8KO mice in particular began to show significant hyperphagia during the dark phase (Figure 27). Food intake was measured for 5 days in Slc12a8KO mice and control wild-type (WT) littermates during the light (9:00 AM-6:00 PM) and dark (6:00 PM-9:00 AM) phases (n=8 mice per genotype, 6-7 month old females, analyzed by unpaired t-test). However, Slc12a8KO mice did not show any difference in fat and lean mass compared to wild-type controls (Figure 28). Fat and lean mass were measured using a whole-body NMR instrument in Slc12a8KO and control wild-type littermates (n=8 mice per genotype, 8-10 month old females). The hyperphagia shown was not due to intestinal malabsorption, since there was no difference in fecal lipid content between control and Slc12a8KO mice (Figure 29). Total lipid content, expressed as a percentage of the starting stool volume extracted, was measured in feces collected between 9 and 10 am from Slc12a8KO mice and control wild-type littermates ( n = 8 mice per genotype, female, 6–8 months of age).

[0336] [Example 13] This example demonstrates increased respiration and activity in Slc12a8KO mice.

[0337] Interestingly, Slc12a8KO mice showed a moderate increase in oxygen consumption (VO2, Fig. 30), energy expenditure (Fig. 31) and total locomotor activity (Fig. 32) during the dark phase, providing an explanation for their hyperphagic phenotype without weight gain. These mice also showed a significantly lower respiratory exchange ratio (RER, Fig. 33), suggesting higher fatty acid utilization during the light phase, providing an additional explanation for the maintenance of normal fat mass, although no differences in plasma fatty acid levels were detected between control and Slc12a8KO mice (not shown).

[0338] [Example 14] This example demonstrates enhanced glucose metabolism in Slc12a8 knockout mice.

[0339] After 16 hours of fasting, female Slc12a8KO mice (n=8 mice per genotype, 6-8 month old females, analyzed by unpaired t-test) showed higher glucose (Figure 34), insulin (Figure 35), and triglyceride (Figure 36) levels. Without being limited by theory, all these phenotypes together suggest hypothalamic dysfunction.

[0340] [Example 15] This example shows impairment of glucagon-like peptide 2 receptor (GLP-2R) in Slc12a8 knockout mice.

[0341] GLP-2R is a 33 amino acid peptide derived from proglucagon produced by enteroendocrine L cells. Plasma GLP2-levels were measured by ELISA in Slc12a8KO mice and control wild-type littermates after 24 hours fasting (9 am to 9 am) and then 6 hours refeeding (10 am to 4 pm) (n=8 mice for each genotype, 7-8 month old females). Slc12a8KO mice showed significantly lower plasma levels of GLP-2 in response to 6 hours refeeding after a 24 hour fast, whereas control mice showed a significant increase in plasma GLP-2 levels upon refeeding (Figure 37).

[0342] [Example 16] This example demonstrates disruption of expression in the arcuate nucleus in Slc12a8 knockout mice.

[0343] Chop (DNA damage-inducible transcript 3) and Socs3 (suppressor of cytokine signaling 3) are upregulated in response to endoplasmic reticulum (ER) stress and the resulting leptin resistance. Chop and Socs3 mRNA expression levels were measured in the hypothalamic arcuate nucleus of Slc12a8KO mice and control wild-type littermates during the dark phase (9:00-10:00 pm) (Chop, n=3 mice per genotype; Socs3, n=4 mice per genotype, 8-10 month old females, analyzed by unpaired t-test). Consistent with this lack of valid GLP-2 stimulation, the arcuate nucleus of Slc12a8KO mice showed enhanced mRNA expression levels (Figure 38). Without being limited by theory, these results suggest a dysfunction of the arcuate neurons (Williams, KW, et al., Cell Metab., 20, 471-482, 2014). The mRNA expression levels of Pepck (phosphoenolpyruvate carboxykinase) and G6pc (glucose-6-phosphatase catalytic subunit) were measured in the livers of ad libitum-fed Slc12a8KO mice and control wild-type littermates (n=5 mice for each genotype, 8-10 month old females, analyzed by unpaired t-test). These gluconeogenic genes were found to be upregulated in Slc12a8KO mice (Figure 39). Without being limited by theory, these data provide evidence of dysfunction of globular neurons, explaining the higher glucose and insulin levels in fasted Slc12a8KO mice.

[0344] [Example 17] This example shows the diurnal mRNA expression profile of PMCH in Slc12a8 knockout mice.

[0345] PMCH (promelanin-concentrating hormone) mRNA expression levels were measured in the lateral hypothalamus (LH) of Slc12a8KO mice and control wild-type littermates during the light (9:00-10:00 AM) and dark (9:00-10:00 PM) phases (n=3 mice for each genotype, 8-10 month old females, analyzed by unpaired t-test). The diurnal mRNA expression profile of PMCH was abnormal in the lateral hypothalamus of Slc12a8KO mice (Figure 40), showing little change in expression between the light and dark phases, whereas expression of PMCH in wild-type littermates was significantly increased during the dark phase. Without being limited by theory, loss of the NMN transporter globally affects hypothalamic function. No abnormalities were detected in the expression of genes encoding major neuropeptides and their receptors in other hypothalamic nuclei of Slc12a8KO mice (not shown). Without being limited by theory, these findings demonstrate a novel function for the Slc12a8 NMN transporter, possibly regulating proper inter-tissue communication between the gut and the hypothalamus, possibly via NMN itself and GLP-2.

[0346] [Example 18] This example shows NAD+ and Slc12a8 concentrations in the small intestine.

[0347] It is well established that NAD+ content decreases with age in many tissues (Yoshino, J., et al., Cell Metab., 2018, 27: 513-528, 2018). NAD+ levels were measured in jejunum and ileum collected during the dark phase (9:00 pm to 10:00 pm) from 2- and 24-month-old female B6 mice. NAD+ content decreased with age in the jejunum and ileum, but the difference did not reach statistical significance in the jejunum (Figure 43, n=4 mice per age, analyzed by unpaired t-test). Slc12a8 mRNA expression was measured in jejunum and ileum collected during the dark phase (9:00 pm to 10:00 pm) from 2- and 24-month-old female B6 mice (n=4 mice per age, analyzed by unpaired t-test). Consistent with the observed decrease in NAD+, expression of Slc12a8 was significantly upregulated in the aged ileum (Figure 44). These results indicate that upregulation of Slc12a8 correlates with the decrease in NAD+ in the gastrointestinal tract.

[0348] [Example 19] This example shows the effect of Slc12a8 gut knockdown on glucose metabolism in mice.

[0349] We orally gavaged 2- and 24-month-old mice with lentivirus carrying control firefly luciferase (fLuc) shRNA and Slc12a8 shRNA and examined their feeding behavior. Food intake was measured for 4 days in 2- and 24-month-old intestinal Slc12a8 knockdown female B6 mice (shSlc12a8) and control mice (shfLuc) during the light (9 am to 6 pm) and dark (6 pm to 9 am) phases. Interestingly, gut-specific Slc12a8 knockdown caused a significant decrease in food intake during the dark phase in old mice, but had no effect on food intake in young mice (Figure 45, n=6 mice each for 2-month-old intestinal Slc12a8 knockdown and control female B6 mice, and n=9 mice each for 24-month-old intestinal Slc12a8 knockdown and control female B6 mice, analyzed by ANOVA with Tukey's test). Fasting glucose levels were measured in 2-month-old or 24-month-old intestinal Slc12a8 knockdown female B6 mice (shSlc12a8) and control mice (shfLuc) after 24-hour fasting (Figure 46, n=6 mice each for 2-month-old intestinal Slc12a8 knockdown and control female B6 mice, and n=9 mice each for 24-month-old intestinal Slc12a8 knockdown and control female B6 mice, analyzed by unpaired t-test). Thus, Slc12a8 knockdown reduced fasting glucose levels in old mice, but not in young mice (Figure 46). Without being limited by theory, these results indicate that expression of Slc12a8 affects the production of NAD+ in the digestive tract.

[0350] [Example 20] This example shows that gut-specific Slc12a8 knockdown significantly increases circulating levels of glucagon-like peptide 1 (GLP-1).

[0351] GLP-1 is a proglucagon-derived appetite suppressant peptide produced by enteroendocrine L cells. Plasma GLP-1 levels were measured by ELISA in 2-month-old or 24-month-old intestinal Slc12a8 knockdown female B6 mice (shSlc12a8) and control mice (shfLuc) after 24-hour fasting followed by 6-hour refeeding (10:00 am to 4:00 pm). Slc12a8 knockdown increased circulating levels of GLP-1 in response to 6-hour refeeding after 24-hour fasting in old mice but not in young mice (Figure 47, n=6 mice each for 2-month-old intestinal Slc12a8 knockdown and control female B6 mice + and n=9 mice each for 24-month-old intestinal Slc12a8 knockdown and control female B6 mice, analyzed by unpaired t-test). Without being limited by theory, these results indicate that secretion of GLP-1 is affected by Slc12a8 activity.

[0352] [Example 21] This example shows the effect of Slc12a8 knockdown on the aged ileum.

[0353] It is well known that GLP-producing L cells are enriched in the ileum (Yoon, MJ, et al., Cell Metab., 2015, 21, 706-717). Therefore, we investigated the expression of Slc12a8 and GAPDH in the ileum. Western blots of Slc12a8 and GAPDH were performed in ileal lysates from intestinal Slc12a8 knockdown female B6 mice (shSlc12a8) and control mice (shfLuc) at 2 months of age (Figure 48) or 24 months of age (Figure 49). Figures 48-49 show representative Western blots (left panels) and the corresponding bar graphs show Slc12a8 protein levels normalized to ileal GAPDH protein levels (right panels). Antiserum against the first 17 amino acids of the N-terminal domain of Slc12a8 was used (n=6 each for 2-month-old intestinal Slc12a8 knockdown and control mice, and n=4-5 each for 24-month-old intestinal Slc12a8 knockdown and control mice, analyzed by unpaired t-test). Although the levels of Slc12a8 protein were significantly decreased in the ileum of young and aged Slc12a8 knockdown mice (Figures 48-49), only aged Slc12a8 knockdown ileum showed a significant decrease in NAD+ levels (Figure 50), confirming the physiological importance of upregulation of Slc12a8 in maintaining NAD+ homeostasis in the aged ileum. Figure 50 shows NAD+ levels in the ileum of intestinal Slc12a8 knockdown (shSlc12a8) and control female B6 mice (shfLuc) at 2 or 24 months of age (n=6 mice for intestinal Slc12a8 knockdown and control female B6 mice at 2 months of age, and n=9 mice each for intestinal Slc12a8 knockdown and control female B6 mice at 24 months of age, analyzed by unpaired t-test). Without being limited by theory, these results indicate that upregulation of Slc12a8 is required to maintain NAD+ homeostasis in the aged ileum.

[0354] [Example 22] This example shows the effect of Slc12a8 knockdown on GLP-1 levels in cultured ileum.

[0355] We also performed ex vivo culture of ileum from aged fLuc control and Slc12a8 knockdown mice. GLP-1 levels were measured by ELISA in the supernatants of ex vivo ileum explants from 24-month-old intestinal Slc12a8 knockdown female B6 mice (shSlc12a8) and control mice (shfLuc) cultured at 37°C for 2 hours (n=4 mice, analyzed using unpaired t-test). Figure 51 shows that aged Slc12a8-knockdown ileum secreted 3-fold higher levels of GLP-1 compared to those from aged fLuc control ileum. Ex vivo cultured colons from aged fLuc control and Slc12a8 knockdown mice showed no difference in GLP-1 secretion. GLP-1 levels were measured by ELISA in the supernatants of ex vivo ileal explants from 12-month-old Slc12a8KO female mice (Slc12a8KO) and control wild-type littermates (WT) cultured for 2 hours at 37°C (n=4 mice, analyzed by unpaired t-test). NAD+ levels were also measured in these ileal samples from 12-month-old Slc12a8KO female mice (Slc12a8KO) and control wild-type littermates (WT) (n=3 mice for WT and n=4 for Slc12a8KO, analyzed by unpaired t-test). All values ​​are presented as mean±SEM. *p<0.05, **p<0.01. Figure 52 shows increased GLP-1 secretion and decreased NAD+ in ex vivo cultured ileum from 12-month-old control and Slc12a8KO mice. Without being limited by theory, these examples indicate that Slc12a8 is responsible for the decrease in NAD+ in the gut of aged mice.

[0356] [Example 23] This example shows the effect of SIRT1 and SIRT6 inhibitors on ex vivo cultured ileum.

[0357] The contribution of SIRT1 (Yoon, MJ, et al., Cell Metab., 2015, 21, 706-717) and SIRT6 (Jiang, H., et al., Nature, 2013, 496, 110-113) to the observed increase in GLP-1 secretion was investigated. Ex vivo cultured wild-type ileum was treated with SIRT1 and SIRT6 inhibitors. GLP-1 levels were measured by ELISA in the supernatants of ex vivo ileal explants from 24-month-old female B6 mice cultured for 4 hours with 0.2% DMSO, 20 μM EX527 or 20 μM STK665401 (n=3 mice, analyzed by ANOVA using Tukey's test). Only the potent SIRT1-specific inhibitor EX527, but not the SIRT6-specific inhibitor STK665401, was able to enhance GLP-1 secretion from the ileum (Figure 53). Without being limited by theory, these results indicate that the enhanced GLP-1 secretion observed in aged Slc12a8 knockdown ileum is likely to involve reduced SIRT1 activity due to a significant decrease in NAD+.

[0358] When introducing elements of the present invention or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0359] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0360] As various changes can be made in the products and methods described above without departing from the scope of the invention, all matter contained in the above description and shown in the accompanying drawings is intended to be interpreted in an illustrative and not a limiting sense. Furthermore, the present invention includes the following aspects. 1. A method of treating an age-related symptom in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one additional compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharmaceutically acceptable salt thereof, and any combination thereof, wherein the age-related symptom comprises at least one symptom selected from the group consisting of loss of insulin sensitivity, loss of insulin secretion, loss of insulin action and secretion, impaired memory function, decreased ocular function, retinal degeneration, decline, and any combination thereof. 2. The method of claim 1, wherein the age-related symptoms include loss of insulin sensitivity. 3. The method according to item 1 or 2, wherein the age-related symptoms include loss of insulin secretion. 4. The method according to any one of items 1 to 3, wherein the age-related symptoms include loss of insulin action and secretion. 5. The method according to any one of items 1 to 4, wherein the aging-related symptoms include memory function disorders. 6. The method according to any one of items 1 to 5, wherein the aging-related symptoms include a decline in ocular function. 7. The method according to any one of items 1 to 6, wherein the aging-related symptom includes retinal degeneration. 8. The method according to any one of items 1 to 7, wherein the aging-related symptoms include a decline in function. 9. The method of claim 8, wherein the decline in function is selected from the group consisting of loss of appetite, low glucose levels, muscle weakness, malnutrition, loss of appetite due to aging, and any combination thereof. 10. A method of treating a medical condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of nicotinamide mononucleotide (NMN) and at least one compound selected from the group consisting of nicotinic acid, niceritrol, tocopherol nicotinate, β-pyridylcarbinol, inositol hexanicotinate, any ester thereof, any pharmaceutically acceptable salt thereof, and any combination thereof, wherein the medical condition comprises at least one selected from the group consisting of muscle disease, type 2 diabetes, obesity, and any combination thereof. 11. The method of claim 10, wherein the medical condition comprises a muscular disorder. 12. The method according to paragraph 10 or 11, wherein the muscle disease is selected from the group consisting of muscle weakness, muscle atrophy, muscle wasting, muscle weakness, and any combination thereof. 13. The method according to any one of items 10 to 12, wherein the muscle disease is selected from the group consisting of sarcopenia, dynapenia, cachexia, muscular dystrophy, dystonia, spinal muscular atrophy, myopathy, and any combination thereof. 14. The method of claim 13, wherein the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, and any combination thereof. 15. The method of claim 13 or 14, wherein the muscle tone disorder is selected from the group consisting of myotonic dystrophy, myotonia congenita, paramyotonia congenita, and any combination thereof. 16. The method according to any one of clauses 13 to 15, wherein the myopathy is selected from the group consisting of Bethlem myopathy, congenital fiber type imbalance, progressive fibrous dysplasia, hyperthyroid myopathy, hypothyroid myopathy, minicore myopathy, multicore myopathy, myotubular myopathy, nemaline myopathy, periodic paralysis, hypokalemic myopathy, hyperkalemic myopathy, and any combination thereof. 17. The method according to any one of items 10 to 16, wherein the muscle disease is selected from the group consisting of acid maltase deficiency, carnitine deficiency, carnitine palmityltransferase deficiency, debranching enzyme deficiency, lactate dehydrogenase deficiency, mitochondrial myopathy, myoadenylate deaminase deficiency, phosphorylase deficiency, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, and any combination thereof. 18. The method according to any one of items 10 to 17, wherein the muscle disease is selected from the group consisting of sarcopenia, dynapenia, cachexia, and any combination thereof. 19. The method according to item 18, wherein the muscle disease is sarcopenia. 20. The method of any one of paragraphs 10 to 19, wherein the medical condition comprises type 2 diabetes. 21. The method of any one of items 10 to 20, wherein the medical condition includes obesity. 22. The method of any one of paragraphs 1 to 21, wherein the at least one additional compound comprises nicotinic acid, any ester thereof, or any pharma- ceutically acceptable salt thereof. 23. The ester or pharma- ceutically acceptable salt of nicotinic acid is represented by the structural formula (I):

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Claims

1. A method for screening candidate compounds to identify compounds that promote NMN transport, the method comprising: (a) contacting the candidate compound with a cell expressing an NMN transporter protein or a proteoliposome containing an NMN transporter protein; and (b) detecting a change in the expression or activity of the NMN transporter protein in the cell or a change in the activity of the NMN transporter protein in the proteoliposome; wherein a change in the expression or activity of the NMN transporter protein in the cell or a change in the activity of the NMN transporter protein in the proteoliposome after contact with the candidate compound indicates that the candidate compound regulates the transport of NMN, wherein the NMN transporter protein comprises Slc12a8 protein.

2. The method of claim 1, wherein the Slc12a8 protein comprises an amino acid sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 12 (mouse Slc12a8 protein), SEQ ID NO: 13 (mouse Slc12a8 mutant A protein), SEQ ID NO: 14 (mouse Slc12a8 mutant B protein), or SEQ ID NO: 15 (human Slc12a8 protein).

3. (i) determining the expression or activity of the NMN transporter protein in the cell expressing the NMN transporter protein after contact with the candidate compound; The method of claim 1 or 2, further comprising (ii) comparing the expression or activity of the NMN transporter protein after contact with the candidate compound with that in a cell that does not express the NMN transporter protein or in a cell in which expression or activity of the NMN transporter protein has been inhibited.

4. The method of claim 1 , wherein the cells that do not express the NMN transporter protein comprise cells of an Slc12a8 knockout animal.

5. (i) the activity of the NMN transporter protein in the proteoliposomes containing the NMN transporter protein after contact with the candidate compound. The method of claim 1 or 2, further comprising (ii) comparing the activity of the NMN transporter protein after contact with the candidate compound with that in proteoliposomes that do not contain the NMN transporter protein, or in proteoliposomes in which the activity of the NMN transporter protein has been inhibited.

6. The method of claim 5 , wherein the proteoliposomes that do not contain the NMN transporter protein comprise proteoliposomes from cells of an Slc12a8 knockout animal.

7. The method of any one of claims 1 to 6, wherein the cells comprise mammalian fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells, or the proteoliposomes comprise proteoliposomes derived from mammalian fibroblasts, intestinal cells, pancreatic cells, hepatic cells, adipocytes, neurons, or glial cells.

8. the cells comprise mammalian cells or the proteoliposomes comprise proteoliposomes derived from mammalian cells or mammalian cell lines; wherein said mammalian cell or mammalian cell line comprises a cDNA encoding a Slc12a8 protein, wherein said cDNA (a) a cDNA encoding SEQ ID NO:12 (mouse Slc12a8 protein), SEQ ID NO:13 (mouse Slc12a8 mutant A protein), SEQ ID NO:14 (mouse Slc12a8 mutant B protein), or SEQ ID NO:15 (human Slc12a8 protein); (b) a cDNA encoding a protein having at least 90%, at least 95%, or at least 99% homology to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; or (c) a cDNA sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with the GenBank reference sequence: NM_134251 (SEQ ID NO: 1) or Slc12a8 human full-length cDNA (SEQ ID NO: 11).

9. The method of any one of claims 1 to 8, wherein the proteoliposomes comprise proteoliposomes derived from a mammalian cell or mammalian cell line containing Slc12a8 cDNA.

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