Recruiting the contribution of DLG1 to enhance insulin signaling pathway

Abstract

The present disclosure pertains to compositions that are suitable for use in modulating cellular glucose uptake. The compositions may include one or more of the following compounds: (1) adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof (compound 1); (2) DLG1, a nucleic acid encoding DLG1, or combinations thereof (compound 2); and / or (3) a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof (compound 3). The present disclosure also pertains to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to the subject.

Description

TITLERECRUITING THE CONTRIBUTION OF DLG1 TO ENHANCE INSULIN SIGNALING PATHWAYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 661,183, filed on June 18, 2024. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF13368.P07 IWO.xml”. The date of the creation of the Sequence Listing is June 18, 2025. The size of the Sequence Listing is 15,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.BACKGROUND

[0003] Current compositions and methods for treating or preventing metabolic disorders suffer from numerous limitations. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to compositions that are suitable for use in modulating cellular glucose uptake. In some embodiments, the compositions of the present disclosure include one or more of the following compounds: (1) adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof (compound 1); (2) DLG1, a nucleic acid encoding DLG1, or combinations thereof (compound 2); and / or (3) a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof (compound 3).

[0005] In additional embodiments, the present disclosure pertains to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to the subject. The methods of the present disclosure may be used to treat or prevent various metabolic disorders. For instance, in some embodiments, the metabolic disorder includes, without limitation, prediabetes, diabetes type 1, diabetes type 2, insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, fatty liver disease, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, lipodystrophy, insulin receptor gene (INSR) mutation syndromes, or combinations thereof.DRAWINGS

[0006] FIGS. 1A-1C show that Insparin’s (Insp) action on glycemic control is dependent on DLG1 protein. FIG. 1A is a western blot showing a knockdown of DLG1 with siRNA transfection and subsequent downregulation of pAKT. FIG. IB is a quantitative analysis of DLG1 showing a 47% reduction with siRNA. N=3 / group. FIG. 1C is a quantitative analysis of pAKT after Insparin treatment. Insparin increases cellular pAKT expression in the presence of DLG1 , which is attenuated when DLG1 is knocked down. N=3 / group. Data are presented as mean ± SEM. Welch’s t-test or one-way ANOVA: Bars sharing the same letters are not statistically significant.

[0007] FIG. 2 shows data indicating that DLG1 knockdown reduces cellular glucose uptake (first vs third bars), and Insparin cannot increase glucose uptake when DLG1 is knocked down (first vs fourth bar).

[0008] FIG. 3 provides data showing that Insparin’s (Insp) actions on cellular glucose uptake is dependent on DLG1. In particular, murine pre-adipocyte 3T3-L1 cells treated with Insp show significantly reduced cellular glucose uptake in the presence of siRNA mediated knock down of DLG1 gene expression (fourth bar) compared with cells expressing DLG1 (second bar). N=3 / group; Data are presented as mean±SEM. One-way ANOVA: Bars sharing the same letters are not statistically significant.

[0009] FIG. 4 provides a proposed mechanism of the Insparin-DLGl interaction in regulating the PI3K / AKT / GLUT4 pathway.DETAILED DESCRIPTION

[0010] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0011] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0012] Metabolic disorders present significant public health challenges. For instance, diabetes is a serious chronic disease, which needs better glycemic control in presence of insulin resistance or insufficiency. However, current compositions and methods for treating or preventing metabolic disorders, such as insulin resistant diabetes, suffer from numerous limitations. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0013] Compositions for modulating cellular glucose uptake

[0014] In some embodiments, the present disclosure pertains to novel compositions. In some embodiments, the compositions of the present disclosure are suitable for use in modulating cellular glucose uptake. In some embodiments, the compositions of the present disclosure include one or more of the following compounds: (1) adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof (compound 1); (2) DLG1, a nucleic acid encoding DLG1, or combinations thereof (compound 2); and / or (3) a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof (compound 3). As set forth in more detail herein, the compositions of the present disclosure can have numerous combinations and embodiments.

[0015] Compound combinations

[0016] The compositions of the present disclosure can include various combinations of compounds. For instance, in some embodiments, the compositions of the present disclosure include compound 1. In some embodiments, the compositions of the present disclosure include compound 2. In some embodiments, the compositions of the present disclosure include compound 3. In some embodiments, the compositions of the present disclosure include compounds 1 and 2. In some embodiments, the compositions of the present disclosure include compounds 1 and 3. In some embodiments, the compositions of the present disclosure include compounds 2 and 3. In some embodiments, the compositions of the present disclosure include compounds 1, 2 and 3.

[0017] Compound 1

[0018] In the present disclosure, compound 1 generally refers to adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof. The compositions of the present disclosure can include various forms of compound 1.

[0019] In some embodiments, compound 1 includes an adenovirus-36 E4orfl protein. In some embodiments, the adenovirus-36 E4orfl protein includes SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the adcnovirus-36 E4orfl protein includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.

[0020] In some embodiments, compound 1 includes an adenovirus-36 E4orfl protein. In some embodiments, the adenovirus-36 E4orfl protein includes SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 65% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the adcnovirus-36 E4orfl protein includes a sequence with at least 75% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 99% sequence identity to SEQ ID NO: 4.

[0021] In some embodiments, compound 1 includes an adenovirus-36 E4orfl protein. In some embodiments, the adenovirus-36 E4orfl protein includes SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 65% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 75% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 85% sequence identity to SEQ ID NO: 7. In some embodiments, the adcnovirus-36 E4orfl protein includes a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 99% sequence identity to SEQ ID NO: 7.

[0022] In some embodiments, compound 1 includes an adenovirus-36 E4orfl protein. In some embodiments, the adenovirus-36 E4orfl protein includes SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 65% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 75% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 85% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the adenovirus-36 E4orfl protein includes a sequence with at least 99% sequence identity to SEQ ID NO: 8.

[0023] In some embodiments, compound 1 includes a nucleic acid encoding Adenovirus-36 E4orfl. In some embodiments, the nucleic acid includes SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0024] In some embodiments, the nucleic acid encoding Adenovirus-36 E4orfl includes SEQ ID NO: 3. In some embodiments, the nucleic acid encoding Adenovirus-36 E4orfl includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.

[0025] Compound 2

[0026] In the present disclosure, compound 2 generally refers to DLG1, a nucleic acid encoding DLG1, or combinations thereof. The compositions of the present disclosure can include various forms of compound 2.

[0027] In some embodiments, compound 2 includes a DLG1 protein. In some embodiments, the DGL1 protein includes SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the DGL1 protein includes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0028] In some embodiments, compound 2 includes a nucleic acid sequence encoding DLG1. In some embodiments, the DGL1 nucleic acid sequence includes SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 65% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 75% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the DGLI nucleic acid sequence includes a sequence with at least 99% sequence identity to SEQ ID NO: 6.

[0029] Compound 3

[0030] In the present disclosure, compound 3 generally refers to a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof. The compositions of the present disclosure can include various forms of compound 3.

[0031] In some embodiments, compound 3 includes a DLG1 agonist. In some embodiments, the DLG1 agonist includes, without limitation, lipopolysaccharides, E3 Ubiquitin ligase, or combinations thereof.

[0032] In some embodiments, compound 3 includes a nucleic acid encoding a DLG1 agonist. For instance, in some embodiments, compound 3 includes a nucleic acid encoding lipopolysaccharides, E3 Ubiquitin ligase, or combinations thereof.

[0033] Forms

[0034] The compounds of the present disclosure may be in various forms within a composition. For instance, in some embodiments, the compounds of the present disclosure may be in the form of one or more proteins. In some embodiments, the proteins may be in recombinant form. In some embodiments, the proteins may be in purified form.

[0035] In some embodiments, the compounds of the present disclosure may be in the form of one or more nucleic acids. In some embodiments the nucleic acids may be in the form of RNA, DNA, messenger RNA, or combinations thereof. In some embodiments, the nucleic acids may be in the form of DNA. In some embodiments, the nucleic acids may be in an expression vector, such as a plasmid.

[0036] In some embodiments, the compounds are associated with one or more delivery agents. In some embodiments, the delivery agents include a particle, In some embodiments, the particle includes, without limitation, a viral particle, a nanoparticle, a lipid-based particle, a liposome, or combinations thereof. In some embodiments, the delivery agent is a nanoparticle.

[0037] In some embodiments, individual compounds in a composition may be associated with the same particles. In some embodiments, individual compounds in a composition may be associated with different particles.

[0038] In some embodiments, the composition includes compound 1 and compound 2. In some embodiments, compound 1 is associated with a first delivery agent and compound 2 is associated with a second delivery agent.

[0039] In some embodiments, the composition includes compound 1 and compound 3. In some embodiments, compound 1 is associated with a first delivery agent and compound 3 is associated with a second delivery agent.

[0040] The compositions of the present disclosure may include additional materials. For instance, in some embodiments, the compositions of the present disclosure also include at least one solubilizing agent. In some embodiments, the solubilizing agent includes, without limitation, polyethylene glycol, glycerin, propylene glycol, ethanol, sorbitol, polyoxyethylated glycerides, polyoxyethylated oleic glycerides, polysorbates, sorbitan monooleate, hydroxypropyl-beta- cyclodextrin (HPCD), polyoxyl 40 hydrogenated castor oil, polyoxyl hydroxystearates, or combinations thereof.

[0041] Methods of modulating cellular glucose uptake

[0042] Additional embodiments of the present disclosure pertain to methods of modulating cellular glucose uptake by associating cells with a composition of the present disclosure. In some embodiments, the composition increases cellular glucose uptake. In some embodiments, the composition increases cellular glucose uptake independently of proximal insulin signaling pathways. In some embodiments, the composition causes an increase in pAKT / AKT ratios. In some embodiments, the composition causes reduction in endogenous insulin secretion, while improving or maintaining systemic glucose levels.

[0043] The methods of the present disclosure may be utilized to modulate cellular glucose uptake in various cells. For instance, in some embodiments, the cells are fat cells, muscle cells, or liver cells. In some embodiments, the composition causes an increase in cellular glucose uptake in the fat cells, muscle cells, or reduces glucose output from liver cells.

[0044] In some embodiments, the compositions of the present disclosure may be associated with cells in vitro. In some embodiments, the compositions of the present disclosure may be associated with cells in vivo in a subject. In some embodiments, the in vivo association includes administering the composition to the subject. In some embodiments, the administration occurs by a method that includes, without limitation, intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, intramuscular administration, oral administration, intratumor administration, or combinations thereof.

[0045] In some embodiments, the compositions of the present disclosure may be used to treat or prevent a metabolic disorder in the subject. In additional embodiments, the present disclosure pertains to methods of treating or preventing a metabolic disorder in a subject by administering a composition of the present disclosure to the subject.

[0046] The methods of the present disclosure may be used to treat or prevent various metabolic disorders. For instance, in some embodiments, the metabolic disorder includes, without limitation, prediabetes, diabetes type 1, diabetes type 2, insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, lipodystrophy, insulin receptor gene (INSR) mutation syndromes (e.g., Rabson Mendenhall and / or Donahue Syndromes), or combinations thereof. In some embodiments, the metabolic disorder includes insulin resistance. In some embodiments, the metabolic disorder includes, without limitation, prediabetes, diabetes type 1, diabetes type 2, insulin resistance, or combinations thereof. In some embodiments, the metabolic disorder includes diabetes. In some embodiments, the diabetes includes diabetes type 1. In some embodiments, the diabetes includes diabetes type 2.

[0047] The compositions of the present disclosure can be used to treat or prevent metabolic disorders through various mechanisms. For instance, in some embodiments, the compositions of the present disclosure reduce endogenous insulin levels in the blood. In some embodiments, the compositions of the present disclosure improve blood glucose levels by reducing high levels of glucose in the blood, reducing the rise in glucose levels or the duration of glucose elevation expected after eating food, or reducing glucose output from the liver. In some embodiments, the compositions of the present disclosure prevent an increase in blood insulin levels.

[0048] In some embodiments, the compositions of the present disclosure reduce production and secretion of insulin while improving blood glucose levels, thereby preventing damage to pancreatic cells that make insulin. In some embodiments, the compositions of the present disclosure reduce fat accumulation in the liver or reduce accumulated lipid from the liver. In some embodiments, the compositions of the present disclosure prevent accumulation of fat in the liver.

[0049] The methods of the present may be used to treat or prevent metabolic disorders in various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal includes, without limitation, a horse, a rabbit, a mouse, a rat, a pig, a sheep, a cow, a dog, or a cat. In some embodiments, the non-human mammal is a domestic animal, such as a dog or a cat.

[0050] In some embodiments, the subject is suffering from a metabolic disorder. In some embodiments, the subject is vulnerable to a metabolic disorder.

[0051] Additional Embodiments

[0052] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0053] Example 1. Role of DLG1 in enhancing insulin signaling pathway

[0054] Diabetes is a serious chronic disease, which needs better glycemic control in presence of insulin resistance or insufficiency. This requires a better understanding of systemic glucose disposal and molecular participants of pathways leading to cellular glucose uptake. Activation of AKT / PKB by phosphorylation is a key step in insulin signaling leading to cellular glucose uptake mediated by glucose transporters (e.g., Glut 4). DLG1 (discs large homolog 1), a scaffolding protein, contributes to Glut4 membrane translocation [1], Moreover, the E4orfl protein of some adenoviruses activates AKT by recruiting DLG1 [2].

[0055] The E4orfl proteins of these adenoviruses are not reported to influence cellular glucose uptake. On the other hand, the E4orf 1 protein of a human adenovirus-36 (Insparin) binds to DLG1 and increases cellular glucose uptake via a Ras-mediated activation of AKT [3] [4], Activation of AKT is a marker for upregulating cellular glucose uptake[5]. These findings suggest that DLG1 is a crucial component in metabolic regulation.

[0056] DLG1 plays a role in signaling pathways by interacting with and regulating the activity of signaling molecules, such as receptors, kinases, and ion channels. Furthermore, it can modulate the localization, activity, and downstream effects of these signaling proteins. While DLG1 remains associated with the plasma membrane, its crucial role involves organizing protein complexes and signaling pathways at the cell membrane and junctions.

[0057] The interaction between Insparin and DLG 1 forms a complex that aids in the recruitment of PI3K to the plasma membrane[6]. This recruitment triggers the activation of AKT, which in turn, through its downstream pathway, results in the translocation of Glucose transporter 4 (GLUT4) to the cell membrane, facilitating glucose uptake. Hence, Applicant investigated if DLG1 could be recruited to activate AKT and potentially upregulate glucose uptake.

[0058] Example 1,1. Methods

[0059] Murine 3T3-L1 mbx cells grown in DMEM media containing FBS and pen-strep were exposed to DLG1 siRNA or scrambled siRNA as control. Each group was further exposed for 5h to pulsin, a lipid-based transfection agent, with or without Insparin. Post-treatment, protein lysates were collected. Western blot analysis determined relative protein abundance of Insparin, DLG1, and pAKT. Abundance of pAKT was normalized to total AKT and other proteins were normalized to GAPDH.

[0060] Example 1,2. Results

[0061] As expected, Insparin was present only in cells transfected with it. Also, DLGl-siRNA knocked down DLG1 abundance by 47%. Surprisingly, in the absence of Insparin, DLG1 knockdown reduced pAKT abundance by 50% (FIG. 1C, bars 2 & 3), indicating its hitherto unknown contribution to AKT activation. This important finding underscores the role of DLG1 in upregulating the AKT pathway, which is crucial for cellular glucose uptake. In presence of intact DLG1, Insparin increased pAKT abundance two-fold (FIG. 1C, bar 1) compared to cells with DLG1 knockdown with or without Insparin (FIG. 1C, bars 3 & 4). Addition of Insparin to DLG1 knocked down cells could not increase pAKT activation.

[0062] Moreover, additional data included in FIG. 2 shows that, in addition to pAKT activation (as set forth in FIGS. 1A-1C), DLG1 is required for glucose uptake. In particular, the data in FIG. 2 shows that DLG1 knockdown reduces cellular glucose uptake (first vs. third bars), and Insparin cannot increase glucose uptake when DLG1 is knocked down (first vs. fourth bars).

[0063] The observations in FIG. 2 are particularly important because DLG1 uses E4orfls of some adenoviruses to upregulate pAKT, but that does not lead to increased glucose uptake [2], However, when DLG1 uses Ad36 E4orfl, it upregulates pAKT and increases glucose uptake. Such results are surprising and unexpected.

[0064] The results shown in FIG. 3 indicate that Insparin treatment of murine 3T3-L1 cells significantly increases cellular glucose uptake compared with untreated control cells. (FIG. 3; second bar vs. first bar). However, Insparin’s ability to significantly improve glucose uptake in these cells is compromised when DLG1 expression is reduced via siRNA transfection (FIG. 3; fourth bar vs. second bar). These observations support the role of Insparin as an agonist for DLG1- mediated activation of the distal insulin signaling pathway via pAKT leading to increased cellular glucose uptake. These observations also affirm that Insparin’s action on cellular glucose uptake is dependent on DLG1 protein.

[0065] Example 1,3. Summary

[0066] In sum, the results show that Insparin increases pAKT two-fold in the presence of intact DLG1, but not in cells with DLG1 knockdown, indicating its dependency on DLG1 for AKT activation. The results also show that DLG1 -dependent activation of AKT by Insparin leads to increased cellular glucose uptake. Additionally, the results show that knocking down DLG1 with siRNA reduces Insparin mediated glucose uptake, indicating that DLG1 is necessary for Insparin- mediated glucose uptake.

[0067] The experimental results set forth in this Example suggest that intact DLG1 contributes to cellular glucose uptake via AKT activation. The results also suggest that increased AKT activation and possibly an increase in cellular glucose uptake can be achieved by recruiting DLG1 with Insparin. Additionally, the results suggest that intact DLG1 is needed for Insparin to upregulate AKT activation. Moreover, DLG1 -dependent AKT activators, which upregulate cellular glucose uptake could be a new class of anti-diabetic agents. Moreover, as a DLG1 -dependent AKT activator, Insparin could be developed as a new class of drug for treating insulin resistance and diabetes.

[0068] Without being bound by theory, FIG. 4 illustrates a proposed mechanism of Insparin binding to DLG1 and activation of the PI3K / AKT / GLUT4 pathway in 3T3-L1 preadipocytes. Upon Insparin binding to DLG1, the DLGl-Inspaiin complex initiates a signaling cascade that upregulates PI3K, leading to subsequent activation of AKT. Activated AKT promotes the translocation of GLUT4 to the cell membrane, enhancing glucose uptake.

[0069] Example 1,4, References1. Inoue, M., et al., Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering. Molecular biology of the cell, 2006. 17(5): p. 2303-2311.2. Kumar, M., K. Kong, and R.T. Javier, Hijacking Discs Large 1 for Oncogenic Phosphatidylinositol 3-Kinase Activation in Human Epithelial Cells Is a Conserved Mechanism of Human Adenovirus E4-ORF1 Proteins. Journal of virology, 2014.3. Shastri, A. A., et al., E4orfl: A protein for enhancing glucose uptake despite impaired proximal insulin signaling. PLoS One, 2018. 13(12): p. e0208427.4. Dhurandhar, E.J., et al., E4orfl: A Novel Ligand that Improves Glucose Disposal in Cell Culture. PLoS ONE, 2011. 6(8): p. e23394.5. Tonks, K.T., et al., Impaired Akt phosphorylation in insulin-resistant human muscle is accompanied by selective and heterogeneous downstream defects. Diabetologia, 2013. 56(4): p. 875-85.6. Kong, K., et al., The human adenovirus E4-ORF1 protein subverts discs large 1 to mediate membrane recruitment and dysregulation of phosphatidylinositol 3-kinase. PLoS pathogens, 2014. 10(5): p. el004102.

[0070] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the ail without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

WHAT IS CLAIMED IS:

1. A composition suitable for use in modulating cellular glucose uptake, wherein the composition comprises one or more of the following compounds:(1) Adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof (compound 1);(2) DLG1, a nucleic acid encoding DLG1, or combinations thereof (compound 2);(3) a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof (compound 3); or combinations thereof.

2. The composition of claim 1 , wherein the composition comprises compounds 1 and 2.

3. The composition of claim 1, wherein the composition comprises compound 2.

4. The composition of claim 1, wherein the composition comprises compounds 1 and 3.

5. The composition of claim 1, wherein the one or more compounds are associated with one or more delivery agents.

6. The composition of claim 5, wherein the delivery agent is a nanoparticle.

7. The composition of claim 5, wherein the composition comprises compound 1 and compound2, wherein compound 1 is associated with a first delivery agent, and wherein compound 2 is associated with a second delivery agent.

8. The composition of claim 5, wherein the composition comprises compound 1 and compound3, wherein compound 1 is associated with a first delivery agent, and wherein compound 3 is associated with a second delivery agent.

9. The composition of claim 1, wherein compound 1 comprises an adenovirus-36 E4orfl protein.

10. The composition of claim 9, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 2 or a sequence with at least 95% sequence identity to SEQ ID NO: 2.

11. The composition of claim 9, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 4 or a sequence with at least 95% sequence identity to SEQ ID NO: 4.

12. The composition of claim 9, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 7 or a sequence with at least 95% sequence identity to SEQ ID NO: 7.

13. The composition of claim 9, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 8 or a sequence with at least 95% sequence identity to SEQ ID NO: 8.

14. The composition of claim 1, wherein compound 1 comprises a nucleic acid encoding Adenovirus-36 E4orfl.

15. The composition of claim 14, wherein the nucleic acid comprises SEQ ID NO: 1 or a sequence with at least 95% sequence identity to SEQ ID NO: 1.

16. The composition of claim 14, wherein the nucleic acid comprises SEQ ID NO: 3 or a sequence with at least 95% sequence identity to SEQ ID NO: 3.

17. A method of treating or preventing a metabolic disorder in a subject, wherein the method comprises: administering a composition to the subject, wherein the composition comprises one or more of the following compounds:(1) adenovirus-36 E4orfl protein, a nucleic acid encoding an adenovirus-36 E4orfl protein, or combinations thereof (compound 1);(2) DLG1, a nucleic acid encoding DLG1, or combinations thereof (compound 2);(3) a DLG1 agonist, a nucleic acid encoding a DLG1 agonist, or combinations thereof (compound 3); or combinations thereof.

18. The method of claim 17, wherein the metabolic disorder is selected from the group consisting of prediabetes, diabetes type 1, diabetes type 2, insulin resistance, hyperinsulinemia, hyperglycemia, metabolic syndrome, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steato-hepatitis (NASH), liver dysfunction characterized by fatty liver and / or insulin resistance, polycystic ovary syndrome, lipodystrophy, insulin receptor gene (INSR) mutation syndromes, or combinations thereof.

19. The method of claim 17, wherein the metabolic disorder is selected from the group consisting of prediabetes, diabetes type 1, diabetes type 2, insulin resistance, or combinations thereof.

20. The method of claim 17, wherein the administering occurs by a method selected from the group consisting of intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, intramuscular administration, oral administration, intratumor administration, and combinations thereof.

21. The method of claim 17, wherein the subject is a human being.

22. The method of claim 17, wherein the subject is suffering from the metabolic disorder.

23. The method of claim 17, wherein the subject is vulnerable to the metabolic disorder.

24. The method of claim 17, wherein the composition comprises compounds 1 and 2.

25. The method of claim 17, wherein the composition comprises compound 2.

26. The method of claim 17, wherein the composition comprises compounds 1 and 3.

27. The method of claim 17, wherein the one or more compounds are associated with one or more delivery agents.

28. The method of claim 27, wherein the delivery agent is a nanoparticle.

29. The method of claim 28, wherein the composition comprises compound 1 and compound 2, wherein compound 1 is associated with a first delivery agent, and wherein compound 2 is associated with a second delivery agent.

30. The method of claim 28, wherein the composition comprises compound 1 and compound 3, wherein compound 1 is associated with a first delivery agent, and wherein compound 3 is associated with a second delivery agent.

31. The method of claim 17, wherein compound 1 comprises an adenovirus-36 E4orfl protein.

32. The method of claim 31, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 2 or a sequence with at least 95% sequence identity to SEQ ID NO: 2.

33. The method of claim 31, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 4 or a sequence with at least 95% sequence identity to SEQ ID NO: 4.

34. The method of claim 31, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 7 or a sequence with at least 95% sequence identity to SEQ ID NO: 7.

35. The method of claim 31, wherein the adenovirus-36 E4orfl protein comprises SEQ ID NO: 8 or a sequence with at least 95% sequence identity to SEQ ID NO: 8.

36. The method of claim 15, wherein compound 1 comprises a nucleic acid encoding Adenovirus-36 E4orfl.

37. The method of claim 36, wherein the nucleic acid comprises SEQ ID NO: 1 or a sequence with at least 95% sequence identity to SEQ ID NO: 1.

38. The method of claim 36, wherein the nucleic acid comprises SEQ ID NO: 3 or a sequence with at least 95% sequence identity to SEQ ID NO: 3.

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