Ribosomal remodeling in diabetes

Abstract

This disclosure relates to ribosomal remodeling due to high glucose conditions. In some embodiments, translation of proteins in pancreatic beta-cells is altered based on the ribosomal remodeling. Means of increasing the percentage of large ribosomal subunit protein P1 (RPLP1) associated with ribosomes and / or decreasing the percentage of soluble RPLP1 in a cell to counteract effects of high glucose conditions in diabetes are disclosed.

Description

RIBOSOMAL REMODELING IN DIABETESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 517,267 filed August 2, 2023, entitled Ribosomal Remodeling in Diabetes, and designated by Attorney Docket No. 01123-0015-60US. The entire content of the foregoing application is incorporated herein by reference in its entirety including all text, tables and drawings.SUPPORT

[0002] This invention was made with government support under Research Grant No. 5DP1 DK119141 awarded by NIH / National Institute of Diabetes and Digestive and Kidney Diseases. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy created on July 25, 2024, is named “01123-0015-00PCT-ST26.xml” and is 74,137 bytes in size.FIELD

[0004] This disclosure relates to ribosomal remodeling due to high glucose conditions. In some embodiments, translation of proteins in pancreatic beta-cells is altered based on the ribosomal remodeling. Means of increasing the percentage of Large Ribosomal Subunit Protein Pl (RPLP1) associated with ribosomes and / or decreasing the percentage of soluble RPLP1 in a cell to counteract effects of high glucose conditions in diabetes are disclosed.BACKGROUND

[0005] Diabetes results from pancreatic beta-cell failure to secrete sufficient insulin to regulate glucose homeostasis. Progressive decline in beta-cell function occurs in the setting of hyperglycemia during the progression from early appearance of autoantibodies to frank type 1 diabetes, and during the evolution from compensated insulin resistance to type 2 diabetes (1, 2). In type 1 diabetes, intensive insulin therapy that restores normal glycemic levels increases stimulated C-peptide levels, a reflection of improved insulin biosynthesis and preserved beta-cell function (3). In patients with newly diagnosed type 2 diabetes, intensive short-term insulin therapy improves beta-cell function and long-term glycemic control (4, 5). These observations support the hypothesis that glucose toxicity contributes to the decline in insulin production in diabetes.

[0006] Pancreatic beta-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair beta-cell insulin secretion have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown.

[0007] In response to an acute physiological rise in glucose, proinsulin mRNA translation rapidly increases within 30 to 60 minutes, without change in insulin mRNA abundance (6). This translational regulation requires sequences predicted to form a stem-loop structure within the 5 ’-untranslated region of the insulin mRNA that bind to protein factors in a glucose-dependent fashion (7). In addition to insulin, glucose acutely upregulates translation of proteins involved in glucose metabolism, insulin processing, secretory granule biogenesis,and insulin exocytosis, without causing an equivalent increase in total protein synthesis (8- 11). Given that newly synthesized insulin is preferentially released initially, this mRNA translational program supports physiological increases in glucose-stimulated insulin secretion (GSIS) following brief exposures to high glucose (12).

[0008] By contrast, persistently elevated glucose impairs GSIS and insulin translation.Among obese subjects, GSIS decreases with increasing plasma glucose area under the curve in a 3-hour oral glucose tolerance test, without change in insulin sensitivity (13). Ex vivo incubation of isolated human or rodent islets over one week in media containing high glucose also impairs GSIS and inhibits translation of proinsulin (14, 15). The impact of sustained high glucose on GSIS in cadaveric human islets is apparent as early as 2 days following exposure to high glucose, a time point at which impairment is reversible, associated with only modest transcriptomic changes, and without evidence for ER stress (16). Sustained exposure of insulinoma cells and isolated islets to high concentrations of glucose and saturated fatty acids to model the metabolic stress of type 2 diabetes increases translation of JUND, a transcriptional regulator of beta-cell apoptosis, and induces ER stress (17, 18). However, it is not known whether sustained high glucose alone initiates programmatic regulation of translation prior to induction of ER stress.

[0009] Given the importance of translational regulation in GSIS, the impact of sustained glucose elevation was studied on genome-wide beta-cell mRNA translation. Using complementary high-throughput approaches in a MIN6 model and validating findings in primary isolated islets ex vivo and in an in vivo model of hyperglycemia, chronic glucose excess was shown to coordinately downregulate translation of genes that function in metabolism-coupled insulin secretion.

[0010] The present disclosure adds to a growing body of evidence that the mammalian proteome is strongly influenced by mRNA-specific rates of translation (40), a process that can rapidly regulate protein abundance independent of new transcription or RNA decay.

[0011] Selective mRNA translation often leverages unique features within untranslated regions of mRNAs, trans-acting mRNA binding proteins, and / or selection of specific translation initiation or elongation factors (50). Although the widely held dogma is that all ribosomes are compositionally and functionally similar (51), growing evidence supports the idea that ribosomes with varying stoichiometry of select ribosomal proteins, known as heterogeneous ribosomes, direct functionally distinct programs of mRNA translation (52-56). To date, examples of heterogeneous ribosomes have been identified as stable features of distinct cell types or tissues and are presumed to be generated during ribosome biogenesis in the nucleolus.

[0012] Disclosed herein is the impact of sustained high glucose on the composition of actively translating ribosomes (polysomes) in beta-cells. As described in this disclosure, RPLP1 dissociates from ribosomes of high glucose-treated cells. Thus, exposure of pancreatic beta-cells to sustained high glucose, not only decreases translation of key genes for beta-cell function but also dynamically remodels actively translating ribosomes. RPLP1 is known to be phosphorylated on serine residues 101 and 104 (Sacco F, et al. (2016) Nat Commun. 7: 13250). As described in this disclosure, mutation of serine 101 and serine 104 to alanine, which renders them non-phosphorylatable, decreases RPLP1 association with translating ribosomes. Methods disclosed herein can be used to increase association of RPLP1 in ribosomes and / or to decrease soluble RPLP1 outside of ribosomes to prevent maladaptive changes in mRNA translation.

[0013] Targeted ribosome remodeling for therapeutic effect on protein expression could also be applied to diabetes as well as other disorders. This approach could therefore serve as a platform technology to address many human diseases.SUMMARY

[0014] High-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells as described herein elucidate the genome-wide impact of sustained high glucose on beta-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin but also genes related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these genes was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins.

[0015] In some embodiments, a translational regulatory circuit during beta-cell glucose toxicity impairs expression of proteins with critical roles in beta-cell function, and the present methods inhibit this circuit.

[0016] As described herein, interventions to increase large ribosomal subunit protein Pl (RPLP1) association with ribosomes could be used to improve production of key proteins in beta-cell insulin secretion. Such a strategy could be used to enhance insulin secretion in vivo during the development or progression of type 1 or type 2 diabetes. In some embodiments, treatments to increase RPLP1 association with ribosomes improves the function of beta-cells or iPSC-generated beta-like cells used for cell replacement therapies. In some embodiments, modification of these cells to increase RPLP1 phosphorylation prior to implantation increases glucose-stimulated secretion of insulin. Exemplary embodiments are listed below:

[0017] Embodiment 1. A method of treating or preventing diabetes in a subject in need thereof comprising increasing the percentage of, ribosome-associated RPLP1 or decreasing the percentage of soluble RPLP1 in a cell of the subject comprising phosphorylating RPLP1, inhibiting dephosphorylation of RPLP1, and / or expressing an RPLP1 mutant comprising one or more phosphomimetic amino acids, thereby increasing the percentage of ribosome- associated RPLP1 and / or decreasing the percentage of soluble RPLP1 in the subject.

[0018] Embodiment 2. The method of embodiment 1, wherein the subject has Type I (Tl) or Type II (T2) diabetes.

[0019] Embodiment 3. The method of embodiment 1, wherein the subject is at risk for T2 diabetes. For example, in some cases the subject may have pre-diabetes such as an HbAlc level of 5.8-6.5% or 5.8-6.4%, or may have a family history of Tl or T2 diabetes.

[0020] Embodiment 4. The method of any one of embodiments 1-3, wherein increasing the percentage of ribosomes containing RPLP1 increases translation of one or more proteins encoded by a gene selected from INS, SCGN, IDH2, VPS41, SLC2A1, IGF2, SLC30A8, and PFKFB3.

[0021] Embodiment 5. The method of any one of embodiments 1-3, wherein the method comprises administering to the subject a kinase activator or phosphatase inhibitor that phosphorylates RPLP1 and / or dephosphorylates RPLP1.

[0022] Embodiment 6. The method of any one of embodiments 1-3, wherein the RPLP1 phosphorylation is at serine 101 and / or serine 104.

[0023] Embodiment 7. The method of any one of embodiments 1-3, wherein expressing an RPLP1 mutant comprising one or more phosphomimetic amino acids is performed by gene editing of RPLP1.

[0024] Embodiment 8. The method of embodiment 7, wherein the gene editing is performed with a system for gene editing.

[0025] Embodiment 9. The method of embodiment 8, wherein the system for gene editing comprises a CRISPR / Cas9 system, zinc-finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, or group one intron encoded endonuclease (GHEE).

[0026] Embodiment 10. The method of any one of embodiments 7-9, wherein the RPLP1 mutant comprises one or more phosphomimetic amino acid mutations at an amino acid that is a serine in wildtype RPLP1.

[0027] Embodiment 11. The method of embodiment 10, wherein the serine in wildtypeRPLP1 is serine 101 and / or serine 104.

[0028] Embodiment 12. The method of any one of embodiments 1-4 or 7-11, wherein the one or more phosphomimetic amino acid mutation is an aspartic acid or glutamic acid.

[0029] Embodiment 13. The method of any one of embodiments 1-12, wherein the cell is a pancreatic beta-cell.

[0030] Embodiment 14. The method of any one of embodiments 1-13, wherein the method increases the levels of insulin within or released by the cell.

[0031] Embodiment 15. The method of any one of embodiments 5, 6, or 14, wherein the cell is a pancreatic beta-cell, and the subject is treated with a kinase activator or phosphatase inhibitor.

[0032] Embodiment 16. The method of any one of embodiments 1-4 or 7-15, wherein the cell is an induced pluripotent stem cell (iPSC) or pancreatic beta-cell in culture and gene editing is performed in vitro, and the cell is introduced into the subject after the gene editing or where the cell is an iPSC or pancreatic beta-cell in a subject and gene editing is performed invivo using a delivery system that selectively delivers the gene editing system to the iPSC or pancreatic beta-cell.

[0033] Embodiment 17. The method of embodiment 15 or embodiment 16, wherein insulin levels in the subject are increased and / or glucose levels in the subject are decreased.

[0034] Embodiment 18. A method of treating or preventing diabetes in a subject in need thereof comprising preparing iPSCs and / or pancreatic beta-cells in vitro., treating said iPSCs or pancreatic beta-cells with an agent for gene editing of RPLP1, wherein the gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell; and transplanting the treated iPSCs or pancreatic beta-cells into the subject, wherein the transplanting increases insulin levels in the subject and / or decreases glucose levels in the subject. In some cases, the subject has T1 or T2 diabetes, or alternatively is at risk for T2 diabetes. For example, in some cases the subject may have pre-diabetes such as an HbAlc level of 5.8-6.5% or 5.8-6.4%, or may have a family history of T1 or T2 diabetes.

[0035] Embodiment 19. A method of reducing the incidence of hyperglycemia or immune attack on pancreatic beta-cells in a subject in need thereof comprising: preparing iPSCs and / or pancreatic-beta-cells in vitro., treating said iPSCs or pancreatic beta-cells with an agent for gene editing of RPLP1, wherein the gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell; and transplanting the treated iPSCs or pancreatic beta-cells into the subject, wherein the transplanting increases insulin levels in the subject and / or decreases glucose levels in the subject. In some cases, the subject has T1 or T2 diabetes, or alternatively is at risk for T2 diabetes. For example, in some cases the subject may have pre-diabetes such as an HbAlc level of 5.8-6.5% or 5.8- 6.4%, or may have a family history of T1 or T2 diabetes.

[0036] Embodiment 20: The method of embodiment 18 or 19, wherein the RPLP1 mutant comprises one or more phosphomimetic amino acid mutations at an amino acid that is a serine in wildtype RPLP1.

[0037] Embodiment 21 : The method of embodiment 20, wherein the serine in wildtype RPLP1 is serine 101 and / or serine 104.

[0038] Embodiment 22: The method of any one of embodiments 18-21, wherein the one or more phosphomimetic amino acid mutations is an aspartic acid or glutamic acid.

[0039] Embodiment 23. A composition comprising (1) an iPSC or pancreatic beta-cell and (2) a system for gene editing of RPLP1.

[0040] Embodiment 24. The composition of embodiment 23, wherein the system for gene editing is a CRISPR / Cas9 system, zinc-finger nuclease, TALEN, meganuclease, or GHEE.

[0041] Embodiment 25. The composition of embodiment 23 or embodiment 24, wherein the system for gene editing is capable of introducing a phosphomimetic amino acid at positions serine 101 and / or serine 104.

[0042] Embodiment 26. The composition of embodiment 25, wherein the phosphomimetic amino acid mutation is an aspartic acid or glutamic acid.

[0043] Additional objects and advantages are set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0044] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0045] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figures 1 A-H show that sustained high glucose decreases basal insulin translation in isolated rat islets. Figure 1 A shows an embodiment of a general workflow showing hand- picked islets from adult Sprague-Dawley rats that were cultured for 4 days in media containing 5.5 mM (solid gray bars) or 16.7 mM (hatched gray bars) glucose (GLU). Following 1 hour rest in 2.8 mM GLU, GSIS quantified at 2.8 mM (basal, B) and 16.7 mM (stimulated, S) GLU. Figure IB shows the results of GSIS normalized by DNA with stimulation (stim) index quantified as stimulatory / basal secretion. Figure 1C shows Insulin content normalized to DNA. Figure ID shows qPCR quantification of beta-cell markers Ucn3, Mafa, and Pdxl relative to 18S rRNA. Figure IE shows cells pulse labeled with O- propargyl-puromycin (OPP) and analyzed by SDS-PAGE and showing total protein quantified by Coomassie stain (left) and newly synthesized protein quantified by clickaddition of Alexa-647 (right). Representative images with quantification. Figure IF shows metabolic labeling with OPP, click-biotin addition and streptavidin pulldown of nascent proteins with immunoblot analysis of newly synthesized proinsulin (proINS) with tubulin control. Figure 1G shows representative immunoblots and quantification for ER stress markers with thapsigargin (THAP)-treated control. Means ± standard error (SE) for n = 3 - 4 independent experiments. *’Padj < 0.05 by 2-way ANOVA with Bonferroni post-hoc correction (IB [GSIS], 1D,1 G).#, P < 0.05 by unpaired t-test (IB [stim index], 1C, IE) or by ratio-paired t-test (IF), ns, not significant.

[0047] Figures 2A-2I show sustained high glucose decreases insulin synthesis in MIN6 cells. Figure 2A shows an embodiment of a general workflow showing MIN6 cells incubated in media containing 5.5 mM (solid gray bars) or 25 mM (hatched gray bars) GLU for 24 hours. Following 1 hour rest in 2.8 mM GLU, GSIS was quantified at 2.8 mM (B) and 16.8 mM (S) GLU. Figure 2B shows the results of GSIS normalized by cell number with stim index quantified as stimulatory / basal secretion. Figure 2C shows Insulin content per 106cells. Figure 2D GSIS normalized to cellular insulin content. Figure 2E shows GSIS normalized by cell number following incubation in 5.5 mM GLU, 25 mM GLU, or 5.5 mM GLU with 19.5 mM mannitol (MAN, open bars). Figure 2F shows qPCR quantification of beta-cell markers Ucn3, Mafa, and Pdxl relative to 18S rRNA. Figure 2G shows OPP pulse labeling and SDS- PAGE with total protein quantified by Coomassie stain and newly synthesized protein quantified by click-addition of Alexa-647. Representative images with quantification. Figure 2H shows OPP labeling, click-biotin addition, and streptavidin pulldown of nascent proteins with immunoblot for newly synthesized proINS and tubulin control. Figure 21 shows Representative immunoblots and quantification for ER stress markers with tunicamycin (TN)-treated control. Means ± SE for n = 3 - 4 independent experiments. *, Padj < 0.05 by 2- way ANOVA with Bonferroni post-hoc correction (2B [GSIS], 2D, 2E, 21).#, P < 0.05 by unpaired t-test (2B [stim index], 2C, 2G) or by ratio-paired t-test (2H). ns, not significant.

[0048] Figures 3 A-3I show sustained high glucose treatment has genome-wide impact on translation. Figure 3 A shows an embodiment of a workflow showing MIN6 cells incubated in media containing 5.5 mM or 25 mM GLU for 24 hours and analyzed by ribosome profiling using RNA sequence analysis of ribosome protected footprints (RPFs, translatome) and total RNA (transcriptome). Figure 3B shows RPF read lengths with boxes indicating 25thto 75thpercentiles, line in middle of box is median, and whiskers indicating smallest to largestvalues. Figure 3C shows distribution of reads to coding sequence (CDS, hatched bars), 5’UTR (open bars), 3’UTR (solid bars) for RNAs and RPFs. Figure 3D shows triplet periodicity of RPFs near CDS start and stop. Figure 3E, 3F, and 3G show volcano plots of - log FDR vs. Iog2 fold change (FC), calculated for 25 mM vs. 5.5 mM GLU for RNA (3E, dotted line FDR = 0.01), RPF (3F, dotted line FDR = 0.1) and translation efficiency (TE = RPF / RNA, 3G, dotted line FDR = 0.1). Figure 3H and 31 show representative Reactome gene sets over-represented at a significance threshold FDR < 0.05 as upregulated (3H) and downregulated (31) by 25 vs. 5.5 mM glucose, n = 8 independent samples / condition.

[0049] Figures 4A-4G show sustained high glucose treatment has genome-wide impact on nascent proteome. Figure 4A shows representative workflow in which MIN6 cells were incubated for 24 hours in media containing 5.5 mM or 25 mM GLU followed by nascent proteomics analysis. Figure 4B shows surrogate variable PCA analysis. Figure 4C shows volcano plot of -log FDR v. log2FC, calculated for 25 vs. 5.5 mM GLU. Figure 4D shows correlation analysis of nascent proteomics Z-scores vs. RPF z-scores. Figure 4E and 4F show overlap of proteins upregulated (4E) or downregulated (4F) by 25 mM vs. 5 mM GLU in both TE and nascent proteomics datasets, -log FDR > 1 and log2FC > 20%. Figure 4G shows representative RPF gene coverage plots for SCGN and IDH2, showing no evidence for new upstream open reading frames or pausing in 25 mM GLU. n = 8 independent samples per condition. CDS, coding sequence.

[0050] Figures 5A-5B show translational regulation by sustained high glucose impacts protein abundance in MIN6 cells. MIN6 cells incubated for 24 h in media containing 5.5 mM (solid gray bars) or 25 mM (hatched gray bars) GLU. Figure 5A shows qPCR quantification of polysome and total RNA for Insl, Ins2, Scgn, Slc2a2, Pfkfb3, Slc30a8, Vps41, Idh2, and Igf2, relative to 18S rRNA. Actb and Tubgl as controls. Means ± SE for n = 3 - 4 independentexperiments. Figure 5B shows immunoblot of cell lysates for SCGN, SLC2A2, PFKFB3, SLC30A8, VPS41, IDH2, and IGF2. Tubulin loading control. Tubulin for SLC2A2 panel and SLC30A8 panel are identical, since they were the same lanes on the gel. Representative blots with quantification of means ± SE for n = 3 - 7 independent experiments. *, P < 0.05 by unpaired t-test (5 A) or ratio-paired t-test (5B).

[0051] Figures 6A-6B show regulation by sustained high glucose impacts protein abundance in rat islets. Primary rat islets incubated for 4 days in media containing 5.5 mM (solid gray bars) or 16.7 mM (hatched gray bars) GLU. Figure 6A shows qPCR quantification of ribosome-associated and total RNA for Insl, Ins2, Scgn, Slc2a2, Pfkfb3, Slc30a8, Vps41, Idh2, andlgf2, relative to 18S rRNA. Actb and Tubgl as controls. Means ± SE for n = 3 - 4 rats. Figure 6B shows representative immunoblots of islet lysates for SCGN, SLC2A2, PFKFB3, SLC30A8, VPS41, IDH2, and IGF2. Tubulin loading control. Tubulin for SLC2A2 panel and SLC30A8 panel are identical, since they were same lanes on the gel. Quantification of means ± SE for n = 5 - 6 rats. *, P < 0.05 by unpaired t-test (6A) or ratio-paired t-test (6B).

[0052] Figures 7A-7F show translational regulation by sustained high glucose impacts protein abundance in human islets. Figure 7A shows work flow in which human cadaveric islets were cultured for 2 days in media containing 5.5 mM (solid gray bars) or 20 mM (hatched gray bars) GLU, followed by 1 hour rest in 2.8 mM GLU, and GSIS quantified at 2.8 mM (B) and 16.7 mM (S) GLU. Figure 7B shows GSIS normalized by DNA with stim index quantified as stimulatory / basal secretion. Figure 7C shows insulin content normalized to DNA. Figure 7D shows GSIS normalized to cellular insulin content. Figure 7E shows qPCR quantification of ribosome-associated and total RNA for INS, SCGN, SLC2A1, PFKFB3, SLC30A8, VPS41, IDH2, IGF2, and SLC2A2, relative to 18S rRNA. TUBG1 as control. Figure 7F shows representative immunoblots of islet lysates for INS, SCGN,SLC2A1, PFKFB3, SLC30A8, and VPS41. Tubulin as control. Tubulin for SCGN panel and VPS41 panel are identical, since they were the same lanes on the gel. Quantification of means ± SE for n = 5 donors. *, Padj< 0.05 by pre-planned paired t-test (Bonferroni post-hoc correction, 7B [GSIS], D). #, P < 0.05 by unpaired t-test (7B [stim index], 7C), paired t-test (7E), or by ratio-paired t-test (7F). ns, not significant.

[0053] Figures 8A-8D show translational regulation by hyperglycemia impacts protein abundance in partial pancreatectomy model of hyperglycemia. Figure 8 A shows workflow in which islets were isolated from Sprague-Dawley rats 10 weeks after sham or 90% pancreatectomy (PX) surgery. Figure 8B shows fed blood glucose. Means ± SE for n = 21 sham rats; n = 29 PX rats. Figure 8C shows qPCR quantification of ribosome-associated and total islet RNA for Insl, Ins2, Scgn, Slc2a2, and Slc30a8, relative to 18S rRNA. Tubgl as control for sham (solid gray bars) vs. PX (hatched gray bars). Means ± SE for n = 5 - 7 samples, each pooled from 2-3 rats. Figure 8D shows representative immunoblots of islet lysates for INS, SCGN, SLC2A2, and SLC30A8 with tubulin as control. Tubulin for INS panel, SCGN panel, and SLC30A8 panel are identical, since they were the same lanes on the gel. Quantification of means ± SE for n = 5 samples, each pooled from 2-3 rats. *, P < 0.05 by unpaired t-test (B, C, and D). ns, not significant.

[0054] Figures 9A and 9B show that sustained high glucose decreases RPLP1 on translating ribosomes. MIN6 cells treated with 25 mM vs. 5.5 mM glucose (GLU) for 24 hours. Actively translating ribosomes isolated by sucrose density gradient fractionation. Figure 9A shows core ribosomal proteins quantified by tandem mass tag (TMT)-labeled proteomics. Figure 9B shows immunoblot analysis of RPLP1 relative to other core ribosomal proteins in total cell lysates and actively translating polysomes with quantification. Means ± SE for n = 6 per condition. *, P <0.05.

[0055] Figures 10A and 10B show that sustained high glucose causes RPLP1 to dissociate from actively translating polysomes to a soluble pool. MIN6 cells were transduced with a lentivirus expressing wild type FLAG-tagged RPLP1. Following 24-hour treatment with 5.5 vs. 25 mM glucose (GLU), RPLP1 was assessed by immunoblotting of polysome (10A) and soluble fractions (10B). RPL27 and tubulin served as controls in polysome and soluble fractions, respectively. Means ± SE for n = 3 per condition. *, P < 0.05.

[0056] Figure 11 shows that mutation of serine 101 and serine 104 to the unphosphorylatable amino acid alanine mimics the effect of sustained high glucose on RPLP1 polysome association. MIN6 cells were transduced with lentiviruses expressing wild type FLAG-tagged RPLP1 or RPLP1S1O1A / S1O4A. Anti -FLAG immunoblot was used to assess quantity of epitopetagged wild type or mutant RPLP1 in lysate and in actively translating polysomes with RPL27 as a control. Means ± SE for n = 3 per condition. *, P < 0.05

[0057] Figure 12 shows a model of how RPLP1 localization is regulated by phosphorylation. RPLP1, an exchangeable acidic ribosomal protein, binds to the stalk of the large subunit.RPLP1 undergoes reversible phosphorylation of two serine residues (S101 and SI 04) near its carboxyl terminal. Soluble RPLP1 is unphosphorylated, whereas phosphorylation drives ribosome association. As described herein, sustained high glucose can cause RPLP1 to dissociate from ribosomes.

[0058] DESCRIPTION OF THE SEQUENCES

[0055] A listing of certain sequences referenced herein is provided in Table 5 below. A sequence listing is also included in this application, as referenced above.DESCRIPTION OF CERTAIN EMBODIMENTSI. Ribosomal remodeling in diabetes

[0056] Ribosomes in eukaryotes are comprised of approximately 80 core ribosomal proteins and four ribosomal RNAs (rRNAs) (57). In some embodiments, diversity of ribosomes, and their function, is regulated by diversity and modifications of rRNA. In addition, ribosomal protein composition and modifications can also impact ribosomal diversity and function.

[0057] As described herein, “ribosomal remodeling” refers to a change in protein composition within a ribosome, such as a change in stoichiometry of various ribosomal proteins. In other words, the term ribosomal remodeling can be used to describe changes in ribosomal protein abundances within ribosomes. In some embodiments, ribosomal remodeling allows for cell-specific differences in ribosomal composition. In some embodiments, ribosomal remodeling is a mechanism for dynamic control of translation of mRNAs in response to changing environmental stimuli such as nutrients. As such, ribosomal remodeling can have regulatory effects on translation, leading to differences in expression of proteins within a cell.

[0058] In some embodiments, pharmacological or genetic interventions to drive proteins onto or off the ribosome for ribosomal remodeling can be used to effect changes in protein expression and cellular functions. In some embodiments, ribosomal proteins may dynamically associate and dissociate from ribosomes during ribosomal remodeling. In some embodiments, post-translational modifications of ribosomal proteins leads to ribosomal remodeling.A. RPLP1 composition in ribosomes

[0059] Ribosome-associated ribosomal protein Pl (also known as large ribosomal subunit protein Pl or RPLP1, UniProt entry P05386) is an acidic protein that binds to the flexible P-stalk of the large ribosomal subunit, which forms part of the GTPase-associated center where translation factors engage and where GCN2 interacts to regulate translation in response to nutrient stress (58, 59). Unlike most core ribosomal proteins, RPLP1 exchanges between ribosome-bound and soluble protein pools (60). When bound to the ribosome, RPLP1 is phosphorylated on two serine residues (S101 and SI 04) near its carboxyl terminus, whereas soluble RPLP1 is dephosphorylated (61-64). Moreover, phosphorylation is required for and drives ribosome association of RPLP1 (65-67).

[0060] The amino acid sequence of RPLP1 is shown here, with underlining of serine 101 and 104:MAS VSELACIYSALILHDDEVTVTEDKIN ALIK AAGVNVEPFWPGLFAKALANVNIG SLICNVGAGGPAPAAGAAPAGGPAPSTAAAPAEEKKVEAKKEESEESDDDMGFGLF D (SEQ ID NO: 1).

[0061] The present disclosure shows changes in RPLP1 associated with ribosomes in high glucose conditions (see, for example, Figures 9A and 9B). In some embodiments, hyperglycemic conditions may increase the percentage of soluble RPLP1 and / or decrease the amount of ribosome-associated RPLP1 (Figure 10). Described herein are means of blocking ribosomal remodeling based on RPLP1, such as with kinase activators or phosphatase inhibitors. Also described are means of blocking this ribosomal remodeling with gene editing of RPLPl.

[0062] In some embodiments, ribosomal remodeling occurs in pancreatic beta-cells. In some embodiments, the presence ofRPLPl within ribosomes in pancreatic beta-cells is altered due to the process of ribosomal remodeling. In some embodiments, association of RPLPl with the ribosome increases insulin translation and secretion in pancreatic beta-cells. In some embodiments, phosphorylation of RPLPl leads the protein to associate with the ribosome anddephosphorylation leads the protein to dissociation from the ribosome. In some embodiments, association of RPLP1 with the ribosome can be measured using isolation of actively translating ribosomes by sucrose density gradient fractionation (as shown in Figure 9A). In some embodiments, targeting posttranslational modification of RPLP1 affects its incorporation into ribosomes, which could have therapeutic implications with respect to insulin secretion.II. Methods of treating or preventing diabetes

[0063] In some embodiments, diabetes in a patient is treated or prevented by regulating RPLP1. In some embodiments, the subject has Type I (Tl) or Type II (T2) diabetes. In some embodiments, the subject is at risk for T2 diabetes.

[0064] The term “treating” or “treatment,” as used herein, covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of type 1 diabetes subjects may comprise alleviating hyperglycemia as compared to a time point prior to administration or reducing the subject’s need for exogenous insulin administration.

[0065] The term “preventing” or “prevention,” as used herein, covers any administration or application of a therapeutic for disease in a subject for the purpose of blocking the occurrence of one or more symptoms of the disease. For example, preventing diabetes may include administration or application of a therapeutic in a subject with a family history of diabetes type 1, wherein the incidence of hyperglycemia or immune attack on pancreatic beta-cells associated with type 1 diabetes is reduced by the administration.

[0066] In each embodiment of the invention, the subject treated is a mammal. In one embodiment, the mammal is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In embodiment, the subject is a human subject.

[0067] Glucose levels in the blood are normally tightly regulated to maintain an appropriate source of energy for cells of the body. Dysregulation of blood sugar must be ameliorated to maintain health and longevity, and therapies that are fast acting are especially desired. Such fast acting therapies allow subjects to monitor blood glucose in real time and immediately self-medicate themselves to bring glucose levels within normal limits. Dosing with exogenous insulin is one example of a fast-acting glucose modulator that has allowed subjects with diabetes to maintain relatively normal lifestyles. Described herein is a noninsulin fast-acting compound that regulates blood glucose levels in real-time.

[0068] Insulin and glucagon are principal hormones that regulate blood glucose levels. In response to an increase in blood glucose, such as after a meal, insulin is released from betacells of the pancreas. Insulin regulates the metabolism of carbohydrates and fats by promoting uptake of glucose from the blood into fat and skeletal muscle. Insulin also promotes fat storage and inhibits the release of glucose by the liver. Regulation of insulin levels is a primary means for the body to regulate glucose in the blood.

[0069] When glucose levels in the blood are decreased, insulin is no longer released and instead glucagon is released from the alpha cells of the pancreas. Glucagon causes the liver to convert stored glycogen into glucose and to release this glucose into the bloodstream. Thus, insulin and glucagon work in concert to regulate blood glucose levels.

[0070] In one embodiment, treatment of diabetes mellitus is to administer a composition to a subject to lower blood glucose.

[0071] Hyperglycemia refers to an increased level of glucose in the blood as compared to the levels normally seen in a healthy subject. Hyperglycemia can be associated with high levels of sugar in the urine, frequent urination, and increased thirst. Diabetes mellitus refers to a medical state of hyperglycemia.

[0072] The American Diabetes Association (ADA) suggests that fasting plasma glucose (FPG) levels of 100 mg / dL to 125 mg / dL or HbAlc levels of 5.7% to 6.4% may be considered hyperglycemia and may indicate that a subject is at high risk of developing diabetes mellitus (i.e. prediabetes, see ADA Guidelines 2015).

[0073] The ADA states that a diagnosis of diabetes mellitus may be made in a number of ways. A diagnosis of diabetes mellitus can be made in a subject displaying an HbAlc level of >6.5%, an FPG levels of >126 mg / dL, a 2-hour plasma glucose of >200 mg / dL during an OGTT, or a random plasma glucose level >200 mg / dL in a subject with classic symptoms of hyperglycemia.

[0074] Diabetes mellitus can be classified as Type 1 or Type 2. Type 1 diabetes mellitus (previously known as insulin-dependent diabetes or juvenile diabetes) is an autoimmune disease characterized by destruction of the insulin-producing beta-cells of the pancreas. Classic symptoms of Type 1 diabetes mellitus are frequent urination, increased thirst, increased hunger, and weight loss. Subjects with Type 1 diabetes mellitus are dependent on administration of insulin for survival.

[0075] Type 2 diabetes mellitus is a metabolic disease characterized by a relative decrease in insulin levels and / or a phenotype of insulin resistance. Insulin resistance refers to when cells of the body no longer respond appropriately to insulin. The risk of Type 2 diabetes mellitus is increased in individuals who are obese or who have a sedentary lifestyle.

[0076] In the absence of regulation of glucose levels in subjects with diabetes, a range of serious complications may be seen. These include atherosclerosis, kidney disease, stroke, nerve damage, and blindness.

[0077] A method of treating diabetes mellitus comprising administering a composition is encompassed. In one embodiment, the method comprises lowering blood glucose levels in the diabetic subject to below about 200 mg / dL, 150 mg / dL, 100 mg / dL, or about 125 mg / dL.

[0078] In some embodiments, treatment of diabetes is increasing insulin levels in the subject after administering a composition.

[0079] In some embodiments, administering a composition causes a decrease in blood glucose levels such that levels are less than 200 mg / dL.

[0080] In some embodiments, the subject treated with a composition has Type 1 diabetes mellitus. In some embodiments, the diabetic subject treated has a relative decrease in insulin levels as compared to a healthy control subject. In some embodiments, the subject treated has decreased beta-cell mass as compared to a healthy control subject. In some embodiments, the decrease in beta-cell mass in a subject is due to an autoimmune disease.

[0081] In some embodiments, the subject treated has diabetes mellitus based on diagnosis criteria of the American Diabetes Association. In some embodiments, the subject with diabetes mellitus has an HbAlc level of >6.5%. In some embodiments, the subject with diabetes mellitus has fasting plasma glucose (FPG) levels of >126 mg / dL. In some embodiments, the subject with diabetes mellitus has a 2-hour plasma glucose of >200 mg / dL during an oral glucose tolerance test (OGTT). In some embodiments, the subject with diabetes mellitus has a random plasma glucose level >200 mg / dL or 11.1 mmol / L. In some embodiments, the subject with diabetes mellitus has a random plasma glucose level >200 mg / dL or 11.1 mmol / L with classic symptoms of hyperglycemia. In some embodiments,administering an agent as described herein improves one or more of these markers of diabetes.

[0082] In some embodiments, treating diabetes in a subject reduces the subject’s HbAlc level to 6.5% or less. In some embodiments, treating diabetes in a subject reduces the subject’s FPG levels to 126 mg / dL or less. In some embodiments, treating diabetes in a subject reduces the subject’s 2-hour plasma glucose during an OGTT to 200 mg / dL or less. In some embodiments, after treatment, a subject with diabetes mellitus has a random plasma glucose level of 200 mg / dL or less or 11.1 mmol / L or less without classic symptoms of hyperglycemia after treating diabetes in the subject.

[0083] In some embodiments, a method herein is performed to prevent diabetes and / or to reduce the incidence of hyperglycemia or immune attack on pancreatic beta-cells associated with type 1 diabetes in the subject. In some embodiments, the subject’s HbAlc level is 6.5% or less when diabetes is prevented. For example, in some embodiments, the subject has an HbAlc level of 5.8-6.5% or of 5.8-6.4%. In some embodiments, the subject’s FPG levels are 126 mg / dL or less when diabetes is prevented. In some embodiments, the subject’s the subject’s 2-hour plasma glucose during an OGTT is 200 mg / dL or less when diabetes is prevented. In some embodiments, a subject with diabetes mellitus has a random plasma glucose level of 200 mg / dL or less or 11.1 mmol / L or less without classic symptoms of hyperglycemia when diabetes is prevented. For example, in some cases, incidence of hyperglycemia or immune attack on pancreatic beta-cells associated with type 1 diabetes is reduced in the subject, and / or the subject’s HbAlc level remains 6.5% or less following administration.A. Changes in RPLP1 in high glucose conditions

[0084] As described herein, translation of a number of proteins critical for insulin secretion by pancreatic beta-cells is decreased by high glucose conditions (see, for example, Table 2 below) and leads to decreased steady-state abundance of these proteins (see, for example, Figures 5, 6, and 7). Such a decrease in expression of insulin synthesis / secretion genes could initiate conditions wherein initial high glucose levels are exacerbated by deficiencies in insulin synthesis or secretion. As described herein, decreases in the percentage of RPLP1 in ribosomes and / or increases in percentage of soluble RPLP1 could lead to these detrimental changes in protein translation.

[0085] In some embodiments, a method of treating or preventing diabetes in a subject in need thereof comprises increasing the percentage of ribosome-associated RPLP1 or decreasing the percentage of soluble RPLP1 in a cell of the subject. In some embodiments, this method comprises phosphorylating RPLP1, inhibiting dephosphorylation of RPLP1, and / or expressing an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell, thereby increasing the percentage of ribosome-associated RPLP1 and / or decreasing the percentage of soluble RPLP1 in the cell and / or in the subject. In some embodiments, increasing the percentage of ribosome-associated RPLP1 or decreasing the percentage of soluble RPLP1 in the subject is in comparison to the same subject before the treating. In some embodiments, increasing the percentage of ribosome-associated RPLP1 or decreasing the percentage of soluble RPLP1 in the cell is in comparison to the cell before the treating. In some embodiments, high glucose cellular conditions in a subject with diabetes leads to a low percentage of RPLP1 associated with the ribosome and / or a high level of soluble RPLP1 in the subject, and a treatment described herein can reverse these effects of high glucose. Insome embodiments, association of RPLP1 with the ribosome can be measured using isolation of actively translating ribosomes by sucrose density gradient fractionation.

[0086] In some embodiments, increased RPLP1 phosphorylation at serine 101 and / or serine 104 of SEQ ID NO: 1 increases incorporation of RPLP1 into ribosomes as compared to RPLP1 that is not phosphorylated at serine 101 and / or serine 104. In some embodiments, increased RPLP1 phosphorylation at serine 101 and / or serine 104 of SEQ ID NO: 1 decreases levels of soluble RPLP1 in cells as compared to the same cells before the treatment. In some embodiments, increased RPLP1 phosphorylation at serine 101 and / or serine 104 of SEQ ID NO: 1 in a pancreatic beta-cell changes expression of one or more proteins in the beta-cell as compared to the expression before treatment. In some embodiments, the change in expression is in one or more proteins associated with insulin production and / or secretion. In any of these embodiments, inhibiting dephosphorylation of RPLP1 may have the same effect as increasing phosphorylation of RPLP1.

[0087] In some embodiments, increasing the percentage of ribosomes containing RPLP1 increases translation of one or more proteins encoded by a gene selected from INS (gene encoding insulin; Gene ID: 3630), SCGN (gene encoding secretagogin; GENE ID: 10590), IDH2 (gene encoding isocitrate dehydrogenase; GENE ID: 3418), VPS41 (gene encoding VPS41 subunit of HOPS complex: GENE ID: 27072), SLC2A1 (solute carrier family 2 member 1; GENE ID: 6513), IGF2 (insulin like growth factor; GENE ID: 3481), SLC30A8 (solute carrier family 30 member 8; 169026), and PFKFB3 (6-phosphofructo-2- kinase / fructose-2,6-biphosphatase 3; GENE ID: 5209). In some embodiments, the increased translation of the one or more proteins is in comparison to the same cell before being treated with an agent as described herein.

[0088] In some embodiments, administering a kinase activator or phosphatase inhibitor increases levels of phosphorylated RPLP1 in a cell, as compared to levels of phosphorylated RPLP1 before the administering. In some embodiments, administering a kinase activator or phosphatase inhibitor decreases levels of dephosphorylated RPLP1 in a cell, as compared to levels of dephosphorylated RPLP1 before the administering. In some embodiments, administering a system for gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids. In some embodiments, the administering of the kinase activator or phosphatase inhibitor or of the system for gene editing can be by a buccal, enteral, inhalable, infused, intramuscular, intrathecal, intravenous, nasal, ophthalmic, oral, otic, rectal, subcutaneous, sublingual, topical, or transdermal route. In some embodiments, the administering of a gene editing system, for example, may be to a cell in vitro.

[0089] In some embodiments, the cell of the subject is a pancreatic beta-cell. In some embodiments, the method increases the levels of insulin within or released by the cell as compared to levels before administering of an agent described herein. As such, the method can improve symptoms or diabetes and / or decrease blood sugar levels as compared to before the administering.B. Methods of Regulating RPLP1 Composition in Ribosomes by Kinases and Phosphatases

[0090] Kinases and phosphatases are druggable targets, and pharmacologically activating the kinase responsible for RPLP1 phosphorylation or inhibiting the phosphatase that dephosphorylates the protein would be an approach to increase RPLP1 association with the ribosome.

[0091] In some embodiments, RPLP1 phosphorylation is increased by treatment of a cell with a kinase activator. In some embodiments, RPLP1 phosphorylation is increased bytreatment of a cell with a phosphatase inhibitor. In some embodiments, the increase in RPLP1 phosphorylation is in comparison to the same cell before treatment. In any of these embodiments, inhibiting dephosphorylation of RPLP1 may have the same effect as increasing phosphorylation of RPLP1.

[0092] In some embodiments, RPLP1 phosphorylation is measured using radiolabeling or Western blot or immunoprecipitation with a phosphospecific antibody or mass spectrometry.

[0093] In some embodiments, the RPLP1 phosphorylation is at serine 101 of SEQ ID NO: 1. In some embodiments, the RPLP1 phosphorylation is at serine 104 of SEQ ID NO: 1. In some embodiments, the RPLP1 phosphorylation is at both serine 101 and serine 104 of SEQ ID NO: 1.

[0094] In some embodiments, increasing RPLP1 phosphorylation at serine 101 and / or 104 by treatment of a cell with a kinase activator or phosphatase inhibitor increases the percentage of RPLP1 associated with the ribosome as compared to the same cell before treatment. In some embodiments, increasing RPLP1 phosphorylation at serine 101 and / or 104 by treatment of a cell with a kinase activator or phosphatase inhibitor increases the percentage of RPLP1 associated with the ribosome as compared to the same cell before treatment.

[0095] In some embodiments, increasing RPLP1 phosphorylation or inhibiting dephosphorylation of RPLP1 at serine 101 and / or 104 by treatment with a kinase activator or phosphatase inhibitor leads to a change in expression of one or more protein in the cell. In some embodiments, the one or more protein is associated with insulin synthesis and / or secretion. In some embodiments, increasing RPLP1 phosphorylation or inhibiting dephosphorylation of RPLP1 at serine 101 and / or 104 by treatment with a kinase activator or phosphatase inhibitor increases translation of one or more proteins encoded by a gene selected from INS, SCGN, IDH2, VPS41, SLC2A1, IGF2, SLC30A8, and PFKFB3. In someembodiments, the increased translation of one or more proteins is in relation to the level of translation of the same protein in the same cell before treatment with an agent described herein.

[0096] In some embodiments, the cell treated is a pancreatic beta-cell, and the subject is treated with a kinase activator or phosphatase inhibitor.

[0097] In some embodiments, insulin levels in the subject are increased and / or glucose levels in the subject are decreased by the treatment in comparison to the levels in the patient before treatment. As such, the subject may be able to reduce or eliminate treatment with other agents for diabetes management, such as insulin or other pharmacologic therapy.C. Methods of Regulating RPLP1 Composition in Ribosomes by Gene Editing

[0098] In some embodiments, an RPLP1 mutant is prepared comprising one or more phosphomimetic amino acids. As used herein, a “phosphomimetic amino acid” refers to an amino acid substitution in a protein that mimics a phosphorylated protein. In some embodiments, a phosphomimetic amino acid has the same characteristics as the phosphorylated wildtype protein (without the substitution). A RPLP1 mutant comprising a phosphomimetic amino acid may have the phosphomimetic acid at a position that can be phosphorylated physiologically, such as a serine, tyrosine, or threonine.

[0099] In some embodiments, the phosphomimetic amino acid is at position S101 and / or position S104 of SEQ ID NO: 1, and the characteristics of RPLP1 comprising the phosphomimetic amino acid is the same or similar to the characteristics of wildtype RPLP1 that is phosphorylated at that position. In some embodiments, the one or more phosphomimetic amino acid mutation is an aspartic acid or glutamic acid. In some embodiments the RPLP1 mutant comprises a S101D or S104D substitution in SEQ ID NO: 1.In some embodiments the RPLP1 mutant comprises a S101E or S104E substitution in SEQ ID NO: 1. In some embodiments, the RPLP1 mutant comprises a S101D or S1014D substitution together with a S101E or S104E substitution.

[0100] In some embodiments, expression of a RPLP1 mutant comprising one or more phosphomimetic amino acids is performed by gene editing of RPLP1. In some embodiments, the gene editing is performed with a system for gene editing.

[0101] A wide range of gene editing systems are well-known in the art (68 and 69). In some embodiments, a system for gene editing comprises a CRISPR / Cas9 system, zinc-finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, or group one intron encoded endonuclease (GHEE), However, the present methods are not limited to these specific gene editing systems, as one skilled in the art would be well-aware that new systems from gene editing are being rapidly developed.

[0102] In some embodiments, the cell treated is an iPSC or pancreatic beta-cell in culture and gene editing is performed in vitro. In some embodiments, the treated cell is introduced into the subject after the gene editing. In some embodiments, the iPSC or pancreatic beta-cell is taken from the patient, treated with a system for gene editing in vitro, and reintroduced to the patient. In some embodiments, an iPSC or pancreatic beta-cell from a donor is treated with a system for gene editing in vitro and introduced to the patient. In other words, the iPSC or pancreatic beta-cell may be used for a homologous transplant (i.e., the donor of the cell and the patient treated are the same) or the iPSC or pancreatic beta-cell may be used for a heterologous transplant (i.e., the donor of the cell and the patient treated are the different).

[0103] In some embodiments, the cell is an iPSC or pancreatic beta-cell in a subject and gene editing is performed in vivo using a delivery system that selectively delivers thegene editing system to the iPSC or pancreatic beta-cell. A number of different means to target a delivery system to iPSC or pancreatic beta-cells are known in the art (68). For example, a virus that targets iPSCs or pancreatic beta-cells may be used to deliver a gene editing system. In this way, the gene editing system may be targeted in vivo the iPSC or pancreatic beta-cell.

[0104] In some embodiments, a method of treating or preventing diabetes in a subject in need thereof comprises preparing iPSCs and / or pancreatic beta-cells in vitro., treating said iPSCs or pancreatic beta-cells with an agent for gene editing of RPLP1, wherein the gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell; and transplanting the treated iPSCs or pancreatic beta-cells into the subject, wherein the transplanting increases insulin levels in the subject and / or decreases glucose levels in the subject in comparison to the levels in the subject before the transplanting.

[0105] In some embodiments, an RPLP1 mutant comprises one or more phosphomimetic amino acid mutations at an amino acid that is a serine in wildtype RPLP1. In some embodiments, the serine in wildtype RPLP1 is serine 101 and / or serine 104. In some embodiments, the one or more phosphomimetic amino acid mutation is an aspartic acid or glutamic acid.III. Compositions comprising a system for gene editing

[0106] Also described herein are compositions comprising (1) an iPSC or pancreatic beta-cell and (2) a system for gene editing of RPLP1. In some embodiments, the system for gene editing is a CRISPR / Cas9 system, zinc-finger nuclease, TALEN, meganuclease, or GIIEE, although any gene editing system may be used. In some embodiments, the system for gene editing is capable of introducing a phosphomimetic amino acid at positions serine 101and / or serine 104. In some embodiments, the phosphomimetic amino acid mutation is an aspartic acid or glutamic acid.EXAMPLESExample 1. Sustained high glucose impairs basal insulin translation

[0107] Chronic high glucose impairs glucose-stimulated insulin translation and secretion in human and rodent islets (14, 15). To determine whether sustained high glucose affects basal insulin translation, chronic high glucose exposure was modeled by incubating isolated rat islets in media containing 16.7 mM glucose versus 5.5 mM glucose. Islets were then rested in media with 2.8 mM glucose for 1 hour prior to assaying for GSIS (Figure 1 A). Compared to low glucose, islets incubated in high glucose for 4 days had increased basal insulin secretion and diminished response to stimulatory glucose with a 69% decrease in stimulation index, without impact on total islet insulin content (Figures 1B-1C). Shorter incubations in high glucose did not decrease GSIS (not shown). Exposure to high glucose over 4 days did not cause dedifferentiation or transdifferentiation, as expression of beta-cell identity genes was similar between the two glucose conditions (Figure ID). Although global protein synthesis was unchanged between the glucose conditions, sustained high glucose decreased the rate of insulin synthesis (Figure 1E-1F).

[0108] Pancreatic beta-cells are particularly susceptible to increased ER stress and prolonged ER stress is detrimental to beta-cell function (19, 20). However, treatment with high glucose over 4 days did not increase the phosphorylation of PERK or expression of ATF4, well-established upstream regulators of ER stress (Figure 1G).

[0109] Thus, 4 days exposure to sustained high glucose decreased basal translation of insulin prior to suppression of global protein synthesis, compromised beta-cell identity, or sustained engagement of the unfolded protein response pathway.Example 2. MIN6 cells model chronic high glucose effects on islet insulin translation

[0110] Pancreatic islets are micro-organs that consist of several cell types including glucagon-containing a-cells, somatostatin-containing 6-cells, and polypeptide-producing PP- cells in addition to insulin-producing beta-cells. To delineate the effects of chronic high glucose specifically on beta-cells and to identify a system that more readily provides sufficient material for high-throughput analyses, chronic high glucose exposure was modeled in early passage MIN6 insulinoma cells that support robust GSIS (21). These cells are typically propagated in 25 mM glucose to sustain rapid cell growth but can be maintained for limited periods in lower glucose with slower growth. Following incubation for 24 hours in 5.5 mM glucose, stimulatory glucose caused a 10-fold increase in insulin secretion (Figure 2A-2B). Basal insulin secretion did not increase in MIN6 cells maintained in high glucose, contrary to observations in islets, but similar to islets, the stimulation index was decreased by 59% by high glucose. Although high glucose decreased insulin content of MIN6 cells, the impact of sustained high glucose on GSIS remained significant even when secretion was calculated as a percent of insulin content (Figure 2C-2D). This did not reflect a general response to increased osmolarity, since incubation in low glucose media supplemented with mannitol did not recapitulate the effect of sustained high glucose (Figure 2E). As observed in rat islets, beta-cell identity markers were similar in both glucose conditions, and sustained high glucose specifically decreased insulin translation without affecting global protein synthesis or inducing ER stress (Figure 2F-2I). Taken together, these results demonstrate that 24-hour treatment of MIN6 cells with 25 mM glucose vs 5.5 mM glucose largely models the effects of sustained high glucose treatment of primary rodent islets.Example 3. Broad impact of sustained high glucose on gene-specific mRNA translation based on ribosomal profiling

[0111] Incubation of MIN6 cells or islets in high glucose media supplemented with high concentrations of the saturated fatty acid palmitate induces ER stress and has profound effects on mRNA translation (17, 18). To determine the genome-wide effects of sustained high glucose alone on beta-cell mRNA translation in the absence of ER stress, the translatome of MIN6 cells in low vs. high glucose was evaluated by ribosome profiling. This RNA-sequencing method is based on the principle that more efficiently translated mRNAs are associated with more ribosomes and therefore generate more ribosome protected footprints (RPFs) upon nuclease digestion. Both RPF and total RNA libraries were sequenced from cells following treatment for 24 hours with 25 mM vs. 5.5 mM glucose (Figure 3A). As observed in other ribosome profiling studies of mammalian cells (22), peak RPF fragment sizes were 30-35 base pairs, RPFs were enriched for open reading frames of genes compared to mRNAs, and RPF sequences showed triplet periodicity (Figure 3B-3D). Sustained high glucose had substantial impact on the transcriptome (Figure 3E), consistent with prior studies (16), and on the translatome (Figure 3F). To identify genes for which glucose treatment specifically altered mRNA translation regulation, the translation efficiency (TE) was calculated as the ratio of normalized RPF reads to normalized total mRNA reads per gene (Figure 3G). This measure accounts for changes in transcription and enables identification of genes for which changes in translation do not simply parallel changes in mRNA abundance. Using FDR < 0.1, sustained high glucose was found to up-regulate 3393 genes and down- regulate 3382 genes (data not shown).

[0112] Among genes for which TE was up-regulated by chronic high glucose, pathways related to chromatin organization, RNA splicing, translation, deubiquitination, andM phase of cell cycle were over-represented (Figure 3H). Among genes for which TE was down-regulated by chronic high glucose, pathways important for beta-cell function including insulin processing, ER-to Golgi transport, glucose metabolism, and TCA cycle were over- represented (Figure 31).

[0113] Thus, independent of ER stress or change in global protein synthesis rates, sustained high glucose had genome-wide effects on translation. Moreover, in addition to insulin, sustained high glucose down-regulated translation of other genes required for metabolism-coupled insulin secretion.Example 4. Nascent proteomics is an independent measure of glucose-altered translation

[0114] Ribosome profiling analysis in MIN6 cells identified a large number of genes for which translation was affected by sustained high glucose based on the ratio of RPFs to total mRNA. To corroborate and filter these results, an orthogonal method of translation analysis was used in which nascent peptides were pulse labeled with the methionine analogue azidohomoalanine (AHA), click biotin conjugated, and enriched by streptavidin pulldown up front of mass spectrometry-based proteomics (Figure 4A). Principle component analysis revealed distinct patterns of nascent protein synthesis under the different glucose treatment conditions (Figure 4B). Sustained high glucose significantly affected new peptide synthesis for many genes, and this correlated well with abundance of RPF sequence (Figure 4C-4D).

[0115] To identify high confidence glucose-driven translation changes, the overlap between nascent proteomics and ribosome profiling TE was evaluated, focusing on genes for which changes in both analyses met FDR < 0.1 and 20% log2 fold. Based on these criteria, 207 genes were upregulated by sustained high glucose and 183 genes were downregulated (Figures 4E-4F, Table 1).

[0116] Given that sustained high glucose down-regulates GSIS and insulin translation, a focus was on down-regulated proteins known to function in insulin production and metabolism-coupled insulin secretion (Table 2). RPF gene coverage analysis revealed no evidence that translational downregulation resulted from selection of new upstream open reading frames or pausing under sustained high glucose conditions (Fig. 4G).

[0117] SCGN (secretagogin) enhances second phase insulin secretion, and its knockdown impairs GSIS (23, 24). VPS41, a component of the homotypic function and vacuole protein sorting complex, and SLC30A8, which transports zinc into insulin granules, are both required for optimal GSIS (25, 26). SLC2A2, the plasma membrane glucose transporter in rodent beta-cells, and IDH2, which functions in reductive flux of glutamine to citrate in the mitochondria, are critical for metabolism-coupled insulin secretion (27, 28). While PFKFB3 and IGF2 were not detected in nascent proteomics, they were significantly downregulated by high glucose in ribosome profiling and were included in further analyses given established roles in potentiating insulin secretion (29, 30).Example 5. Validation of translational changes and impact on steady state protein abundance

[0118] To confirm these chronic glucose-induced translation changes, the ratio of mRNA associated was quantified with actively translating polysomes relative to total mRNA as a measure of TE in MIN6 cells incubated in 25 vs. 5.5 mM glucose for 24 hours. Sustainedhigh glucose decreased TE of Insl, Ins2, Scgn, Slc2a2, Pfkfb3, Slc30a8, Vps41, Idh2, and Igf2 (Figure 5A). Actb and Tubl were unchanged by high glucose, consistent with lack of change in nascent protein. To determine whether changes in TE impacted protein levels, steady-state protein abundance was measured in lysates of MIN6 cells treated with 25 vs. 5.5 mM glucose. Sustained high glucose significantly decreased cellular content of SCGN, SLC2A2, PFKFB3, SLC30A8, VPS41, IDH2, and IGF2 (Figure 5B).

[0119] Although MIN6 cells were an important tool for technically challenging high- throughput discovery studies, these cells replicate rapidly, grow dispersed in cell culture, and lack complex cellular make-up and architecture of islets. The findings were next validated in isolated rat islets using the conditions as in Figure 1 A established above that impair glucose stimulated insulin translation and secretion. Given the large amount of tissue needed to collect actively translating polysomes and limited number of islets, ribosome-associated (rather than polysome) mRNA relative to total mRNA was quantified as a measure of TE. Sustained high glucose decreased TE for Insl, Ins2, Scgn, Slc2a2, Pfkfb3, Slc30a8, Vps41, Idh2, and Igf2, without affecting TE of Actb and Tubgl (Figure 6A). As observed in MIN6 cells, this led to significantly decreased steady-state protein abundance for SCGN, SLC2A2, PFKFB3, VPS41, IDH2, and IGF2 and a trend for decrease in SLC30A8 that did not reach significance (Figure 6B). Collectively, the results in primary islets and in MIN6 cells confirm findings from ribosome profiling and provide evidence that translational regulation has a meaningful impact on the beta-cell proteome.

[0120] To investigate the clinical relevance of the observed glucose effects on translation, TE and protein abundance was analyzed following incubation of cadaveric human islets in 20 vs. 5.5 mM glucose (Figure 7A). Exposure of human islets to high glucose for 2 days was sufficient to increase basal insulin secretion and decrease stimulation index (Figures7B and 7D), consistent with previous reports (16), and decreased insulin content (Figure 7C). TE, as assessed by ribosome-associated / total mRNA was decreased for Ins, Scgn, Pfkfb3, and Vps41 (Figure 7E). For Slc30a8, trend for decreased TE was not statistically significant and TE for Idh2 and Igf2 were unchanged. Although translation of Slc2a2 was unchanged, TE for Slc2al, the main plasma membrane glucose transporter in human islets (31), was decreased. Consistent with findings in rodent islets, decreased TE led to decreased steady-state protein abundance for INS, SCGN, SLC2A1, PFKFB3, and SLC30A8, and (Figure 7F). VPS41 protein, however, was unchanged.

[0121] To extend these findings to an in vivo model of hyperglycemia, partial (90%) pancreatectomy (PX) or sham surgery was performed in adult male rats (Figure 8A). Despite partial regeneration during the initial weeks of recovery, PX animals have sustained mild hyperglycemia and show selective loss of glucose-stimulated insulin secretion at 10 weeks post-surgery (32). As expected, PX rats had modest, but significantly elevated, fed blood glucose compared to sham animals (Figure 8B). Islets isolated 10-weeks after PX demonstrated decreased TE for highly expressed genes including Insl, Ins2, Scgn, Slc2a2, and Slc30a8 compared to sham with no effect on TE of Tubgl control (Figure 8C). For genes expressed at lower levels (Vps41, Idh2, Pfkfb3, and Igf2), recovery of mRNAs was insufficient to quantify TE. Steady-state protein levels were decreased for INS, SCGN SLC2A2, and SLC30A8 proteins (Figure 8D). Thus, sustained exposure to high glucose in a pathophysiologically relevant setting suppressed translation of key mRNAs required for metabolism-coupled insulin secretion and abundance of their encoded proteins.

[0122] In summary, sustained high glucose selectively impairs mRNA translation of genes that serve critical roles at almost every step of glucose-metabolism coupled insulin secretion in pancreatic beta-cells. These nutrient-induced translation changes are coincidentwith impaired GSIS following prolonged exposure of cultured insulinoma cells or isolated islets ex vivo to high glucose and in the setting of 10 weeks of systemic hyperglycemia induced by partial pancreatectomy. These results show that programmatic dysregulation of beta-cell mRNA translation is a manifestation of glucose toxicity prior to the onset of ER stress or impairment of global translation. Translational downregulation decreases steady state levels of these proteins, which serve important roles in metabolism-coupled insulin secretion and optimal beta-cell function.

[0123] Beta-cells leverage structural and functional specializations for coupling glucose metabolism to robust insulin peptide production and secretion. The insulin mRNA is highly abundant, and ER and Golgi are extensive in beta-cells (33, 34). Moreover, translation of insulin and genes involved in insulin processing and secretory granule biogenesis is rapidly and coordinately upregulated when glucose is acutely increased from basal to stimulatory concentrations (8, 9). Recently, high throughput studies have shown that acute exposure to high glucose selectively upregulates translation of hundreds of beta-cell mRNAs (10, 11). The observations that mRNAs encoding proteins related to insulin processing, exocytosis, and glucose metabolism are enriched in polysomes and that production of these peptides is increased by acute glucose provide evidence that translation of functionally related proteins is coordinately regulated for optimal beta-cell function under physiological conditions. These data provide new evidence that concerted beta-cell translational regulation occurs in the pathophysiological setting of chronic high glucose exposure. Further, sustained high glucose conditions that impair GSIS are associated with translational downregulation of mRNAs required for metabolism-coupled insulin secretion. Moreover, these translational changes result in decreased protein abundance, which likely contributes to decreased secretory function.

[0124] Beta-cells synthesize up to a million proinsulin protein molecules per minute(35), creating a challenge for proper folding and processing of nascent proteins in the ER. Not surprisingly, prolonged exposure to high glucose can lead to ER-stress that activates the unfolded protein response to decrease total mRNA translation (36). The present experimental design incorporated ex vivo treatment of islets and MIN6 cells with glucose at concentrations and for durations that did not increase ER stress markers in order to model early nutrient- induced changes. Consistent with lack of engagement of the PERK-eIF2 alpha arm of ER- stress, total mRNA translation was unchanged under conditions in which translation of mRNAs involved in glucose-coupled insulin secretion was suppressed (37). It is also not surprising that ATF4 and JUND, proteins whose translation is increased under glucolipotoxic conditions that induce ER stress, were not upregulated in this study (17, 18). These results indicate that programmatic alterations in translation of specific mRNAs occurs prior to ER stress during the progression of beta-cell dysfunction.

[0125] Ribosome profiling has emerged as a powerful method for assessing mRNA translation in species ranging from yeast to human (38). However, TE calculated as the ratio of RPFs to RNA is an indirect measure of translation that could be confounded by increased RPFs resulting from translation pausing. The present strategy to also use nascent proteomics provided an approach to filter ribosome profiling results for translation changes that resulted specifically in synthesis of new proteins. As expected, only a fraction of newly synthesized proteins reflected altered TE, since nascent proteomics also includes changes in translation that result from increased mRNA abundance (39). These findings from MIN6 cell studies were validated in primary rat islets in which significant decreases in protein abundance for translationally down-regulated genes were demonstrated. In the rat partial pancreatectomy model, sustained mild hyperglycemia in vivo led to decreases in translation and proteinabundance for the majority of these genes for which mRNA is highly abundant. Inability to quantify less abundant RNAs was likely a consequence of the present experimental design to analyze islets immediately upon isolation without overnight recovery of the islets in order to capture the impact of in vivo glycemia. Human islet analyses largely phenocopied observations in rodent studies with several notable exceptions. First, the observation that TE for Slc2al, but not Slc2a2, was significantly decreased in human islets is likely attributable to differences in the glucose transporters utilized in these different species (Glut2 / Slc2a2 in rodent and GLUT1 / Slc2al in human beta-cells) (31). Second, absence of change in Idh2 and Igf2 translation in human islets may reflect species differences in regulation of insulin secretion, as their role has been best characterized in rodents (28, 30).

[0126] Regulatory steps following transcription play an important role in determining gene expression, and simultaneous RNA sequencing and proteomic analyses combined with metabolic labeling of macromolecules provides evidence that mRNA-specific translation rates are a major determinant of the cellular proteome (40). Moreover, the development of high throughput tools for discovery of coordinated mRNA-specific translation has advanced the understanding of how environmental cues shape gene expression. The present disclosure provides insights into nutrient-driven translational regulation that alters the abundance of proteins important for insulin secretion in settings of beta-cell dysfunction.Example 6. Sustained high glucose causes RPLP1 dissociation from actively translating ribosomes

[0127] To characterize the impact of sustained high glucose on the composition of actively translating ribosomes (polysomes) in beta-cells, quantitative TMT -labeled proteomics was used to analyze the stoichiometry of ribosomal proteins in polysomes isolatedfrom MIN6 cells treated with 25 mM vs 5.5 mM glucose for 24 hours. Compared to other core ribosomal proteins, RPLP1 was reduced by 40% in ribosomes from high glucose-treated cells, whereas other core ribosomal proteins were unchanged (Figures 9A and 9B). Proteomic findings were confirmed in independent samples of high vs. low glucose-treated cells. Thus, exposure of pancreatic beta-cells to sustained high glucose, not only decreases translation of key genes for beta-cell function, but also dynamically remodels actively translating beta-cell ribosomes and decreases RPLP1 association with actively translating ribosomes.

[0128] RPLP1 is one of the acidic proteins that binds to periphery of the ribosome on the flexible P-stalk of the large ribosomal subunit. In contrast to most core ribosomal proteins that assemble co-transcriptionally onto ribosomal RNAs in the nucleolus and are degraded if not incorporated into nascent ribosomes, RPLP1 joins the nascent ribosome in late steps of biogenesis in the cytoplasm, where it is present in a soluble pool that exchanges with the ribosome-bound RPLP1 (Tsurugi K, Ogata K. (1985) J Biochem. 98(6): 1427-31). Data herein suggest a model in which glucose toxicity causes remodeling of the ribosome structure that mediates mRNA-specific translational dysregulation in the setting of hyperglycemia.

[0129] To test whether altered stoichiometry of RPLP1 on polysomes reflects exchange of RPLP1 between ribosomes and a soluble pool, polysome and soluble fractions of MIN6 cells expressing amino-terminal FLAG-tagged RPLP1 were analyzed following treatment with 5.5 vs. 25 mM glucose for 24 hours. Sustained high glucose-induced decrease in polysome-associated FLAG-RPLP1 was associated with an increase in soluble FLAG- RPLP1 (Figures 10A and 10B). These findings support a model in which sustained high glucose stimulates RPLP1 exchange between the polysomes and a soluble pool.Example 7. Phosphorylated amino acid residues of RPLP1 are critical for association with translating ribosomes.

[0130] Phosphorylation of RPLP1 has been shown to regulate association of RPLP1 with the ribosome (MacConnell WP, Kaplan NO. (1982) J Biol Chem. 257(10):5359-66; Hasler P, et al. (1991) J Biol Chem. 266(21): 13815-20), and phosphoproteomic analysis of MIN6 cells has shown that RPLP1 is phosphorylated near its carboxyl terminus on serine 101 and serine 104 (Sacco F, et al. (2016) Nat Commun. 7: 13250). To test whether serine 101 and serine 104 are critical for RPLP1 ribosome association, wild type FLAG-RPLP1 and non- phosphorylatable mutant FLAG-RPLP1S101A / S104A were assessed for polysome association. The mutant demonstrated decreased association with polysomes (Figure 11). These findings are consistent with a model in which phosphorylation at serine 101 and / or serine 104 promotes ribosome association, whereas conditions that cause dephosphorylation of these residues, such as hyperglycemia, cause RPLP1 to dissociate from ribosomes (Figure 12).

[0131] There is growing evidence that heterogeneous ribosomes with varying stoichiometry of select ribosomal proteins (RPs) occur in different tissues or developmental contexts and direct functionally distinct programs of mRNA translation (Shi Z, et al. (2017) Mol Cell. 67(l):71-83; Segev N, et al. (2018) J Cell Biol. 217(1): 117-126; Erratum in: J Cell Biol. (2018) 217(3): 1155; Mageeney CM, et al. (2019) Mol Biol Cell. 30(17):2240-2253). To date, known examples of ribosome heterogeneity are stable characteristics of different cell types. The present disclosure provides new evidence that the composition of actively translating ribosomes in pancreatic beta-cells is dynamically modified by the nutrient environment.

[0132] The remodeling of beta-cell ribosomes under glucotoxic conditions that dysregulate mRNA-specific translation is likely to contribute to beta-cell dysfunction in the setting of hyperglycemia, because the proteins whose translation is altered are critical for glucose-stimulated insulin secretion. The finding that RPLP1 mutations that remove sites of phosphorylation similarly decrease RPLP1 ribosome association indicates that dephosphorylation of RPLP1 is a likely driver of its dissociation from ribosomes in the setting of nutrient excess. Further, regulation of post-translational modifications of RPLP1, such as by phosphorylating RPLP1, inhibiting dephosphorylation of RPLP1, and / or expressing an RPLP1 mutant comprising one or more phosphomimetic amino acids as described herein, may be able combat the effects of high glucose conditions on pancreatic beta-cells to prevent their dysfunction.

[0133] A variety of materials and methods were used to generate the data shown inFigures 1-11. Table 3 provides a list of reagents used in these studies.Rodent Islets

[0134] Islets were isolated from 7-8-week-old male Sprague-Dawley rats (TaconicBiosciences) by collagenase digestion followed by density gradient centrifugation as previously described (41). Islets were hand-picked and cultured overnight in RPMI 1640 containing 11 mM glucose, 10% FCS, 100 units / ml penicillin, 100 pg / ml streptomycin at 37C with 5% CO2. For analysis of GSIS, RNA or protein, media was changed to RPMI media containing either 5.5 mM or 16.7 mM glucose for 4 days.Human islets

[0135] Islets from cadaveric nondiabetic donors (ages 30-50) (Table 4) were obtained from Prodo Labs and cultured overnight in RPMI 1640 containing 5.5 mM glucose, 10%FCS, 100 units / ml penicillin, 100 g / ml streptomycin at 37°C with 5% CO2. The following day, islets were incubated in media containing either 5.5 mM or 16.7 mM glucose for 2 days prior to analysis of GSIS, RNA or protein.Cell lines

[0136] Low passage MIN6 cells (generously provided by Dr. Jun-ichi Miyazaki) were cultured in DMEM containing 25 mM glucose, 15% FBS, 0.1 mM P-mercaptoethanol, 100 units / ml penicillin and 100 g / ml streptomycin at 37°C with 5% CO2. For analyses of translation, MIN6 cells were incubated in DMEM media containing 5.5 mM or 25 mM glucose for 24 hours prior to analysis of GSIS, RNA or protein. 293T cells were obtained from ATCC CRL-3216).GSIS

[0137] Following incubations at different glucose concentrations, 10-12 islets (-150 pm diameter) in triplicate or MIN6 cells ( 105 / 35 mm well) in duplicate were washed and incubated with Krebs Ringer Bicarbonate HEPES buffer (KRBH: 137 mM NaCl, 4.8 mMKC1, 1.2 mM KH2PO4, 1.2 mM MgSO4-7H2O, 2.5 mM CaCl2.2H2O, 5mM NaHCO3, 16 mM HEPES, 0.1% BSA) containing 2.8 mM glucose for 1 hour. Following media change, cells were successively incubated in KRBH containing 2.8 and 16.7 (islet) or 16.8 mM (MIN6) glucose, each for 1 hour. Media was collected for insulin quantification by Ultra Sensitive Mouse Insulin ELISA (Crystal Chem). Insulin was normalized to DNA content for islets (CyQuant cell proliferation kit, Fisher Scientific C7026) and to cell number for MIN6 cells.RT-qPCR quantification of mRNA

[0138] Total RNA was isolated from cell lysates, sucrose density gradient fractions, or ribosome pellets using Trizol or Trizol-LS reagents (Invitrogen) and Direct-zol RNA miniprep kit (Zymo Research). RNA recovered from sucrose density gradient fractions was treated with 600 units / ml heparinase (NEB P0735S, 20 minutes, room temperature [RT]). 500 ng to 1 pg RNA was reverse transcribed using iScript cDNA synthesis kit (Biorad). RT- qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Biorad). RNA abundance was calculated according to the AACT method relative to 18S rRNA. Primers are listed in Table 5.Quantification of nascent peptides using OPP

[0139] During the last 2 hours of incubations in different media, 20 pM O-propargyl- puromycin (OPP, Click Chemistry Tools) was added to the media. Cells were lysed with RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5 % sodium deoxycholate, 1% NP-40, 0.1% SDS) containing cOmplete EDTA-free protease inhibitor cocktail (Sigma). Protein was quantified using bicinchoninic acid assay (BCA, Pierce). For in-gel quantification of total nascent peptides, 100-300 pg protein were used for copper-catalyzed cycloaddition reactions using the Click-iT Plus Alexa Fluor 647 Picolyl Azide Toolkit (Fisher Scientific). Proteins were separated using NuPAGE 10% Bis-Tris gels and gels were fixed with 10% acetic acid, 50% methanol. Total nascent peptides were detected by in-gel imaging at 647 pm and total protein was detected by far red epi imaging after staining with 0.1% Coomassie brilliant blue (BioRad ChemiDoc MP Imaging System). For detection of nascent insulin and tubulin, 1 mg of cell lysate was used for cycloaddition reactions containing 5% SDS, 500 pM Biotin azide, 5 mM DTT, 0.5 mM TBTA, and 5 mM CuSCU (1.5 hours, RT). Biotinylated protein was precipitated using methanol / chloroform, re-suspended in RIPA buffer, and incubated overnight, 4°C with high-capacity streptavidin agarose beads (Pierce). Beads were washed twice sequentially with RIPA, 1 M KC1, 0.1 M Na2COs, 2 M Urea in 50 mM Hepes, RIPA and eluted with 2x Laemmli buffer. Proteins were separated using NuPAGE 10%, Bis-Tris gels and immunoblotting was used to detect nascent proteins.Immunoblotting

[0140] Islets were lysed by sonication in buffer containing 5 mM EDTA, 7 M urea, 2 M thiourea, 100 mM sodium fluoride, 100 mM pyrophosphate, 10 mM orthovanadate, 50 mM PMSF, 1 pg / ml aprotinin (Pierce 78432), and 1% Triton. MIN6 cells were lysed in RIPA buffer containing complete protease inhibitor cocktail. 10-20 pg protein was separated on 10% NuPAGE Bis-Tris gels, transferred to PVDF membranes, blocked with 5% BSA, andblotted for the indicated proteins. Alexa Fluor-coupled (Invitrogen) secondary antibodies or HRP-coupled secondary antibodies (Cell Signaling Technologies) and chemiluminescent substrates (Biorad) were used for detection with a ChemiDoc MP Imaging System (Biorad). For ER-stress controls, islets were treated with 1 pM thapsigargin for 6 hours and MIN6 cells were treated with 5pg / mL tunicamycin for 3 hours. Antibodies and dilutions are listed inTable 6.Ribosome profiling

[0141] MIN6 cells incubated in media with 5.5 mM or 25 mM glucose for 24 hours were treated with 100 pg / ml of cycloheximide for 5 minutes. Ribosome profiling was performed as previously described (42) except that 1 U / 20 x 106cells RNase 1 (ThermoScientific) was used and rDNA depletion was performed using biotinylated rDNA sequences (38). Input RNA was extracted using TRIzol (Invitrogen) and Direct-zol RNA miniprep kit. RNA libraries were generated using polyA enrichment, and Kapa stranded mRNA Hyper Prep (Illumina). RPF and RNA libraries were sequenced using Illumina NS500 single-end 75 bp reads. Data analyses employed the XPRESSyourself pipeline (43). Briefly, trimmed reads were aligned to the genome (Ensemble release version 102) with the two-pass option that removes rRNA alignments and PCR duplicates and counts reads that map to the exons or truncated coding sequences of the longest transcripts of protein-coding genes. XPRESSpipe was used for quality control analyses (RPF coverage, length and periodicity) and to obtain normalized quantification of RNA, RPF counts and TE defined as ratio of RPF to RNA. Differential expression and differential TE were performed using DESeq2 (44). Pathway analysis was performed by testing over-representation of genes with differential TE in the Reactome gene sets from MSigDB using the pre-ranked CAMERA method in the limma package with the function cameraPR (45).Sucrose Density Gradient Fractionation of Polysomes

[0142] 5-50% sucrose gradients were generated using a BioComp Gradient Master IP from 5% and 50% sucrose solutions in sucrose buffer (10 Mm Tris pH 7.2, 60 mM KC1, 10 mM MgCh, 1 mM DTT, and 0.1 mg / ml heparin). MIN6 cells were treated with 100 pg / ml of cycloheximide for 5 minutes. Cells were lysed with ribosome profiling lysis buffer and layered onto gradients. Following centrifugation (SW41T, 222,200 x g, 3 hours, 4°C), fractions were collected using a BR-188 Density Gradient Fractionation System (Brandel). RNA was extracted from combined polysome fractions with TRIzol LS, and cleanup used Direct-zol RNA miniprep kit.AHA nascent proteomics

[0143] MIN6 cells were incubated in media with 5.5 mM or 25 mM glucose for 24 hours. During the last 2.5 hours, cells were changed to methionine-free media for 30 minutes, washed with PBS and then incubated in methionine-free media containing 250 pM AHA for 2 hours. Cells were collected, lysed in RIPA buffer containing cOmplete EDTA-free protease inhibitor cocktail, and proteins were quantified by BCA. 2 mg protein per condition was reduced with 15 mM DTT (1 hour, RT), alkylated with 20 mM iodoacetamide (20 minutes, dark, RT), quenched with 10 mM DTT (15 minutes, dark, RT), precipitated using methanol / chloroform, and resuspended in 50 mM HEPES, 150 mM NaCl, 2% SDS pH 7.2. Copper-catalyzed cycloaddition of biotin was performed with 1 mg of protein by addition of 100 uM TBTA, 1 mM sodium ascorbate, 1 mM copper sulfate, 100 uM biotin-alkyne (2 hours, RT). Proteins were precipitated to remove excess biotin-alkyne, re-suspended in 2% SDS, 5 mM DTT, and diluted with RIPA buffer to final SDS to < 0.5%. Samples were mixed with 10 ul of high-capacity streptavidin beads (overnight, RT) and then washed twice sequentially with RIPA, 1 M KC1, 0.1 M Na2COs, 2 M Urea in 50 mM Hepes, RIPA, and PBS pH 7.4. Tryptic digest, TMT labeling, separation into 6 fractions and LC-MS3 analysis was performed as described (46). MS2 spectra were searched using the COMET algorithm against a Uniprot composite database derived from the mouse proteome, exogenous sequence, known contaminants, and reverse sequences. Peptide spectral matches were filtered to a 1% FDR using the target-decoy strategy combined with linear discriminant analysis. The proteins from the 6 runs were filtered to a <1% FDR. At least 2 unique peptides were required for identification, and proteins were quantified only from peptides with a summed SN threshold of >150. Protein intensity was log2 transformed, missing values imputed, and data was normalized such that all samples had the same median abundance (47). Limma wasused to perform linear modeling and moderated t-tests, with adjustment for surrogate variable analysis as previously described (48, 49).Polysome proteomics

[0144] Proteins were isolated from combined polysome fractions, precipitated, digested with trypsin, TMT labeled, and fractionated prior to LC-MS3 analysis as described (An and Harper Nat Cell Biol 2018;20(2): 135-43). Alternatively, 10-20 pg protein was separated on 10% NuPAGE Bis-Tris gels, transferred to PVDF membranes, blocked with 5% BSA, and immuoblotted.Partial pancreatectomy

[0145] Six-week-old Sprague-Dawley (-100 g) male rats underwent 90% pancreatectomy or sham surgery as previously described (32). Under anesthesia with ketamine / xylazine, pancreatic tissue was removed by gentle abrasion with cotton-tipped applicators, leaving a small remnant 1-2 mm from the common bile duct and extending to the first loop of the duodenum. For sham surgery, the pancreas was disengaged from the mesentery but not removed. Post-operatively, body weights and morning fed glucose values were measured weekly. 10 weeks following surgery, islets were isolated as above and immediately lysed for analysis of total and ribosome-associated RNA and protein expression analysis.Isolation of ribosome-associated mRNA from rat and human islets

[0146] Following ex vivo incubation of rat and human islets with low or high glucose or immediately following isolation of islets from sham and PX rats, islets were treated with 100 pg / ml of cycloheximide for 5 minutes, washed with ice-cold PBS, lysed with ribosome profiling buffer. One-third of the sample was collected for total RNA isolation and the remaining material was centrifuged through a IM sucrose cushion to collect pelletedribosome-associated mRNA (435,400 x g, 1 hour, 4°C). Total and ribosome-associated mRNA were isolated using TRIzol (Invitrogen) and l-bromo-3 -chloropropane (Sigma) using phase separation method.Lentiviral expression of epitope-tagged wild type and mutant RPLP1

[0147] Lentiviral plasmids containing FLAG-tagged RPLP1WTor RPLP1S1O1 / 1O4Awere custom synthesized by VectorBuilder. 293T cells were transfected with 2 pg ready-to- use lentiviral packaging mix (Cellecta) and 2 pg lentiviral plasmids expressing FLAG-tagged RPLP1WTor RPLP1S1O1 / 1O4Ausing lipofectamine™ and plus™ reagent (ThermoFisher Scientific), according to standard manufacturer’s protocol. Supernatant containing lentivirus were collected on day 2 and day 3 post-transfection and filtered using 0.45 pM Nalgene syringe filters (ThermoFisher Scientific). Transduction of MIN6 cells with lentivirus was done by incubating cells with viral supernatant and 0.8 pg / ml polybrene (Santa Cruz Biotechnology).Statistics

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[0149] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0150] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:

1. A method of treating or preventing diabetes in a subject in need thereof comprising increasing the percentage of ribosome-associated RPLP1 or decreasing the percentage of soluble RPLP1 in a cell of the subject comprising:(a) phosphorylating RPLP1,(b) inhibiting dephosphorylation of RPLP1, and / or(c) expressing an RPLP1 mutant comprising one or more phosphomimetic amino acids, thereby increasing the percentage of ribosome-associated RPLP1 and / or decreasing the percentage of soluble RPLP1 in the subject and treating or preventing diabetes.

2. The method of claim 1, wherein the subject has Type I (Tl) or Type II (T2) diabetes.

3. The method of claim 1, wherein the subject is at risk for T2 diabetes.

4. The method of any one of claims 1-3, wherein increasing the percentage of ribosomes containing RPLP1 increases translation of one or more proteins encoded by a gene selected from INS, SCGN, IDH2, VPS41, SLC2A1, IGF2, SLC30A8, and PFKFB3.

5. The method of any one of claims 1-3, wherein the method comprises administering to the subject a kinase activator or phosphatase inhibitor that phosphorylates RPLP1 and / or dephosphorylates RPLP1.

6. The method of any one of claims 1-3, wherein the RPLP1 phosphorylation is at serine 101 and / or serine 104.

7. The method of any one of claims 1-3, wherein expressing an RPLP1 mutant comprising one or more phosphomimetic amino acid mutations is performed by gene editing ofRPLPl.

8. The method of claim 7, wherein the gene editing is performed with a system for gene editing.

9. The method of claim 8, wherein the system for gene editing comprises a CRISPR / Cas9 system, zinc-finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, or group one intron encoded endonuclease (GIIEE).

10. The method of any one of claims 7-9, wherein the RPLP1 mutant comprises one or more phosphomimetic amino acid mutations at an amino acid that is a serine in wildtype RPLP1.

11. The method of claim 10, wherein the serine in wildtype RPLP1 is serine 101 and / or serine 104.

12. The method of any one of claims 1-4 or 7-11, wherein the one or more phosphomimetic amino acid mutations is an aspartic acid or glutamic acid.

13. The method of any one of claims 1-12, wherein the cell is a pancreatic beta-cell.

14. The method of any one of claims 1-13, wherein the method increases the levels of insulin within or released by the cell.

15. The method of any one of claims 5, 6, or 14, wherein the cell is a pancreatic beta-cell, and the subject is treated with a kinase activator or phosphatase inhibitor.

16. The method of any one of claims 1-4 or 7-15, wherein:(a) the cell is an iPSC or pancreatic beta-cell in culture and gene editing is performed in vitro, and the cell is introduced into the subject after the gene editing; or(b) the cell is an iPSC or pancreatic beta-cell in a subject and gene editing is performed in vivo using a delivery system that selectively delivers the gene editing system to the iPSC or pancreatic beta-cell.

17. The method of claim 15 or claim 16, wherein insulin levels in the subject are increased and / or glucose levels in the subject are decreased.

18. A method of treating or preventing diabetes in a subject in need thereof comprising:(a) preparing iPSCs and / or pancreatic-beta-cells in vitro,(b) treating said iPSCs or pancreatic beta-cells with an agent for gene editing of RPLP1, wherein the gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell; and(c) transplanting the treated iPSCs or pancreatic beta-cells into the subject, wherein the transplanting increases insulin levels in the subject and / or decreases glucose levels in the subject.

19. A method of reducing the incidence of hyperglycemia or immune attack on pancreatic beta-cells in a subject in need thereof comprising:(a) preparing iPSCs and / or pancreatic-beta-cells in vitro,(b) treating said iPSCs or pancreatic beta-cells with an agent for gene editing of RPLP1, wherein the gene editing causes expression of an RPLP1 mutant comprising one or more phosphomimetic amino acids in the cell; and(c) transplanting the treated iPSCs or pancreatic beta-cells into the subject, wherein the transplanting increases insulin levels in the subject and / or decreases glucose levels in the subject.

20. The method of claim 18 or 19, wherein the RPLP1 mutant comprises one or more phosphomimetic amino acid mutations at an amino acid that is a serine in wildtype RPLP1.

21. The method of claim 20, wherein the serine in wildtype RPLP1 is serine 101 and / or serine 104.

22. The method of any one of claims 18-21, wherein the one or more phosphomimetic amino acid mutations is an aspartic acid or glutamic acid.

23. A composition comprising (1) a iPSC or pancreatic beta-cell and (2) a system for gene editing of RPLP1.

24. The composition of claim 23, wherein the system for gene editing is a CRISPR / Cas9 system, zinc-finger nuclease, TALEN, meganuclease, or GHEE.

25. The composition of claim 23 or claim 24, wherein the system for gene editing is capable of introducing one or more phosphomimetic amino acids at positions serine 101 and / or serine 104.

26. The composition of claim 25, wherein the one or more phosphomimetic amino acid mutation is an aspartic acid or glutamic acid.

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