Application of sulfated glycogen synthase kinase-3 in prevention and treatment of liver insulin resistance

Abstract

The invention relates to application of sulfated glycogen synthase kinase-3 in prevention and treatment of liver insulin resistance. Specifically, the invention provides an application of a hyposulfated GSK-3beta inhibitor in preparation of a medicine, and the medicine is used for preventing or treating insulin resistance; the invention further discloses application of the GSK-3beta knockout reagent in preparation of a medicine. The medicine is used for preventing or treating insulin resistance.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine. Specifically, it relates to the use of hyposulfonated glycogen synthesis kinase-3β (Gsk-3β) in the prevention and treatment of hepatic insulin resistance. Background Technology

[0002] Hepatic insulin resistance (HIR) refers to a pathological state in which the liver's sensitivity to insulin is reduced, making it unable to effectively promote glucose uptake by peripheral tissues or effectively inhibit intrahepatic glucose production, ultimately leading to elevated blood glucose levels. HIR has been proven to be one of the important contributing factors to non-alcoholic fatty liver disease, diabetes, and cardiovascular disease. Its typical characteristics are reduced liver glycogen synthesis capacity and abnormally enhanced gluconeogenesis.

[0003] With the transformation of modern lifestyles and the improvement of residents' living standards, these metabolic diseases, due to their highly interconnected pathogenesis, often present with multiple co-occurring symptoms in clinical practice, significantly increasing the difficulty of treatment and becoming a major public health problem threatening public health. Existing research indicates that although the pathogenesis of HIR is not fully understood, the abnormal accumulation of reactive oxygen species (ROS) in the liver is considered one of the key factors driving the development of HIR.

[0004] Glycogen synthase kinase 3 (GSK-3) is a highly conserved family of protein kinases. In humans, two different genes encode two isoforms: GSK-3α and GSK-3β, which are structurally similar but functionally different. Current research confirms that insulin signaling can mediate phosphorylation of GSK-3β at the Ser9 site (p-GSK-3β) via AKT, inactivating this protein kinase and subsequently activating hepatic glycogen synthase 2 (Gys2), promoting hepatic glycogen synthesis. However, insulin-induced p-GSK-3β can be reactivated, a process that inhibits hepatic glycogen synthesis and further exacerbates hepatocellular carcinoma (HIR). Therefore, exploring potential intervention targets around the reactivation mechanism of p-GSK-3β opens up new directions for HIR prevention and treatment research. Summary of the Invention

[0005] The inventors discovered that in primary hepatocytes and liver organoids induced by palmitic acid (PA) insulin resistance, as well as in a mouse model of hepatic insulin resistance (HIR) induced by a high-fat diet (HFD), elevated levels of reactive oxygen species (ROS), decreased glycogen synthesis in hepatocytes, and enhanced gluconeogenesis were observed. They also confirmed that excessive ROS accumulation in hepatocytes can convert inactive p-GSK-3β into its active form through hyposulfonation modification of the GSK-3β cysteine ​​site.

[0006] To address the limitations of current HIR prevention and treatment strategies, the purpose of this disclosure is to provide a new HIR prevention and treatment strategy that targets GSK-3β sulfonation modification. This strategy aims to reverse the HIR process by inhibiting this target, thus opening up new avenues for the treatment and prevention of HIR.

[0007] While not confined to a specific theory, insulin signaling can be transduced to GSK-3β and mediated to phosphorylate it in insulin-resistant hepatocytes. However, high levels of intracellular ROS selectively attack phosphorylated GSK-3β (p-GSK-3β) rather than unphosphorylated GSK-3β, inducing p-GSK-3β hyposulfonation modification. This reactivated p-GSK-3β plays a crucial role in the development and progression of hepatitis-related inflammatory response (HIR). Therefore, blocking or inhibiting this active form of p-GSK-3β can prevent it from phosphorylating GYS2, thereby promoting glycogen synthesis. Simultaneously, it can activate the AKT-mediated FoxO1 inhibitory pathway, inhibiting gluconeogenesis and ultimately improving HIR.

[0008] In view of the above-mentioned needs in the art, this application provides an inhibitor of hyposulfonated GSK-3β. The inhibitor of hyposulfonated GSK-3β is capable of inhibiting, reducing, interfering with, inactivating, or blocking the undesirable hyposulfonation of GSK-3β. Hyposulfonation refers to the oxidation of the sulfhydryl group (-SH) of cysteine ​​(Cys) on GSK-3β to hyposulfonic acid (-SOH). Therefore, the term "inhibitor of hyposulfonated GSK-3β" herein refers to any molecule capable of achieving any of the following: - Reduce the expression level of GSK-3β or sulfonated GSK-3β, such as by knocking it out; - Inhibit, reduce, interfere with, inactivate or block the activity of GSK-3β or sulfonated GSK-3β; - Inhibits or blocks the hyposulfonation of GSK-3β.

[0009] The "inhibitors of hyposulfonated GSK-3β" in this article can be small molecule drugs, antibodies, or RNA drugs.

[0010] In some embodiments, the use of inhibitors of hyposulfonated GSK-3β in the preparation of medicaments for the prevention or treatment of insulin resistance is provided.

[0011] In some embodiments, the inhibitor of hyposulfonated GSK-3β is selected from: glutathione ethyl ester or a pharmaceutically acceptable salt thereof, 1-aminobenzotriazole, N-acetylcysteine ​​or a pharmaceutically acceptable salt thereof, GSK-3β knockout reagent, and vectors expressing GSK-3β variants.

[0012] In some specific embodiments, the inhibitor of sulfonated GSK-3β is glutathione ethyl ester (GEE) or its acetate form. The acetate form of GEE improves water solubility and stability.

[0013] In some specific embodiments, the inhibitor of the hyposulfonated GSK-3β is 1-aminobenzotriazole (ABT).

[0014] In some specific embodiments, the inhibitor of hyposulfonated GSK-3β is N-acetylcysteine ​​or its pharmaceutically acceptable salt (such as sodium N-acetylcysteine ​​or lysine N-acetylcysteine).

[0015] In some embodiments, the inhibitor of hyposulfonated GSK-3β is a GSK-3β knockout agent. A knockout agent is a molecule or vector tool that specifically targets the GSK-3β gene, causing it to be silenced, deleted, or permanently inactivated in cells or animals. Unlike small molecule inhibitors, it does not directly bind to GSK-3β, but rather achieves a reduction in GSK-3β levels or loss of function at the gene, transcription, or genomic level. Types that may be mentioned include, but are not limited to: CRISPR-Cas9 knockout agents (which target and cleave GSK-3β genomic DNA to achieve permanent gene knockout), shRNA (which silences GSK-3β mRNA to achieve stable gene knockdown), and siRNA knockout agents (e.g., capable of transiently silencing GSK-3β).

[0016] In some specific implementations, the GSK-3β knockout agent is a CRISPR-Cas9 GSK-3β knockout vector.

[0017] As an exemplary implementation, the CRISPR-Cas9 GSK-3β knockout vector can be a plasmid vector, with structural elements including a Cas9 expression cassette, an sgRNA expression cassette (U6 or H1 promoter), a selection marker Puro (puromycin), GFP or Neo, etc.; for example, pSpCas9(BB)-2A-Puro (PX459)-sgGSK3β.

[0018] As another exemplary implementation, the CRISPR-Cas9 GSK-3β knockout vector can be a lentiviral vector, an adeno-associated virus (AAV) vector, or a dual-vector / co-expression system (such as Cas9 and sgRNA on two separate vectors) to improve knockout efficiency or tissue-specific expression; for example, lentiCRISPRv2-sgGSK3β. The target site for GSK-3β is not limited to a specific sequence and can be selected by technicians according to standard procedures, such as targeting highly conserved coding regions in the GSK-3β exon.

[0019] In one specific implementation, for the CRISPR-Cas9 GSK-3β knockout vector, lentiCRISPRv2 is used as the backbone to carry a specific sgRNA targeting the GSK-3β exon and a Cas9 nuclease. Stable GSK-3β knockout cell lines are constructed through lentiviral infection and puromycin screening.

[0020] In some specific implementations, the GSK-3β knockout agent is a siRNA that targets GSK-3β. siRNA is a double-stranded small interfering RNA of approximately 21-23 nt in length that specifically recognizes and degrades GSK-3β mRNA through the RNA interference pathway, thereby reducing GSK-3β protein expression.

[0021] As an example implementation, the siRNA targeting GSK-3β comprises: - The sense strand is homologous to the GSK-3β mRNA sequence; - Antisense strand: complementary pairing with GSK-3β mRNA; The length is typically 21-23 nucleotides; optional modifications such as 2'-O-methylation and phosphate thiocarbamate modification can be added to improve stability and reduce off-target effects and immune stimulation. The target site of GSK-3β is not limited to a specific sequence and can be selected by technicians according to standard procedures.

[0022] The preparation methods for siRNA are conventional in the field, such as: chemical synthesis of siRNA; vector expression form (shRNA), shRNA expressed by plasmid / lentivirus, which is cleaved into siRNA by Dicer in cells; and transfection complex form, siRNA + liposome transfection reagent (such as Lipofectamine) to form a nanocomplex, which improves the efficiency of cell entry.

[0023] In some specific implementations, the GSK-3β knockout agent is a shRNA targeting GSK-3β. shRNA is a short hairpin RNA that specifically degrades GSK-3β mRNA and inhibits its protein expression via the RNA interference (RNAi) pathway, thereby achieving targeted gene silencing of GSK-3β. A typical shRNA structure includes: - The sense strand is a 19–25 nt sequence complementary to the target sequence of GSK-3β mRNA; - Loop / hairpin loops are typically 3–9 nt of non-complementary sequences; - Antisense strand: the reverse complementary sequence of the justice strand; - The terminal termination structure, usually a series of Ts (commonly six Ts), serves as a transcription termination signal for RNA polymerase III.

[0024] shRNA can only be stably expressed in cells when loaded into a vector. An exemplary vector structure includes: a promoter (such as U6 or H1), an shRNA expression cassette (the aforementioned sense + loop + antisense + TTTTTT), selection markers (such as PuroR, GFP, mCherry, Neo, etc.), and a vector backbone.

[0025] In some specific embodiments, the inhibitor of hyposulfonated GSK-3β is selected as a vector expressing a GSK-3β variant. Hyposulfonated GSK-3β is a modification at a specific site; this site can be specifically mutated to prevent hyposulfonation, thus achieving a specific inhibition of hyposulfonated GSK-3β. The GSK-3β variant mutates the Cys residue at position 178 to Ala or Ser. Mutating Cys (cysteine) to Ser (serine) or Ala (alanine) does not significantly affect the overall protein structure and basic activity.

[0026] As an example, a suitable vector type for mammalian cells is a conventional eukaryotic expression plasmid vector (for transient transfection), such as pcDNA3.1, pcDNA3.4, pCMV-6, pCAGGS, and pEF1α-IRES. The GSK-3β C178A / C178S coding sequence is inserted at the multiple cloning site, and Flag / Myc / HA tags can be added for easy detection.

[0027] As another example, the specific vector type suitable for mammalian cells is a lentiviral expression vector, such as pLenti-CMV-MCS, pLenti-EF1α-MCS, pLVX-IRES-Puro, and pCDH-CMV-MCS-EF1α-Puro; selection markers such as Puromycin and GFP can be added.

[0028] As another example, a specific vector type suitable for mammalian cells is the adeno-associated virus (AAV) vector, such as serotypes like AAV2, AAV8, and AAV9. In one specific embodiment, the vector used is AAV8.

[0029] In some specific implementations, the drug also includes pharmaceutically acceptable excipients.

[0030] For small molecule drugs, the excipients may be mentioned as follows: Solvents or cosolvents (e.g., water for injection, sterile water for injection, ethanol, propylene glycol, polyethylene glycol, glycerol, buffer solutions); osmotic regulators (e.g., sodium chloride, glucose, mannitol, sorbitol); protectants or fillers (e.g., mannitol, trehalose, sucrose, maltose, lactose, dextran); diluents or fillers (e.g., microcrystalline cellulose, lactose, starch, pregelatinized starch, mannitol); binders (e.g., hydroxypropyl methylcellulose (HPMC), povidone (PVP), starch paste). Disintegrants (such as sodium carboxymethyl starch, crospovidone, low-substituted hydroxypropyl cellulose); lubricants (such as magnesium stearate, talc, micronized silica gel); coating materials (such as HPMC); stabilizers or antioxidants (such as vitamin C, vitamin E, citric acid, EDTA-2Na, sodium thiosulfate, sodium bisulfite); pH adjusters (such as hydrochloric acid, sodium hydroxide, citric acid, sodium dihydrogen phosphate / disodium hydrogen phosphate); preservatives (such as benzyl alcohol, ethylparaben, benzalkonium chloride).

[0031] By way of example, the inhibitors disclosed herein can be prepared for injection (injection, lyophilized powder for injection), and pharmaceutically acceptable excipients are selected from, but not limited to, water for injection, physiological saline, glucose, phosphate buffer, mannitol, lactose, trehalose, ethanol, propylene glycol, and PEG400.

[0032] Exemplarily, the inhibitors of this disclosure can be formulated for oral administration. Pharmaceutically acceptable excipients are selected from, but are not limited to, microcrystalline cellulose, starch, pregelatinized starch, lactose, mannitol, sucrose, magnesium stearate, talc (lubricant), crospovidone, sodium carboxymethyl starch (disintegrant), and hydroxypropyl methylcellulose (HPMC).

[0033] For nucleic acid drugs, pharmaceutically acceptable excipients may include: nucleic acid delivery carriers, such as, but not limited to, liposomes, cationic lipids, lipid nanoparticles (LNPs), polymer carriers (such as polyethyleneimine (PEI), polylysine, poly-β-amino esters), and viral carriers (such as lentiviruses, AAVs); solvents and suspension media, such as water for injection, sterile water for injection, physiological saline, 5% glucose injection, phosphate buffer, and citrate buffer; osmotic pressure regulators, such as sodium chloride, glucose, mannitol, sorbitol, and trehalose; lyophilization protectants, such as mannitol, trehalose, sucrose, maltose, dextran, and lactose; stabilizers and antioxidants, such as mannitol, trehalose, sucrose, polyethylene glycol (PEG), EDTA-2Na (metal ion chelating agent), citric acid, and disodium hydrogen phosphate; and preservatives, such as benzyl alcohol, parabens, and benzalkonium chloride.

[0034] In some embodiments, the inhibitor of hyposulfonated GSK-3β disclosed herein can achieve any of the following or combinations: increase hepatic glycogen synthesis, decrease gluconeogenesis, decrease the expression level of phosphoenolpyruvate carboxykinase 1, decrease the expression level of glucose-6-phosphatase catalytic subunit, improve glucose tolerance, improve insulin tolerance, and improve pyruvate tolerance. Attached Figure Description

[0035] Figures 1A to 1C Inhibitors targeting hyposulfonated Gsk-3β showed that they could effectively increase palmitic acid (PA)-induced glycogen synthesis and reduce gluconeogenesis in primary hepatocytes of an insulin-resistant model.

[0036] Figure 2 Procedure for hyperinsulin-euglycemic clamp.

[0037] Figures 3A to 3C The study showed that inhibitors targeting hyposulfonated Gsk-3β could effectively increase liver glycogen synthesis and reduce gluconeogenesis in HIR mice induced by a high-fat diet.

[0038] Figures 4A to 4C Measurement of GIR, HGP, and GDR.

[0039] Figure 5A The inhibitor ABT alleviates HFD-induced HIR in mice.

[0040] Figure 5B Specific point mutations can effectively block the development of HIR.

[0041] Figures 6A to 6C Inhibitors targeting hyposulfonated Gsk-3β have been shown to effectively increase glycogen synthesis and reduce gluconeogenesis in PA-induced human liver organoids. Detailed Implementation

[0042] Experimental materials HEK293T human embryonic kidney cells were purchased from the China Center for Type Culture Collection (Beijing, China); male wild-type C57BL / 6 mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). All animals were housed under sterile conditions at the animal facilities of the Chinese Academy of Medical Sciences. All studies involving mice were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (ACUC-A02-2023-092). Mice were housed in a 12-hour light / dark cycle and fed a normal diet or HFD.

[0043] Human liver specimens used in this embodiment were obtained from individuals who underwent liver surgery and liver biopsy at the First Affiliated Hospital of Nanjing Medical University. Samples from patients with viral infections (e.g., hepatitis B or C virus), autoimmune hepatitis, excessive alcohol consumption, alcoholism, or drug- or toxin-induced hepatic steatosis or steatohepatitis were excluded. All study protocols involving human samples were approved by the First Affiliated Hospital of Nanjing Medical University (2024-SR-538). All protocols adhered to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all individuals or their families.

[0044] Example 1. An inhibitor targeting hyposulfonated Gsk-3β (GEE) effectively increased glycogen synthesis and decreased gluconeogenesis in primary hepatocytes in a palmitic acid (PA)-induced insulin resistance model. 1. Experimental Procedure Primary hepatocytes were isolated from mice using a two-step collagenase perfusion method, at a concentration of 1.6 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. After cell attachment, cells were pretreated with 1 mM GEE for 2 hours (control group treated with PBS), followed by stimulation with 500 μM PA for 3 hours, and then incubated with 100 nM insulin for 30 minutes. The culture medium was discarded, and the cells were washed twice with PBS, with the last wash completely removing the PBS. The following assays were then performed: cells from one well were collected using a cell scraper, and glycogen synthesis levels were measured using a cuvette method; total RNA was extracted from the other wells using 1 mL of TRIZOL reagent, and after reverse transcription, the expression levels of gluconeogenesis-related genes Pck1 phosphoenolpyruvate carboxykinase 1 and G6pc glucose-6-phosphatase catalytic subunits were analyzed by qPCR.

[0045] 2. Experimental Results Compared with the control group, hepatocytes treated with GEE showed increased glycogen levels, indicating increased glycogen synthesis. Figure 1A qPCR analysis showed that GEE treatment reduced the expression of gluconeogenesis-related genes Pck1 and G6pc in hepatocytes. Figure 1B , Figure 1C ).

[0046] Example 2. Inhibitors targeting hyposulfonated Gsk-3β (GEE) can effectively increase the high-fat diet-induced HIR phenomenon. 1. Experimental Methods C57BL / 6 mice were divided into four groups (n=5 per group), with two groups fed a normal diet (ND) and the other two groups fed a high-fat diet (HFD). After 5 weeks of feeding, in the last week, one group of ND mice and one group of HFD mice were intraperitoneally injected with GEE daily, while the remaining two groups of ND and HFD mice were injected with an equal volume of PBS as controls. Subsequently, the hyperinsulin-eukaryotic clamp technique—considered the "gold standard" for assessing systemic insulin sensitivity—was used to establish a hyperinsulinemia state through continuous insulin infusion and dynamically regulate the glucose infusion rate to maintain glycemic stability. Key parameters included glucose infusion rate (GIR), glucose disposal rate (GDR), and hepatic glucose production rate (HGP), which reflect the degree of hepatic and systemic insulin resistance. Combined with isotope tracing technology, tissue-specific glucose uptake and endogenous glucose production capacity could be precisely assessed, thereby revealing the mechanisms of insulin resistance in greater depth. The improvement of insulin resistance was evaluated by comparing the GIR, GDR, and HGP of mice in each group, and further verified by glucose tolerance test, insulin tolerance test, and pyruvate tolerance test.

[0047] 2. Experimental Results The hyperinsulin-eukaryotic clamp results showed that the HGP in the experimental group of mice was significantly reduced. Figure 3A ) decrease, GIR ( Figure 3B ) and GDR ( Figure 3C All levels of insulin increased significantly, and insulin resistance was significantly alleviated. Glucose, insulin, and pyruvate tolerance tests demonstrated that insulin resistance was significantly suppressed in the inhibitor group mice.

[0048] Example 3. Inhibitors targeting hyposulfonated Gsk-3β (gsk3b knockout) alleviate insulin resistance C57BL / 6 mice were divided into four groups (n=5 per group). All mice were injected via tail vein with AAV8 virus carrying a shRNA sequence targeting GSK-3β (AAV-shGSK-3β) or control virus (AAV-shNC, meaningless random sequence).

[0049] Three weeks after injection, once GSK-3β expression in hepatocytes was stably knocked down, two groups of AAV-shGSK-3β mice and one group of AAV-shNC mice were fed a high-fat diet (HFD), while the remaining group of AAV-shNC mice continued to be fed a normal diet (ND). After five consecutive days of feeding, all mice underwent subsequent experimental testing, and hepatic insulin sensitivity was assessed using the hyperinsulinemia-eukemia clamp technique.

[0050] In the knockout group (AAV-shGSK-3β) mice, HGP decreased, GIR and GDR increased, and insulin resistance was significantly alleviated. Figures 4A to 4C ).

[0051] Example 4. An inhibitor targeting hyposulfonated Gsk-3β (ABT) can alleviate insulin resistance in model mice. Twenty C57BL / 6 mice were randomly divided into four groups (n=5 per group). Two groups were fed a normal diet (ND), and the other two groups were fed a high-fat diet (HFD). After 5 weeks of feeding, during the last week, one group of ND mice and one group of HFD mice were administered ABT (50 μg / g, prepared with PBS, freshly prepared and used, gavage volume approximately 200 μL / mouse) by gavage daily. The other two groups of ND and HFD mice were administered an equal amount of PBS by gavage as a control.

[0052] Hyperinsulin-eukaryotic clamp results showed that treatment with the inhibitor ABT significantly alleviated HFD-induced HIR in mice. Figure 5A ).

[0053] Example 5. Overexpression of gsk3b (GSK-3β variant) that cannot be sulfonated First, specific knockdown of GSK-3β in hepatocytes was achieved by injecting AAV8 virus (AAV-shGSK-3β) carrying a shRNA sequence targeting GSK-3β into C57BL / 6 mice via tail vein injection.

[0054] After the knockdown efficiency stabilized, plasmid DNA (20 μg each) carrying the coding sequences of wild-type GSK-3β (GSK-3β-WT) or the C178A mutant (GSK-3β-C178A) was dissolved in sterile saline (total volume 8-10% of mouse body weight) and rapidly injected into the mice via the tail vein within 5-7 seconds using a hydrodynamic gene delivery method. The control group received an equal volume of empty plasmid. Highly efficient transient expression of the target gene was achieved in liver tissue 24-48 hours after injection. Mice were then fed a high-fat diet for 5 days to establish an early HIR model. Hepatic insulin sensitivity was subsequently assessed using the hyperinsulinemia-eukopenia clamp technique.

[0055] Mice that knocked out and overexpressed the C178 site mutation did not develop HIR despite being treated with HFD.

[0056] The results confirmed that specific intervention at the Cys178 site can effectively block the development of HIR and avoid the potential side effects of broad-spectrum GSK-3β knockout, providing important evidence for translational research on this target. Figure 5B ).

[0057] Example 6. Inhibitors targeting hyposulfonated Gsk-3β (GEE) can effectively increase PA-induced glycogen synthesis and reduce gluconeogenesis in human liver organoids. Primary hepatocytes were isolated from adult liver tissue using a two-step collagenase perfusion method. Cell suspensions were collected, and pre-prepared Matrigel gel was mixed with the cell suspension at a 1:3 ratio. Cell density was adjusted based on cell counting results to achieve a density of 1 × 10⁶ cells per 12-well plate. 4 Hepatocytes / well. Then, 20 μL of a mixture of different concentrations was dropped into the center of each well to form a spherical shape. After standing for a short time to allow it to solidify slightly, the well was carefully inverted into a cell incubator. After 1 hour, 500 μL of culture medium was added to each well. The growth and morphological changes of the liver organoids were observed daily under a microscope, and the culture medium was changed every 2–3 days. After 12 days of 3D culture, siRNA transfection was performed: 25 nM siGSK-3β or negative control siRNA was mixed with RNAiMAX (Invitrogen) in Opti-MEM containing 10% normal FBS (Gibco) to form a transfection complex, which was then added to the organoids. Subsequently, 500 μM palmitic acid (PA) was added to both the experimental and control groups for 3 hours. The culture medium was discarded, and the cells were washed twice with PBS, with the PBS completely discarded on the last wash. Glycogen synthesis levels were measured using the cuvette method, and the expression of gluconeogenesis-related genes Pck1 and G6pc was analyzed using qPCR. The organoid culture medium was replaced with a sugar-free medium, and the amount of glucose released from the organoids into the supernatant was detected using the cuvette method.

[0058] Experimental results: Compared with the control group, the organoid group treated with GEE showed increased glycogen synthesis after PA treatment. Figure 6A ); Gluconeogenesis-related genes decreased ( Figure 6B This study demonstrated that inhibiting Gsk-3β targeting the hyposulfonation site effectively suppressed insulin resistance in organoids.

Claims

1. Use of an inhibitor of hyposulfonated GSK-3β in the preparation of a medicament for the prevention or treatment of insulin resistance.

2. In the use according to claim 1, the inhibitor of hyposulfonated GSK-3β is selected from: glutathione ethyl ester or its pharmaceutically acceptable salt, 1-aminobenzotriazole, N-acetylcysteine ​​or its pharmaceutically acceptable salt, GSK-3β knockout reagent, and vector expressing GSK-3β variants.

3. The use according to claim 1, wherein the treatment is selected from: Increase liver glycogen synthesis, Reduce gluconeogenesis Reduce the expression level of phosphoenolpyruvate carboxykinase 1 Reduce the expression level of the glucose-6-phosphatase catalytic subunit; Improve glucose tolerance Improve insulin tolerance, Improves pyruvate tolerance.

4. According to claim 2, the GSK-3β knockout reagent is selected from: CRISPR-Cas9 GSK-3β knockout vector, siRNA targeting GSK-3β, and shRNA targeting GSK-3β.

5. The use according to claim 2, wherein the GSK-3β variant has a mutated Cys residue at position 178 to Ala or Ser, compared to wild-type GSK-3β.

6. The use according to claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients.

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