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 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 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.

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 hyperinsulinemia models via the insulin/phosphoinositide 3-kinase/Foxo1 pathway and is correlated with adiponectin sensitivity.Adiponectin/Acrp30 (1-4) is a hormone secreted by adipocytes, which acts as an antidiabetic (5-12) and antiatherogenic (8, 12, 13) adipokine. This insulin-sensitizing effect of adiponectin appears to be mediated by an increase in fatty acid oxidation via activation of the 5Ј-AMP-activated protein kinase (AMPK) 1 (10, 11) and peroxisome proliferator-activated receptor-␣ (5, 6, 12). Very recently, we have reported the cloning of complementary DNAs encoding adiponectin receptors AdipoR1 and -R2 by expression cloning (14). AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. AdipoR1 and -R2 are predicted to contain seven transmembrane domains (14) but to be structurally and functionally distinct from G-protein-coupled receptors (15-17). AdipoR1 and -R2 serve as receptors for globular and full-length adiponectin and mediate increased AMPK (10, 11), peroxisome proliferator-activated receptor-␣ ligand activities (12), and fatty acid oxidation and glucose uptake by adiponectin (14).It has not yet been determined whether the expressions of AdipoR1 and -R2 are altered in physiological and pathophysiological states. To address these questions, we first studied the expressions of AdipoR1 and -R2 during fasting and refeeding. We also analyzed the expressions of Ad...

We examined the effects of activation of peroxisome proliferator-activated receptor (PPAR)␣, PPAR␥, and both of them in combination in obese diabetic KKAy mice and investigated the mechanisms by which they improve insulin sensitivity. PPAR␣ activation by its agonist, Wy-14,643, as

We previously reported that brain-derived neurotrophic factor (BDNF) regulates both food intake and blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-lepr N eurotrophins are important regulators in the embryogenesis, development, and functioning of nervous systems (1,2). At present, four neurotrophins are known: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, and NT-4 / N T-5 (3-6) in mammals. BDNF, discovered long after NGF, enhances the survival and differentiation of several classes of neurons in the central and peripheral nervous systems, including motoneurons and sensory neurons. BDNF and its receptor, TrkB, are widely expressed in a variety of neurons through the embryonic, postnatal, and adult stages (7-10). These results and the many activities of BDNF described in in vitro cell cultures and lesioned animal studies (11,12) indicate that BDNF is likely to have multiple functions.In addition to the efficacy of BDNF in neurological disorders, we previously found that BDNF reduces food intake and blood glucose concentration in rodent obese diabetic models, such as C57BL/KsJ-d b/d b mice (13,14). To eliminate the effect of reduced food intake on the regulation of glucose metabolism, we evaluated the hypoglycemic effect of BDNF in d b/d b mice using the conventional pair-feeding protocol in which the amount of food provided to each pair-fed mouse was the same as the average amount of food eaten by the BDNF-treated mice during the preceding 24-h period (13,14). H o w e v e r, because such hyperphagic diabetic mice given a vehicle ate all of the food over a period of several hours, leaving them in a fasting condition until the next feeding, this protocol was inappropriate to study the effect of antidiabetic agents on glucose metabolism by mechanisms other than appetite alteration.In this study, to overcome these drawbacks, we designed a novel pellet pair-feeding apparatus and evaluated the effect of Received for publication 28 April 1999 and accepted in revised form 1 8 November 1999. B D N F, brain-derived neurotrophic factor; ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor; NT, neurotrophin; PBS, phosphate-buffered saline; PPA R-, peroxisome proliferator-a c t i v a t e d r e c e p t o r-; STZ, streptozotocin.

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.

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