2024 Volume 72 Issue 10 Pages 845-855
Obesity is a global medical issue that can be effectively treated by relieving adipose inflammation and subsequent insulin resistance. Diosgenin (DIOS) has various effects as a steroidal saponin in inflammatory disorders. This study explored the effects and mechanism of DIOS on adipose inflammation and insulin sensitivity, both in silico and in vivo. The high-fat diet-induced obesity model in C57BL/6 mice was divided into five groups: normal chow (NC), high-fat diet (HFD), HFD with atorvastatin 10 mg/kg (AT), HFD with DIOS 100 mg/kg (DIOS 100), and HFD with DIOS 200 mg/kg (DIOS 200). Each group underwent an oral intervention for seven weeks. DIOS significantly suppressed weight gain in the body, liver, and epididymal fat pads. Additionally, it significantly improved fasting glucose and insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR), and oral glucose tolerance test results, and reduced the proportion of total and M1 adipose tissue macrophages. Significant changes were shown in mRNA expression of janus kinase 2 (JAK2), insulin receptor (INRS), insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), all of which exhibited high binding affinity in the in silico. Safety indices, including aspartate aminotransferase (AST), alanine transaminase (ALT), and creatinine level indicated the preventive effects of DIOS. In conclusion, DIOS improves insulin resistance and obesity-associated inflammation via the PI3K/Akt signaling pathway.
Obesity, characterized by excessive adipose tissue accumulation, and it considerably increases the risk of developing chronic metabolic disorders such as type 2 diabetes mellitus resulting in notable morbidity and mortality worldwide.1) Obesity is recognized for its association with chronic inflammation in different tissues, marked by the release of inflammatory adipokines, typically originating from adipose tissue.2) Moreover, inflammation is a contributing factor in the onset of insulin resistance. Once initiated, inflammation and insulin resistance can mutually worsen each other.3) Insulin resistance is another important underlying characteristic of metabolic health issues.4) Despite the implementation of various therapeutic approaches, the prevalence of obesity continues to increase, as demonstrated by the Center for Disease Control and Prevention.
This study aimed to elucidate the therapeutic effect and mechanism of diosgenin (DIOS), a naturally occurring steroidal saponin known to be effective against metabolic disorders associated with obesity and insulin resistance.5,6) DIOS, found in various plants such as Solanum and Dioscorea,7) has been actively studied for its anti-inflammatory effects. Wang et al. reported that DIOS significantly inhibited the interleukin-1β (IL-1β)-stimulated expression of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human osteoarthritis chondrocytes.8) Another study reported that DIOS suppressed the secretion of tumor necrosis factor-α (TNF-α), IL-1β, and interleukin-6 (IL-6) by enhancing the expression of secretory leukocyte peptidase inhibitor (SLPI), glucocorticoid-induced leucine zipper (GILZ), and mitogen-activated protein kinase phosphatase 1 (MKP-1) in ovalbumin-induced asthmatic mice and primary tracheal epithelial cells.9) In studies by Fang et al.10) and Liu et al.,11) the effects of DIOS on the insulin signaling pathway were investigated in HepG2 cells and endothelial cells, respectively. However, there have been no studies examining the insulin signaling pathway in a mouse model after oral administration of DIOS. Although several studies have investigated the therapeutic advances of DIOS in managing insulin sensitivity and inflammation, this is the first study to demonstrate that it improves insulin sensitivity in high-fat diet (HFD)-induced obese mice by regulating the insulin signaling pathway.
Preliminary in silico research was conducted to identify common targets between DIOS and disease-associated genes, setting the stage for subsequent investigations. Through in silico studies, we confirmed that DIOS is effective not only in reducing inflammation and increasing insulin sensitivity, but also that these effects are likely mediated through the regulation of the insulin signaling pathway. Subsequently, an 11-week intervention was conducted on an HFD-induced metabolic disorder model to investigate the role of DIOS in obesity-associated inflammation and insulin resistance. This investigation was carried out at the cellular level, focusing on the phenotype of macrophages, and at the mRNA level, by examining gene expression in both the liver and adipose tissues.
Using 90 overlapping genes between insulin resistance and DIOS, we performed Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with DAVID (http://david.ncifcrf.gov/). The results indicated a high correlation with the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (Akt) signaling pathway, insulin resistance, and the insulin signaling pathway (Fig. 1). We selected specific genes for molecular docking studies based on the premise that DIOS is likely to impact the PI3K/Akt pathway.
(a) Biological process (BP); (b) cellular component (CC); (c) molecular function (MF); (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
The molecular docking analysis provided the results of the binding affinity between DIOS and the target genes: TNF-α, janus kinase 2 (JAK2), p38 alpha in complex with compound 75 (MCP33) for mitogen-activated protein kinase 14 (MAPK14/p38), insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), insulin receptor substrate 2 (IRS-2), PI3K, and Akt. The binding activity values for TNF-α, JAK2, MAPK14/p38, INSR, IRS-1, IRS-2, PI3K, and Akt were found to be −10.2, −10.0, −9.6, −9.6, −7.8, −10.4, −8.7, and −8.5 kcal/mol, respectively (Figs. 2a–h). All docking scores fell below −7, indicating strong docking activity.12)
(a) TNF-α with DIOS (PDB ID: 2AZ5); (b) JAK2 with DIOS (PDB ID: 4FVQ); (c) MAPK14 (p38) with DIOS (PDB ID: 2NPQ); (d) INSR with DIOS (PDB ID: 1IRK); (e) IRS-1 with DIOS (PDB ID: 5U1M); (f) IRS-2 with DIOS (PDB ID: 3FQX); (g) PI3K with DIOS (PDB ID:8TSA); (h) Akt with DIOS (PDB ID:4GV1). The left image represents the full docking state; and the right image shows the close view of the white box in the left image.
After 11 weeks, the HFD group demonstrated significant increases in dietary intake, body weight, liver weight, and epididymal fat weight compared to the NC group. Despite comparable food intake in the HFD group, DIOS effectively suppressed the significant increase in body weight, epididymal fat pad, and liver weight compared with the HFD group. We performed a two-way ANOVA with Bonferroni's multiple comparison test on the weekly weight changes, and the p-value was less than 0.001 (Fig. 3a). Both the DIOS100 and DIOS200 groups exhibited significantly suppressed mean changes in body weight from baseline to week 11 compared to the HFD group (24.7 ± 3.01 vs. 32.06 ± 1.01 g, p < 0.05, and 19.94 ± 2.20 vs. 32.06 ± 1.01 g, p < 0.01, respectively) (Fig. 3b).
(a) Body weight per week. (b) Weight gain. (c) Weight of epididymal fat pads. (d) Weight of liver. (e) Daily calorie intake. data are presented as mean ± standard error of the mean (S.E.M.); # p < 0.05, ### p < 0.001 compared to NC. * p < 0.05, ** p < 0.01 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5).
The DIOS 200 group significantly suppressed epididymal fat pad weight increase, compared to the HFD group (1.26 ± 0.13 vs. 1.88 ± 0.17 g, p < 0.05). However, the epididymal fat weight did not differ significantly between the DIOS 100 and HFD groups (Fig. 3c). DIOS 100 and DIOS 200 significantly suppressed weight gain in the liver as compared with HFD (1.10 ± 0.13 vs. 2.16 ± 0.32 g, p < 0.05 and 0.93 ± 0.09 vs. 2.16 ± 0.32 g, p < 0.01, respectively) (Fig. 3d).
Effects of DIOS on Oral Glucose Tolerance Test (OGTT) and Insulin SensitivityAn OGTT was conducted to investigate the effects of DIOS on glucose metabolism. We performed a two-way ANOVA with Bonferroni’s multiple comparison test on the OGTT, and the p-value was less than 0.05 (Fig. 4a). Glucose levels at 60, 90, and 120 min were improved in the DIOS 100 group compared to those in the HFD group (60, 90, and 120 min, p < 0.05). Glucose levels were consistently lower in the DIOS 200 group than in the HFD group at all time points, including fasting glucose levels, throughout the test (0, 30, 60, and 90 min, p < 0.01; 120 and 180 min, p < 0.05) (Figs. 4a, b).
(a) OGTT. (b) Fasting glucose level. (c) Area under the curve of oral glucose tolerance test. (d) HOMA-IR. (e) Fasting insulin level. Data are described as mean ± S.E.M.; ## p < 0.01, ### p < 0.001 compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5); HOMA-IR, homeostatic model assessment for insulin resistance.
The evaluation of glucose tolerance using the area under the curve (AUC) demonstrated reduced AUC values for both the DIOS 100 and DIOS 200 groups in comparison to the HFD group, with significant differences between the groups (48741.00 ± 1248.87 vs. 66546.00 ± 5999.11 mg·min/dL, p < 0.05; 42498 ± 1801.45 vs. 66546 ± 5999.11 mg·min/dL, p < 0.01, respectively) (Fig. 4c).
The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) of the DIOS 100 and DIOS 200 groups significantly improved compared to the HFD group (20.84 ± 4.09 vs. 40.01 ± 4.60 units, p < 0.05, and 15.17 ± 1.73 vs. 40.01 ± 4.60 units, p < 0.001, respectively) (Fig. 4d).
The fasting insulin concentrations in both the DIOS 100 and DIOS 200 groups were significantly lower than that in the HFD group in a dose-dependent manner (1.91 ± 0.27, 1.65 ± 0.22 vs. 2.91 ± 0.33 ng/mL, p < 0.05) (Fig. 4e). Additionally, DIOS 200 demonstrated a similar mean fasting insulin level to that of the NC group (1.65 ± 0.22 vs. 1.37 ± 0.20 ng/mL, p > 0.05) (Fig. 4e).
Effects of DIOS on Lipid ProfileIn this study, we assessed the effect of DIOS on lipid metabolism. Low-density lipoprotein (LDL) levels demonstrated a significant difference with DIOS 100 and DIOS 200 compared to the HFD group (26.40 ± 4.78 vs. 54.40 ± 8.38 mg/dL, p < 0.05 and 20.80 ± 1.66 vs. 54.40 ± 8.38 mg/dL, p < 0.01, respectively) (Fig. 5a). However, the levels of high-density lipoprotein (HDL), total cholesterol (TC), free fatty acids, and phospholipids were not significantly different between the DIOS and HFD groups (Figs. 5b–e).
(a) LDL cholesterol. (b) HDL cholesterol. (c) Total cholesterol. (d) Free fatty acid. (e) Phospholipid. Data are described as mean ± S.E.M. # p < 0.05, ## p < 0.01 compared with NC, * p < 0.05, ** p < 0.01 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5); LDL, Low-density lipoprotein; HDL, High-density lipoprotein; TC, Total cholesterol.
DIOS 100 and DIOS 200 exhibited dose-dependent preventive effects on aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevation compared to the HFD group (DIOS 100: AST, 98.4 ± 8.95 vs. 182.4 ± 22.30 IU/L, p < 0.01; ALT, 43.80 ± 10.76 vs. 180.4 ± 19.09 IU/L, p < 0.001; DIOS 200: AST, 97.4 ± 10.46 vs. 182.4 ± 22.30 IU/L, p < 0.01; and ALT, 36.2 ± 7.89 vs. 180.4 ± 19.09 IU/L, p < 0.001, respectively) (Figs. 6a, b). Both the DIOS 100 and DIOS 200 groups demonstrated no significant differences in mean creatinine levels compared to the HFD group (Fig. 6c).
(a) AST. (b) ALT. (c) Creatinine level. Data are described as mean ± S.E.M.; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with NC, ** p < 0.01, *** p < 0.001 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5); AST, Aspartate aminotransaminase; ALT, Alanine aminotransaminase.
The proportion of ATMs among CD45+ cells was significantly higher in the HFD group than the NC group. However, DIOS 100 and DIOS 200 groups markedly reduced the proportion of ATMs compared to the HFD group (39.55 ± 2.72 vs. 67.88 ± 0.08%, p < 0.001; and 39.29 ± 4.38 vs. 67.88 ± 0.08%, p < 0.01) (Fig. 7a).
(a) Percentage of adipose tissue macrophages. (b) Percentage of CD11c+ ATMs. Data are described as mean ± S.E.M.; ## p < 0.01 compared with NC, ### p < 0.001 compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with HFD; NC, Normal Chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5).
The percentage of CD11c+ ATMs was significantly higher in the HFD group than the NC group. However, both DIOS 100 and DIOS 200 groups demonstrated a significant decline in the percentage of CD11c+ ATMs compared to the HFD group (DIOS 100, 40.79 ± 4.01 vs. 61.51 ± 3.66%, p < 0.05; and DIOS 200, 38.45 ± 2.47 vs. 61.51 ± 3.66%, p < 0.01) (Fig. 7b).
Regulation of DIOS on Gene ExpressionComparing the DIOS 200 group to the HFD group, significant gene expression downregulation in liver tissue was noted for TNF-α, F4/80, JAK2, and MAPK14/p38 (TNF-ɑ, 4.85 ± 0.91 vs. 9.97 ± 0.50, p < 0.01; F4/80, 2.73 ± 0.59 vs. 5.82 ± 0.82, p < 0.05; JAK2, 2.15 ± 0.12 vs. 4.12 ± 0.46, p < 0.01; and MAPK14, 0.79 ± 0.09 vs. 1.54 ± 0.21, p < 0.05) (Figs. 8a–d). Furthermore, gene expressions of INSR, IRS-1, IRS-2, PI3K, and AKT were all significantly higher in the DIOS 200 group compared to the HFD group (INSR, 0.77 ± 0.06 vs. 0.38 ± 0.08, p < 0.01; IRS-1, 0.88 ± 0.04 vs. 0.63 ± 0.08, p < 0.05; and IRS-2, 0.75 ± 0.05 vs. 0.46 ± 0.04, p < 0.05; PI3K, 0.88 ± 0.01 vs. 0.67 ± 0.06, p < 0.05; AKT, 0.89 ± 0.02 vs. 0.67 ± 0.06, p < 0.05) (Figs. 8e–i).
(a) TNF-ɑ. (b) F4/80. (c) JAK2. (d) MAPK14/p38. (e) INSR. (f) IRS-1. (g) IRS-2. (h) PI3K. (i) AKT. Data are described as mean ± S.E.M.; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with NC, * p < 0.05 compared with HFD, ** p < 0.01 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5); TNF-ɑ, Tumor necrosis factor-ɑ; JAK2, Janus kinase 2; INSR, Insulin receptor; MAPK14, Mitogen-activated protein kinase 14; IRS-1, Insulin receptor substrate 1; and IRS-2, Insulin receptor substrate 2; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B.
In the DIOS 100 group, a significant regulatory effect on the gene expressions of the liver tissue, including TNF-ɑ, F4/80, JAK2, INSR, and PI3K, was observed compared to the HFD group (TNF-ɑ, p < 0.01; F4/80, p < 0.05; JAK2, p < 0.05; INSR p < 0.05; and PI3K p < 0.05) (Figs. 8a–c, e, h). Conversely, MAPK14 (p38), IRS-1, IRS-2, and AKT expressions demonstrated no significant between-group differences.
In adipose tissue, TNF-α and F4/80 gene expressions in both DIOS groups exhibited significant decreases compared to the HFD group in a dose-dependent manner (TNF-α, 7.06 ± 0.47, 5.85 ± 0.82 vs. 9.43 ± 0.87, p < 0.05; F4/80, 5.98 ± 0.63, 5.37 ± 1.09 vs. 8.71 ± 0.64, p < 0.05) (Figs. 9a, b).
(a) TNF-ɑ. (b) F4/80. (c) JAK2. (d) MAPK14/p38. (e) INSR. (f) IRS-1. (g) IRS-2. Data are described as mean ± S.E.M.; ### p < 0.001 compared with NC, * p < 0.05 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5); TNF-ɑ, Tumor necrosis factor-ɑ.
In the liver tissue, the lipid droplet area was significantly increased in the HFD group compared to that in the NC group. However, with DIOS 100 and DIOS 200, the percentage of lipid droplets in the liver tissue decreased to 4.06 and 8.76%, respectively, showing significant reductions compared to the HFD group (both p < 0.01) (Figs. 10a, b).
(a) Histological image of liver and epididymal fat tissue. (b) Lipid droplets in the liver. (c) Size of adipocyte in the epididymal fat tissue. Data are described as mean ± S.E.M.; ### p < 0.001 compared with NC, * p < 0.05, ** p < 0.01 compared with HFD; NC, Normal chow (n = 5); HFD, High-fat diet (n = 5); DIOS 100, HFD + Diosgenin 100 mg/kg/d (n = 5); DIOS 200, HFD + Diosgenin 200 mg/kg/d (n = 5); AT, HFD + atorvastatin 10 mg/kg/d (n = 5).
The adipocytes in the epididymal fat pads were significantly larger in the HFD group than in the NC group. However, DIOS 200 effectively suppressed the enlargement of adipocyte size compared to the HFD group (2723.62 ± 10.02 vs. 4076.46 ± 287.65 µm2, p < 0.05) (Figs. 10a, c).
This study aimed to explore the molecular and genetic mechanisms underlying DIOS in obesity-related inflammation and insulin resistance, with comprehensive experimental results offering insights into the potential of DIOS in addressing metabolic disorders. In the liver, it improves the JAK2-INSR-IRS1/2-PI3K and Akt expression, promotes hepatic insulin signaling, and alleviates hepatic glucose production and insulin resistance. It was also indicated that inflammation was alleviated by improving TNF-α and F4/80 levels in the liver and adipose tissue. These findings differ from those of previous studies that have suggested that DIOS inhibits lipid accumulation in hepatocytes by activating AMPK13) or exerts its anti-inflammatory effect by regulating lipoxygenase activity,14) in terms of elucidating the genetic mechanisms related to the insulin signaling pathway.
Based on KEGG analysis, conducted using common target genes of DIOS and insulin resistance, pathways related to inflammation and insulin signaling such as the PI3K–Akt signaling pathway, insulin signaling pathway, AMPK signaling pathway, and adipocytokine signaling pathway were identified. Subsequently, molecular docking using frequently observed genes derived from these pathways revealed strong binding affinities between DIOS and key target genes associated with inflammation (TNF-α, F4/80, and MAPK14/p38) and insulin signaling pathway (JAK2, INSR, IRS-1, IRS-2, PI3K, and Akt). These findings substantiate the potential of DIOS to modulate crucial pathways involved in metabolic regulation. The results from the in silico and in vivo experiments specifically studied the weight of the body, liver, and epididymal fat; glucose metabolism; lipid profile; ATMs; and mRNA expression of related genes in the liver and adipose tissue.
In this in vivo study, the administration of a DIOS regimen improved various weight-related outcomes, hepatic lipid accumulation, and adipocyte size in the epididymal fat pad. Despite similar daily calorie intake between the DIOS and HFD groups, or even slightly higher daily calorie intake in the DIOS 100 group than in the HFD group, without statistical significance, a noticeable inhibition of weight gain was observed in the DIOS group in a dose-dependent manner. Additionally, the weights of the liver and epididymal adipose tissue, the major insulin-sensitive organs,15) were significantly lower than those in the HFD group. These findings are consistent with the histological analysis results, which revealed suppressed hepatic lipid accumulation and adipocyte enlargement in the epididymal fat pads. Furthermore, all glycemic metabolism indicators, including all time points of the OGTT, fasting blood glucose, fasting insulin,16) and HOMA-IR,17) improved. Insulin resistance prolongs hyperglycemia after glucose ingestion because insulin secretion is delayed and insulin-induced hepatic glucose uptake is impaired. Delayed insulin secretion, in turn, leads to higher peak blood glucose concentrations, and excessive insulin secretion damages beta cell function.18,19) Considering the fact, the result suggests that DIOS has the potential to improve glucose tolerance, alleviate insulin resistance, and indirectly prevent beta cell dysfunction.
The pathogenesis of obesity is multifactorial, but adipocyte expansion induces a chronic low-grade inflammatory state, which is coupled with lipotoxic insulin signaling and insulin resistance.20,21) One key feature of obesity-induced inflammation is the infiltration of adipose tissue and the liver by immune cells such as M1 macrophages.3,22) Therefore, we examined the percentage of ATMs and the proportion of M1 among ATMs, using F4/80 and CD11c in adipose tissue as ATM-specific markers, and useful surface markers to differentiate M1.23) Significant alleviation was observed in DIOS groups. TNF-α is a highly secreted proinflammatory cytokine released from activated M1 macrophages that triggers inflammation and insulin resistance in adipose tissue.22) p38 kinase can be activated by insulin signaling and cytokines such as TNF-α and p38 play a pivotal role in macrophage-mediated inflammation, being involved in the manifestation of pro-inflammatory mediators like TNF-α and IL-6.24) It is reported that TNF-α in diet-induced obese mice further aggravates insulin resistance and glucose intolerance25) by interfering with insulin receptor signaling at the degree of IRS molecules.26) As TNF-α and p38 are crucial factors in obesity-associated inflammation and insulin resistance, we examined the TNF-α, F4/80, and p38 mRNA levels in the liver and adipose tissue to elucidate the mechanism of the anti-inflammatory effect of DIOS. DIOS inhibited the mRNA expression of these inflammatory factors, except p38, in adipose tissue. These results suggest that DIOS can suppress inflammatory changes in the obese liver and adipose tissues, which is consistent with previous studies.14,27)
To elucidate the mechanism of DIOS on obesity-related insulin resistance, we investigated the INSR, IRS-1, IRS-2, JAK2, PI3K, and Akt mRNA expression. The critical pathways linking IRS proteins to the metabolic actions of insulin are the PI3K and Akt pathways.28) Irregularities in insulin signaling, with reduced tyrosine phosphorylation of IRS families such as IRS-1 and IRS-2 by the insulin receptor, are associated with impaired insulin-induced glycogen synthesis and glucose production down-regulation in the liver.29) Under normal conditions, activation of insulin and IGF-1 receptors by their respective ligands initiates the recruitment of receptor substrates, including IRS-1 and IRS-2, promoting the PI3K–Akt pathway. Since Akt mediates most of the metabolic consequences of insulin by regulating glucose transport, gluconeogenesis, and glycogen synthesis,28) increased INSR, IRS-1, IRS-2, and PI3K activation could potentially facilitate insulin signaling cascades. JAK2, known to regulate various physiological processes, including inflammation, erythropoiesis, and thrombopoiesis,30) serves as an intermediary for insulin-like effects by facilitating IRS phosphorylation and activating downstream mediators such as PI3K and Akt.31) While limited studies have provided insight into its mechanism, it has been discovered that the absence of macrophage JAK2 in diet-induced obesity led to a decrease in inflammation and insulin resistance.30,32) Furthermore, when considering the reported results that mice with hepatocyte-specific-JAK2 deletion were protected from HFD-induced steatohepatitis and insulin resistance,33) along with the observation that JAK2 might attenuate the downstream signaling of Akt,31) it can be concluded that inhibiting JAK2 could potentially mitigate insulin resistance. DIOS treatment significantly enhanced the INSR, IRS-1, IRS-2, PI3K, and Akt activation in liver tissue compared to the HFD group, but also notably reduced JAK2 expression in liver tissue. These findings suggest that DIOS enhances IRS1/2-PI3K-Akt signaling pathway activation, ultimately leading to a reduction in insulin resistance.
This study had a few limitations. Contrary to prior research,34,35) our study did not show an improvement in lipid markers including cholesterol and triglycerides, which was attributed to differences in the duration of drug administration or dosage. Further studies are required to confirm these contradictory results. Further investigation of Kupffer cells, which serve as indicators of inflammatory changes in the liver tissue, is necessary. Despite these limitations, our study demonstrated that DIOS regulates the expression of genes related to insulin signaling pathway and inflammatory response.
In an in silico study, the examination of pathways involving genes overlapping between DIOS and insulin resistance demonstrated the potential to improve the insulin signaling pathway. Based on these findings, in vivo experiments revealed that DIOS improved insulin resistance in insulin-sensitive tissues such as the liver and adipose tissue. This effect appears to be associated with an improvement in insulin mediated PI3K/Akt signaling pathway in the liver and a reduction in ATM-mediated inflammatory responses through adipocyte proliferation inhibition.
In order to identify the common targets of DIOS and insulin resistance, we searched for DIOS targets using SwissTargetPrediction (http://swisstargetprediction.ch/) and obtained the insulin resistance targets from GeneCards (http://genecards.org/). The data from GO enrichment analysis and KEGG pathway analysis were obtained by analyzing overlapped genes using DAVID (http://david.ncifcrf.gov/), accessed on 16 October 2022. Subsequently, we visualized the data using R Studio. From the KEGG data, we selected the pathways highly associated with insulin resistance and inflammation, and compiled their gene lists for ClueGO analysis in Cytoscape.
In Silico Study of Molecular Docking of DIOSThe three-dimensional structure of DIOS was downloaded from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/), while the structures of proteins, including TNF-α (PDB ID: 2AZ5), JAK2 (PDB ID: 4FVQ), INSR (PDB ID: 1IRK), MAPK14 (PDB ID: 2NPQ), IRS-1 (PDB ID: 5U1M), IRS-2 (PDB ID: 3FQX), PI3K (PDB ID: 8TSA), and Akt (PDB ID: 4GV1) were provided by PDB database (http://rcsb.org), accessed on November 15, 2022. Before molecular docking, unnecessary protein domains were eliminated, and hydrogenation was carried out using the Biovia Discovery Studio Visualizer. Molecular docking was performed in PyRx to confirm binding affinity, and the resulting simulated binding status was visualized using PyMOL.
In Vivo Study on the Effects of DIOS on MetabolismAnimals and Experimental DesignAll animal procedures were approved by the Kyung Hee Medical Animal Research Ethics Committee (Approval No. 21-012-01). Male C57BL/6 mice (6-week-old, Central Lab Animals, Inc., Korea), were housed under conditions of 40–70% humidity and 12-h day-night cycles throughout the experiment. During the initial seven days, all mice were acclimated with free access to water and standard rodent chow. After the acclimation period, the mice were divided into five groups: normal chow (NC), control (HFD), atorvastatin 10 mg/kg (AT), and two DIOS treatment groups (DIOS 100 and DIOS 200) that received 100 and 200 mg/kg of DIOS, respectively. The HFD, AT, DIOS 100, and DIOS 200 groups were fed on the HFD, comprising 60% of calories from fat for 11 consecutive weeks. Starting from the 4th week, the AT and DIOS groups were orally administered their respective atorvastatin and DIOS doses daily, whereas the NC and HFD groups were administered normal saline. Each group received their respective treatments for 7 weeks. DIOS (CAS RN: 512-04-9) was purchased from Tokyo Chemical Industry (https://www.tcichemicals.com/KR/ko/) (Lot Number: 5JWYM-BI).
Weight Measurement of Body, Liver, and Fat TissueThroughout the 11-week experiment, individual body weights were measured once a week, starting on the first day of the study. The measurements were performed using an electronic scale (CAS 2.5D; Seoul, Korea).
At the end of the 11 weeks, epididymal fat pad and liver weights were measured using the same electronic scale (CAS 2.5D, Seoul, Korea).
OGTT and Insulin Resistance MeasurementTo investigate the effects of DIOS on hyperglycemia, an OGTT was performed at 10 weeks. The test involved fasting for 14 h, followed by the examination of blood glucose levels at 0, 30, 60, 120, and 180 min after orally administering 2g/kg of glucose. Blood glucose levels were measured using a blood glucose sensor with test strips (Accu-Check Performance; Australia). Insulin levels were measured using an ultrasensitive mouse insulin ELISA kit Crystal Chem, Inc., U.S.A. The determination of insulin resistance was done using the HOMA-IR formula, which is calculated as follows: HOMA-IR = Fasting blood glucose (mg/dL) × Fasting insulin (µg/mL) × 0.0717225161669606.
Lipid ProfileAt 11 weeks, serum lipid profiles including TC, LDL, HDL, phospholipid, and non-esterified free fatty acid were assessed using an ELISA kit (Cusabio Technology LLC, U.S.A.) taken from the heart.
Safety OutcomesBlood samples were collected from the hearts of mice at week 11 to assess liver and renal function. Subsequently, the collected samples were centrifuged at 3000 rpm for 20 min. The resulting supernatants were stored at −40 °C. Biochemical analyses to measure AST, ALT, and serum creatinine levels were conducted using Cusabio Technology LLC, U.S.A.
Fluorescence-Activated Cell Sorting (FACS) of ATMThe number of cells in the Stromal Vascular Fraction of adipose tissue was quantified using a cellometer (Nexcelom Bioscience LLC, U.S.A.), and each sample was standardized to a concentration of 106 cells. FcBlock (BD Pharmingen) was added to each sample at a ratio of 1 : 100. Fluorophore-conjugated antibodies, CD45-APC Cy7 (BioLegend) and F4/80-APC (BioLegend), were added to induce the reaction under light-shielding conditions. After washing each sample with 2% fetal bovine serum/phosphate-buffered saline (FBS/PBS) solution, they were centrifuged at 1500 rpm and transferred to FACS tubes for analysis using the FACS Canto (BD Biosciences, U.S.A.). The percentage of macrophages expressing CD45 (+) and F4/80 (+) was assessed using the FlowJo software (Tree Star, Inc., U.S.A.).
Real-Time Quantitative RT-PCRRNA was isolated from liver and epididymal fat tissue samples using RNA Isolation IITM (ZYMO Research, CA, U.S.A.). For cDNA synthesis, 1 µg of RNA isolated from tissues was used using Advantage RT for PCR Kit (Clontech, U.S.A.). The relative ex-pression levels of mRNA encoding the TNF-α, F4/80, JAK2, MAPK14, INSR, IRS-1, IRS-2, PI3K, and Akt. Primers, dH2O 2x SYBR reaction buffer and cDNA were mixed together to perform PCR amplification using 7900HT Fast real-time PCR System (Applied Biosystems®, U.S.A.).
Histological InvestigationThe epididymal fat tissues and the liver tissues were embedded in paraffin and sliced into 4 µm thick sections. The sections were mounted on gelatin-coated slides. The slides were dewaxed in xylene and rehydrated using a series of ethanol solutions with concentrations of 100, 95, 80, and 70%, and distilled water. Hematoxylin and eosin staining was performed on tissue sections, and images were captured using a high-resolution camera-mounted optical microscope (Olympus BX-51; Olympus Optical, Tokyo, Japan). The size of adipocytes in the epididymal fat and liver tissues was evaluated using the Image J software.
Statistical AnalysisData were analyzed using the GraphPad PRISM 5 software (GraphPad Software Inc., San Diego, CA, U.S.A.). Group differences were assessed using one-way ANOVA followed by Tukey’s post-hoc test. Data are presented as mean ± standard error (S.E.). Statistical significance was set at a p-value less than 0.05. In the comparisons between the NC and HFD groups, statistical significance was denoted by number signs (#), whereas in the comparisons between the HFD and DIOS groups, asterisks (*) were used (e.g., # or * for p < 0.05, ## or ** for p < 0.01, ### or *** for p < 0.001).
This research was supported by a Grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: RS-2020-KH087720).
The authors declare no conflict of interest.
