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We explored the beneficial effects of GW7647, a peroxisome proliferator activated receptor α (PPARα) agonist, and metformin, an anti-diabetic drug on an advanced nonalcoholic steatohepatitis (NASH) model in rodents and investigated the possible mechanisms involved. Mice were fed control chow or a choline-deficient l-amino acid-defined diet containing 45% fat (HF-CDAA). The mice fed HF-CDAA diets for 16 weeks were divided into four groups: the no treatment (HF-CDAA), HF-CDAA containing 1000 mg/kg metformin, HF-CDAA containing 10 mg/kg GW7647, and HF-CDAA with both metformin and GW7647 groups. Metformin alone slightly deteriorated the aspartate and alanine aminotransferase (AST/ALT) values, whereas co-treatment with GW7647 and metformin greatly suppressed liver injury and fibrosis via activation of the AMP-activated protein kinase (AMPK) pathway. Further study revealed that co-treatment decreased the expression of inflammatory-, fibrogenesis-, and endoplasmic reticulum (ER) stress-related genes and increased the oxidized nicotinamide adenine dinucleotide (NAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio, suggesting the superiority of co-treatment due to restoration of mitochondrial function. The additive benefits of a PPARα agonist and metformin in a HF-CDAA diet-induced advanced NASH model was firstly demonstrated, possibly through restoration of mitochondrial function and AMPK activation, which finally resulted in suppression of hepatic inflammation, ER stress, then, fibrosis.

We evaluated the effects of NT-702 (parogrelil hydrochloride, NM-702, 4-bromo-6-[3-(4-chlorophenyl) propoxy]-5-[(pyridine-3-ylmethyl) amino] pyridazin-3(2H)-one hydrochloride), a selective phosphodiesterase 3 inhibitor, on the asthmatic response in guinea pigs. NT-702 at a concentration of 1 x 10(-7)M elevated the cyclic adenosine monophosphate content in prostaglandin E(2)-treated guinea pig tracheal smooth muscle cells. Leukotriene (LT) D(4)- and histamine-induced contraction of isolated guinea pig tracheal strips was inhibited by NT-702, with EC(50) values of 3.2 x 10(-7) and 2.5 x 10(-7)M, respectively. In an in vivo study, NT-702 suppressed LTD(4)-induced bronchoconstriction and the ovalbumin-induced immediate asthmatic response in guinea pigs through its bronchodilating effect. Furthermore, NT-702 also suppressed the ovalbumin-induced late asthmatic response, airway hyperresponsiveness, and the accumulation of inflammatory cells in the bronchoalveolar lavage fluid. These results suggest that NT-702 has an anti-inflammatory effect as well as a bronchodilating effect and might be useful as a novel potent therapeutic agent for the treatment of bronchial asthma, a new type of agent with both a bronchodilating and an anti-inflammatory effect.

Heterozygous peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-deficient (PPARgamma(+/-)) mice were protected from high-fat diet-induced insulin resistance. To determine the impact of systemic reduction of PPAR-gamma activity on beta-cell function, we investigated insulin secretion in PPARgamma(+/-) mice on a high-fat diet. Glucose-induced insulin secretion in PPARgamma(+/-) mice was impaired in vitro. The tissue triglyceride (TG) content of the white adipose tissue, skeletal muscle, and liver was decreased in PPARgamma(+/-) mice, but it was unexpectedly increased in the islets, and the increased TG content in the islets was associated with decreased glucose oxidation. Administration of a PPAR-gamma agonist, pioglitazone, reduced the islet TG content in PPARgamma(+/-) mice on a high-fat diet and ameliorated the impaired insulin secretion in vitro. Our results demonstrate that PPAR-gamma protects islets from lipotoxicity by regulating TG partitioning among tissues and that a PPAR-gamma agonist can restore impaired insulin secretion under conditions of islet fat accumulation.

Mice lacking the p85alpha regulatory subunit of phosphoinositide (PI) 3-kinase (Pik3r1(-/-)) showed increased glucose uptake in white adipose tissue (WAT) and skeletal muscle due to increased phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3] production and on a normal diet had a body weight and fat mass similar to wild-type mice. After 3 months on a high-fat diet, Pik3r1(-/-) mice still had increased insulin sensitivity and better glucose tolerance than wild-type mice, but showed markedly greater increases in body weight and WAT mass than wild-type mice. On the normal diet, serum leptin levels of Pik3r1(-/-) mice were significantly higher than in wild-type mice as a result of increased leptin secretion from adipocytes, presumably due to the increased PtdIns(3,4,5)P3 production in adipocytes. Leptin (5 microg/g body wt per day) caused a reduction in food intake and decrease in body weight by the wild-type mice as well as Pik3r1(-/-) mice, suggesting Pik3r1(-/-) mice having leptin sensitivity similar to wild-type mice. The slightly increased serum leptin compensated for the increased glucose uptake by adipocytes in Pik3r1(-/-) mice, thereby preventing adiposity on the normal diet. On the high-fat diet, leptin (5 microg/g body wt per day) failed to decrease food intake or body weight in either genotype, indicating that both genotypes had indeed become severely leptin resistant. Consequently, leptin secretion was unable to sufficiently compensate for the severe leptin resistance caused by the high-fat diet, thereby failing to prevent obesity in Pik3r1(-/-) mice. Our findings suggest that primary increase in serum leptin on the normal diet play a role in the protection from adiposity in Pik3r1(-/-) mice.

Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARgamma and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARgamma by PPARgamma agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARgamma activity by heterozygous PPARgamma deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARgamma deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARgamma deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor alpha, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARgamma deficiency and PPARgamma agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, we established embryonic fibroblast cells with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1 2/2 ), IRS-2 deficient (IRS-2 2/2 ), and IRS-1 IRS-2 double deficient (IRS-1 2/2 IRS-2 2/2 ), from mouse embryos of the corresponding genotypes. The abilities of IRS-1 2/2 cells and IRS-2 2/2 cells to differentiate into adipocytes are approximately 60 and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1 2/2 IRS-2 2/2 cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein a (C/EBPa) and peroxisome proliferator-activated receptor g (PPARg) is severely decreased in IRS-1 2/2 IRS-2 2/2 cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1 2/2 IRS-2 2/2 cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1 2/2 IRS-2 2/2 cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPa and PPARg, a result which is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1 2/2 IRS-2 2/2 double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. Our results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPa and PPARg expression and adipocyte differentiation. Recently there has been a dramatic increase in the prevalence of obesity attributable to excessive white adipose tissue both in Western countries and in Japan. Because adipocytes play a critical role in energy balance, understanding the molecular mechanisms of adipocyte differentiation may provide clues to strategies for the prevention and treatment of obesity. The mechanisms of adipocyte differentiation have been ex

Arachidonic acid (AA) at 0.2 mM enhances glucose uptake through increased levels of glucose transporter (GLUT) 1 protein in 3T3-L1 adipocytes. Since AA is a precursor of prostaglandins (PGs), we investigated the effect of PGs on glucose consumption in 3T3-L1 cells. Among several PGs, only prostaglandin F(2)alpha (PGF(2)alpha) enhanced glucose consumption in 3T3-L1 cells treated with dexamethasone (DEX), 3-isobutyl-1-methyl-xanthine (IBMX), and insulin. To study the mechanism of PGF(2)alpha-enhanced glucose consumption, we investigated the effect of PGF(2)alpha on glycerol-3-phosphate dehydrogenase (GPDH) activity, triglycerides (TGs) content, and the expression of GLUT1 protein. PGF(2)alpha suppressed GPDH activity and did not increase the expression of GLUT1 protein in 3T3-L1 cells treated with DEX, IBMX, and insulin. These results suggest that AA-stimulated glucose uptake is not through the effect of PGF(2)alpha. Our results indicate that PGF(2)alpha is a unique regulator of adipocyte differentiation (suppression) and glucose consumption (enhancement) in 3T3-L1 cells.

Background— The acetylcholine-activated K + current ( I K,ACh ) is a novel candidate for atrial-specific antiarrhythmic therapy. The present study investigates the involvement of I K,ACh in atrial fibrillation (AF) using NTC-801, a novel potent and selective I K,ACh blocker. Methods and Results— The effects of NTC-801, substituted 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro- 2H -benzopyran-3-ol, on I K,ACh and other cardiac ionic currents ( I Na , I CaL , I to , I Kur , I Kr , I Ks , I Kl , I KATP , and I f ) and on atrial and ventricular action potentials were examined in vitro. NTC-801 potently inhibited carbachol-induced I K,ACh in guinea pig atrial cells and the GIRK1/4 current in Xenopus oocytes with IC 50 values of 5.7 and 0.70 nmol/L, respectively. NTC-801 selectively inhibited I K,ACh >1000-fold over other cardiac ionic currents. NTC-801 (10 to 100 nmol/L) reversed the action potential duration (APD 90 ) shortened by carbachol or adenosine in atrial cells, whereas it did not affect APD 90 at 100 nmol/L in ventricular cells. Antiarrhythmic effects of NTC-801 were evaluated in 3 AF models in vivo. NTC-801 significantly prolonged atrial effective refractory period without affecting ventricular effective refractory period under vagal nerve stimulation. NTC-801 dose-dependently converted AF to normal sinus rhythm in both vagal nerve stimulation–induced (0.3 to 3 μg · kg −1 · min −1 IV) and aconitine-induced (0.01 to 0.1 mg/kg IV) models. In a rapid atrial pacing model, NTC-801 (3 μg · kg −1 · min −1 IV) significantly decreased AF inducibility with a prolonged atrial effective refractory period that was frequency-independent. Conclusions— A selective I K,ACh blockade induced by NTC-801 exerted anti-AF effects mediated by atrial-selective effective refractory period prolongation. These findings suggest that I K,ACh may be important in the development and maintenance of AF.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin.

Adiponectin is an anti-diabetic and anti-atherogenic hormone that is exclusively secreted from fat cells. Serum adiponectin levels are reduced in obese patients and obese model mice, despite increased adipose tissue mass. Elucidation of the mechanism(s) by which plasma adiponectin levels are decreased in obese and diabetic patients would provide insight into the cause of obesity-induced diabetes and the development of therapeutic advances. In the present study, the regulation of adiponectin secretion was investigated using 3T3-L1 adipocytes and a diabetic-/obese-mouse model. A novel insulin sensitizer, IkappaB kinase beta (IKKbeta) inhibitor, ameliorated insulin resistance and up-regulated plasma levels of adiponectin without producing a significant change in body weight in KKAy mice that were fed a high-fat diet. The IKKbeta inhibitor cancelled the TNFalpha-mediated down-regulation of adiponectin secretion and simultaneously up-regulated the phosphorylation of Akt in 3T3-L1 adipocytes. Using dominant-negative mutants of Akt or PKClambda (downstream effectors of phosphoinositide 3-kinase), insulin-stimulated Akt activity was found to be important in the regulation of adiponectin secretion by insulin in 3T3-L1 adipocytes. These observations suggest that "insulin-stimulated Akt activity in adipocytes" may play an important role in the regulation of adiponectin secretion.

Adiponectin/Acrp30 is a hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic adipokine. We reported previously that AdipoR1 and -R2 serve as receptors for adiponectin and mediate increased fatty acid oxidation and glucose uptake by adiponectin. In the present study, we examined the expression levels and roles of AdipoR1/R2 in several physiological and pathophysiological states such as fasting/refeeding, obesity, and insulin resistance. Here we show that the expression of AdipoR1/R2 in insulin target organs, such as skeletal muscle and liver, is significantly increased in fasted mice and decreased in refed mice. Insulin deficiency induced by streptozotocin increased and insulin replenishment reduced the expression of AdipoR1/R2 in vivo. Thus, the expression of AdipoR1/R2 appears to be inversely correlated with plasma insulin levels in vivo. Interestingly, the incubation of hepatocytes or myocytes with insulin reduced the expression of AdipoR1/R2 via the phosphoinositide 3-kinase/Foxo1-dependent pathway in vitro. Moreover, the expressions of AdipoR1/R2 in ob/ob mice were significantly decreased in skeletal muscle and adipose tissue, which was correlated with decreased adiponectin binding to membrane fractions of skeletal muscle and decreased AMP kinase activation by adiponectin. This adiponectin resistance in turn may play a role in worsening insulin resistance in ob/ob mice. In conclusion, the expression of AdipoR1/R2 appears to be inversely regulated by insulin in physiological and pathophysiological states such as fasting/refeeding, insulin deficiency, and hyper-insulinemia models via the insulin/phosphoinositide 3-kinase/Foxo1 pathway and is correlated with adiponectin sensitivity.

We previously reported that insulin receptor substrate-2 (IRS-2)-deficient mice develop diabetes as a result of insulin resistance in the liver and failure of beta-cell hyperplasia. In this study we introduced the IRS-2 gene specifically into the liver of Irs2(-/-) mice with adenovirus vectors. Glucose tolerance tests revealed that the IRS-2 restoration in the liver ameliorated the hyperglycemia, but the improvement in hyperinsulinemia was only partial. Endogenous glucose production (EGP) and the rate of glucose disappearance (Rd) were measured during hyperinsulinemic-euglycemic clamp studies: EGP was increased 2-fold in the Irs2(-/-) mice, while Rd decreased by 50%. Restoration of IRS-2 in the liver suppressed EGP to a level similar to that in wild-type mice, but Rd remained decreased in the Adeno-IRS-2-infected Irs2(-/-) mice. Irs2(-/-) mice also exhibit obesity and hyperleptinemia associated with impairment of hypothalamic phosphatidylinositol 3-kinase activation. Continuous intracerebroventricular leptin infusion or caloric restriction yielded Irs2(-/-) mice whose adiposity was comparable to that of Irs2(+/+) mice, and both the hyperglycemia and the hyperinsulinemia of these mice improved with increased Rd albeit partially. Finally combination treatment consisting of adenovirus-mediated gene transfer of IRS-2 and continuous intracerebroventricular leptin infusion completely reversed the hyperglycemia and hyperinsulinemia in Irs2(-/-) mice. EGP and Rd also became normal in these mice as well as in mice treated by caloric restriction plus adenoviral gene transfer. We therefore concluded that a combination of increased EGP due to insulin signaling defects in the liver and reduced Rd due to obesity accounts for the systemic insulin resistance in Irs2(-/-) mice.

Obesity, a state of increased adipose tissue mass, is a major cause for type 2 diabetes, hyperlipidemia, and hypertension, resulting in clustering of risk factors for atherosclerosis. Heterozygous PPARgamma knockout mice and KKA(y) mice administered with a PPARgamma antagonist were protected from high-fat diet-induced adipocyte hypertrophy and insulin resistance. Moderate reduction of PPARgamma activity prevented adipocyte hypertrophy, thereby diminution of TNFalpha, resistin, and FFA and upregulation of adiponectin and leptin. These alterations led to reduction of tissue TG content in muscle/liver, thereby ameliorating insulin resistance. Insulin resistance in the lipoatrophic mice and KKA(y) mice were ameliorated by replenishment of adiponectin. Moreover, adiponectin transgenic mice ameliorated insulin resistance and diabetes, but not the obesity of ob/ob mice. Furthermore, targeted disruption of the adiponectin gene caused moderate insulin resistance and glucose intolerance. In muscle, adiponectin activated AMP kinase and PPARgamma pathways, thereby increasing beta-oxidation of lipids, leading to decreased TG content, which ameliorated muscle insulin resistance. In the liver, adiponectin also activated AMPK, thereby downregulating PEPCK and G6Pase, leading to decreased glucose output from the liver. In conclusion, PPARgamma plays a central role in the regulation of adipocyte hypertrophy and insulin sensitivity. The upregulation of the adiponectin pathway by PPARgamma may play a role in the increased insulin sensitivity of heterozygous PPARgamma knockout mice, and activation of adiponectin pathway may provide novel therapeutic strategies for obesity-linked disorders such as type 2 diabetes and metabolic syndrome.

Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states.

Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-alpha. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-alpha ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.

The CBP protein (cAMP response element binding protein (CREB) binding protein) is a co-activator for several transcription factors with a wide range of important biological functions, such as sterol regulatory element binding proteins (SREBPs), CCAAT/enhancer-binding proteins (C/EBPs), nuclear receptors (including peroxisome proliferator-activated receptors, PPARs), and signal transducers and activators of transcription (STATs). In contrast to these individual transcription factors, the biological roles of CBP are poorly understood. CBP enhances transcriptional activities via histone acetylation and the recruitment of additional co-activators such as SRC (steroid coactivator)-1 (ref. 9). To identify its physiological functions using a loss-of-function mutant, we analyzed CBP-deficient mice. As Crebbp null mice (Crebbp-/-) died during embryogenesis, we used Crebbp+/- mice. Unexpectedly, Crebbp+/- mice showed markedly reduced weight of white adipose tissue (WAT) but not of other tissues. Despite this lipodystrophy, Crebbp+/- mice showed increased insulin sensitivity and glucose tolerance and were completely protected from body weight gain induced by a high-fat (HF) diet. We observed increased leptin sensitivity and increased serum adiponectin levels in Crebbp+/- mice. These increased effects of insulin-sensitizing hormones secreted from WAT may explain, at least in part, the phenotypes of Crebbp+/- mice. This study demonstrates that CBP may function as a 'master-switch' between energy storage and expenditure.

Insulin receptor substrate (IRS)-2(-/-) mice develop diabetes because of insulin resistance in the liver and failure to undergo beta-cell hyperplasia. Here we show by DNA chip microarray analysis that expression of the sterol regulatory element-binding protein (SREBP)-1 gene, a downstream target of insulin, was paradoxically increased in 16-week-old IRS-2(-/-) mouse liver, where insulin-mediated intracellular signaling events were substantially attenuated. The expression of SREBP-1 downstream genes, such as the spot 14, ATP citrate-lyase, and fatty acid synthase genes, was also increased. Increased liver triglyceride content in IRS-2(-/-) mice assures the physiological importance of SREBP-1 gene induction. IRS-2(-/-) mice showed leptin resistance; low dose leptin administration, enough to reduce food intake and body weight in wild-type mice, failed to do so in IRS-2(-/-) mice. Interestingly, high dose leptin administration reduced SREBP-1 expression in IRS-2(-/-) mouse liver. Thus, IRS-2 gene disruption results in leptin resistance, causing an SREBP-1 gene induction, obesity, fatty liver, and diabetes.

Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.

Nature 423, 762–769 (2003). In this Letter, Fig. 1 is an illustration of the sorting procedure, rather than an original data set, which we did not explicitly describe. Because the x-axis was used for FACS analysis of both FITC- and PE-labelled cells, only the gated population was shown; these data were extracted from the analyses and inserted into a FACS profile.