Thioredoxin regulates adipogenesis through thioredoxin-interacting protein (Txnip) protein stability

Txnip (thioredoxin-interacting protein) is a critical mediator of metabolism and adipogenesis in vivo. The mechanisms of action of Txnip are believed to operate at least in part by inhibiting the redox signaling functions of thioredoxin. We tested here whether Txnip suppressed adipogenesis by inhibiting thioredoxin and discovered a reversal of roles; Txnip inhibits adipogenesis directly, and thioredoxin binding regulates Txnip by enhancing Txnip protein stability. Unlike Txnip, a Txnip mutant that cannot bind thioredoxin (C247S) did not prevent adipocyte differentiation, but was degraded more quickly by proteasomal targeting. Finding that endogenous Txnip protein is also rapidly degraded at the onset of adipogenesis suggested that Txnip degradation is required for adipocyte differentiation. Thioredoxin overexpression stabilized Txnip protein levels to inhibit adipogenesis, and adipogenic stimulants such as insulin promoted Txnip-thioredoxin dissociation to the more labile free Txnip state. As an α-arrestin protein, Txnip has two C-terminal tail PPXY motifs that mediate E3 ubiquitin ligase binding and Txnip protein stability. Mutating the PPXY motifs prevented Txnip degradation, even when thioredoxin binding was lost, and restored the ability of C247S Txnip to inhibit adipogenesis. These studies present a novel reconsideration of Txnip-thioredoxin signaling by showing that thioredoxin regulates the intrinsic function of Txnip as an inhibitor of adipogenesis through protein stabilization.     

SIRT4 Represses Peroxisome Proliferator-Activated Receptor α Activity To Suppress Hepatic Fat Oxidation

Sirtuins are a family of protein deacetylases, deacylases, and ADP-ribosyltransferases that regulate life span, control the onset of numerous age-associated diseases, and mediate metabolic homeostasis. We have uncovered a novel role for the mitochondrial sirtuin SIRT4 in the regulation of hepatic lipid metabolism during changes in nutrient availability. We show that SIRT4 levels decrease in the liver during fasting and that SIRT4 null mice display increased expression of hepatic peroxisome proliferator-activated receptor α (PPARα) target genes associated with fatty acid catabolism. Accordingly, primary hepatocytes from SIRT4 knockout (KO) mice exhibit higher rates of fatty acid oxidation than wild-type hepatocytes, and SIRT4 overexpression decreases fatty acid oxidation rates. The enhanced fatty acid oxidation observed in SIRT4 KO hepatocytes requires functional SIRT1, demonstrating a clear cross talk between mitochondrial and nuclear sirtuins. Thus, SIRT4 is a new component of mitochondrial signaling in the liver and functions as an important regulator of lipid metabolism.     

Quantifying the contribution of the liver to glucose homeostasis: a detailed kinetic model of human hepatic glucose metabolism

Despite the crucial role of the liver in glucose homeostasis, a detailed mathematical model of human hepatic glucose metabolism is lacking so far. Here we present a detailed kinetic model of glycolysis, gluconeogenesis and glycogen metabolism in human hepatocytes integrated with the hormonal control of these pathways by insulin, glucagon and epinephrine. Model simulations are in good agreement with experimental data on (i) the quantitative contributions of glycolysis, gluconeogenesis, and glycogen metabolism to hepatic glucose production and hepatic glucose utilization under varying physiological states. (ii) the time courses of postprandial glycogen storage as well as glycogen depletion in overnight fasting and short term fasting (iii) the switch from net hepatic glucose production under hypoglycemia to net hepatic glucose utilization under hyperglycemia essential for glucose homeostasis (iv) hormone perturbations of hepatic glucose metabolism. Response analysis reveals an extra high capacity of the liver to counteract changes of plasma glucose level below 5 mM (hypoglycemia) and above 7.5 mM (hyperglycemia). Our model may serve as an important module of a whole-body model of human glucose metabolism and as a valuable tool for understanding the role of the liver in glucose homeostasis under normal conditions and in diseases like diabetes or glycogen storage diseases.     

The interaction of thioredoxin with Txnip. Evidence for formation of a mixed disulfide by disulfide exchange

The thioredoxin system plays an important role in maintaining a reducing environment in the cell. Recently, several thioredoxin binding partners have been identified and proposed to mediate aspects of redox signaling, but the significance of these interactions is unclear in part due to incomplete understanding of the mechanism for thioredoxin binding. Thioredoxin-interacting protein (Txnip) is critical for regulation of glucose metabolism, the only currently known function of which is to bind and inhibit thioredoxin. We explored the mechanism of the Txnip-thioredoxin interaction and present evidence that Txnip and thioredoxin form a stable disulfide-linked complex. We identified two Txnip cysteines that are important for thioredoxin binding and showed that this interaction is consistent with a disulfide exchange reaction between oxidized Txnip and reduced thioredoxin. These cysteines are not conserved in the broader family of arrestin domain-containing proteins, and we demonstrate that the thioredoxin-binding property of Txnip is unique. These data suggest that Txnip is a target of reduced thioredoxin and provide insight into the potential role of Txnip as a redox-sensitive signaling protein.     

Oxidative stress and thioredoxin-interacting protein promote intravasation of melanoma cells

Although intravasation may be a critical rate-limiting step in the metastatic cascade, the role of oxidative stress in intravasation is unknown. We tested the hypothesis that reactive oxygen species (ROS), regulated by thioredoxin interacting protein (Txnip) through the action of thioredoxin (Trx), influence human SK-MEL-28 melanoma cell reverse (basolateral-to-apical) transendothelial migration (TEM) in vitro as a model for intravasation. Reverse transendothelial migration was dose-dependently induced by hydrogen peroxide 2.4-fold for 0.1 microM (P < 0.01) and 3.9-fold for 1 microM (P < 0.001) vs. control, and this effect was blocked by the antioxidant N-acetylcysteine. Overexpression of Txnip by infecting melanoma cells with adenovirus increased TEM 3-fold vs. control (P < 0.001), and this increase was blocked by N-acetylcysteine, indicating a redox-sensitive mechanism. Conversely, thioredoxin overexpression blocked hydrogen peroxide-induced TEM. Exposure to ultraviolet-A radiation increased ROS 1.8-fold (P < 0.01), and this was accompanied by a 45% reduction (P < 0.05) in thioredoxin activity and an 11.4-fold (P < 0.001) increase in Txnip gene expression. These data suggest that TEM of melanoma cells during intravasation is in part mediated by ROS-sensitive cellular signaling cascades, may be controlled by Txnip and its interaction with thioredoxin that in turn modulates cellular levels of oxidative stress, and may be initiated by ultraviolet-A induction of this cascade.     

Adenovirus-mediated expression of caveolin-1 in mouse liver increases plasma high-density lipoprotein levels

Caveolae are 50-100 nm plasma membrane invaginations, which function in cell signaling and transcytosis, as well as in regulating cellular cholesterol homeostasis. These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins. Recent studies have indicated that caveolae may be involved in the regulation of cellular cholesterol efflux to HDL, as well as selective uptake mediated by SR-BI. In the present study, we have determined the effect of caveolin-1 overexpression in mouse liver on plasma lipoprotein metabolism. We evaluated this effect using an adenovirus-mediated gene delivery system. C57BL/6J mice were injected with adenoviruses encoding either caveolin-1 (Adcav-1) or green fluorescent protein (AdGFP) together with a transactivator adenovirus (AdtTA). We found that, after adenovirus injection, caveolin-1 was overexpressed in hepatocytes. Moreover, the recombinant protein was localized to the plasma membrane. We also found that caveolin-1 overexpression induced a marked change in the lipoprotein profile of injected animals. In caveolin-1 overexpressing animals, plasma HDL-cholesterol levels were found to be approximately 2-fold elevated, as compared with control animals. To determine the effect of caveolin-1 on SR-BI-mediated selective uptake, we infected murine hepatocytes in culture with an adenoviral vector carrying the caveolin-1 cDNA or GFP as a control protein. We show that, in primary cultures of hepatocytes, caveolin-1 inhibits DiI-HDL uptake mediated by SR-BI. This result would mechanistically explain the increased plasma HDL-cholesterol levels we observed in caveolin-1 adenovirus-injected animals. In addition, caveolin-1 expression increased the secretion of apolipoprotein A-I in cultured hepatocytes and increased apolipoprotein A-I plasma levels in mice. Our study therefore demonstrates an important role for caveolin-1 in regulating HDL metabolism.     

Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene

Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp-/- mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a approximately 10-fold increase in hepatic triglyceride content while L-Fabp-/- mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp-/- mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum beta-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp-/- mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPAR alpha target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp-/- mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp-/- mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp-/- mice that involves targeting of substrate for use in triglyceride metabolism.     

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

Hepatic Overexpression of a Constitutively Active Form of Liver Glycogen Synthase Improves Glucose Homeostasis

In this study, we tested the efficacy of increasing liver glycogen synthase to improve blood glucose homeostasis. The overexpression of wild-type liver glycogen synthase in rats had no effect on blood glucose homeostasis in either the fed or the fasted state. In contrast, the expression of a constitutively active mutant form of the enzyme caused a significant lowering of blood glucose in the former but not the latter state. Moreover, it markedly enhanced the clearance of blood glucose when fasted rats were challenged with a glucose load. Hepatic glycogen stores in rats overexpressing the activated mutant form of liver glycogen synthase were enhanced in the fed state and in response to an oral glucose load but showed a net decline during fasting. In order to test whether these effects were maintained during long term activation of liver glycogen synthase, we generated liver-specific transgenic mice expressing the constitutively active LGS form. These mice also showed an enhanced capacity to store glycogen in the fed state and an improved glucose tolerance when challenged with a glucose load. Thus, we conclude that the activation of liver glycogen synthase improves glucose tolerance in the fed state without compromising glycogenolysis in the postabsorptive state. On the basis of these findings, we propose that the activation of liver glycogen synthase may provide a potential strategy for improvement of glucose tolerance in the postprandial state.     

Deletion of CaMKK2 from the liver lowers blood glucose and improves whole-body glucose tolerance in the mouse

Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2(floxed) mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content.     

Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression

Leptin has been shown to improve insulin sensitivity and glucose metabolism in obese diabetic ob/ob mice, yet the mechanisms remain poorly defined. We found that 2 d of leptin treatment improved fasting but not postprandial glucose homeostasis, suggesting enhanced hepatic insulin sensitivity. Consistent with this hypothesis, leptin improved in vivo insulin receptor (IR) activation in liver, but not in skeletal muscle or fat. To explore the cellular mechanism by which leptin up-regulates hepatic IR activation, we examined the expression of the protein tyrosine phosphatase PTP1B, recently implicated as an important negative regulator of insulin signaling. Unexpectedly, liver PTP1B protein abundance was increased by leptin to levels similar to lean controls, whereas levels in muscle and fat remained unchanged. The ability of leptin to augment liver IR activation and PTP1B expression was also observed in vitro in human hepatoma cells (HepG2). However, overexpression of PTP1B in HepG2 cells led to diminished insulin-induced IR phosphorylation, supporting the role of PTP1B as a negative regulator of IR activation in hepatocytes. Collectively, our results suggest that leptin acutely improves hepatic insulin sensitivity in vivo with concomitant increases in PTP1B expression possibly serving to counterregulate insulin action and to maintain insulin signaling in proper balance.     

Hyperglycemia promotes oxidative stress through inhibition of thioredoxin function by thioredoxin-interacting protein

Increased intracellular reactive oxygen species (ROS) contribute to vascular disease and pro-atherosclerotic effects of diabetes mellitus may be mediated by oxidative stress. Several ROS-scavenging systems tightly control cellular redox balance; however, their role in hyperglycemia-induced oxidative stress is unclear. A ubiquitous antioxidative mechanism for regulating cellular redox balance is thioredoxin, a highly conserved thiol reductase that interacts with an endogenous inhibitor, thioredoxin-interacting protein (Txnip). Here we show that hyperglycemia inhibits thioredoxin ROS-scavenging function through p38 MAPK-mediated induction of Txnip. Overexpression of Txnip increased oxidative stress, while Txnip gene silencing restored thioredoxin activity in hyperglycemia. Diabetic animals exhibited increased vascular expression of Txnip and reduced thioredoxin activity, which normalized with insulin treatment. These results provide evidence for the impairment of a major ROS-scavenging system in hyperglycemia. These studies implicate reduced thioredoxin activity through interaction with Txnip as an important mechanism for vascular oxidative stress in diabetes mellitus.     

Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.     

Thioredoxin-interacting protein deficiency disrupts the fasting-feeding metabolic transition

Through a positional cloning approach, the thioredoxin-interacting protein gene (Txnip) was recently identified as causal for a form of combined hyperlipidemia in mice (Bodnar, J. S., A. Chatterjee, L. W. Castellani, D. A. Ross, J. Ohmen, J. Cavalcoli, C. Wu, K. M. Dains, J. Catanese, M. Chu, S. S. Sheth, K. Charugundla, P. Demant, D. B. West, P. de Jong, and A. J. Lusis. 2002. Positional cloning of the combined hyperlipidemia gene Hyplip1. Nat. Genet. 30: 110-116). We now show that Txnip-deficient mice in the fed state exhibit a metabolic profile similar to fasted mice, including increased levels of plasma ketone bodies and free fatty acids, decreased glucose, and increased hepatic expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and acyl-CoA oxidase. Dramatic differences in the expression of key metabolic enzymes were also observed in other tissues, and the fat-to-muscle ratio of Txnip-deficient mice was increased by approximately 40%. We demonstrate an effect of Txnip on the redox status, as the Txnip-deficient mice in the fed state had a significant increase in the ratio of NADH to NAD(+). Surprisingly, we observed that Txnip-deficient mice and wild-type mice had similar levels of thioredoxin activity, suggesting that the effects of Txnip deficiency may be mediated in part by other interactions. These results indicate a role for Txnip in the metabolic response to feeding and the maintenance of the redox status.     

Enhanced gluconeogenesis and hepatic insulin resistance in insulin-like growth factor binding protein-1 transgenic mice

Fasting hyperglycemia is observed in transgenic mice which overexpress insulin-like growth factor binding protein-1. In an attempt to understand the mechanisms underlying this observation we have examined glycogenolysis and gluconeogenesis in isolated hepatocytes from wild-type and transgenic mice. Glucose production from pyruvate was significantly less responsive to inhibition by insulin in hepatocytes from transgenic mice compared to hepatocytes from wild-type mice. Serum from transgenic mice resulted in more glucose production by hepatocytes than serum from wild-type mice. Serum alanine was increased while serum lactate was significantly reduced in transgenic mice compared to wild-type mice. Serum free fatty acids and beta-hydroxybutyrate were similar in both groups of mice. These data suggest that fasting hyperglycemia is due to enhanced gluconeogenesis, hepatic insulin resistance and increased serum gluconeogenic substrate in transgenic mice.     

The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.