Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin.

Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, 113-8655, Japan.
Journal of Biological Chemistry (Impact Factor: 4.65). 11/2003; 278(41):40352-63. DOI: 10.1074/jbc.M300365200
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo.
    Biochemical and Biophysical Research Communications 04/2014; · 2.41 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The interest in adiponutrin stems from adiponutrin variant I148M, which is strongly associated to non-alcoholic fatty liver disease. Adiponutrin has to date been considered to be solely an intracellular protein, with a role in lipid metabolism in liver and adipose tissue. However, a physiologically relevant role for adiponutrin has not been found. The aim of this study was to investigate the presence of adiponutrin in human plasma, a new facet of adiponutrin research. We demonstrate that adiponutrin is present in plasma as disulfide-bond dependent multimers, estimated to circulate at a concentration of 1.25-4 nM. Experiments reveal that adiponutrin is released from HepG2 cells in the presence of oleate. The presence of adiponutrin in plasma makes it accessible for clinical investigations and use as a potential biomarker for metabolic disease.
    Biochemical and Biophysical Research Communications 03/2014; · 2.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Adiponectin (APN), the most abundant adipocyte-secreted adipokine regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but still not clearly understood. In the present study, we found APN knockout (APN-KO) mice fed on a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared to wild-type (WT) mice. APN intracerebroventricular (ICV) infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), epinephrine and norepinephrine serum levels, and osteoclast numbers, while increasing osteoblast osteogenic markers expression and trabecular bone mass in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART) and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions, but decreased hypothalamic cannabinoid receptor (CB)-1 expression. Treatment of immortalized mouse neurons with APN, demonstrated that APN-mediated effects on TPH2, CART and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation and promote osteoblastic commitment of BMSC.
    AJP Endocrinology and Metabolism 04/2014; · 4.51 Impact Factor