Obesity, hyperlipidemia, and insulin resistance are common forerunners of type 2 diabetes mellitus. We have identified the human winged helix/forkhead transcription factor gene FOXC2 as a key regulator of adipocyte metabolism. Increased FOXC2 expression, in adipocytes, has a pleiotropic effect on gene expression, which leads to a lean and insulin sensitive phenotype. FOXC2 affects adipocyte metabolism by increasing the sensitivity of the beta-adrenergic-cAMP-protein kinase A (PKA) signaling pathway through alteration of adipocyte PKA holoenzyme composition. Increased FOXC2 levels, induced by high fat diet, seem to counteract most of the symptoms associated with obesity, including hypertriglyceridemia and diet-induced insulin resistance--a likely consequence hereof would be protection against type 2 diabetes.

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.

Epithiospecifier protein (ESP), a ferrous ion dependent protein, has a potential role in regulating the release of elemental sulphur, nitriles, isothiocyanates and cyanoepithioalkanes from glucosinolates. Two classes of ESP polypeptides were purified with molecular masses of 39 and 35 kDa, and we show that the previously reported instability was conditionally dependent. The 39 kDa polypeptide was made up of two distinct isozymes (5.00, 5.14) whilst several were present for the 35 kDa form of ESP (5.40^5.66). An anti-ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and strongly with a polypeptide corresponding to the 35 kDa ESP form in Crambe abyssinica, but did not detect any ESP in Sinapis alba or Raphanus sativus. A cytochrome P-450 mediated iron dependent epoxidation type mechanism is suggested for ESP.z 2000 Federation of European Biochemical Societies.

Insulin resistance, obesity, and diabetes are characterized by hyperglycemia, hyperinsulinemia, and hyperleptinemia and are associated with increased risk of atherosclerosis. In an effort to understand how this occurs, we have investigated whether these factors cause disregulation of cholesterol ester metabolism in J774.2 macrophages. Raising glucose levels alone was sufficient to increase uptake of acetylated low density lipoprotein but did not stimulate synthesis of cholesterol esters. In the presence of high glucose, both insulin and leptin increased the rate of cholesterol ester synthesis, although they did not further increase uptake of acetylated low density lipoprotein. However, in the presence of high glucose both insulin and leptin caused a significant increase in the activity of acyl-CoA: cholesterol O-acyltransferase (ACAT) combined with a significant reduction in the level of hormone-sensitive lipase (HSL). Because ACAT is the main enzyme responsible for cholesterol ester synthesis and HSL contributes significantly to neutral cholesterol ester hydrolase activity, this suggests that glucose primes the J774.2 cells so that in the presence of high insulin or leptin they will store cholesterol esters. This contrasts with 3T3-L1 adipocytes, where HSL activity and expression are increased by insulin in high glucose conditions. These findings may provide an explanation for the observation that in conditions characterized by hyperglycemia, hyperleptinemia, and hyperinsulinemia, triglyceride lipolysis in adipocytes is increased while hydrolysis of cholesterol esters in macrophages is decreased, contributing to foam cell formation.Atherosclerosis-related disorders are the principle cause of death in the Western world, and insulin resistance, type-2 diabetes, and obesity are well recognized risk factors for coronary heart disease. Individuals in these categories have a 2-3-fold increased risk of developing macrovascular heart disease (1-5). Insulin resistance, type-2 diabetes, and obesity are characterized by hyperleptinemia, hyperinsulinemia, and hyperglycemia, suggesting these may be acting as proatherogenic factors (6 -8). Indeed, there is good evidence that a range of growth factors and cytokines can act to promote atherogenesis (9), and the importance of glucose levels is shown by the fact that there is a very strong positive correlation between glycated hemoglobin levels and mortality from cardiovascular disease, even in non-diabetic subjects (10). However, the molecular mechanisms underlying the increased rate of atherogenesis in these groups is poorly understood, and little is known about the specific roles of leptin, insulin, and glucose in this process.The first lesion of atherosclerosis (fatty streak or type-1 lesion) is characterized by an accumulation in the intima of the vascular epithelia of lipid-laden macrophage foam cells (11,12). Cholesterol cannot efflux from the cell when esterified, but the bulk (ϳ75%) of the lipid in foam cells is in the form of cholesteryl esters (CEs) 1 (11,12). Regulat...

The suppressors of cytokine signaling (SOCS) family is thought to act largely as a negative regulator of signaling by cytokines and some growth factors. Surprisingly, the SOCS-6 transgenics had no significant defects in the cytokine signaling and hematopoietic system but displayed significant improvements in glucose metabolism. Insulin stimulation of Akt/protein kinase B was also potentiated. Biochemical analysis showed that, after insulin stimulation, SOCS-6 interacted with the monomeric p85 subunit of class-Ia phosphoinositide (PI) 3-kinase but not with p85/p110 dimers. Furthermore, SOCS-6 expression is transiently increased by serum and insulin in normal fibroblasts. However, both the mRNA and protein of SOCS-6 were rapidly degraded after induction by insulin. The degradation of the SOCS-6 protein was partially inhibited by a proteasome inhibitor, suggesting a proteasome-mediated degradation mechanism. In contrast, SOCS-6-associated p85 was not degraded and could be recruited to the newly synthesized SOCS-6 molecules in the presence of insulin, suggesting that SOCS-6 expression and its interaction with p85, but not the degradation, is regulated by insulin. The phenotype of SOCS-6 transgenic mice bears a striking resemblance to p85 knock-out mouse models in which glucose metabolism stimulated by insulin is significantly improved despite reduced activation of PI 3-kinase. This suggests that monomeric p85 might play a physiologically important role in attenuating signaling through PI 3-kinase-dependent pathways in unstimulated cells. Therefore, our results indicate that SOCS-6 may provide a dynamically regulated mechanism by which insulin can transiently overcome the negative effects that p85 monomers have on signaling via PI 3-kinase-dependent signaling pathways.

We have reported recently that mice overexpressing the forkhead/winged helix transcription factor FOXC2 are lean and show increased responsiveness to insulin due to sensitization of the ␤-adrenergic cAMP-PKA -7), and the Fox 1 nomenclature (Forkhead box) has now been adopted for all chordate forkhead genes (www.biology. pomona.edu/fox.html) (8). Among these are several forkhead related activators (FREACs) cloned from human (9 -13) that all share the minimum requirement for a 7-bp core binding motif (RTAAAYA). One of these factors, FOXD1 (FREAC4, FKHL8), has expression restricted to kidney, the central nervous system testis, and is regulated by Ets-1 and p53 in kidney-derived cell lines (11,14). FOXC2 (FREAC11, FKHL14, MFH-1) (15, 16) is restricted to adipocytes in adults (1), whereas the prenatal form is important for development. Mice lacking Foxc2 die during embryogenesis or perinatally and exhibit aortic arch and skeletal defects (17, 18). Instead, overexpression of FOXC2 in adipose tissue has been important in understanding its function in adult mice. FOXC2 transgenic mice develop a phenotype characterized by a high sensitivity to insulin and partial resistance to diet-induced obesity (1). This effect is partly due to up-regulation of ␤-adrenergic receptors and PKA type I␣ that lower the threshold for activation of PKA by cAMP, resulting in a hypersensitive ␤-adrenergic pathway.Activation of the cAMP-dependent protein kinase (PKA) proceeds by a concerted reaction in which binding of the intracellular second messenger cAMP to the regulatory subunit dimer (R 2 ) in a positive, cooperative fashion results in dissociation and activation of two catalytic (C) subunits (reviewed in Ref. 19). Targeted disruption of the RII␤ regulatory subunit gene in mice leads to a lean phenotype with elevated levels of uncoupling protein 1 and increased metabolic rate due to a shift in the PKA composition from PKA II␤ (RII␤ 2 C 2 ) to type I␣ (RI␣ 2 C 2 ) holoenzyme (20 -22). The effect of this regulatory subunit shift was shown to lower the threshold for PKA activation by cAMP and to modulate lipolysis (23). The RII␤ knockout phenotype resembles that of the FOXC2 transgenic mice (1) and supports the notion that regulation of PKA isozyme composition, particularly RII␤ versus RI␣, is important for hormonal responsiveness and cAMP sensitivity.

In germinating seedlings of Brassica napus glucosinolate levels decrease and are potentially degraded to nitriles by a myrosinase. Little is known about the metabolism of glucosinolate aglycone products and the objective of this work was to investigate nitrilase activity and carry out a purification of the enzyme from seedlings of B. napus. A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid was purified to apparent homogeneity from seedlings of B. napus. The protein has a molecular mass of approximately 420 kDa made up of 38 kDa subunits. The pI of the native protein was found to be 4.6. Under denaturing conditions on an isoelectric focusing (IEF) gel a major and minor protein was observed with pI in the range of 5.4‐5.9, suggesting the presence of isoforms. Apart from the potential role of the nitrilase in indole‐3‐acetic acid (IAA) synthesis a developmental study with seedlings indicates that the increase in activity observed may be linked to the in vivo degradation of glucosinolates.

Changes in glucose levels are known to directly alter gene expression. A number of previous studies have found that these effects are in part mediated by modulating the levels and the activity of transcription factors. We have investigated an alternative mechanism by which glucose might regulate gene expression by modulating levels of a transcriptional repressor. We have focused on Id2, which is a protein that indirectly regulates gene expression by sequestering certain transcription factors and preventing them from forming functional dimers. Id2 targets include the class A basic helix-loop-helix transcription factors and the sterol regulatory element-binding protein (SREBP)-1. We demonstrate that increases in glucose levels cause a rapid increase in levels of Id2 in J774.2 macrophages, and a number of lines of evidence indicate that this is via the hexosamine pathway because 1) the effect of glucose requires glutamine; 2) the effect of glucose is mimicked by low levels of glucosamine; 3) the effect of glucose is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT); and 4) adenoviral mediated overexpression of GFAT increases levels of Id2. We go on to show that increases in Id2 can have functional effects on metabolic genes, because Id2 blocked the SREBP-1-induced induction of hormone-sensitive lipase (HSL) promoter activity, whereas Id2 alone does not modulate activity of the HSL promoter. In summary, these studies define a new mechanism by which glucose uses the hexosamine pathway to regulate gene expression by increasing levels of a transcriptional repressor. E proteins; diabetes; atherosclerosis IT IS WELL RECOGNIZED that changes in glucose levels have direct effects of gene expression in a range of tissues (49). Gene expression events induced by normal physiological fluctuations in glucose levels play a critical role in the maintenance of proper glucose and lipid homeostasis. These effects have been thoroughly investigated mostly in liver, where changes in glucose levels play an important role in switching off gluconeogenesis and switching on glycolytic pathways (15, 17). Glucose also plays a key role in regulating the expression and release of insulin in the ␤-cells of the pancreas (45), and the insulin released subsequently plays an important role in the regulation of metabolic genes (17). However, persistent hyperglycemia associated with diabetes is known to contribute to a wide range of gene expression events associated with the development of diabetic complications (5). More recently, we and others (19,21,41,44) have shown that glucose has direct effects on genes controlling cholesterol ester metabolism in macrophages, and these findings have suggested a possible link between hyperglycemia and foam cell formation. The mechanisms by which glucose regulates gene expression in macrophages have not been characterized.A number of mechanisms have been implicated in glucosemediated regulation of gene expression (5). The hexosamine pathway has drawn particular attentio...