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 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/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

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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Background: Human serum adiponectin exists in 3 multimer forms: high molecular weight (HMW), middle molecular weight, and low molecular weight (LMW), with some of the latter bound to albumin (Alb)-LMW. Some studies have suggested that adiponectin crosses the blood-brain barrier and plays a central role in energy homeostasis. Methods: To determine cerebrospinal fluid (CSF) adiponectin at extremely low concentrations, we modified the protocol of the ELISA system used to assay serum adiponectin. The 3 multimers of adiponectin were measured separately by pretreating CSF with 2 proteases. We measured the CSF adiponectin concentrations in anonymous human samples (n ‫؍‬ 19). The molecular sizes of adiponectin in CSF pretreated with proteases or untreated were determined by use of native PAGE and immunoblotting. Results: The ELISA system measured adiponectin in the range of 1.0 -167 g/L. The between-assay imprecision estimates (CVs) were 6%-17% for the 3 forms. The mean total CSF adiponectin concentration (7.2 g/L) was ϳ1/ 1000 of the mean concentration in serum. Unlike serum adiponectin, the LMW and Alb-LMW forms predominated in all of the CSF samples. Immunoblotting analysis revealed that most LMW forms were bound to Alb, although the HMW form was detected in some samples. Conclusions: The modified ELISA system measures the 3 multimers separately and is sufficiently sensitive to measure adiponectin in CSF. © 2007 American Association for Clinical ChemistryAdiponectin is an adipocyte-derived adipokine (1 ) with multiple functions, including antidiabetic (2 ), antiatherogenic (3 ), and antiinflammatory actions. Although the adiponectin target organs are the liver, muscles, and blood vessels, some studies have suggested that adiponectin has central effects on energy homeostasis. The intracerebroventricular administration of adiponectin in normal mice led to dose-dependent decreases in the body weight without substantial inhibition of food intake. Furthermore, intravenous adiponectin injection induced a Ͼ3-fold increase in cerebrospinal fluid (CSF) adiponectin concentration (4 ). In contrast, a study of humans revealed that the adiponectin concentration in CSF made up only 0.1% of the serum adiponectin concentration, suggesting that adiponectin does not cross the blood-brain barrier (5 ). Hence, controversy remains as to whether adiponectin can cross the blood-brain barrier under physiological conditions.Adiponectin in human blood exists as 3 multimers with distinct molecular sizes: trimeric low molecular weight (LMW), hexameric middle molecular weight (MMW), and high molecular weight (HMW) forms (6 ). Some of the LMW adiponectin exists as albumin (Alb)-bound forms (Alb-LMW) (7 ). The molecular weights of adiponectin affect the strength of their metabolic actions. Several studies have suggested that HMW adiponectin and the ratio of HMW adiponectin to total adiponectin are more closely associated with insulin sensitivity and metabolic syndrome than is total adiponectin (8, 9 ). ELISAs for measuring the 3 multimers separate...