Dual roles of adiponectin/Acrp30 in vivo as an anti-diabetic and anti-atherogenic adipokine

Genome-wide scanning is a powerful tool to identify susceptible chromosome loci, however, individual chromosomal regions still have many candidate genes. Although cDNA microarray analysis provides valuable information for identifying genes involved in pathogenesis, expression levels of many genes are changed. A novel approach for identification of therapeutic targets is the combination of genome-wide scanning and the use of DNA chips, as shown in Fig. (1). Using DNA chips, we screened for secreted molecules, the expressions of which were changed in adipose tissues from mice rendered insulin resistance. Decreased expression of one of these molecules, adiponectin/Acrp30, correlates strongly with insulin resistance. Interestingly, recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where adiponectin gene is located. Decreasing serum adiponectin levels are associated with increased risk for type 2 diabetes. Interestingly, adiponectin was decreased in insulin resistant rodent models both of obesity and lipoatrophy, and replenishment of adiponectin ameliorated their insulin resistance. Moreover, adiponectin transgenic mice ameliorated insulin resistance and diabetes Adiponectin knockout mice showed insulin resistance and glucose intolerance. In muscle and liver, adiponectin activated AMP kinase and PPARalpha pathways thereby increasing beta-oxidation of lipids, leading to decreased TG content, which ameliorated insulin resistance under a high-fat diet. Despite similar plasma glucose and lipid levels on an apoE deficient background, adiponectin transgenic apoE deficient mice showed amelioration of atherosclerosis, which was associated with decreased expressions of class A scavenger receptor and tumor necrosis factor alpha. Finally, cDNA encoding adiponectin receptors (AdipoR1 and R2) have been identified by expression cloning, which facilitates the understanding of molecular mechanisms of adiponectin actions and obesity-linked diseases such as diabetes and atherosclerosis and the designing of novel antidiabetic and anti-atherogenic drugs with AdipoR1 and R2 as molecular targets.     

The potential insulin sensitizing and glucose lowering effects of a novel indole derivative in vitro and in vivo.

Thiazolidinediones (TZDs) such as rosiglitazone are antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma agents improve diabetes by increasing insulin sensitivity and enhancing the differentiation of preadipocytes into adipocytes. The present study aimed to identify if 1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-3-indoleacetitic acid (GY3), a newly synthesized indole compound, could enhance adipocytes differentiation and insulin sensitivity. The results showed that both GY3 and rosiglitazone significantly increased the lipid accumulating of 3T3-L1 adipocytes induced by isobutylmethylxanthine, dexamethasone and insulin mixture, but GY3 (not rosiglitazone) failed to increase the lipid accumulation when induced by insulin alone. In addition, GY3- or rosiglitaozne-induced protein expression of GLUT4 and adiponectin was determined by Western blot analysis. GY3 activated PPARalpha weakly but did not affect PPARgamma, while rosiglitazone activated PPARgamma significantly, suggesting different mechanisms between GY3 and rosiglitazone on adipocyte differentiation. Furthermore, both GY3 and rosiglitazone enhanced the adiponectin and insulin pathway proteins expression and adiponectin secretion in mature adipocytes, but only GY3 not rosiglitazone elevated gene expression of leptin and resistin. Both GY3 and rosiglitazone enhanced glucose consumption in HepG2 cells especially in the presence of insulin. In the in vivo study, GY3 decreased serum glucose and insulin in db/db mice, indicating the insulin sensitizing effect might contribute to its antidiabetic mechanism. Altogether, these results suggest that GY3 could improve insulin resistance and lower glucose level, GY3 and its derivatives might be developed as a substitution therapy for diseases with insulin resistance.     

Antidiabetic effect of a novel non-thiazolidinedione PPAR γ/α agonist on ob/ob mice

AIM: To study whether T33, a new synthesized non-thiazolidinedione (TZD) peroxisome proliferator-activated receptor (PPAR) gamma/alpha dual agonist has an antidiabetic effect on ob/ob mice. METHODS: Ob/ob mice were treated with 4 mg/kg or 8 mg/kg T33 by gavage for 20 d. Blood glucose levels were measured regularly. An oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT) were preformed on d 8 and d 12, respectively. The levels of insulin, triglyceride and free fatty acid (FFA) in the serum were measured at the end of administration. The intramuscular and liver triglyceride content was also determined. RESULTS: T33 reduced the hyperglycemia, hyperinsulinemia and hyperlipidemia of the ob/ob mice. The OGTT and ITT showed that the insulin resistance state of the ob/ob mice was obviously ameliorated after T33 treatment. After 20 d treatment with 8 mg/kg T33, the triglyceride content in the gastrocnemius muscle decreased significantly. T33 did not have any effect on triglyceride content in the liver, whereas rosiglitazone significantly increased the hepatocyte lipid deposition. CONCLUSION: The PPARgamma/alpha dual agonist T33 has antidiabetic and insulin-sensitizing effects in ob/ob mice. It has the potential to be a new therapeutic candidate for the treatment of type 2 diabetes.     

The role of PPARgamma in the onset of type 2 diabetes

We have investigated the role of PPARgamma and its related genes on the pathogenesis of type 2 diabetes. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet, whose phenotypes were abrogated by PPARgamma agonist treatment. Moreover, a Pro12Ala polymorphism in the human PPARgamma 2 gene, which has been reported to cause a reduction in the PPARgamma activity, was associated with decreased risk of type 2 diabetes in Japanese. The protective effect of this polymorphism against type 2 diabetes has been confirmed by meta-analysis using reports in various ethnic groups. Taken together with these results, PPARgamma is a thrifty gene mediating type 2 diabetes both in mice and humans. Indeed, pharmacological inhibitors of PPARgamma/RXR ameliorates high-fat diet-induced insulin resistance in animal models of type 2 diabetes.     

Phosphatase and tensin homolog deleted on chromosome 10 suppression is an important process in peroxisome proliferator-activated receptor-gamma signaling in adipocytes and myotubes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) activation enhances insulin sensitivity in type 2 diabetes mellitus. However, downstream mediators of PPARgamma activation in adipocytes and myotubes, the most important cell types involved in glucose homeostasis, remained unclear. Here we show by using two synthetic PPARgamma agonists (rosiglitazone and KR-62776, a novel PPARgamma agonist) that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a key downstream mediator of PPARgamma signaling. The PPARgamma agonists down-regulated PTEN expression, resulting in glucose uptake increase in differentiated 3T3-L1 adipocytes and C2C12 skeletal muscle cells. In both cells, PTEN knockdown increased glucose uptake, whereas overexpression abolished the agonist-induced effects. The effects of PPARgamma agonists on PTEN expression and glucose uptake disappeared by pretreatment with a PPARgamma antagonist or by knockdown of PPARgamma expression. In vivo treatment of the agonists to C57BL/6J-ob/ob mice resulted in the reduction of PTEN level in both adipose and skeletal muscle tissues and decreased plasma glucose levels. Thus, these results suggest that PTEN suppression is a key mechanism of the PPARgamma-mediated glucose uptake stimulation in insulin-sensitive cells such as adipocytes and skeletal muscle cells, thereby restoring glucose homeostasis in type 2 diabetes.     

Central role of the adipocyte in insulin resistance

Mechanisms of insulin resistance in subjects at risk for type 2 diabetes remain to be elucidated. Insulin acts slowly in vivo, but rapidly in vitro, suggesting that the pathway insulin traverses from B-cell to insulin sensitive tissue may be altered in diabetes. An important component of that pathway is transport of insulin across the capillary endothelium. Several groups have demonstrated that insulin resistance may result from reduced capillary permeability to insulin--it remains to be determined whether reduced permeability contributes to insulin resistance in any stage leading to type 2 diabetes. Interestingly, the transport of insulin across the endothelial barrier not only limits the rate of insulin to stimulate glucose uptake by skeletal muscle, but appears also to determine the rate at which insulin suppresses liver glucose output. Because the liver circulation is fenestrated, it is not possible that insulin transport into the liver is the rate determining step for suppression of liver glucose output. An alternative hypothesis was considered--that insulin is transported into an extrahepatic tissue. A "second signal" is generated by the extrahepatic tissue, the signal is released into the blood, and the signal in turn controls hepatic glucose output. Several lines of evidence suggest that the second signal is free fatty acids (FFA): 1) There is a strong correlation between FFA and liver glucose output under a variety of experimental conditions. 2) If FFA are maintained at basal concentrations during insulin administration, glucose output fails to decline. 3) If FFA are reduced independent of insulin administration, glucose output is reduced. These three points support the concept that insulin, by regulating adipocyte lipolysis, controls liver glucose production. Thus, the adipocyte is a critical mediator between insulin and liver glucose output. Evidence that FFA also suppress skeletal muscle glucose uptake and insulin secretion from the B-cell supports the overall central role of the adipocyte in the regulation of glycemia. Insulin resistance at the fat cell may be an important component of the overall regulation of glycemia because of the relationships between FFA and glucose production, glucose uptake, and insulin release. It is possible that insulin resistance at the adipocyte itself can be a major cause of the dysregulation of carbohydrate metabolism in the prediabetic state.     

Improvement of hyperglycaemia and metabolic syndromes in type 2 diabetic KKAy mice by oral treatment with [meso-tetrakis(4-sulfonatophenyl) porphyrinato]oxovanadium(IV)(4-) complex

Recently, we reported that [meso-tetrakis(4-sulfonatophenyl)porphyrinato]oxovanadium(IV)(4-), VO(tpps), shows in-vitro insulin-mimetic and in-vivo anti-diabetic activity in streptozotocin (STZ)-induced type 1 diabetic mice. This result prompted us to examine its ability in type 2 diabetic model KKA(y) mice with insulin resistance. We studied the in-vivo anti-diabetic activity of VO(tpps), compared with that of vanadium(IV) oxide sulfate, VS, as control. Both compounds were orally administered at doses of 5-10 mg (0.1-0.2 mmol) V/kg body weight to the KKA(y) mice for 28 days. VO(tpps) normalized the hyperglycaemia within 15 days, while VS lowered the blood glucose concentration only by a small degree. In addition, metabolic syndromes characterized by insulin and leptin resistance were significantly improved in VO(tpps)-treated KKA(y) mice compared with those treated with VS. The improvement in diabetes was validated by oral glucose tolerance test and decrease in HbA(1c) concentration. Based on these observations, VO(tpps) is proposed to be an orally active oxovanadium(IV)-porphyrin complex for treating not only type 2 diabetes but also metabolic syndromes in animals.     

Molecular activation of PPARgamma by angiotensin II type 1-receptor antagonists

OBJECTIVE AND DESIGN: Elevated blood pressure and insulin resistance are strongly associated in patients. We explored the potential for the anti-hypertensive angiotensin II type 1-receptor (ATR(1)) antagonists to improve insulin sensitivity through modulation of the nuclear receptor PPARgamma, in vitro and in vivo compared to the potent insulin sensitizer, rosiglitazone. METHODS: PPARgamma modulation by ATR(1) antagonists was measured first by direct recruitment of PGC-1, followed by trans-activation reporter assays in cells, and promotion of adipogenesis in fibroblast and pre-adipocyte cell lines. Improvement of insulin sensitivity was measured as changes in levels of glucose, insulin, and adiponectin in ob/ob mice. RESULTS: Telmisartan, candesartan, irbesartan, and losartan (but not valsartan or olmesartan) each served as bona fide PPARgamma ligands in vitro, with EC(50) values between 3 and 5 micro mol/l. However, only telmisartan, and to a lesser extent candesartan, resulted in significant PPARgamma agonism in cells. In vivo, although rosiglitazone significantly lowered both glucose (33%, p<0.01) and insulin (61%, p<0.01) levels and increased expression of adiponectin (74%, p<0.001), sartan treatment had no effect. CONCLUSIONS: Many members of the sartan family of ATR(1) antagonists are PPARgamma ligands in cell-free assays but their modulation of PPARgamma in cells is relatively weak. Furthermore, none appear to improve insulin sensitivity in a rodent model under conditions where other insulin sensitizers, including rosiglitazone, do. These results question whether reported effects of sartans on insulin sensitivity may be through other means, and should guide further efforts to develop dual agents to treat hypertension and insulin resistance.     

Fenretinide ameliorates insulin resistance and fatty liver in obese mice

Fenretinide (FEN), a ligand of retinol binding protein 4 (RBP4), has been suggested as a measure to reduce insulin resistance and its associated disorders such as obesity, and fatty liver by reducing serum RBP4. We investigated whether there is another possible mechanism by which fenretinide reduces insulin resistance and fatty liver in genetically obese (ob/ob) mice. Male obese mice fed a high-fat diet (45% of calories from fat) were divided into two groups (n=13 each). One (FEN) received fenretinide (20 mg/kg body weight, intraperitoneally) and the other (O) received vehicle three times weekly for 24 d. C57BL/6J mice fed a normal-fat diet (16% of calories from fat) were used as a control (C; n=13). No changes in fat weight and serum leptin level could be observed in FEN mice. Lower plasma RBP4 was observed in FEN mice compared with O mice. Fenretinide improved whole-body insulin sensitivity based on glucose and insulin tolerance tests and the homeostasis model assessment of insulin resistance. Fenretinide decreased the plasma lipid (triglyceride, cholesterol, and free-fatty acid) levels, hepatic TG level, and histological steatosis score. The mechanism by which fenretinide prevents fatty liver may be explained by an increased plasma adiponectin level, increased activation of hepatic AMP-activated protein kinase, and the expression of peroxisome proliferator-activated protein-α and peroxisomal acyl-CoA oxidase, which promote fat oxidation. FEN alleviated insulin resistance and fatty liver in obese mice and thus may act as an anti-lipidemic and anti-diabetic drug.     

Antidiabetic and hypolipidemic effects of a novel dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, E3030, in db/db mice and beagle dogs

We investigated the antidiabetic effects of E3030, which is a potent dual activator of peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma, in an animal model of diabetes, C57BL/KsJ-db/db mice (db/db mice), and the lipidemic effects of E3030 in beagle dogs, whose PPARalpha and PPARgamma transactivation responses to E3030 were similar to those of humans. E3030 activated human PPARalpha, mouse PPARalpha, dog PPARalpha, human PPARgamma, mouse PPARgamma, and dog PPARgamma with EC(50) values of 65, 920, 87, 34, 73, and 34 nM, respectively, in the chimeric GAL4-PPAR receptor transactivation reporter assay. In db/db mice orally administered E3030 decreased blood glucose, triglyceride (TG), non-esterified fatty acids (NEFA), and insulin levels and increased blood adiponectin levels during a 14-day experimental period. Significant effects on blood glucose and adiponectin levels were observed at a dose of 3 mg/kg or greater. Furthermore, significant effects on blood TG, NEFA, and insulin levels were observed at doses of 1 mg/kg or more. An oral glucose tolerance test (OGTT) performed on Day 15 showed that E3030 at 3 mg/kg improved glucose tolerance in this model. Fourteen days of oral treatment with E3030 at a dose of 0.03 mg/kg or greater showed remarkable TG- and non high-density lipoprotein (non-HDL) cholesterol-lowering effects in beagle dogs. These results were similar to those observed for the PPARalpha agonist fenofibrate. E3030 also reduced apo C-III levels on Days 7 and 14, and elevated lipoprotein lipase (LPL) levels on Day 15. These results indicate that the TG- and non-HDL cholesterol-lowering actions of E3030 involve combined effects on reduction of apo C-III and elevation of LPL, resulting in increased lipolysis. The experimental results in animals suggest that E3030 has potential for use in the treatment of various aspects of metabolic dysfunction in type 2 diabetes, including dyslipidemia, hyperglycemia, hyperinsulinemia, and impaired glucose disposal.     

Improvement of insulin resistance by removal of systemic hydrogen peroxide by PEGylated catalase in obese mice

Insulin resistance, a condition in which insulin action is impaired, is one of the characteristic features of type 2 diabetes. Excessive amounts of reactive oxygen species (ROS) interfere with the insulin signaling pathway, which leads to the progression of insulin resistance. To examine whether removal of systemic hydrogen peroxide is effective in improving insulin resistance, polyethylene glycol-conjugated catalase (PEG-catalase), a derivative with a long circulation half-life, was repeatedly injected into leptin-deficient ob/ob or high fat diet-induced obese mice for 16 or 10 consecutive weeks, respectively. Although ob/ob mice gradually gained weight with time irrespective of the treatment, repeated intraperitoneal injections of PEG-catalase significantly reduced glucose levels in the fed state. Glucose and insulin tolerance tests also showed PEG-catalase significantly improved glucose tolerance and insulin sensitivity in ob/ob mice, respectively. Similar but less marked results were obtained in the diet-induced obese mice. Treatment of 3T3-L1 adipocytes with glucose oxidase (GO) increased lipid hydroperoxide formation and reduced insulin-stimulated Akt phosphorylation. Addition of catalase or PEG-catalase significantly inhibited the GO-induced changes in adipocytes. These findings indicate that systemic removal of hydrogen peroxide by PEG-catalase activates the insulin signaling pathway and improves insulin resistance in obese mice.     

The insulin-sensitizing role of the fat derived hormone adiponectin

Adiponectin is an insulin-sensitizing hormone whose blood concentration is reduced in obesity and type 2 diabetes. Administration of recombinant adiponectin in rodents increases glucose uptake and increases fat oxidation in muscle, reduces fatty acid uptake and hepatic glucose production in liver, and improves whole body insulin resistance. The exact receptor and signaling systems are unknown, however, recent studies suggest adiponectin activates AMPK, a putative master metabolic regulator. Thus, excitement surrounds the potential for adiponectin, or a homologue of adiponectin, as pharamacotherapy agents for patients suffering from the metabolic syndrome and more particularly for individuals with insulin resistance and type 2 diabetes.     

Role of PPAR- gamma agonist thiazolidinediones in treatment of pre-diabetic and diabetic individuals: a cardiovascular perspective

The peroxisome proliferator-activated receptors (PPARs) are nuclear fatty acid receptors, which contain a type II zinc finger DNA binding motif and a hydrophobic ligand binding pocket. These receptors are thought to play an important role in metabolic diseases such as obesity, insulin resistance, and coronary artery disease. Three subtypes of PPAR receptors have been described: PPARalpha, PPARdelta/beta, and PPARgamma. PPARalpha is found in the liver, muscle, kidney, and heart. In the liver, its role is to up-regulate genes involved in fatty acid uptake (beta-oxidation and omega-oxidation). PPARdelta/beta is involved in fatty acid oxidation in muscle. PPARgamma has high expression in fat, low expression in the liver, and very low expression in the muscle. The thiazolidinediones (TZD) are synthetic ligands of PPARgamma. By activating a number of genes in tissues, PPARgamma increases glucose and lipid uptake, increases glucose oxidation, decreases free fatty acid concentration, and decreases insulin resistance. There is a sound rationale for the use of TZDs in patients with type 2 diabetes mellitus and promising preliminary data in patients with patients with pre-diabetes. In patients with type 2 diabetes, thiazolidinediones had been shown to decrease mean HbA(1c)by 1.5% and lower HbA(1c) to less than 7% in 30% of patients. Decreased muscle insulin resistance primarily mediates the glucose lowering effect. In addition, there are several nonhypoglycemic effects of TZDs which may be beneficial to both diabetics and patients with pre-diabetes. These include effects on lipid metabolism, blood pressure, endothelial function, atherosclerotic plaque, coagulation, and albuminuria. In a pilot study, we recently demonstrated that insulin sensitizers such as thiazolidinediones appear to be associated with better clinical outcomes compared to insulin providers in diabetic patients presenting with acute coronary syndromes. In another study, we showed that the prediabetic state is a marker for worse prognosis in patients with acute coronary syndromes. In this article, we review the existing literature on the effectiveness of PPAR-gamma agonists in patients with either overt diabetes or a prediabetic state.     

Acanthopanax senticosus reverses fatty liver disease and hyperglycemia in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) is common in obesity. However, weight reduction alone does not prevent the progression of NAFLD to end-stage disease associated with the development of cirrhosis and liver disease. In a previous experiment, 50% ethanol extract of Acanthopanax senticosus stem bark (ASSB) was found to reduce body weight and insulin resistance in high fat diet-induced hyperglycemic and hyperlipidemic ICR mice. To evaluate the anti-steatosis action of ASSB, insulin-resistant ob/ob mice with fatty livers were treated with ASSB ethanol extract for an 8 week-period. ASSB ethanol extract reversed the hepatomegaly, as evident in reduction of % liver weight/body weight ratio. ASSB ethanol extract also specifically lowered circulating glucose and lipids, and enhanced insulin action in the liver. These changes culminated in inhibition of triglyceride synthesis in non-adipose tissues including liver and skeletal muscle. Gene expression studies confirmed reductions in glucose 6-phosphatase and lipogenic enzymes in the liver. These results demonstrate that ASSB ethanol extract is an effective treatment for insulin resistance and hepatic steatosis in ob/ob mice by decreasing hepatic lipid synthesis.     

The effect of 1,3-diaryl-[1H]-pyrazole-4-acetamides on glucose utilization in ob/ob mice

This article provides evidence of a new class of compounds, 1,3-diaryl-[1H]-pyrazole-4-acetamides, initially identified from their ability to increase glucose transport in an adipocyte and muscle cell line and ultimately demonstrating dramatic glucose lowering in ob/ob mice, a diabetic animal model. The lead compound, 1, possessed some behavioral-like effects which were removed by structural variation during the course of this investigation. Specifically, 11g (R1 = meta-CF(3), Ar2 = 4'biphenyl, R3 = diethylamide) illustrated the potency of this series with ED(50) values for glucose lowering in ob/ob mice of 3.0 mg/kg/day. Concomitant with its effect on glucose lowering, 11g also caused a 50% reduction in insulin levels consistent with an agent that increases whole body insulin sensitivity. 11g showed favorable pharmacokinetic data with acceptable absorption, negligible metabolism, and good duration of action. 11g demonstrated no appreciable adipogenic effect through PPAR gamma agonism, a characteristic of the thiazolidinediones (TZD), and so represents a potentially new class of agents for the treatment of diabetes.