Hironori Waki

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.

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.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

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.

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

Corrigendum (2004)10.1038/nature03091Adiponectin (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-α. 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-α ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.

Impaired Multimerization of Human Adiponectin Mutants Associated with Diabetes MOLECULAR STRUCTURE AND MULTIMER FORMATION OF ADIPONECTIN

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 and AdipoR1 regulate PGC-1α and mitochondria by Ca2+ and AMPK/SIRT1

Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca2+ influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca2+ concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1α expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.

Inhibition of RXR and PPARγ ameliorates diet-induced obesity and type 2 diabetes

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 leptins 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.

NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer.

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation.

TLE3 Is a Dual-Function Transcriptional Coregulator of Adipogenesis

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.