Carbohydrate Response Element Binding Protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

Glucocorticoids, which are well established to regulate body fat mass distribution, adipocyte lipolysis, hepatic gluconeogenesis, and hepatocyte VLDL secretion, are speculated to play a role in the pathology of metabolic syndrome. Recent focus has been on the activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is capable of regenerating, and thus amplifying, glucocorticoids in key metabolic tissues such as liver and adipose tissue. To determine the effects of global 11beta-HSD1 inhibition on metabolic syndrome risk factors, we subcutaneously injected Western-type diet-fed hyperlipidemic mice displaying either moderate or severe obesity (LDLR-/- and Agouti;LDLR-/-, respectively) with the nonselective 11beta-HSD inhibitor carbenoxolone for 4 weeks. Body composition throughout the study, end-point fasting plasma analyses, and extent of hepatic steatosis and atherosclerosis were assessed. This route of treatment led to high levels of carbenoxolone detected in liver and fat and resulted in decreased weight gain due to reduced body fat mass in both mouse models. However, only Agouti;LDLR-/- mice showed an effect of 11beta-HSD1 inhibition on fasting insulin and plasma lipids, coincident with a reduction in VLDL due to mildly increased VLDL clearance and dramatically decreased hepatic triglyceride production. Agouti;LDLR-/- mice also showed the greatest effect of the drug on reducing atherosclerotic lesion formation. These findings indicate that subcutaneous injection of an 11beta-HSD1 inhibitor allows for the targeting of the enzyme in not only liver, but also adipose tissue and attenuates many metabolic syndrome risk factors, with more pronounced effects in cases of severe obesity and hyperlipidemia. Key words: 11 beta hydroxysteroid dehydrogenase, agouti, obese, low density lipoprotein receptor deficient.

11beta-Hydroxysteroid dehydrogenase type I (11beta-HSD1), an NADPH-dependent reductase, functions in intact cells to convert inactive 11-keto metabolites of glucocorticoids into biologically active glucocorticoids. The enzyme is thus capable of amplifying glucocorticoid action in tissues in which it is expressed. In the experiments presented here, we show that 11beta-HSD1 is expressed in the murine thymus and that expression increases from late fetal development to maximal levels in the adult thymus. Quantitative real time-PCR, immunoblots, and assays of enzymatic activity reveal adult thymic expression of 11beta-HSD1 mRNA and protein at levels approximately 6-7% of those observed in liver. Immunofluorescence experiments show that the enzyme is expressed in the medullary thymocytes and thymocytes present at the corticomedullary junction. These experiments extend our recognition of 11beta-HSD1 expression in cells of the immune system and lend support to the notion that glucocorticoid signaling and amplification of those signals by regeneration of active glucocorticoids from inactive 11-keto metabolites might impact intrathymic T cell development and the establishment of the immune repertoire.

Despite a clear association between obesity, insulin resistance and atherosclerosis in humans, to date, no animal models have been described in which insulin resistance is associated with atherosclerotic lesion burden. Using two mouse models of obesity-induced hyperlipidemia:leptin deficient (ob/ob) mice on an apolipoprotein E deficient (apoE(-/-)) or low density lipoprotein receptor deficient (LDLR(-/-)) background, we sought to determine metabolic parameters most closely associated with atherosclerotic lesion burden. Total plasma cholesterol (TC) levels in ob/ob;apoE(-/-) mice and ob/ob;LDLR(-/-) mice were indistinguishable (682+/-48 versus 663+/-16, respectively). Analysis of lipoprotein profiles showed that cholesterol was carried primarily on VLDL in the ob/ob;apoE(-/-) mice and on LDL in the ob/ob;LDLR(-/-) mice. Plasma triglycerides (TG) were 55% lower (P<0.001), non-esterified fatty acids (NEFA) were 1.5-fold higher (P<0.01), and insulin levels were 1.7-fold higher (NS) in ob/ob;apoE(-/-) mice compared to ob/ob;LDLR(-/-) mice. Other parameters such as body weight, fat pad weight, and glucose levels were not different between the groups. Aortic sinus lesion area of ob/ob;apoE(-/-) mice was increased 3.2-fold above ob/ob;LDLR(-/-) mice (102,455+/-8565mum(2)/section versus 31,750+/-4478mum(2)/section, P<0.001). Lesions in ob/ob;apoE(-/-) mice were also more complex as evidenced by a 7.7-fold increase in collagen content (P<0.001). Atherosclerotic lesion area was positively correlated with body weight (P<0.005), NEFA (P=0.007), and insulin (P=0.002) levels in the ob/ob;LDLR(-/-) mice and with insulin (P=0.014) in the ob/ob;apoE(-/-) mice. In contrast, lesion burden was neither associated with TC and TG, nor with individual lipoprotein pools, in either animal model. These data provide a direct demonstration of the pathophysiologic relevance of hyperinsulinemia, NEFA, and increased body weight to atherosclerotic lesion formation.