Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions.

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.

Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake.

Adiponectin has been shown to stimulate fatty acid oxidation and enhance insulin sensitivity through the activation of AMP-activated protein kinase (AMPK) in the peripheral tissues. The effects of adiponectin in the central nervous system, however, are still poorly understood. Here, we show that adiponectin enhances AMPK activity in the arcuate hypothalamus (ARH) via its receptor AdipoR1 to stimulate food intake; this stimulation of food intake by adiponectin was attenuated by dominant-negative AMPK expression in the ARH. Moreover, adiponectin also decreased energy expenditure. Adiponectin-deficient mice showed decreased AMPK phosphorylation in the ARH, decreased food intake, and increased energy expenditure, exhibiting resistance to high-fat-diet-induced obesity. Serum and cerebrospinal fluid levels of adiponectin and expression of AdipoR1 in the ARH were increased during fasting and decreased after refeeding. We conclude that adiponectin stimulates food intake and decreases energy expenditure during fasting through its effects in the central nervous system.

Development of a new class of benzoylpyrrole-based PPARα/γ activators.

Starting with a subtle blood glucose-lowering effect of a TGF-ß inhibitor, we designed and synthesized a series of benzoylpyrrole-based carboxylic acids as PPARs activators. Among these compounds, 10sNa exhibited favorable blood glucose-lowering effect without body weight gain. We assume that the beneficial effect of 10sNa is attributed to not only its compound PPARα agonistic activity but also its PPARγ partial agonistic activity.

Analysis of extracellular vesicles as a potential index for monitoring differentiation of neural lineage cells from induced pluripotent stem cells.

To improve cell production efficacy, it is important to evaluate cell conditions during culture. Extracellular vesicles (EVs) secreted from various cells are involved in stem cell differentiation. As EVs carry information about their source cells, we hypothesized that they may serve as a noninvasive index of cell conditions. We evaluated changes in EV morphology, concentration, and microRNA (miRNA) and protein expression in culture supernatants during the differentiation of induced pluripotent stem cells (iPSCs) into neural lineage cells, for application in regenerative medicine for Parkinson's disease. We observed EVs (50-150 nm) in culture supernatants of iPSCs and differentiated cells. The EVs expressed the exosome markers CD63, CD81, and CD9. Throughout differentiation, the EV concentration in the supernatants decreased, and EV miRNA and protein expression changed substantially. Especially, miR-106b, involved in neural stem cell differentiation and normal brain development, was considerably downregulated. CD63 expression correlated with the CORIN-positive cell rate, which is an index of differentiation. Thus, EV concentration and miRNA and protein expression may reflect the differentiation status of iPSCs. These findings pave the way for the development of novel and sensitive cell culture monitoring methods.

Peroxisome proliferator-activated receptor gamma agonist rosiglitazone increases expression of very low density lipoprotein receptor gene in adipocytes.

Apolipoprotein E (apoE) and its receptor, very low density lipoprotein receptor (VLDLR), are involved in fat accumulation in adipocytes. Here, we investigated the effect of a peroxisome proliferator-activated receptor (PPAR) gamma agonist, rosiglitazone, on regulation of VLDLR expression both in white adipose tissue (WAT) of obese mice and in cultured adipocytes. Furthermore, to determine whether rosiglitazone directly regulates transcription of the VLDLR gene, we carried out luciferase assay with a reporter gene containing mouse VLDLR promoter region, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Four-day treatment with rosiglitazone increased the expression of VLDLR in WAT of ob/ob mice. Moreover, rosiglitazone increased the expression of VLDLR in cultured adipocytes. The PPAR-responsive element (PPRE)-directed mutagenesis analyses revealed that the PPRE motif in the VLDLR promoter region plays a significant role in transcriptional activation of the VLDLR gene in adipocytes. In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that endogenous PPARgamma directly binds to this functional PPRE motif in the VLDLR promoter region. We also investigated the effects of rosiglitazone on insulin sensitivity and lipid accumulation in both ob/ob mice and apoE-deficient ob/ob mice. Rosiglitazone ameliorated insulin sensitivity in both ob/ob mice and apoE-deficient ob/ob mice, possibly through decreasing the expression of monocyte chemoattractant protein-1 (MCP-1), increasing the expression of superoxide dismutase 1 (SOD1) in WAT, and increasing plasma adiponectin concentration. In ob/ob mice, body weight and WAT weight were significantly higher in the mice treated with rosiglitazone than those treated with vehicle. However, in apoE-deficient ob/ob mice, no significant difference in body weight or WAT weight was observed between the vehicle-treated group and the rosiglitazone-treated group. Moreover, rosiglitazone did not increase body weight and WAT weight in VLDLR-deficient mice. These findings indicate that rosiglitazone directly increases VLDLR expression, thereby enhancing apoE-VLDLR-dependent lipid accumulation in adipocytes.

Intermittent administration of brain-derived neurotrophic factor (BDNF) ameliorates glucose metabolism and prevents pancreatic exhaustion in diabetic mice.

We previously demonstrated that repetitive administration of brain-derived neurotrophic factor (BDNF) ameliorates glucose metabolism and energy expenditure in obese diabetic db/db mice. However, we have not evaluated in detail the effect of single or intermittent BDNF administration on glucose metabolism in a diabetic animal model. The objectives of this study were to examine the dose-response effect and dosing interval of BDNF administration in db/db mice and to evaluate the effect of intermittent BDNF administration on pancreatic function in db/db mice. We evaluated the dose-response effect of BDNF by single administration in db/db mice. First, single administration of BDNF greater than 70 mg/kg significantly reduced blood glucose concentration one day after administered, and the BDNF effect was maintained for 6 d. Next, the effects of BDNF administered twice a week at 4, 10, 25, and 62.5 mg/kg on blood glucose concentration, and the effects of BDNF administered once a week at 10, 20, 30, 50, and 70 mg/kg on blood glucose concentration were examined in db/db mice. In the intermittent treatment studies, BDNF dose-dependently ameliorated glucose metabolism by not only the twice-a-week administration but also the once-a-week administration. Lastly, because BDNF reduces the food intake of obese hyperphagic diabetic mice, the effects of BDNF administered once or twice a week on the blood glucose concentration and plasma and pancreatic insulin concentrations in db/db mice were compared with those of the vehicle under pair-fed conditions. Under pair-fed conditions, the intermittent administration of BDNF (25 mg/kg, twice a week, or 50 mg/kg, once a week) significantly reduced the blood glucose concentration and increased the plasma and pancreatic insulin concentrations compared with those in the pair-fed vehicle-treated db/db mice. This indicates that the prolonged hypoglycemic effect of BDNF is not simply due to the reduction of food intake. In conclusion, we demonstrated that the intermittent administration of BDNF ameliorates glucose metabolism and prevents pancreatic exhaustion in obese diabetic mice. These findings indicate that BDNF may have potential as a unique hypoglycemic agent for the treatment of diabetes at a fundamental level with good patient compliance.

Peroxisome proliferator-activated receptor (PPAR)alpha activation increases adiponectin receptors and reduces obesity-related inflammation in adipose tissue: comparison of activation of PPARalpha, PPARgamma, and their combination.

We examined the effects of activation of peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, and both of them in combination in obese diabetic KKAy mice and investigated the mechanisms by which they improve insulin sensitivity. PPARalpha activation by its agonist, Wy-14,643, as well as PPARgamma activation by its agonist, rosiglitazone, markedly improved insulin sensitivity. Interestingly, dual activation of PPARalpha and -gamma by a combination of Wy-14,643 and rosiglitazone showed increased efficacy. Adipocyte size in Wy-14,643-treated KKAy mice was much smaller than that of vehicle- or rosiglitazone-treated mice, suggesting that activation of PPARalpha prevents adipocyte hypertrophy. Moreover, Wy-14,643 treatment reduced inflammation and the expression of macrophage-specific genes in white adipose tissue (WAT). Importantly, Wy-14,643 treatment upregulated expression of the adiponectin receptor (AdipoR)-1 and AdipoR2 in WAT, which was decreased in WAT of KKAy mice compared with that in nondiabetic control mice. Furthermore, Wy-14,643 directly increased expression of AdipoRs and decreased monocyte chemoattractant protein-1 expression in adipocytes and macrophages. Rosiglitazone increased serum adiponectin concentrations and the ratio of high molecular weight multimers of adiponectin to total adiponectin. A combination of rosiglitazone and Wy-14,643 increased both serum adiponectin concentrations and AdipoR expression in WAT. These data suggest that PPARalpha activation prevents inflammation in WAT and that dual activation of PPARalpha and -gamma enhances the action of adiponectin by increasing both adiponectin and AdipoRs, which can result in the amelioration of obesity-induced insulin resistance.

Protective effect of brain-derived neurotrophic factor on pancreatic islets in obese diabetic mice.

We have previously demonstrated that brain-derived neurotrophic factor (BDNF) ameliorates glucose metabolism and energy expenditure in obese diabetic db/db mice. In the present study, the effect of BDNF treatment on pancreatic islets of db/db mice was examined, using vehicle-treated pair-fed db/db mice as controls. Brain-derived neurotrophic factor (10 mg/kg) or vehicle was subcutaneously administered to male db/db mice for 4 weeks. The food intake of vehicle-treated db/db mice was restricted and precisely synchronized with that of BDNF-treated db/db mice using a pellet pair-feeding apparatus because BDNF decreases food intake in hyperphagic mice. Repetitive administration of BDNF significantly lowered the blood glucose concentration compared with pair-fed vehicle-treated db/db mice. The pancreatic insulin and glucagon concentrations were measured in db/db mice to evaluate the effect of BDNF on the pancreas. Although the insulin concentration in the pancreas of pair-fed vehicle-treated db/db mice was lower than in nondiabetic control +m/+m mice, it was higher in BDNF-treated db/db mice than in vehicle-treated pair-fed db/db mice and comparable to the concentration in +m/+m mice. The glucagon concentration in the pancreas of vehicle-treated pair-fed db/db mice was higher than in +m/+m mice, and BDNF partially decreased the glucagon concentration in the pancreas of db/db mice compared with vehicle. Histologic analyses of pancreatic sections were performed to characterize the mechanism through which BDNF modulates the hormonal concentration in the pancreas of db/db mice. Although there were no significant differences in the number and total area of islets between the BDNF- and vehicle-treated groups, immunostaining with an anti-insulin antibody indicated that the islet beta-cell area in BDNF-treated db/db mice was larger than that in vehicle-treated pair-fed db/db mice. Furthermore, immunostaining with an antiglucagon antibody indicated that BDNF normalized the delocalization of non-beta cells in islets of db/db mice. Electron microscopic images of beta cells indicated a decrease in secretory granules in vehicle-treated pair-fed db/db mice; this change was reversed in BDNF-treated db/db mice and reached a level comparable to that found in +m/+m mice. These findings suggest that BDNF prevents exhaustion of the pancreas in diabetic mice by maintaining the histologic cellular organization of beta cells and non-beta cells in pancreatic islets and restoring the level of insulin-secreting granules in beta cells.

Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1.

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.

Brain-derived neurotrophic factor (BDNF) prevents the development of diabetes in prediabetic mice.

We previously reported that peripheral injection of brain-derived neurotrophic factor (BDNF) exhibits hypophagic and hypoglycemic effects in obese hyperglycemic animals, indicating its antiobesity and antidiabetic effects. Since previous studies were focused on the effect of BDNF on overt diabetic animals with severe hyperglycemia, there was no evidence whether BDNF is effective or not for the development of diabetes in prediabetic animal models. Therefore, we evaluated the effect of BDNF on preventing the development of diabetes in db/db mice. First, we characterized age-related changes in the pathophysiology of diabetes in db/db mice. We chose 8 week-old db/db mice as the early diabetic stage (early intervention study) and 4 week-old db/db mice as the prediabetic stage (prevention study). Next, we examined the effects of BDNF on the progression of diabetes in early diabetic db/db mice. In the early intervention study using 8 week-old db/db mice, intermittent treatment with BDNF prevented the deterioration in hyperglycemia. Lastly, we examined the preventive effects of BDNF on the development of diabetes in prediabetic db/db mice. In the prevention study using 4 week-old db/db mice, treatment with BDNF prevented the age-related increase in blood glucose concentration. These results showed for the first time that BDNF prevents the development of diabetes in prediabetic db/db mice.

Selective purification and characterization of adiponectin multimer species from human plasma.

Adiponectin is an adipocyte-derived hormone and known to form several species of multimer, however, the precise components of each multimer have not been fully determined. We purified each multimer adiponectin selectively from human plasma and characterized them by affinity columns using anti-adiponectin, gelatin, or anti-albumin antibody and gel filtration. We found that adiponectin exists as four species of multimers in human plasma. According to their migrating mobility and N-terminal amino acid analysis, we defined them as a trimer, albumin-binding trimer, hexamer, and HMW. Low pH shifted HMW to hexamer, raising the possibility that HMW is a 12 mer or larger multimer. We also showed that HMW had the highest binding activity to the membrane fractions of C2C12 myocytes and activated AMPK most potently. Our results indicate that adiponectin forms diverse multimer species and at least some of the functional properties are dependent on a multimer status.

Nuclear receptors as targets for drug development: molecular mechanisms for regulation of obesity and insulin resistance by peroxisome proliferator-activated receptor gamma, CREB-binding protein, and adiponectin.

Obesity is defined as increased mass of adipose tissue, conferring a higher risk of cardiovascular and metabolic disorders such as diabetes, hyperlipidemia, and coronary heart disease. To investigate the role of transcriptional factors, which are involved in adipocytes differentiation and adiposity, we have generated peroxisome proliferator-activated receptor (PPAR) gamma or CREB-binding protein (CBP)-deficient mice by gene targeting. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Heterozygous CBP-deficient mice showed increased insulin sensitivity and were completely protected from body weight gain induced by a high-fat diet. PPARgamma or CBP deficiency results in increased effects of hormones such as adiponectin and leptin. Adiponectin was decreased in obesity and lipoatrophy, and replenishment of adiponectin ameliorated insulin resistance. Moreover, adiponectin-deficient mice showed insulin resistance and atherogenic phenotype. Finally, cDNA encoding adiponectin receptors (AdipoR1/R2) have been identified by expression cloning. The expression of AdipoR1/R2 appears to be inversely regulated by insulin in physiological and pathophysiological states such as fasting/refeeding, insulin deficiency, and hyperinsulinemia models, and it is correlated with adiponectin sensitivity. These results facilitate the understanding of molecular mechanisms of adiponectin actions and obesity-linked diseases such as diabetes and atherosclerosis and propose the molecular targets for anti-diabetic and anti-atherogenic drugs.

Hepatocyte growth factor protects functional and histological disorders of HgCl(2)-induced acute renal failure mice.

Hepatocyte growth factor (HGF) enhances proliferation of renal epithelial cells as well as hepatocytes. HGF accelerates recovery from acute renal failure (ARF) in animal models. However, pharmacological profiles of HGF including its action mechanism has not been studied in detail. An HgCl(2)-induced ARF mouse was used in this study to evaluate the efficacy of HGF. Single administrations of recombinant human HGF or vehicle were given to ARF mice 30 min after HgCl(2) injection. Renal function was monitored by measuring serum creatinine, blood urea nitrogen and creatinine clearance. In the ARF mice, there was a deterioration of renal function biochemical parameters and histological evidence of renal damage including acute tubular necrosis of proximal tubules. These were both significantly ameliorated by a single HGF administration. The effect of HGF was noticeable in the early phase of ARF (1 day after onset) when there was no histological evidence of increased labeling indexes in renal tubular epithelial cells. Western blot analysis of the c-Met/HGF receptor showed that tyrosine phosphorylation was enhanced immediately after HGF administration indicating direct activation of renal epithelial cells. HGF prevented increase of apoptotic nuclei with DNA fragmentation in renal epithelial cells which suggests cytoprotective activity of HGF on renal epithelial cells in the ARF mice.

Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity.

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.

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects.

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.