Ablation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vascular endothelial cells enhances insulin sensitivity by reducing visceral fat and suppressing angiogenesis.

The phosphatidylinositol 3-kinase signaling pathway in vascular endothelial cells is important for systemic angiogenesis and glucose metabolism. In this study, we addressed the precise role of the 3-phosphoinositide-dependent protein kinase 1 (PDK1)-regulated signaling network in endothelial cells in vivo, using vascular endothelial PDK1 knockout (VEPDK1KO) mice. Surprisingly, VEPDK1KO mice manifested enhanced glucose tolerance and whole-body insulin sensitivity due to suppression of their hepatic glucose production with no change in either peripheral glucose disposal or even impaired vascular endothelial function at 6 months of age. When mice were fed a standard diet at 6 months of age and a high-fat diet at 3 months of age, hypertrophy of epididymal adipose tissues was inhibited, adiponectin mRNA was significantly increased, and mRNA of MCP1, leptin, and TNFα was decreased in the white adipose tissue of VEPDK1KO mice in comparison with controls. Consequently, both the circulating adiponectin levels and the activity of hepatic AMP-activated protein kinase were significantly increased, subsequently enhancing whole-body insulin sensitivity and energy expenditure with increased hepatic fatty acid oxidation in VEPDK1KO mice. These results provide the first in vivo evidence that lowered angiogenesis through the deletion of PDK1 signaling not only interferes with the growth of adipose tissue but also induces increased energy expenditure due to amelioration of the adipocytokine profile. This demonstrates an unexpected role of PDK1 signaling in endothelial cells on the maintenance of proper glucose homeostasis through the regulation of adipocyte development.

Overexpression of KLF15 transcription factor in adipocytes of mice results in down-regulation of SCD1 protein expression in adipocytes and consequent enhancement of glucose-induced insulin secretion.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic ß cells mediated through modulation of oxidative stress.

Diet-induced up-regulation of gene expression in adipocytes without changes in DNA methylation.

The expansion of white adipose tissue (WAT) mass during the development of obesity is mediated in part through an increase in adipocyte size. Although gene expression profiles associated with adipogenesis in vitro and the development of obesity in vivo have been characterized by DNA microarray analysis, the role of chromatin and chromatin-modifying proteins in the regulation of gene expression related to adipocyte hypertrophy has remained unclear. We have now shown that maintenance of C57BL/6J mice on a high-fat diet for 16 weeks resulted in marked up-regulation of the expression of leptin, Mest (mesoderm specific transcript; also known as paternally expressed gene 1, or Peg1), and sFRP5 (secreted frizzled-related protein 5) genes in WAT. Furthermore, the demethylating agent 5-aza-2'-deoxycytidine increased the amount of Mest/Peg1 mRNA, but not that of leptin or sFRP5 mRNAs, in mouse 3T3-L1 adipocytes. However, analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that maintenance of mice on a high-fat diet for various times did not affect the level of methylation at specific CpG sites in the promoter regions of leptin, Mest/Peg1, and sFRP5 genes in WAT. Our results indicate that the diet-induced up-regulation of leptin, Mest/Peg1, and sFRP5 gene expression in WAT during the development of obesity in mice is not mediated directly by changes in DNA methylation.

The Krüppel-like factor KLF15 inhibits transcription of the adrenomedullin gene in adipocytes.

KLF15 (Krüppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides -70 and -29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue.