Effect of fermented cassava tuber on the gene expression of PI3K/Akt signaling and AMPK pathway in STZ-NA-induced diabetic rats

Purpose The downstream insulin signaling, such as phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway, is an important step for skeletal glucose disposal through the translocation of glucose transporter (GLUT)-4. In addition, the master of energy regulator adenosine monophosphate-activated kinase (AMPK) is also involved in GLUT-4 translocation, independent from the PI3K/Akt pathway. Fermented cassava tuber or gatot is a traditional food from Indonesia with antihyperglycemic properties. However, the molecular mechanism leading to this effect is unclear. Therefore, this paper aims to evaluate whether the antidiabetic activity of gatot is through PI3K/Akt dependent or AMPK pathway. Design/methodology/approach Diabetes mellitus was induced in 20 male Wistar rats by intraperitoneal injection of 65 mg/kg body weight streptozotocin and 230 mg/kg body weight nicotinamide. Diabetic rats were randomly allocated into four groups; negative control, positive control (metformin 100 mg/kg body weight), fermented cassava diet replacing 50% of carbohydrate (FC-50) and 100% of carbohydrate (FC-100) in the diet. Serum glucose, insulin and lipid profile were analyzed before and after four weeks of intervention. Genes expression of PI3K subunit alpha, PI3K subunit beta, PI3K regulatory subunit, Akt and AMPK were analyzed using real time polymerase chain reaction (PCR). GLUT-4 protein expression was performed using immunohistochemistry. Findings There is a significant difference (p = 0.000) in serum glucose, insulin, total cholesterol, triglyceride, high density lipoprotein (HDL)-cholesterol and LDL-cholesterol between groups. Skeletal AMPK gene expression was higher and significantly different between FC-100 (p = 0.006) and healthy control groups. No significant difference was observed in the messenger ribonucleic acid (mRNA) expression of the PI3K/Akt pathway among groups. GLUT-4 expression was highly expressed in a positive control group followed by FC-100. Research limitations/implications This paper did not characterize the bioactive component that is responsible for increasing mRNA expression of AMPK. This paper also did not analyze the phosphorylation of PI3K/Akt and AMPK that are important in activating the protein. Originality/value To the best of the authors’ knowledge, this is the first study that showed the antidiabetic activity of traditional fermented food is through AMPK-dependent activity.

Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach

Type-2 diabetes mellitus (T2DM), the leading global health burden of this century majorly develops due to obesity and hyperglycemia-induced oxidative stress in skeletal muscles. Hence, developing novel drugs that ameliorate these pathological events is an immediate priority. The study was designed to analyze the possible role of Stevioside, a characteristic sugar from leaves of Stevia rebaudiana (Bertoni) on insulin signaling molecules in gastrocnemius muscle of obesity and hyperglycemia-induced T2DM rats. Adult male Wistar rats rendered diabetic by administration of high fat diet (HFD) and sucrose for 60 days were orally administered with SIT (20 mg/kg/day) for 45 days. Various parameters were estimated including fasting blood glucose (FBG), serum lipid profile, oxidative stress markers, antioxidant enzymes and expression of insulin signaling molecules in diabetic gastrocnemius muscle. Stevioside treatment improved glucose and insulin tolerances in diabetic rats and restored their elevated levels of FBG, serum insulin and lipid profile to normalcy. In diabetic gastrocnemius muscles, Setvioside normalized the altered levels of lipid peroxidase (LPO), hydrogen peroxide (H2O2) and hydroxyl radical (OH*), antioxidant enzymes (CAT, SOD, GPx and GSH) and molecules of insulin signaling including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt mRNA levels. Furthermore, Stevioside enhanced glucose uptake (GU) and oxidation in diabetic muscles by augmenting glucose transporter 4 (GLUT 4) synthesis very effectively in a similar way to metformin. Results of molecular docking analysis evidenced the higher binding affinity with IRS-1 and GLUT 4. Stevioside effectively inhibits oxidative stress and promotes glucose uptake in diabetic gastrocnemius muscles by activating IR/IRS-1/Akt/GLUT 4 pathway. The results of the in silico investigation matched those of the in vivo study. Hence, Stevioside could be considered as a promising phytomedicine to treat T2DM.

Athrixia phylicoides tea infusion (bushman tea) improves adipokine balance, glucose homeostasis and lipid parameters in a diet-induced metabolic syndrome rat model

Background Central obesity and insulin resistance are associated with metabolic syndrome (MetS) which is aggravated by diet and sedentary lifestyle. Athrixia phylicoides (AP) is reported by rural communities to have medicinal benefits associated with MetS such as obesity and type 2 diabetes. This study was aimed to investigate the effects of AP on diet-induced MetS in Wistar rats to validate its ethnopharmacological use. Methods AP was profiled for phytochemicals by LC-MS. After induction of MetS with high energy diet (HED), 30 male rats were divided into five treatment groups (n = 6): normal diet control, HED control, HED + AP 50 mg/Kg BW, HED + AP 100 mg/Kg BW and HED + 50 mg/Kg BW metformin. The rats were treated daily for 8 weeks orally after which weight gain, visceral fat, total cholesterol, free fatty acids (FFAs) and adipokine regulation; leptin: adiponectin ratio (LAR) were assessed. Also, glucose homeostatic parameters including fasting blood glucose (FBG), oral glucose tolerance test (OGTT), glucose transporter 4 (GLUT 4), insulin and homeostatic model assessment of insulin resistance (HOMA-IR) were determined. Results Findings showed that AP was rich in polyphenols. The HED control group showed derangements of the selected blood parameters of MetS. AP reversed diet-induced weight gain by reducing visceral fat, total blood cholesterol and circulating FFAs (p ≤ 0.05). Treatment with AP improved adipokine regulation depicted by reduced LAR (p<0.05). Treatment with AP improved parameters of glucose homeostasis as demonstrated by reduced FBG and HOMA-IR (p ≤ 0.05) and increased GLUT 4 (p<0.05). Conclusion Athrixia phylicoides tea infusion was shown to possess anti-obesity and anti-inflammatory properties, improved glucose uptake and reduce insulin resistance in diet-induced MetS in rats which could be attributed to its richness in polyphenols. Therefore, AP could have potential benefits against type 2 diabetes and obesity which are components of MetS validating its ethnopharmacological use.

d-Allulose Ameliorates Skeletal Muscle Insulin Resistance in High-Fat Diet-Fed Rats

Background: d-Allulose is a rare sugar with antiobesity and antidiabetic activities. However, its direct effect on insulin sensitivity and the underlying mechanism involved are unknown. Objective: This study aimed to investigate the effect of d-allulose on high-fat diet (HFD)-induced insulin resistance using the hyperinsulinemic–euglycemic (HE)-clamp method and intramuscular signaling analysis. Methods: Wistar rats were randomly divided into three dietary groups: chow diet, HFD with 5% cellulose (HFC), and HFD with 5% d-allulose (HFA). After four weeks of feeding, the insulin tolerance test (ITT), intraperitoneal glucose tolerance test (IPGTT), and HE-clamp study were performed. The levels of plasma leptin, adiponectin, and tumor necrosis factor (TNF)-α were measured using the enzyme-linked immunosorbent assay. We analyzed the levels of cell signaling pathway components in the skeletal muscle using Western blotting. Results: d-allulose alleviated the increase in HFD-induced body weight and visceral fat and reduced the area under the curve as per ITT and IPGTT. d-Allulose increased the glucose infusion rate in the two-step HE-clamp test. Consistently, the insulin-induced phosphorylation of serine 307 in the insulin receptor substrate-1 and Akt and expression of glucose transporter 4 (Glut-4) in the muscle were higher in the HFA group than HFC group. Furthermore, d-allulose decreased plasma TNF-α concentration and insulin-induced phosphorylation of stress-activated protein kinase/Jun N-terminal kinase in the muscle and inhibited adiponectin secretion in HFD-fed rats. Conclusions: d-allulose improved HFD-induced insulin resistance in Wistar rats. The reduction of the proinflammatory cytokine production, amelioration of adiponectin secretion, and increase in insulin signaling and Glut-4 expression in the muscle contributed to this effect.

Exposure to Perfluoro-Octanoic Acid Associated With Upstream Uncoupling of the Insulin Signaling in Human Hepatocyte Cell Line

Perfluoro–alkyl substances (PFAS) are chemical pollutants with prevalent stability and environmental persistence. Exposure to PFAS, particularly perfluoro-octanoic acid (PFOA), has been associated with increased diabetes-related cardiovascular mortality in subjects residing areas of high environmental contamination, however the exact pathogenic mechanism remains elusive. Here we used HepG2 cells, an in vitro model of human hepatocyte, to investigate the possible role of PFOA exposure in the alteration of hepatic glucose metabolism. HepG2 cells were exposed for 24 hours to PFOA at increasing concentration from 0 to 1000 ng/mL and then stimulated with 100 nm Insulin (Ins). The consequent effect on glycogen synthesis, glucose uptake and Glut-4 glucose transporter translocation was then evaluated by, respectively, Periodic Acid Schiff (PAS) staining, 2-deoxyglucose (2-DG) uptake assay and immunofluorescence. Exposure to PFOA was associated with reduced glycogen synthesis and glucose uptake, at concentration equal or greater than, respectively, 0,1 ng/mL and 10 ng/mL, with parallel impaired membrane translocation of Glut-4 upon Ins stimulation. Western blot analysis showed early uncoupling of Insulin Receptor (InsR) activation from the downstream Akt and GSK3 phosphorylation. Computational docking analysis disclosed the possible stabilizing effect of PFOA on the complex between InsR and GM3 ganglioside, previously shown to be associated with the low grade chronic inflammation-related insulin resistance. Consistently, long term treatment with glucosyl-ceramide synthase inhibitor PDMP was able to largely restore glycogen synthesis, glucose uptake and Glut-4 translocation upon Ins stimulation in HepG2 exposed to PFOA. Our data support a novel pathogenic mechanism linking exposure to PFOA to derangement of hepatocyte cell metabolism.

Study on the Mechanism of Lupenone for Treating type 2 Diabetes by Integrating Pharmacological Evaluation and Network Pharmacology

Background: Lupenone (LUP) is the active ingredient of Rhizoma Musae, which has good anti-diabetes effects, but the underlying mechanism is unclear. In this study, animal experiments combined with network pharmacology were used to explore the mechanism of LUP for treating diabetes. Methods: Type 2 diabetic rats with insulin resistance (IR) were induced by a high-fat diet and streptozotocin. the fasting blood glucose (FBG), index of oxidative stress, blood lipids, and IR-related targets in skeletal muscle and adipose were detected. a network pharmacology-based strategy was also adopted to clarify the mechanism of LUP for treating diabetes by improving IR. Results: LUP decreased the FBG levels and synthesis of glycogen, improved oxidative stress and lipid metabolism disorders, and increased the gene and protein expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, glucose transporter type 4 (GLUT-4) in skeletal muscle and peroxisome proliferator-activated receptor γ, IRS-1, IRS-2, GLUT-4 in adipose. Network pharmacology analysis revealed that LUP improves IR by multiple targets (like INS, TP53, TNF, SRC and ESR1) and signal pathways. Conclusion: These results suggested that the mechanism of LUP for treating diabetes is closely related to improving IR. LUP has the potential to develop as a new drug for the treatment of type 2 diabetes.

Davidones F and G, Two Novel Flavonoids from Sophora davidii (Franch.) Skeels

An unprecedented novel flavanone davidone F (1) with a seven-membered ring side chain, and a novel flavanonol davidone G (2), along with 11 known flavonoids, were isolated from the ethyl acetate fraction of Sophora davidii (Franch.) Skeels. Their planar structures were established by UV, IR, HRESIMS, 1D and 2D NMR data. The relative configurations of 1 and 2 were determined by calculation of NMR chemical shift values, the absolute configuration of 1 and 2 were assigned by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. Moreover, compounds 1–13 were screened for the translocation activity of glucose transporter 4 (GLUT-4), and the fluorescence intensity was increased to the range of 1.56 and 2.79 folds. Compounds 1 and 2 showed moderate GLUT-4 translocation activity with 1.64 and 1.79 folds enhancement, respectively, at a concentration of 20 μg/mL.

Establishment of long-term high-fat diet and low dose streptozotocin-induced experimental type-2 diabetes mellitus model of insulin resistance and evaluation of seed extracts of Syzygium cumini

Introduction: The principal mechanism responsible for reducing blood glucose is through insulin-stimulated glucose transport into skeletal muscle. The transporter protein that mediates this uptake is GLUT-4. A defect in this step is associated with reduced glucose utilization in muscle and adipose tissue, as observed in insulin-resistant type-2 diabetes mellitus (T2DM) patients. This study aimed to develop an experimental T2DM model and evaluate altered glucose transporter type 4 (GLUT-4) levels as a biomarker of insulin resistance. Antidiabetic activities of Syzygium cumini hydro-ethanolic seed extracts (SCE) were also evaluated. Methods: Adult male Wistar albino rats were fed a high-fat diet for 12 weeks and dosed intraperitoneally with streptozotocin (35 mg/kg). After treatment for 21 days, all investigations were done. The homeostasis model of assessment (HOMA) was used for the calculation of insulin resistance (HOMA-IR) and beta-cell function (HOMA-B) index. Diaphragm muscle and retroperitoneal fat were collected for real-time polymerase chain reaction (RT-PCR) studies. Results: A significant increase in fasting blood glucose, HOMA-IR, and serum lipids, and a decrease in serum insulin and HOMA-B were observed in the diabetic group, effects that reversed following pioglitazone and SCE treatment. The diabetic group showed a downregulation of GLUT-4 expression in skeletal muscle while an increase was observed in adipose tissue. Conclusion: A high-fat diet and low dose streptozotocin-induced experimental T2DM model of insulin resistance was developed to screen novel insulin sensitizers. Data generated demonstrated that altered GLUT-4 levels could be used as a biomarker of insulin resistance. Antidiabetic activity of S. cumini hydro-ethanolic seed extract was also confirmed in this study.