High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

C57BL/6J (B6) and AKR/J (AKR) inbred strains of mice develop a comparable degree of obesity when fed a high-fat diet. However, although obese B6 mice are more glucose intolerant, obese AKR mice are more insulin resistant. To understand the basis for these strain differences, we characterized features of adiposity and glucose homeostasis in mice fed a high-fat diet for 8 weeks. The results indicated that despite hyperglycemia and impaired glucose tolerance, B6 mice have lower plasma insulin and are more insulin sensitive than AKR mice. Compared with adipose tissue of AKR mice, adipose tissue of B6 mice contained about threefold higher levels of total membrane-bound GLUT4 protein, whereas in skeletal muscle the levels were similar. Uptake of 2-[14 C]deoxyglucose in vivo was reduced by a high-fat diet in adipose tissue, but not in skeletal muscle. Surprisingly, no significant differences in uptake occurred between the strains, despite the differences in GLUT4; however, glucose flux was calculated to be slightly higher in B6 mice. Higher expression of PEPCK in the liver of B6 mice, under both standard-diet and high-fat-diet conditions, suggests a plausible mechanism for elevated glycemia in these mice. In conclusion, phenotypic variation in insulin resistance and glucose production in the B6 and AKR strains could provide a genetic system for the identification of genes controlling glucose homeostasis. Diabetes

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1␣, Ppar␣, and type 2 deiodinase. Independent validation that PPAR␣ is associated with Ucp1 induction was obtained by treating mice with the PPAR␣ agonist clofibrate, but not from the analysis of PPAR␣ knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPAR␥, PPAR␣, PGC-1␣, and type 2 deiodinase. However, compared to Ucp1 expression, the two-to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

The sodium/myo-inositol cotransporter is a plasma membrane protein responsible for concentrative cellular accumulation of myo-inositol in a variety of tissues. When cells in kidney and brain are exposed to a hyperosmolar salt condition (hypertonicity) due to the operation of urinary concentration mechanism and pathological conditions, respectively, they survive the stress of hypertonicity by raising the cellular concentration of myo-inositol. Transcription of the sodium/myo-inositol cotransporter gene is markedly stimulated in response to hypertonicity, leading to an increase in the activity of the cotransporter, which in turn drives the osmoprotective accumulation of myo-inositol. To understand the molecular mechanisms by which hypertonicity stimulates transcription, we analyzed the 5-flanking region of the cotransporter gene for cis-acting regulatory sequences. We identified five tonicity-responsive enhancers that are scattered over 50 kilobase pairs. All the enhancers are variations of the same type of enhancer interacting with the transcription factor named tonicity-responsive enhancer binding protein. In vivo methylation experiments demonstrated that exposure of cells to hypertonicity increases the binding of tonicity-responsive enhancer binding protein to the enhancer sites, indicating that all of these enhancers are involved in the transcriptional stimulation. We conclude that the sodium/myo-inositol cotransporter gene is regulated by a large region (ϳ50 kilobase pairs) upstream of the gene.The sodium/myo-inositol cotransporter (SMIT) 1 is a plasma membrane protein catalyzing concentrative uptake of myo-inositol (MI) using the electrochemical gradient of sodium across the cell membrane (1). Since two sodium ions are coupled per molecule of MI (2), SMIT can transport MI against a 1,000-fold concentration gradient, i.e. 50 mM in a cell versus 50 M in the plasma. The level of SMIT activity determines the steady-state cellular concentration of MI at the point where uptake and leak balance out.SMIT is most abundantly expressed in the kidney medulla (1, 3), which is hypertonic most of the time because of the operation of the urinary concentration mechanisms. The high level of SMIT expression in the renal medulla is secondary to the hypertonicity of this tissue in that SMIT mRNA abundance changes pari passu with the tonicity of the medulla (3, 4). The changes in mRNA abundance are primarily due to changes in transcription (5). When SMIT is inhibited under hypertonic conditions in cultured cells (6) or in kidneys in vivo (7), cells undergo necrosis demonstrating the importance of maintaining a high level of SMIT activity in a hypertonic environment.How elevated SMIT activity protects the renal cells is explained by the theory of compatible osmolytes (8). It is useful to note that osmolarity inside a mammalian cell is always in equilibrium with interstitial osmolarity because blood facing plasma membranes are highly permeable to water and very compliant mechanically. Immediately after cells are exposed to hypert...