Diabetic KK-Ay Mice Research Articles

Fumosorinone, a novel PTP1B inhibitor, activates insulin signaling in insulin-resistance HepG2 cells and shows anti-diabetic effect in diabetic KKAy mice

Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathways, and its increased activity and expression are implicated in the pathogenesis of insulin resistance. Therefore, the inhibition of PTP1B is anticipated to become a potential therapeutic strategy to treat T2DM. Fumosorinone (FU), a new natural product isolated from insect fungi Isaria fumosorosea, was found to inhibit PTP1B activity in our previous study. Herein, the effects of FU on insulin resistance and mechanism in vitro and in vivo were investigated. FU increased the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, and also reduced blood glucose and lipid levels of type 2 diabetic KKAy mice. FU decreased the expression of PTP1B both in insulin-resistant HepG2 cells and in liver tissues of diabetic KKAy mice. Furthermore, FU increased the phosphorylation of IRβ, IRS-2, Akt, GSK3β and Erk1/2 in insulin-resistant HepG2 cells, as well as the phosphorylation of IRβ, IRS-2, Akt in liver tissues of diabetic KKAy mice. These results showed that FU increased glucose uptake and improved insulin resistance by down-regulating the expression of PTP1B and activating the insulin signaling pathway, suggesting that it may possess antidiabetic properties.

The Green Algal Carotenoid Siphonaxanthin Inhibits Adipogenesis in 3T3-L1 Preadipocytes and the Accumulation of Lipids in White Adipose Tissue of KK-Ay Mice1–3

Background: Siphonaxanthin, a xanthophyll present in green algae, has been shown to possess antiangiogenic and apoptosis-inducing activities.Objective: We evaluated the antiobesity effects of siphonaxanthin by using a 3T3-L1 cell culture system and in diabetic KK-Ay mice.Methods: 3T3-L1 cells were differentiated with or without 5 μmol/L siphonaxanthin, and lipid accumulation and critical gene expressions for adipogenesis were examined. In vivo, 4-wk-old male KK-Ay mice were administered daily oral treatment of 1.3 mg siphonaxanthin for 6 wk and body weight, visceral fat weight, serum variables, and gene expressions involved in lipid metabolism were evaluated.Results: Compared with the other carotenoids evaluated, siphonaxanthin potently inhibited adipocyte differentiation. Siphonaxanthin significantly suppressed lipid accumulation at noncytotoxic concentrations of 2.5 and 5 μmol/L by 29% and 43%, respectively. The effects of siphonaxanthin were largely limited to the early stages of adipogenesis. Siphonaxanthin significantly inhibited protein kinase B phosphorylation by 48% and 72% at 90 and 120 min, respectively. The expressions of key adipogenesis genes, including CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator activated receptor γ (Pparg), fatty acid binding protein 4 (Fabp4), and stearoyl coenzyme A desaturase 1 (Scd1), were elevated by 1.6- to 166-fold during adipogenesis. After 8 d of adipocyte differentiation, siphonaxanthin significantly lowered gene expression of Cebpa, Pparg, Fabp4, and Scd1 by 94%, 83%, 95%, and 90%, respectively. Moreover, oral administration of siphonaxanthin to KK-Ay mice significantly reduced the total weight of white adipose tissue (WAT) by 13%, especially the mesenteric WAT by 28%. Furthermore, siphonaxanthin administration reduced lipogenesis and enhanced fatty acid oxidation in adipose tissue. Siphonaxanthin was observed to highly accumulate in mesenteric WAT, and the accumulation in the mesenteric WAT was almost 2- and 3-fold that in epididymal (P = 0.14) and perirenal (P < 0.05) WAT, respectively.Conclusion: These results provide evidence that siphonaxanthin may effectively regulate adipogenesis in 3T3-L1 cells and diabetic KK-Ay mice.

Dietary cod protein decreases triacylglycerol accumulation and fatty acid desaturase indices in the liver of obese type-2 diabetic KK-Ay mice

This study investigated the effects of cod protein (CP) on the serum, liver, fecal lipid contents and fatty acid (FA) composition in the liver of obese type-2 diabetic KK-Ay mice. Male KK-Ay mice were fed a casein-based semi-purified diet (Control) or a diet with substitution of part of the dietary protein (50%) by CP for 4 weeks. The CP diet significantly decreased the contents of serum triacylglycerol (TAG), serum non-esterified FAs, serum alanine aminotransferase, and liver TAG compared with the Control diet. In addition, dietary CP decreased saturated FA, monounsaturated FA, and the FA desaturase indices (C16:1/C16:0 and C18:1/C18:0) in the liver. The changes in FA desaturase indices induced by the FP diet could be inhibited by stearoyl-CoA desaturase (SCD)-1 activity due to suppression of the SCD-1 mRNA expression level. These results suggest that the intake of CP has a beneficial effect on the liver in the presence of obesity and diabetes.

Beneficial effects of canagliflozin in combination with pioglitazone on insulin sensitivity in rodent models of obese type 2 diabetes.

BackgroundDespite its insulin sensitizing effects, pioglitazone may induce weight gain leading to an increased risk of development of insulin resistance. A novel sodium glucose co-transporter 2 (SGLT2) inhibitor, canagliflozin, provides not only glycemic control but also body weight reduction through an insulin-independent mechanism. The aim of this study was to investigate the combined effects of these agents on body weight control and insulin sensitivity.MethodsEffects of combination therapy with canagliflozin and pioglitazone were evaluated in established diabetic KK-Ay mice and prediabetic Zucker diabetic fatty (ZDF) rats.ResultsIn the KK-Ay mice, the combination therapy further improved glycemic control compared with canagliflozin or pioglitazone monotherapy. Furthermore, the combination significantly attenuated body weight and fat gain induced by pioglitazone and improved hyperinsulinemia. In the ZDF rats, early intervention with pioglitazone monotherapy almost completely prevented the progressive development of hyperglycemia, and no further improvement was observed by add-on treatment with canagliflozin. However, the combination significantly reduced pioglitazone-induced weight gain and adiposity and improved the Matsuda index, suggesting improved whole-body insulin sensitivity.ConclusionsOur study indicates that combination therapy with canagliflozin and pioglitazone improves insulin sensitivity partly by preventing glucotoxicity and, at least partly, by attenuating pioglitazone-induced body weight gain in two different obese diabetic animal models. This combination therapy may prove to be a valuable option for the treatment and prevention of obese type 2 diabetes.

Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain.

Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

Dietary nitrite supplementation improves insulin resistance in type 2 diabetic KKAy mice

BackgroundBecause insulin signaling is essential for endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, the loss of bioavailable NO might be a common molecular mechanism underlying the development of insulin resistance and endothelial dysfunction. Although dietary nitrite acts as a substrate for systemic NO generation, thereby serving as a physiological alternative source of NO for signaling, it is not precisely known how dietary nitrite affects type 2 diabetes mellitus. Here we report the therapeutic effects of dietary nitrite on the metabolic and histological features of KKAy diabetic mice. MethodsKKAy mice were divided into three groups (without nitrite, and with 50 mg/L and 150 mg/L nitrite in drinking water), and two groups of C57BL/6J mice served as controls (without nitrite and with 150 mg/L nitrite in drinking water). After 10 weeks, blood samples, visceral adipose tissues, and gastrocnemius muscles were collected after a 16-hour fast to assess the homeostasis model assessment of insulin resistance (HOMA-IR) levels, the histology of the adipose tissue, insulin-stimulated sequential signaling to glucose transporter 4 (GLUT4), and nitrite and nitrate contents in the muscle using an HPLC system. ResultsKKAy mice developed obesity with enhanced fasting plasma levels of glucose and insulin and exhibited increased HOMA-IR scores compared with the C57BL/6J control mice. Dietary nitrite dose-dependently reduced the size of the hypertrophic adipocytes and TNF-α transcription in the adipose tissue of KKAy diabetic mice, which also restored the insulin-mediated signal transduction, including p85 and Akt phosphorylation, and subsequently restored the GLUT4 expression in the skeletal muscles. ConclusionsThese results suggest that dietary nitrite provides an alternative source of NO, and subsequently improves the insulin-mediated signaling and the metabolic and histological features in KKAy diabetic mice.

The Effects of Chinese Herbal ShenQi Decoction on Insulin Resistance and Lipid Profile in Genetically Diabetic KK-Ay Mice

The Effects of Chinese Herbal ShenQi Decoction on Insulin Resistance and Lipid Profile in Genetically Diabetic KK-Ay Mice

Selenium-enriched exopolysaccharides improve skeletal muscle glucose uptake of diabetic KKAy mice via AMPK pathway.

Selenium-enriched exopolysaccharides (EPS) produced by Enterobacter cloacae Z0206 have been proven to possess effect on reducing blood glucose level in diabetic mice. To investigate the specific mechanism, we studied the effects of oral supply with EPS on skeletal muscle glucose transportation and consumption in high-fat-diet-induced diabetic KKAy mice. We found that EPS supplementation increased expressions of glucose transporter 4 (Glut4), hexokinase 2 (hk2), phosphorylation of AMP-activated kinase subunit α2 (pAMPKα2), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and increased expression of characteristic protein of oxidative fibers such as troponin I and cytochrome c (Cytc). Furthermore, we found that EPS increased glucose uptake and expressions of pAMPKα2 and PGC-1α in palmitic acid (PA)-induced C2C12 cells. However, while EPS inhibited AMPKα2 with interference RNA (iRNA), effects of EPS on the improvement of glucose uptake diminished. These results indicated that EPS may improve skeletal muscle glucose uptake of diabetic KKAy mice through AMPKα2-PGC-1α pathway.

Proteomic Profile in Glomeruli of Type-2 Diabetic KKAy Mice using 2-Dimensional Differential Gel Electrophoresis

BackgroundDiabetic nephropathy (DN) is a leading cause of end-stage renal disease. To search for glomerular proteins associated with early-stage DN, glomeruli of spontaneous type 2 diabetic KKAy mice were analyzed by 2-dimensional differential gel electrophoresis (2D-DIGE).Material/MethodsGlomeruli of 20-week spontaneous type 2 diabetic KKAy mice and age-matched C57BL/6 mice were isolated by kidney perfusion with magnetic beads. Proteomic profiles of glomeruli were investigated by using 2D-DIGE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Western blot analysis was used to confirm the results of proteomics. Immunohistochemical and semi-quantitative analysis were used to confirm the differential expression of prohibitin and annexin A2 in glomeruli.ResultsWe identified 19 differentially expressed proteins – 17 proteins were significantly up-regulated and 2 proteins were significantly down-regulated in glomeruli of diabetic KKAy mice. Among them, prohibitin and annexin A2 were up-regulated and Western blot analysis validated the same result in proteomics. Immunohistochemical analysis also revealed up-regulation of prohibitin and annexin A2 in glomeruli of KKAy mice.ConclusionsOur findings suggest that prohibitin and annexin A2 may be associated with early-stage DN. Further functional research might help to reveal the pathogenesis of DN.

α-Glucosidase inhibitory activity of protein-rich extracts from extruded adzuki bean in diabetic KK-Ay mice

An extrusion process has been widely used for the development of many functional foods. The aim of this study was to assess the effect of the extrusion process on the α-glucosidase inhibitory properties of adzuki bean protein in type 2 diabetes model KK-A(y) mice. The extruded adzuki bean protein (EA) was prepared by adding 1% and 2% into the diet for 42 days. It was found that the fasting blood glucose concentration was significantly decreased with the EA-2 group compared with the control diabetic mice group. In addition, there was a significant decrease in serum triglyceride, blood urea nitrogen levels and increased high density lipoprotein (HDL)-cholesterol. Meanwhile, hepatic lipids were improved and the content of α-dicarbonyl compounds in the kidney were reduced in mice fed with EA. These results suggest that the intake of EA could moderate type 2 diabetes and diabetic complications.

MeOx-TMS derivatization for GC-MS metabolic profiling of urine and application in the discrimination between normal C57BL/6J and type 2 diabetic KK-Ay mice

GC-MS-based protocol using methoximation followed by silylation with BSTFA + 1%TMCS for the analysis of urine metabolites.

Apolipoprotein A-IV Reduces Hepatic Gluconeogenesis through Nuclear Receptor NR1D1

We showed recently that apoA-IV improves glucose homeostasis by enhancing pancreatic insulin secretion in the presence of elevated levels of glucose. Therefore, examined whether apolipoprotein A-IV (apoA-IV) also regulates glucose metabolism through the suppression of hepatic gluconeogenesis. The ability of apoA-IV to lower gluconeogenic gene expression and glucose production was measured in apoA-IV(-/-) and wild-type mice and primary mouse hepatocytes. The transcriptional regulation of Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) by apoA-IV was determined by luciferase activity assay. Using bacterial two-hybrid library screening, NR1D1 was identified as a putative apoA-IV-binding protein. The colocalization and interaction between apoA-IV and NR1D1 were confirmed by immunofluorescence, in situ proximity ligation assay, and coimmunoprecipitation. Enhanced recruitment of NR1D1 and activity by apoA-IV to Glc-6-Pase promoter was verified with ChIP and a luciferase assay. Down-regulation of apoA-IV on gluconeogenic genes is mediated through NR1D1, as illustrated in cells with NR1D1 knockdown by siRNA. We found that apoA-IV suppresses the expression of PEPCK and Glc-6-Pase in hepatocytes; decreases hepatic glucose production; binds and activates nuclear receptor NR1D1 and stimulates NR1D1 expression; in cells lacking NR1D1, fails to inhibit PEPCK and Glc-6-Pase gene expression; and stimulates higher hepatic glucose production and higher gluconeogenic gene expression in apoA-IV(-/-) mice. We conclude that apoA-IV inhibits hepatic gluconeogenesis by decreasing Glc-6-Pase and PEPCK gene expression through NR1D1. This novel regulatory pathway connects an influx of energy as fat from the gut (and subsequent apoA-IV secretion) with inhibition of hepatic glucose production.

Effects and Potential Mechanisms of Pioglitazone on Lipid Metabolism in Obese Diabetic KKAy Mice.

This study aimed to analyze the effects and potential mechanisms of pioglitazone on triglyceride and cholesterol metabolism in obese diabetic KKAy mice. Pioglitazone was orally administered to KKAy mice over 30 days. Compared to C57BL/6J mice, KKAy mice developed obvious insulin resistance, hepatic steatosis, and hyperlipidemia. Pioglitazone treatment resulted in deteriorated microvesicular steatosis and elevated hepatic triglyceride levels, though plasma triglyceride and free fatty acid levels were reduced by the treatment, compared to nontreated KKAy mice. Plasma alanine aminotransferase activities were also significantly increased. Additionally, pioglitazone increased plasma concentrations of total cholesterol, HDL-cholesterol, and LDL-cholesterol but decreased hepatic cholesterol. Gene expression profiling revealed that pioglitazone stimulated hepatic peroxisome proliferator-activated receptor gamma hyperactivity, and induced the upregulation of adipocyte-specific and lipogenesis-related genes but downregulated of genes involved in triglyceride lipolysis and fatty acid β-oxidation. Pioglitazone also regulated the genes expression of hepatic cholesterol uptake and excretion, such as low density lipoprotein receptor (LDL-R) and scavenger receptor type-BI (SR-BI). These results suggested that pioglitazone could induce excessive hepatic triglyceride accumulation, thus aggravating liver steatosis and lesions in KKAy mice. Furthermore, pioglitazone may suppress the clearance of serum cholesterol from the liver predominantly through inhibition of LDL-R and SR-BI expression, thus increasing the plasma cholesterol.

Effects of Ang II receptor blocker irbesartan on adipose tissue function in mice with metabolic disorders.

Recent studies indicate that the functional renin-angiotensin system (RAS) exists in the adipose tissue. The adipose tissue RAS is proposed in the pathophysiology of metabolic disorders. In the present study, we examined therapeutic effects of irbesartan, an angiotensin II (Ang II) type 1 receptor (AT1R)-specific blocker, in genetically obese diabetic KKAy mice, a model of human metabolic disorders without any dietary loading, with our focus on the analysis on possible effect of irbesartan on the adipose tissue. The treatment with irbesartan significantly lowered systolic blood pressure with a concomitant decrease in body weight in KKAy mice. In addition, irbesartan significantly decreased the adipose leptin mRNA expression and tended to decrease IL-6 mRNA expression in the adipose tissue of KKAy mice. Furthermore irbesartan preserved the adipose gene expression of AT1R-associated protein (ATRAP), an endogenous inhibitory molecule of tissue AT1R signaling, with a concomitant tendency of up-regulation of adipose tissue ATRAP/AT1R ratio. Collectively, these results suggest that the irbesartan-induced beneficial suppressive effect on the leptin-IL-6 axis in the adipose tissue in KKAy mice is partly mediated by a trend of up-regulation of the adipose ATRAP/AT1R ratio as one of pleiotropic effects of irbesartan.

Effects of the Angiotensin receptor blocker olmesartan on adipocyte hypertrophy and function in mice with metabolic disorders.

In the present study, we examined the therapeutic effects of olmesartan, an angiotensin II (Ang II) type 1 receptor (AT1R)-specific blocker, in genetically obese diabetic KKAy mice, a model of human metabolic disorders with visceral obesity, with a focus on an olmesartan effect on the adipose tissue. Olmesartan treatment (3 mg/kg per day) for 4 weeks significantly lowered systolic blood pressure but did not affect body weight during the study period in KKAy mice. However, there were three interesting findings possibly related to the pleiotropic effects of olmesartan on adipose tissue in KKAy mice: (1) an inhibitory effect on adipocyte hypertrophy, (2) a suppressive effect on IL-6 gene expression, and (3) an ameliorating effect on oxidative stress. On the other hand, olmesartan exerted no evident influence on the adipose tissue expression of AT1R-associated protein (ATRAP), which is a molecule interacting with AT1R so as to inhibit pathological AT1R activation and is suggested to be an emerging molecular target in metabolic disorders with visceral obesity. Collectively, these results suggest that the blood pressure lowering effect of olmesartan in KKAy mice is associated with an improvement in adipocyte, including suppression of adipocyte hypertrophy and inhibition of the adipose IL-6-oxidative stress axis. Further study is needed to clarify the functional role of adipose ATRAP in the pleiotropic effects of olmesartan.

Fecal metabonomic study of a polysaccharide, MDG-1 from Ophiopogon japonicus on diabetic mice based on gas chromatography/time-of-flight mass spectrometry (GC TOF/MS)

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder with systemic complications and has been a worldwide epidemic. Ophiopogon japonicus is a traditional Chinese medicine used to treat diabetes for thousands of years. From our previous work, we know that MDG-1, a water-soluble β-D-fructan polysaccharide from O. japonicas could treat T2DM experimentally. However, MDG-1 is poorly absorbed and its mechanism of action is still unknown. Therefore, a GC TOF/MS-based metabonomic approach in combination with multivariate statistical analysis was performed to investigate the mechanism of MDG-1 in a spontaneous diabetic model. Female diabetic KKay mice (21 weeks old) were randomly divided into a diabetic group (n = 6, gavaged with distilled water) and a MDG-1-Diabetic group (n = 7, gavaged with MDG-1, 300 mg kg(-1)) and female C57BL/6 mice (21 weeks old) were set as controls (n = 6, gavaged with distilled water). After 8-weeks of treatment, feces samples were collected for GC-TOF/MS analysis. Consequently, 12 potential biomarkers were identified, including monosugars (D-tagatose, D-lyxose, D-erythrose, xylo-hexos-5-ulose, 2-deoxy-galactose), butanedioic acid, amino acids (phenylalanine, L-lysine, L-methionine, L-aspartic acid) and purine derivatives (7H-purine, 2'-deoxyinosine). We assume the monosugars and butanedioic acid were the fermentation products of MDG-1 by intestinal microbes and MDG-1 actions against diabetes might be accomplished through the absorbable monosugars and butanedioic acid via suppressing intestinal glucose absorption, enhancing liver glycogenesis, inhibiting glycogenolysis and promoting GLP-1 secretion. Besides, MDG-1 might alleviate diabetes and diabetic nephropathy by reducing 7H-purine and 2'-deoxyinosine. Further omics-driven studies including genomics, proteomics and metabonomics were considered to be carried out to provide direct evidence of gut microbiome contribution to MDG-1 actions.

A tripeptide Diapin effectively lowers blood glucose levels in male type 2 diabetes mice by increasing blood levels of insulin and GLP-1.

The prevalence of type 2 diabetes (T2D) is rapidly increasing worldwide. Effective therapies, such as insulin and Glucagon-like peptide-1 (GLP-1), require injections, which are costly and result in less patient compliance. Here, we report the identification of a tripeptide with significant potential to treat T2D. The peptide, referred to as Diapin, is comprised of three natural L-amino acids, GlyGlyLeu. Glucose tolerance tests showed that oral administration of Diapin effectively lowered blood glucose after oral glucose loading in both normal C57BL/6J mice and T2D mouse models, including KKay, db/db, ob/ob mice, and high fat diet-induced obesity/T2D mice. In addition, Diapin treatment significantly reduced casual blood glucose in KKay diabetic mice in a time-dependent manner without causing hypoglycemia. Furthermore, we found that plasma GLP-1 and insulin levels in diabetic models were significantly increased with Diapin treatment compared to that in the controls. In summary, our findings establish that a peptide with minimum of three amino acids can improve glucose homeostasis and Diapin shows promise as a novel pharmaceutical agent to treat patients with T2D through its dual effects on GLP-1 and insulin secretion.

Selenium-enriched exopolysaccharides produced by Enterobacter cloacae Z0206 alleviate adipose inflammation in diabetic KKAy mice through the AMPK/SirT1 pathway

Polysaccharides belong to a structurally diverse class of macromolecules, with the necessary flexibility for the precise regulatory mechanisms and high capacity for carrying biological information. On the basis of a previous study regarding the administration of selenium-enriched exopolysaccharides (Se-ECZ-EPS) produced by Enterobacter cloacae (E. cloacae) Z0206 which resulted in a reduction of blood glucose levels and showed significant anti-inflammatory and anti-diabetic effects, the present study was conducted to evaluate the effects and mechanism of EPS on the alleviation of fat inflammation in high-fat-diet (HFD) induced-diabetic KKAy mice. The HFD induced-diabetic KKAy mice were gavaged once daily with EPS (0.2 mg/g body weight) or distilled water, while the C57BL/6J mice were gavaged with distilled water. Six weeks later visceral adipose tissue (VAT) was collected for quantified polymerase chain reaction (qPCR) and western blot (WB) analysis. The results showed that following supplementation with EPS, interleukin (IL) 6, IL1β and tumor necrosis factor (TNF) α mRNA expression in VAT were significantly reduced, while Glut4, pAMPK and SirT1 protein expression were markedly increased when compared with KKAy mice gavaged with water. Furthermore, ATGL and HSL mRNA were also significantly decreased. Subsequently, 3T3-L1 adipocytes were treated with insulin to induce insulin resistance to determine the mechanism by which EPS affects inflammation. Following the treatment of adipocytes with 100 nM insulin for 8 h, IL6 and TNFα mRNA expression were significantly increased, while the content of glucose uptake and Glut4 protein expression were significantly decreased. When treated with 100 nM insulin and 0.1 mg/ml EPS, no significant change in IL6 and TNFα mRNA expression or glucose uptake were observed. However, when SirT1‑siRNA or AMPKα1-siRNA was transfected into the 3T3-L1 adipocytes prior to treatment with insulin and EPS, there was a significant increase in IL6 and TNFα mRNA abundance. In conclusion, VAT inflammation and lipolysis in HFD-induced KKAy mice were significantly decreased following EPS usage. Moreover, EPS may alleviate VAT inflammation primarily through the AMPK/SirT1 pathway.

Calcium Channel Blocker Enhances Beneficial Effects of an Angiotensin II AT1 Receptor Blocker against Cerebrovascular-Renal Injury in type 2 Diabetic Mice

Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-Ay mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-Ay mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.

Regulation effects of TZQ-F on adipocyte differentiation and insulin action

Ethnopharmacological relevanceTZQ has been used in traditional Chineses medicine for treating diabetes. Based on the recipe of traditional anti-diabetic formula TZQ, we have developed TZQ-F which has been in phase 2 clinical trails. To study the mechanisms by which TZQ-F ameliorates diabetes, we examined whether treatment with TZQ-F improves hyperinsulinemia, hyperglycemia and obesity in type 2 diabetic KKAy mice and whether this is associated with an improvement of adipocyte differentiation and insulin action. MethodsTZQ-F, fenofibrate, rosiglitazone or distilled water was administered to 7-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Insulin resistance index, body weight and levels of serum blood glucose, leptin, insulin and adiponectin were evaluated. The expression of peroxisome proliferator-activated receptor γ (PPARγ) in skeletal muscle and liver tissues were determined with real-time PCR and western boltting. The mRNA expressions of insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), glucose transporter-1 (Glut-1) and Phosphoenolpyruvate 3-kinases (PI3K) in skeletal muscle and liver tissues were determined with real-time PCR. Histopathology of liver has been observed. ResultsTreatment of TZQ-F for 8 weeks ameliorated hyperglycemia, hyperinsulinemia, hyperleptinemia and hypoadiponectinemia in KKAy mice. TZQ-F also up-regulated expression of PPARγ in liver tissue. However, it had no effect on regulation of expression of PPARγ in muscle tissue. In addition, TZQ-F upregulates InsR, IRS-1, IRS-2, Glut-1, and PI3K mRNA expression. Consistent with the in vivo results, histology study demonstrated that TZQ-F alleviated pathologic changes of the liver induced by high-fat diet. ConclusionsThese results first indicate that TZQ-F can be beneficial for reducing hyperinsulinemia, hyperglycemia and obesity through its potency of regulating adipocyte differentiation and insulin action.