Reduction of JNK1 expression with antisense oligonucleotide improves adiposity in obese mice

To investigate the role of JNK1 in metabolism, male ob/ob and diet-induced obese mice were treated with a JNK1-specific antisense oligonucleotide (ASO) or control ASO at 25 mg/kg or saline twice/wk for 6 and 7 wk, respectively. JNK1 ASO reduced JNK1 mRNA and activity by 65-95% in liver and fat tissues in both models. Compared with controls, treatment with JNK1 ASO did not change food intake but lowered body weight, fat pad weight, and whole body fat content. The treatment increased metabolic rate. In addition, the treatment markedly reduced plasma cholesterol levels and improved liver steatosis and insulin sensitivity. These positive observations were accompanied by the following changes: 1) increased mRNA levels of AR-beta(3) and UCP1 by >60% in BAT, 2) reduced mRNA levels of ACC1, ACC2, FAS, SCD1, DGAT1, DGAT2, and RBP4 by 30-60% in WAT, and 3) reduced mRNA levels of ACC1, FAS, G-6-Pase, and PKCepsilon by 40-70% and increased levels of UCP2 and PPARalpha by more than twofold in liver. JNK1 ASO-treated mice demonstrated reduced levels of pIRS-1 Ser(302) and pIRS-1 Ser(307) and increased levels of pAkt Ser(473) in liver and fat in response to insulin. JNK1 ASO-transfected mouse hepatocytes showed decreased rates of de novo sterol and fatty acid synthesis and an increased rate of fatty acid oxidation. These results indicate that inhibition of JNK1 expression in major peripheral tissues can improve adiposity via increasing fuel combustion and decreasing lipogenesis and could therefore provide clinical benefit for the treatment of obesity and related metabolic abnormalities.     

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1beta and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1beta and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.     

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.     

Increased insulin sensitivity and reduced adiposity in phosphatidylinositol 5-phosphate 4-kinase beta-/- mice

Phosphorylated derivatives of the lipid phosphatidylinositol are known to play critical roles in insulin response. Phosphatidylinositol 5-phosphate 4-kinases convert phosphatidylinositol 5-phosphate to phosphatidylinositol 4,5-bis-phosphate. To understand the physiological role of these kinases, we generated mice that do not express phosphatidylinositol 5-phosphate 4-kinase beta. These mice are hypersensitive to insulin and have reduced body weights compared to wild-type littermates. While adult male mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have significantly less body fat than wild-type littermates, female mice lacking phosphatidylinositol 5-phosphate 4-kinase beta have increased insulin sensitivity in the presence of normal adiposity. Furthermore, in vivo insulin-induced activation of the protein kinase Akt is enhanced in skeletal muscle and liver from mice lacking phosphatidylinositol 5-phosphate 4-kinase beta. These results indicate that phosphatidylinositol 5-phosphate 4-kinase beta plays a role in determining insulin sensitivity and adiposity in vivo and suggest that inhibitors of this enzyme may be useful in the treatment of type 2 diabetes.     

The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.     

Abdominal adiposity and insulin resistance in obese men

We examined the independent relationships among various visceral and abdominal subcutaneous adipose tissue (AT) depots, glucose tolerance, and insulin sensitivity in 89 obese men. Measurements included an oral glucose tolerance test (OGTT), glucose disposal by euglycemic clamp, and abdominal and nonabdominal (e.g., peripheral) AT by magnetic resonance imaging (MRI). OGTT glucose and glucose disposal rates were related (P < 0.05) to visceral AT (r = 0.50 and -0.41, respectively). These observations remained significant (P < 0.05) after control for nonabdominal and abdominal subcutaneous AT, and maximal O(2) consumption (VO(2 max)). Abdominal subcutaneous AT was not a significant correlate (P > 0.05) of any metabolic variable after control for nonabdominal and visceral AT and VO(2 max). Division of abdominal subcutaneous AT into deep and superficial depots and visceral AT into intra- and extraperitoneal AT depots did not alter the observed relationships. Further analysis matched two groups of men for abdominal subcutaneous AT but also for low and high visceral AT. Men with high visceral AT had higher OGTT glucose values and lower glucose disposal rates compared with those with low visceral AT values (P < 0.05). A similar analysis performed on two groups of men matched for visceral AT but also for high and low abdominal subcutaneous AT revealed no statistically different values for any metabolic variable (P > 0.10). In conclusion, visceral AT alone is a strong correlate of insulin resistance independent of nonabdominal and abdominal subcutaneous AT and cardiovascular fitness. Subdivision of visceral and abdominal subcutaneous AT by MRI did not provide additional insight into the relationship between abdominal obesity and metabolic risk in obese men.     

Reduction of low molecular weight protein-tyrosine phosphatase expression improves hyperglycemia and insulin sensitivity in obese mice

To investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-beta subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.     

CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.     

Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in diet-induced obese mice

Exercise promotes weight loss and improves insulin sensitivity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Obesity correlates with increased production of inflammatory cytokines, which in turn, contributes to systemic insulin resistance. To test the hypothesis that exercise mitigates this inflammatory response, thereby improving insulin sensitivity, we developed a model of voluntary exercise in mice made obese by feeding of a high fat/high sucrose diet (HFD). Over four wk, mice fed chow gained 2.3 +/- 0.3 g, while HFD mice gained 6.8 +/- 0.5 g. After 4 wk, mice were subdivided into four groups: chow-no exercise, chow-exercise, HFD-no exercise, HFD-exercise and monitored for an additional 6 wk. Chow-no exercise and HFD-no exercise mice gained an additional 1.2 +/- 0.3 g and 3.3 +/- 0.5 g respectively. Exercising mice had higher food consumption, but did not gain additional weight. As expected, GTT and ITT showed impaired glucose tolerance and insulin resistance in HFD-no exercise mice. However, glucose tolerance improved significantly and insulin sensitivity was completely normalized in HFD-exercise animals. Furthermore, expression of TNF-alpha, MCP-1, PAI-1 and IKKbeta was increased in adipose tissue from HFD mice compared with chow mice, whereas exercise reversed the increased expression of these inflammatory cytokines. In contrast, expression of these cytokines in liver was unchanged among the four groups. These results suggest that exercise partially reduces adiposity, reverses insulin resistance and decreases adipose tissue inflammation in diet-induced obese mice, despite continued consumption of HFD.     

Gradient-enhanced TOCSY experiments with improved sensitivity and solvent suppression

Summary Gradient-enhanced versions of the homonuclear TOCSY experiment are described, with solvent suppression and sensitivity superior to that of a conventional TOCSY experiment. The pulse sequences are constructed by appending a WATERGATE module to a z-filtered TOCSY experiment. Pulsed-field gradients and appropriately phased selective rf pulses are used to maintain precise control of the water magnetization vector. Problems associated with radiation damping and spin-locking of the water magnetization are thus alleviated. The water magnetization is returned to equilibrium prior to each acquisition, which improves water suppression and minimizes signal losses due to saturation transfer.     

Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.     

Amylin improves the effect of leptin on insulin sensitivity in leptin-resistant diet-induced obese mice

Leptin enhances insulin sensitivity in addition to reducing food intake and body weight. Recently, amylin, a pancreatic β-cell-derived hormone, was shown to restore a weight-reducing effect of leptin in leptin-resistant diet-induced obesity. However, whether amylin improves the effect of leptin on insulin sensitivity in diet-induced obesity is unclear. Diet-induced obese (DIO) mice were infused with either saline (S), leptin (L; 500 μg·kg?1·day?1), amylin (A; 100 μg·kg?1·day?1), or leptin plus amylin (L/A) for 14 days using osmotic minipumps. Food intake, body weight, metabolic parameters, tissue triglyceride content, and AMP-activated protein kinase (AMPK) activity were examined. Pair-feeding and weight-matched calorie restriction experiments were performed to assess the influence of food intake and body weight reduction. Continuous L/A coadministration significantly reduced food intake, increased energy expenditure, and reduced body weight, whereas administration of L or A alone had no effects. L/A coadministration did not affect blood glucose levels during ad libitum feeding but decreased plasma insulin levels significantly (by 48%), suggesting the enhancement of insulin sensitivity. Insulin tolerance test actually showed the increased effect of insulin in L/A-treated mice. In addition, L/A coadministration significantly decreased tissue triglyceride content and increased AMPKα2 activity in skeletal muscle (by 67%). L/A coadministration enhanced insulin sensitivity more than pair-feeding and weight-matched calorie restriction. In conclusion, this study demonstrates the beneficial effect of L/A coadministration on glucose and lipid metabolism in DIO mice, indicating the possible clinical usefulness of L/A coadministration as a new antidiabetic treatment in obesity-associated diabetes.     

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.     

MiR-690, an exosomal-derived miRNA from M2-polarized macrophages,improves insulin sensitivity in obese mice

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Caenorhabditis elegans ucp-4 regulates fat metabolism: suppression of ucp-4 expression induced obese phenotype and caused impairment of insulin like pathway

Uncoupling proteins, a family of proton carriers located in the inner mitochondrial membrane, have important functions in energy metabolism and free radical generation that are relevant to mitochondrial function. Five family members have been identified, UCP1-5, that have distinct tissue distributions, and differences and similarities in physiological function. Uncoupling protein 4 (UCP4) is highly expressed and has a unique function in brain. UCP4 appears to be involved with metabolism in neurons and adipocytes, but conclusions on this protein have been controversial. Here, we used Caenorhabditis elegans to explore the functions of ucp-4, particularly in fat metabolism. Our results showed that UCP4 knockdown induced an obese phenotype and impaired the insulin-like pathway, possibly via oxidative stress in C. elegans. This highlights the importance of studying the role of ucp-4 in fat metabolism.     

Kaempferol ameliorates hyperglycemia through suppressing hepatic gluconeogenesis and enhancing hepatic insulin sensitivity in diet-induced obese mice

Obesity-associated insulin resistance (IR) is a major risk factor for developing type 2 diabetes and an array of other metabolic disorders. In particular, hepatic IR contributes to the increase in hepatic glucose production and consequently the development of fasting hyperglycemia. In this study, we explored whether kaempferol, a flavonoid isolated from Gink go biloba, is able to regulate hepatic gluconeogenesis and blood glucose homeostasis in high-fat diet-fed obese mice and further explored the underlying mechanism by which it elicits such effects. Oral administration of kaempferol (50 mg/kg/day), which is the human equivalent dose of 240 mg/day for an average 60 kg human, significantly improved blood glucose control in obese mice, which was associated with reduced hepatic glucose production and improved whole-body insulin sensitivity without altering body weight gain, food consumption or adiposity. In addition, kaempferol treatment increased Akt and hexokinase activity, but decreased pyruvate carboxylase (PC) and glucose-6 phosphatase activity in the liver without altering their protein expression. Consistently, kaempferol decreased PC activity and suppressed gluconeogenesis in HepG2 cells as well as primary hepatocytes isolated from the livers of obese mice. Furthermore, we found that kaempferol is a direct inhibitor of PC. These findings suggest that kaempferol may be a naturally occurring antidiabetic compound that acts by suppressing glucose production and improving insulin sensitivity. Kaempferol suppression of hepatic gluconeogenesis is due to its direct inhibitory action on the enzymatic activity of PC.     

Combined effects of rosiglitazone and conjugated linoleic acid on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed mice

Dysfunctional cross talk between adipose tissue and liver tissue results in metabolic and inflammatory disorders. As an insulin sensitizer, rosiglitazone (Rosi) improves insulin resistance yet causes increased adipose mass and weight gain in mice and humans. Conjugated linoleic acid (CLA) reduces adipose mass and body weight gain but induces hepatic steatosis in mice. We examined the combined effects of Rosi and CLA on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed male C57Bl/6 mice. CLA alone suppressed weight gain and adipose mass but caused hepatic steatosis. Addition of Rosi attenuated CLA-induced insulin resistance and dysregulation of adipocytokines. In adipose, CLA significantly suppressed lipoprotein lipase and fatty acid translocase (FAT/CD36) mRNA, suggesting inhibition of fatty acid uptake into adipose; addition of Rosi completely rescued this effect. In addition, CLA alone increased markers of macrophage infiltration, F4/80, and CD68 mRNA levels, without inducing TNF-alpha in epididymal adipose tissue. The ratio of Bax to Bcl2, a marker of apoptosis, was significantly increased in adipose of the CLA-alone group and was partially prevented by treatment of Rosi. Immunohistochemistry of F4/80 demonstrates a proinflammatory response induced by CLA in epididymal adipose. In the liver, CLA alone induced microsteatotic liver but surprisingly increased the rate of very-low-density lipoprotein-triglyceride production without inducing inflammatory mediator-TNF-alpha and markers of macrophage infiltration. These changes were accompanied by significantly increased mRNA levels of stearoyl-CoA desaturase, FAT/CD36, and fatty acid synthase. The combined administration of CLA and Rosi reduced hepatic liver triglyceride content as well as lipogenic gene expression compared with CLA alone. In summary, dietary CLA prevented weight gain in Rosi-treated mice without attenuating the beneficial effects of Rosi on insulin sensitivity. Rosi ameliorated CLA-induced lipodystrophic disorders that occurred in parallel with rescued expression of adipocytokine and adipocytes-abundant genes.