Use of kidins220 in the preparation of a product for aiding in the diagnosis or treatment of obesity

Abstract

The application discloses application of KIDINS220 in preparation of products for assisting in diagnosis of obesity or treatment of obesity, and belongs to the technical field of biological medicine. The application finds that hybrid mutation of KIDINS220 gene leads to reduction of protein expression of KIDINS220, and then reduces combination with AKT2, and causes reduction of phosphorylation level of AKT signal path; AKT2 is a core kinase of insulin signal in adipose tissue, and disorder of activation of AKT2 can cause reduction of glucose uptake, increase of lipid synthesis, and finally cause obesity. An agent for promoting interaction between KIDINS220 gene or protein and AKT2 and a polypeptide for specifically recognizing and combining KIDINS220 and AKT2 can be used for treatment of obesity. The treatment strategy has higher molecular specificity and safety, and is more suitable for large-scale application of overweight and mild to moderate obesity population.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of KIDINS220 in the preparation of products for the auxiliary diagnosis or treatment of obesity. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Obesity has become a major global health challenge. As a metabolically active endocrine organ, fat plays a central role in energy homeostasis. Insulin resistance is a key aspect of obesity-related metabolic disorders, characterized by a decreased efficiency of insulin in promoting glucose uptake and utilization. The body compensates by secreting excessive insulin, leading to hyperinsulinemia to maintain stable blood glucose levels. During this process, insulin converts blood glucose into fat and stores it in adipocytes. However, excessively high insulin levels inhibit fat breakdown for energy and encourage adipocytes to absorb fat from the blood. Although the role of insulin in energy metabolism has been preliminarily understood, its specific signaling regulatory mechanisms still require further elucidation.

[0004] Currently, obesity is primarily attributed to excessive fat storage caused by an imbalance between energy intake and expenditure. Based on this, existing obesity intervention strategies mainly aim to reduce energy intake (such as dietary control, drug therapy, and bariatric surgery) or increase energy expenditure (such as physical activity). However, traditional methods have significant limitations: adherence to lifestyle modifications is generally low; drug and surgical treatments are not only costly but also require strict clinical indications, making them difficult to apply on a large scale in overweight and mild to moderately obese individuals. Therefore, developing more effective new strategies for obesity prevention and treatment is a crucial issue that urgently needs to be addressed in the current research field. Summary of the Invention

[0005] In view of this, the present invention provides the application of KIDINS220 in the preparation of products for the auxiliary diagnosis or treatment of obesity. The present invention discovers that heterozygous mutations in the KIDINS220 gene lead to decreased protein expression, thereby weakening its binding to AKT2 and causing a decrease in the phosphorylation level of the AKT signaling pathway (reduced AKT activity). AKT2 is a core kinase in insulin signaling in adipose tissue; its activation impairment leads to reduced glucose uptake and increased lipid synthesis, ultimately causing obesity. Detecting the phosphorylation levels of KIDINS220 and AKT2 can aid in the diagnosis of obesity or metabolic diseases. Injection of KIDINS220 mRNA (LNP packaging), AKT2 phosphorylation activators, and KIDINS220 and AKT2-specific binding peptides can all aid in the treatment of obesity.

[0006] In a first aspect, the present invention provides the application of the KIDINS220 gene or protein as an auxiliary diagnostic marker in the preparation of reagents for auxiliary diagnosis of obesity.

[0007] KIDINS220, short for "220kDa protein kinase D interacting substrate," is a multifunctional transmembrane protein that plays a crucial role in signal transduction, phosphate homeostasis regulation, and the development of various diseases.

[0008] Secondly, the present invention provides the application of a reagent for detecting KIDINS220 in the preparation of a reagent for assisting in the diagnosis of obesity, wherein the reagent for detecting KIDINS220 is a reagent for detecting the content of the KIDINS220 gene or protein.

[0009] Low levels of KIDINS220 gene or protein expression indicate a high risk of obesity.

[0010] Preferably, the reagent for detecting KIDINS220 also includes a reagent for detecting AKT2 phosphorylation level.

[0011] Preferably, the reagent for detecting the content of the KIDINS220 gene or protein is selected from one of the following: primers for specifically amplifying the KIDINS220 gene, probes for specifically recognizing the KIDINS220 gene or its transcripts, and antibodies for specifically targeting the KIDINS220 protein.

[0012] Preferably, the test sample is tissue, serum or plasma.

[0013] Preferably, the reagents used to assist in the diagnosis of obesity include, but are not limited to, nucleic acid extraction reagents, polymerase chain reaction reagents, Western blotting reagents, and enzyme-linked immunosorbent assay (ELISA) reagents.

[0014] Thirdly, the present invention provides a kit for diagnosing obesity, comprising a reagent for detecting the content of the KIDINS220 gene or protein, wherein the reagent for detecting the content of the KIDINS220 gene or protein is selected from primers for specifically amplifying the KIDINS220 gene, probes for specifically recognizing the KIDINS220 gene or its transcripts, and antibodies for specifically anti-KIDINS220 protein.

[0015] Fourthly, this invention provides the application of reagents that promote the expression of the KIDINS220 gene or protein in the preparation of products for treating obesity.

[0016] Fifthly, the present invention provides the use of reagents that promote the interaction between the KIDINS220 gene or protein and AKT2 in the preparation of products for treating obesity.

[0017] In a sixth aspect, the present invention provides the use of a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 in the preparation of products for treating obesity.

[0018] In a seventh aspect, the present invention provides a pharmaceutical composition in which the active ingredient comprises at least a substance that promotes the expression and / or activity of the KIDINS220 gene or protein; or, the active ingredient comprises at least a substance that promotes the interaction between KIDINS220 and AKT2.

[0019] Preferably, the inactive components of the pharmaceutical composition can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, topical application, suppositories, and sterile injectable solutions. The aforementioned carriers, excipients, and diluents, and other inactive pharmaceutical components, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0020] Furthermore, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0021] Preferably, the pharmaceutical composition of the present invention can be administered into the body in a known manner. For example, it can be delivered to a specific tissue via intravenous systemic delivery or local injection. Optionally, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0022] Preferably, the drug composition can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees.

[0023] Eighthly, the present invention provides the use of the above-described polypeptide or pharmaceutical composition in the preparation of products for treating obesity.

[0024] In a ninth aspect, the present invention provides the application of reagents for knocking down KIDINS220 expression in the construction of animal obesity models.

[0025] This invention does not impose any special restrictions on the method of knocking down KIDINS220 expression; for example, gene knockout can be performed using CRISPR / CAS9 technology.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention reveals a new mechanism of obesity pathogenesis through the reduction of protein expression, weakening of binding with AKT2, and inhibition of AKT signaling pathway activity by heterozygous mutation of KIDINS220 gene. This provides a key molecular target for pathological research on obesity and lays a theoretical foundation for the development of subsequent precision intervention strategies. Based on this, this invention develops an auxiliary diagnostic system with KIDINS220 gene or protein as the core. It can detect the expression level of KIDINS220 and the phosphorylation status of AKT2 through easily accessible samples such as serum and plasma, so as to realize early screening and assessment of obesity risk. This makes up for the shortcomings of traditional methods that rely on clinical indicators or subjective assessment, and improves the objectivity of diagnosis and early warning capability.

[0027] (2) The present invention directly targets and repairs the KIDINS220-AKT2 signaling pathway by promoting the expression of KIDINS220 or its interaction with AKT2, specific binding peptides and drug compositions, thereby restoring insulin sensitivity and improving abnormal lipid metabolism. Compared with traditional weight loss methods, the treatment strategy of the present invention has higher molecular specificity and safety, and is more suitable for large-scale application in overweight and mild to moderate obese populations.

[0028] (3) The present invention also provides a reagent for knocking down KIDINS220 expression to construct an animal obesity model. This model can reproduce the phenotypes such as weight gain and metabolic abnormalities caused by KIDINS220 signal dysregulation (such as glucose intolerance and adipose tissue weight gain), providing a stable and reliable experimental tool for the study of obesity pathological mechanisms, new drug screening and efficacy verification, and promoting the connection between obesity-related basic research and translational applications. Attached Figure Description

[0029] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0030] Figure 1 The following are the experimental results of the effects of KIDINS220 gene knockout on mouse body weight, insulin sensitivity, and lipid accumulation in adipocytes in Example 1 of this invention; wherein, A is the body weight change curve of KIDINS220 systemic heterozygous knockout mice (KO) and wild-type mice (WT) under normal diet (NCD) or high-fat diet (HFD) conditions; B is the body weight change curve of adipose tissue-specific knockout mice (AKO) and control mice (fl / fl) under NCD or HFD conditions; C is the Western blotting results of KIDINS220 and p-AKT2 in subcutaneous adipose tissue (ing) and visceral adipose tissue (epi) of wild mice after 0, 18, and 36 days of high-fat diet feeding; D is the fasting serum insulin level of mice after 2 months of normal diet feeding; E is the fasting serum insulin level of mice after 2 months of high-fat diet feeding; F is the Western blotting results of KIDINS220, p-AKT2, and total AKT2 in visceral adipose tissue (epi). blotting results; G represents the blood glucose change curves of mice after 2 months of normal diet feeding in glucose tolerance test (GTT) and insulin tolerance test (ITT); H represents the blood glucose change curves of mice after 2 months of high-fat diet feeding in GTT and ITT; I represents the Oil Red O staining results and quantitative analysis of the KIDINS220 knockdown group and the control group after induction of differentiation of primary MEF and SVF cells.

[0031] Figure 2This document presents the correlation between KIDINS220 expression levels in clinical samples and age, glycated hemoglobin, and BMI in Example 2 of this invention, as well as the detection results of insulin signaling pathway proteins in adipose tissue of obese patients. Specifically, A represents the linear regression analysis of peripheral blood KIDINS220 mRNA expression levels with age; B represents the linear regression analysis of KIDINS220 mRNA expression levels with glycated hemoglobin (HbA1c) levels; C represents the linear regression analysis of KIDINS220 mRNA expression levels with body mass index (BMI); and D represents the Western blotting results of KIDINS220, p-IR, IR, p-PDK1, PDK1, p-AKT2, AKT2, p-AS160, AS160, and β-actin in white adipose tissue of normal-weight individuals and obese patients.

[0032] Figure 3 The following are experimental results regarding the molecular mechanism by which KIDINS220 regulates AKT2 phosphorylation and interacts with AKT2 and PDK1 in Example 3 of this invention; wherein, A is the Western blotting detection results of KIDINS220, p-AKT2, total AKT2 and β-actin after transfection with different doses of KIDINS220-Flag; B is the immunoprecipitation detection results after co-transfection of KIDINS220-Flag and AKT2-Myc; C is the immunoprecipitation detection results after co-transfection of KIDINS220-Flag and PDK1-Myc; D is the immunofluorescence colocalization images of KIDINS220 and AKT2 / PDK1 before and after insulin treatment; E is the structural prediction model of the KIDINS220, AKT2 and PDK1 triple complex.

[0033] Figure 4 This is the result of the immunoprecipitation of the interaction between the KIDINS220 cutoff and AKT2 in Example 4 of the present invention.

[0034] Figure 5 The graph shows the therapeutic effects of KIDINS220 overexpression, KIDINS220 peptide, and AKT2 activator on obese mice in Example 5 of this invention. Among them, A is the weight change curve of each group of mice within 13 weeks of administration; B is the comparison of fasting serum insulin levels of each group of mice; C is the blood glucose change curve and area under the curve (AUC) statistical graph of the glucose tolerance test (GTT); D is the blood glucose change curve and area under the curve (AUC) statistical graph of the insulin tolerance test (ITT).

[0035] Figure 6 This is a schematic diagram of the mechanism by which KIDINS220 regulates lipid and glucose metabolism as proposed in this invention; where A represents the regulatory mechanism under normal physiological conditions and B represents the regulatory mechanism under obese conditions. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] The technical solution of the present invention will be further described below with reference to specific embodiments. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the sales company; the reagents, consumables, cells, experimental animals, etc. used in the embodiments can be purchased commercially unless otherwise specified.

[0038] The sequences involved in the following embodiments are shown in Table 1.

[0039] Table 1 Sequence Information

[0040] Example 1 This embodiment aims to verify the causal relationship between KIDINS220 gene expression level and obesity occurrence using multiple mouse models, and to elucidate its impact on the insulin signaling pathway.

[0041] 1. Effect of KIDINS220 systemic heterozygous knockout on mouse body weight KIDINS220 systemic heterozygous knockout mice were constructed using CRISPR / Cas9 technology. + / - The KO group (or KO group for short) was used as a control group (WT group) with wild-type mice from the same littermate. Three-week-old male mice were divided into four groups: WT + normal diet group (WTNCD), WT + high-fat diet group (WT HFD, 60% fat for energy), KO + NCD group (KO NCD), and KO + HFD group (KO HFD), with 12 mice in each group. The weight of the mice was measured and recorded weekly for 15 consecutive weeks.

[0042] The results are as follows Figure 1 As shown in Figure A, it can be seen that the body weight of mice in the KO group was higher than that in the WT group under both NCD and HFD feeding conditions, with the difference being more pronounced under HFD feeding conditions. Furthermore, this difference continued to widen with prolonged feeding time. These results indicate that heterozygous knockout of the KIDINS220 gene leads to a significant increase in mouse body weight, and this effect is more pronounced under a high-fat diet, suggesting that reduced KIDINS220 expression is a contributing factor to obesity.

[0043] 2. Effects of KIDINS220 fat-specific knockout on mouse body weight To clarify the direct role of KIDINS220 in adipose tissue, an adipose tissue-specific knockout mouse (Kidins220) was constructed. ΔWAT (AKO group for short). Specifically, Kidins220-flox mice were crossed with Adipoq-iCre transgenic mice to obtain AKO mice with Kidins220 knocked out in adipocytes, with littermate Kidins220-flox mice as controls (fl / fl group). From 3 weeks of age, mice were fed either NCD or HFD, with 12 mice in each group, and their body weight was monitored weekly.

[0044] The results are as follows Figure 1 As shown in B in the figure. Consistent with the whole-body knockout model, the body weight of mice in the AKO group was significantly higher than that in the fl / fl control group under both NCD and HFD dietary conditions (p<0.01). This result further confirms that the loss of KIDINS220 expression in adipose tissue is sufficient to lead to weight gain, indicating that KIDINS220 plays a key role in weight regulation in adipose tissue.

[0045] 3. Dynamic changes in phosphorylation levels of KIDINS220 and AKT2 in adipose tissue of high-fat diet-induced obese mice Eight-week-old wild-type male C57BL / 6J mice from the same batch were fed HFD for days 0, 18, and 36, respectively. At each time point, three mice were sacrificed, and subcutaneous adipose tissue (ing) and epididymal visceral adipose tissue (epi) were separated. After extracting total protein, the expression level of KIDINS220 protein and the phosphorylation level of AKT2 protein at Ser473 site (p-AKT2) were detected by Western blotting.

[0046] The results are as follows Figure 1 C and Figure 1 As shown in F in the figure, with the extension of HFD feeding time (0 days → 18 days → 36 days), the expression level of KIDINS220 protein in both subcutaneous and visceral adipose tissue showed a gradual decreasing trend. Simultaneously, the p-AKT2 level also showed a consistent decreasing trend, indicating a significant positive correlation between the two. These results suggest that in the process of diet-induced obesity, the downregulation of KIDINS220 expression in adipose tissue is closely related to the inactivation of the AKT2 signaling pathway, suggesting that KIDINS220 may be an upstream regulatory molecule of AKT2.

[0047] 4. Effect of KIDINS220 knockdown on fasting serum insulin levels in mice WT mice and Kidins220 mice were fed a normal diet (NCD) for 2 months and a high-fat diet (HFD) for 2 months, respectively. + / - Six mice were included in each of the three groups: mice, AKO mice, and Kidins220-flox mice. Fasting peripheral blood was collected, serum was separated, and serum insulin levels were detected using an ELISA kit.

[0048] The results are as follows Figure 1 D (NCD group) and Figure 1 As shown in E (HFD group). Under both NCD and HFD conditions, the fasting serum insulin levels of KIDINS220 knockdown mice were significantly higher than those of control mice. This result indicates that KIDINS220 knockdown can lead to hyperinsulinemia in mice, a typical manifestation of insulin resistance.

[0049] 5. Effects of KIDINS220 knockdown on glucose tolerance (GTT) and insulin tolerance (ITT) in mice. Glucose tolerance test (GTT): Mice were fasted overnight (16 hours) and then injected intraperitoneally with glucose solution (2 g / kg body weight). Blood samples were collected from the tail tip at 0, 15, 30, 60, 90, and 120 minutes after injection, and blood glucose concentrations were measured using a glucometer.

[0050] Insulin tolerance test (ITT): Mice were fasted for 4 hours and then injected intraperitoneally with human insulin (0.75 U / kg body weight). Blood glucose concentrations were measured at 0, 15, 30, 60, 90, and 120 minutes after injection.

[0051] WT and Kidins220 infants fed NCD for 2 months and HFD for 2 months, respectively + / - The above experiment was conducted on mice, with 6 mice in each group.

[0052] The results are as follows Figure 1 G (NCD group) and Figure 1 The H (HFD group) is shown in the figure. In the GTT experiment, Kidins220... + / - The blood glucose levels in mice at all time points were higher than those in WT mice, and the area under the blood glucose curve was significantly increased, indicating a decreased glucose clearance capacity. In the ITT experiment, after insulin injection, Kidins220... + / - The decrease in blood glucose in mice was significantly smaller than that in WT mice, indicating reduced sensitivity to exogenous insulin. This result directly demonstrates that KIDINS220 knockdown leads to impaired glucose tolerance and insulin resistance in mice, further supporting the crucial role of KIDINS220 in maintaining normal glucose metabolism and insulin sensitivity.

[0053] 6. Effects of KIDINS220 knockdown on lipid accumulation in primary adipocytes Two primary cell lines were isolated from mice: ① mouse embryonic fibroblasts (MEF, obtained from E13.5-day embryos); ② vascular component of adipose tissue matrix (SVF, obtained from inguinal adipose tissue of 8-week-old mice). These cells were induced to differentiate into mature adipocytes in vitro. KIDINS220 expression in the cells was knocked down by lentivirus-mediated shRNA (sequence shown in SEQ ID NO: 2, targeting KIDINS220), with an empty vector as a control. On day 8 of induced differentiation, Oil Red O staining was performed, and the cells were observed and photographed under a microscope. The dye was then dissolved in isopropanol, and the absorbance was measured at 510 nm for quantification.

[0054] The results are as follows Figure 1 As shown in Figure I. Compared with the control group, MEF and SVF cells with knocked-down KIDINS220 showed significantly deeper Oil Red O staining after differentiation. This cellular-level result further confirms that loss of KIDINS220 function directly promotes lipid storage in adipocytes, revealing the negative regulatory role of KIDINS220 in adipogenesis.

[0055] Example 2 This embodiment aims to verify the correlation between the expression level of the KIDINS220 gene or protein and obesity and abnormal glucose metabolism by analyzing clinical samples from different populations.

[0056] 1. Correlation analysis of peripheral blood KIDINS220 mRNA expression level with age Peripheral blood samples were collected from 60 healthy volunteers aged 0-80 years, divided into 10-year age groups with 6 volunteers per age group. Total RNA was extracted from peripheral blood using the Trizol method and reverse transcribed into cDNA. The relative expression level of KIDINS220 mRNA was detected using real-time quantitative PCR, with β-actin as an internal reference gene. The primer pairs used were as follows: forward primer sequence as shown in SEQ ID NO: 3, and reverse primer sequence as shown in SEQ ID NO: 4. Linear regression analysis was used to analyze the correlation between KIDINS220 expression level and age.

[0057] The results are as follows Figure 2 As shown in A in the figure. Linear regression analysis showed that KIDINS220 mRNA expression level was significantly negatively correlated with age (R0). 2 =0.73, p<0.0001). This indicates that the expression level of KIDINS220 in peripheral blood gradually decreases with age. This result suggests that KIDINS220 expression is age-dependent, indicating that the influence of age should be considered in the diagnosis of obesity, and also provides a potential molecular marker for age-related metabolic decline.

[0058] 2. Correlation analysis between peripheral blood KIDINS220 mRNA expression level and glycated hemoglobin (HbA1c) Sixty peripheral blood samples from the same batch were used. Fasting glycated hemoglobin (HbA1c) levels (expressed as a percentage of total hemoglobin) were measured using high-performance liquid chromatography (HPLC). The HbA1c value of each sample was paired with the KIDINS220 mRNA expression level measured in step 1, and the correlation between the two was analyzed using linear regression.

[0059] The results are as follows Figure 2 As shown in B in the figure. Linear regression analysis showed that KIDINS220 mRNA expression level was significantly negatively correlated with HbA1c level (R0). 2 = 0.62, p < 0.0001). This means that the lower the KIDINS220 expression level, the higher the HbA1c level. HbA1c is an important clinical indicator reflecting the average blood glucose level over the past 2-3 months and is closely related to insulin resistance. The negative correlation between KIDINS220 expression and HbA1c further supports the animal experiment results in Example 1 showing that KIDINS220 knockdown leads to insulin resistance, suggesting that KIDINS220 can serve as an auxiliary diagnostic biomarker for assessing abnormal glucose metabolism.

[0060] 3. Correlation analysis between peripheral blood KIDINS220 mRNA expression level and body mass index (BMI) Using the same batch of 60 samples, the height and weight of each subject were recorded, and the Body Mass Index (BMI) was calculated as: weight (kg) / height (m). 2 The BMI value was paired with the KIDINS220 mRNA expression level measured in step 1, and the correlation between the two was analyzed using linear regression.

[0061] The results are as follows Figure 2 As shown in C in the figure. Linear regression analysis showed that KIDINS220 mRNA expression level was negatively correlated with BMI (R0). 2 = 0.08916, p = 0.0205), the correlation was statistically significant.

[0062] 4. Detection of KIDINS220 protein expression and insulin signaling pathway activity in adipose tissue of obese patients White adipose tissue (WAT) samples were collected from normal-weight individuals (BMI < 24, n = 4) and obese patients (BMI ≥ 28, n = 4) undergoing surgical treatment. Total protein was extracted, and the following parameters were measured using Western blotting: (1) Phosphorylated protein levels: tyrosine phosphorylation level of insulin receptor (IR) (p-IR), phosphorylation level of phosphatidylinositol-dependent protein kinase 1 (PDK1) (p-PDK1), phosphorylation level of AKT2 protein at Ser473 site (p-AKT2), ​​and phosphorylation level of AKT substrate protein AS160 (p-AS160). (2) Total protein levels: IR, PDK1, AKT2, AS160; (3) KIDINS220 protein expression level; (4) Sample loading internal reference: β-actin.

[0063] The corresponding total protein was used as the normalization reference for phosphorylated proteins, and β-actin was used as the consistency control for loading amount.

[0064] The results are as follows Figure 2 As shown in D in the figure. Compared with the normal control group, the expression level of KIDINS220 protein in the adipose tissue of obese patients was significantly downregulated; the level of p-AKT2 (Ser473) was significantly reduced, while the level of total AKT2 did not change significantly; the levels of p-PDK1 and p-AS160 also showed varying degrees of reduction, while the corresponding total protein levels did not change significantly; the level of p-IR was reduced, while the level of total IR did not change significantly.

[0065] This result is highly consistent with the findings in mouse adipose tissue in Example 1, validating the association between downregulated KIDINS220 expression and reduced AKT2 phosphorylation levels in human samples under obese conditions. This indicates that abnormalities in the KIDINS220-AKT2 signaling pathway play a crucial role in the pathogenesis of human obesity, further supporting the feasibility of targeting KIDINS220 for obesity diagnosis and treatment.

[0066] Example 3 This embodiment provides a study on the molecular mechanism of KIDINS220's regulation of AKT2 phosphorylation and their interaction.

[0067] 1. Effect of KIDINS220 overexpression on AKT2 phosphorylation level HEK293T cells were seeded in 6-well plates. When the cell density reached 70-80%, they were transfected with 0 μg, 0.5 μg, and 2.0 μg of the KIDINS220-Flag overexpression plasmid, respectively (the total transfection amount was increased by adding an empty vector). Forty-eight hours after transfection, cell lysates were collected, and Western blotting was used to detect the expression level of the KIDINS220-Flag fusion protein, the phosphorylation level of AKT2 protein at Ser473 (p-AKT2), ​​and the total AKT2 protein level. β-actin was used as an internal control.

[0068] The results are as follows Figure 3 As shown in Figure A, the expression level of KIDINS220 protein increased in a dose-dependent manner with increasing KIDINS220-Flag transfection amount (0→0.5→2.0 μg). Simultaneously, the level of p-AKT2 (Ser473) also increased in a dose-dependent manner, while the total AKT2 protein level remained unchanged. These results indicate that the expression level of KIDINS220 positively regulates the phosphorylation activation of AKT2, suggesting that KIDINS220 is a positive upstream regulator of the AKT2 signaling pathway.

[0069] 2. Validation of the interaction between KIDINS220 and AKT2 and PDK1 via co-precipitation immunoprecipitation (CO-IP) HEK293T cells were transfected into four groups: Group 1 was the empty vector control (without KIDINS220-Flag and AKT2-Myc); Group 2 was transfected with AKT2-Myc plasmid alone; Group 3 was transfected with KIDINS220-Flag plasmid alone; and Group 4 was co-transfected with KIDINS220-Flag and AKT2-Myc plasmids. Cells were lysed 48 hours after transfection, and an equal volume of protein lysis buffer was used for immunoprecipitation with anti-Flag antibody-conjugated magnetic beads (Flag beads). The precipitate complex (IP) and a portion of the cell lysis buffer (Input) were subjected to SDS-PAGE, followed by Western blotting with anti-Flag and anti-Myc antibodies. Another experiment was conducted using the same method, but AKT2-Myc was replaced with PDK1-Myc to detect the interaction between KIDINS220 and PDK1.

[0070] The results are as follows Figure 3 B (KIDINS220 and AKT2) and Figure 3As shown in C (KIDINS220 and PDK1). In the Input section, expression of the corresponding Flag or Myc tag protein was detected in each transfection group, indicating successful transfection and normal protein expression. In the IP (Flag beads drop-down) section: Group 1 (empty vector) and Group 2 (AKT2-Myc or PDK1-Myc transfected alone): No Flag or Myc bands were detected. Group 3 (KIDINS220-Flag transfected alone): Flag bands were detected, but Myc bands were not detected. Group 4 (co-transfected KIDINS220-Flag and AKT2-Myc or PDK1-Myc): Both Flag and Myc bands were detected.

[0071] The above results indicate specific interactions between KIDINS220 and AKT2, and between KIDINS220 and PDK1. Only when KIDINS220-Flag and AKT2-Myc (or PDK1-Myc) are co-expressed can the Myc-tagged protein be co-precipitated by anti-Flag magnetic beads; otherwise, it cannot. This confirms that KIDINS220 can bind to both AKT2 and PDK1, suggesting that KIDINS220 may act as a scaffold protein involved in the assembly and activation of the AKT signaling complex.

[0072] 3. Co-localization analysis of KIDINS220 with AKT2 and PDK1 under insulin stimulation HEK293T cells were seeded in laser confocal microscopy culture dishes. After cell attachment, the cells were treated with 10 μg / mL insulin for 15 seconds (the control group received an equal volume of PBS). After treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then incubated overnight at 4°C with primary antibodies against KIDINS220 (rabbit), AKT2 (mouse), and PDK1 (sheep), respectively. The next day, the cells were incubated with the corresponding Alexa Fluor 488 (green), 555 (red), and 647 (far-red) labeled secondary antibodies for 1 hour at room temperature. After counterstaining the cell nuclei with DAPI, the cells were observed and photographed under a laser confocal microscope.

[0073] The results are as follows Figure 3 As shown in D in the figure. In the untreated group, the fluorescence signals of KIDINS220 and AKT2 / PDK1 were diffusely distributed in the cytoplasm, with weak co-localization signals. After insulin treatment for 15 seconds, the co-localization signals of KIDINS220 and AKT2 / PDK1 were significantly enhanced, as evidenced by an increase in yellow fluorescent spots.

[0074] 4. Structural biology prediction of the complex structure of KIDINS220 with AKT2 and PDK1 Using the HADDOCK protein-protein docking software, based on known domains or homology models of KIDINS220, AKT2, and PDK1, the possible interaction interfaces and complex structures among the three proteins were predicted. Results are as follows: Figure 3 As shown in Figure E, structural prediction reveals that KIDINS220 exhibits complementary interactions with both the PH domain of AKT2 and the kinase domain of PDK1, forming a stable triplet complex. This structural prediction provides structural model support for the molecular mechanism by which KIDINS220 acts as a scaffold protein, simultaneously recruiting AKT2 and PDK1 and promoting AKT2 phosphorylation.

[0075] Example 4 This embodiment provides the identification of the key structural domains of the interaction between KIDINS220 and AKT2.

[0076] To identify the key domains of the KIDINS220-AKT2 interaction, a series of KIDINS220 truncated expression plasmids (all tagged with a flag) were constructed, including: full-length KIDINS220-Flag, truncated KIDINS220(Δ440-1771)-Flag (omitting amino acids 440-1771), truncated KIDINS220(Δ954-1771)-Flag (omitting amino acids 954-1771), truncated KIDINS220(Δ1333-1771)-Flag (omitting amino acids 1333-1771), and truncated KIDINS220(Δ1-439)-Flag (omitting amino acids 1-439). These truncated plasmids were co-transfected with the AKT2-Myc plasmid into HEK293T cells. Forty-eight hours after transfection, cells were lysed, and immunoprecipitation was performed using anti-Flag antibody-conjugated magnetic beads. The expression of Flag and Myc tag proteins in the precipitate (IP) and cell lysate (Input) was detected by Western blotting.

[0077] The results are as follows Figure 4 As shown. In the Input section, all truncated variants and AKT2-Myc were expressed normally. In the IP (anti-Flag pull-down) section: the full-length KIDINS220-Flag and all truncated variants except Δ1-439 could effectively pull down AKT2-Myc, showing a positive Myc band; while the Δ1-439 truncated variant, although it could be expressed normally (positive Flag band), no AKT2-Myc band was detected in its pull-down product.

[0078] The above results indicate that the N-terminal region (amino acids 1-439) of the KIDINS220 protein is a key domain essential for its interaction with AKT2. The absence of this region results in KIDINS220 losing its ability to bind to AKT2. This finding provides a structural basis for designing intervention strategies targeting the KIDINS220-AKT2 interaction.

[0079] Example 5 This embodiment demonstrates that KIDINS220 overexpression, KIDINS220 peptide, or AKT2 activator treatment can improve obesity symptoms and insulin resistance in mice.

[0080] Obese mice induced by a high-fat diet were randomly divided into four groups (n=10): HFD model control group, Kidins220 mRNA group (LNP-mRNA injected via tail vein, 0.5 mg / kg), SEQ ID NO:1 peptide group (50 mg / kg injected via tail vein), and SC79 group (AKT2 specific activator, 20 mg / kg injected intraperitoneally). Administered medication every two days for 13 weeks, while continuing the high-fat diet. The measured parameters included body weight, fasting serum insulin, glucose tolerance (GTT), and insulin tolerance (ITT).

[0081] like Figure 5 As shown in Figure A, from week 1 to week 13, the body weight of mice in the HFD group continuously increased, maintaining the highest level from week 6 onwards. The SC79 group mice showed the slowest body weight gain, reaching their lowest weight at week 13. The body weights of mice in the Kidins220 mRNA group and the SEQ ID NO:1 group fell between the two groups, with their weight curves largely overlapping, both significantly lower than the HFD group.

[0082] ELISA was used to measure serum insulin levels after 3 weeks of feeding a high-fat diet with concurrent drug intervention. Figure 5 As shown in Figure B, the fasting insulin level was highest in the HFD group mice. Compared with the HFD group, the Kidins220 mRNA group, SEQ ID NO:1 group, and SC79 group all showed significant decreases. Among them, the Kidins220 mRNA group had the lowest insulin level (significant**, p<0.01), and the SEQ ID NO:1 group and SC79 group also showed significant decreases (*, p<0.05).

[0083] like Figure 5As shown in Figure C, the GTT blood glucose curves indicate that the HFD group had the highest blood glucose levels at all time points, indicating the worst glucose clearance capacity. The Kidins220 mRNA group had the lowest blood glucose curve, followed by the SEQ ID NO:1 group, with the SC79 group falling in between. The GTT AUC histogram shows that the HFD group had the highest AUC; the Kidins220 mRNA group, SEQ ID NO:1 group, and SC79 group all had significantly lower AUCs (*, p<0.05), with the SC79 group having the lowest AUC.

[0084] Figure 5 The ITT blood glucose curves in the D group showed that after insulin injection, the HFD group experienced the smallest decrease in blood glucose and the worst insulin sensitivity. The SC79 group showed the largest decrease in blood glucose, with the lowest curve; followed by the Kidins220 mRNA group. The ITT AUC histogram showed that the HFD group had the highest AUC; the AUCs of the SEQ ID NO:1 group, the Kidins220 mRNA group, and the SC79 group were all significantly reduced (*, p<0.05), with the SC79 group having the lowest AUC.

[0085] The above results indicate that KIDINS220 overexpression (Kidins220 mRNA) significantly reduced weight gain and fasting insulin levels in obese mice, improved glucose tolerance and insulin sensitivity, demonstrating the potential of restoring KIDINS220 expression to treat obesity. The KIDINS220-derived peptide (SEQ ID NO:1) also improved obesity-related metabolic indicators, suggesting that this peptide may exert its effects by mimicking KIDINS220 function or promoting its binding to AKT2. The AKT2 activator (SC79) directly activated the AKT2 signaling pathway, also effectively improving obesity and insulin resistance, further validating the importance of AKT2 as a downstream effector molecule. Therefore, promoting KIDINS220 expression, promoting AKT2 activation (using the SEQ ID NO:1 peptide), or directly activating AKT2 can all be effective strategies for treating obesity.

[0086] In summary, this invention proposes a schematic diagram of the molecular mechanism by which KIDINS220 regulates lipid and glucose metabolism, as follows: Figure 6 As shown. Under normal physiological conditions ( Figure 6 In the A group, KIDINS220 acts as a scaffold protein, recruiting both PDK1 and AKT2 to form a triplet complex, promoting phosphorylation activation of AKT2 at the Ser473 site. Activated AKT2 further phosphorylates downstream substrates AS160 and mTORC1, promoting GLUT4 translocation to the cell membrane and enhancing glucose uptake; simultaneously, AKT2 signaling inhibits lipolysis. In obese individuals (… Figure 6In the B group, the downregulation of KIDINS220 expression in adipose tissue leads to the inhibition of the formation of the KIDINS220-AKT2-PDK1 complex, a decrease in AKT2 phosphorylation level, and a weakening of downstream signals, ultimately resulting in reduced glucose uptake, insulin resistance, and abnormal fat accumulation.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of KIDINS220 gene or protein as an auxiliary diagnostic marker in the preparation of reagents for auxiliary diagnosis of obesity.

2. The application of a reagent for detecting KIDINS220 in the preparation of reagents for the auxiliary diagnosis of obesity, characterized in that, The reagent used to detect KIDINS220 is a reagent for detecting the content of the KIDINS220 gene or protein.

3. The application as described in claim 2, characterized in that, The reagents for detecting KIDINS220 also include reagents for detecting AKT2 phosphorylation levels.

4. A reagent kit for diagnosing obesity, characterized in that, It contains a reagent for detecting the content of the KIDINS220 gene or protein, wherein the reagent for detecting the content of the KIDINS220 gene or protein is selected from primers for specifically amplifying the KIDINS220 gene, probes for specifically recognizing the KIDINS220 gene or its transcripts, and antibodies for specifically anti-KIDINS220 protein.

5. Application of reagents that promote the expression of the KIDINS220 gene or protein in the preparation of products for treating obesity.

6. Application of reagents that promote the interaction between the KIDINS220 gene or protein and AKT2 in the preparation of products for treating obesity.

7. Application of a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 in the preparation of products for treating obesity.

8. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition contains at least a substance that promotes the expression and / or activity of the KIDINS220 gene or protein; or, the active ingredient of the pharmaceutical composition contains at least a substance that promotes the interaction between KIDINS220 and AKT2.

9. The use of the polypeptide of claim 7 or the pharmaceutical composition of claim 8 in the preparation of a product for treating obesity.

10. Application of reagents that knock down KIDINS220 expression in the construction of animal obesity models.

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