Nuclear localization of p38 MAPK in response to DNA damage.

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.

Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes.

Pancreatic beta-cell loss through apoptosis represents a key factor in the pathogenesis of diabetes; however, no effective approaches to block this process and preserve endogenous beta-cell mass are currently available. To study the role of thioredoxin-interacting protein (TXNIP), a proapoptotic beta-cell factor we recently identified, we used HcB-19 (TXNIP nonsense mutation) and beta-cell-specific TXNIP knockout (bTKO) mice. Interestingly, HcB-19 mice demonstrate increased adiposity, but have lower blood glucose levels and increased pancreatic beta-cell mass (as assessed by morphometry). Moreover, HcB-19 mice are resistant to streptozotocin-induced diabetes. When intercrossed with obese, insulin-resistant, and diabetic mice, double-mutant BTBRlep(ob/ob)txnip(hcb/hcb) are even more obese, but are protected against diabetes and beta-cell apoptosis, resulting in a 3-fold increase in beta-cell mass. Beta-cell-specific TXNIP deletion also enhanced beta-cell mass (P<0.005) and protected against diabetes, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) revealed a approximately 50-fold reduction in beta-cell apoptosis in streptozotocin-treated bTKO mice. We further discovered that TXNIP deficiency induces Akt/Bcl-xL signaling and inhibits mitochondrial beta-cell death, suggesting that these mechanisms may mediate the beta-cell protective effects of TXNIP deficiency. These results suggest that lowering beta-cell TXNIP expression could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta-cell survival.

Txnip balances metabolic and growth signaling via PTEN disulfide reduction.

Thioredoxin-interacting protein (Txnip) inhibits thioredoxin NADPH-dependent reduction of protein disulfides. Total Txnip knockout (TKO) mice adapted inappropriately to prolonged fasting by shifting fuel dependence of skeletal muscle and heart from fat and ketone bodies to glucose. TKO mice exhibited increased Akt signaling, insulin sensitivity, and glycolysis in oxidative tissues (skeletal muscle and hearts) but not in lipogenic tissues (liver and adipose tissue). The selective activation of Akt in skeletal muscle and hearts was associated with impaired mitochondrial fuel oxidation and the accumulation of oxidized (inactive) PTEN, whose activity depends on reduction of two critical cysteine residues. Whereas muscle- and heart-specific Txnip knockout mice recapitulated the metabolic phenotype exhibited by TKO mice, liver-specific Txnip knockout mice were similar to WT mice. Embryonic fibroblasts derived from knockout mice also accumulated oxidized (inactive) PTEN and had elevated Akt phosphorylation. In addition, they had faster growth rates and increased dependence on anaerobic glycolysis due to impaired mitochondrial fuel oxidation, and they were resistant to doxorubicin-facilitated respiration-dependent apoptosis. In the absence of Txnip, oxidative inactivation of PTEN and subsequent activation of Akt attenuated mitochondrial respiration, resulting in the accumulation of NADH, a competitive inhibitor of thioredoxin NADPH-reductive activation of PTEN. These findings indicate that, in nonlipogenic tissues, Txnip is required to maintain sufficient thioredoxin NADPH activity to reductively reactivate oxidized PTEN and oppose Akt downstream signaling.

The physiological and molecular regulation of lipoprotein assembly and secretion.

Triglycerides are insoluble in water and yet are transported at milligram per millilitre concentrations in the bloodstream. This is made possible by the ability of the liver and intestine to assemble lipid-protein emulsions (i.e. lipoproteins), which transport hydrophobic molecules. The assembly of triglyceride-rich lipoproteins requires the coordination of protein and lipid synthesis, which occurs on the cytoplasmic surface of the endoplasmic reticulum (ER), and their concerted assembly and translocation into the luminal ER secretory pathway as nascent lipoprotein particles. The availability of lipid substrate for triglyceride production and the machinery for lipoprotein assembly are highly sensitive to nutritional, hormonal, and genetic modulation. Disorders in lipid metabolism or an imbalance between lipogenesis and lipoprotein assembly can lead to hyperlipidemia and/or hepatic steatosis. We selectively review recently-identified machinery, such as transcription factors and nuclear hormone receptors, which provide new clues to the regulation of lipoprotein secretion.

Coordinate transcriptional repression of liver fatty acid-binding protein and microsomal triglyceride transfer protein blocks hepatic very low density lipoprotein secretion without hepatosteatosis.

Unlike the livers of humans and mice, and most hepatoma cells, which accumulate triglycerides when treated with microsomal triglyceride transfer protein (MTP) inhibitors, L35 rat hepatoma cells do not express MTP and cannot secrete very low density lipoprotein (VLDL), yet they do not accumulate triglyceride. In these studies we show that transcriptional co-repression of the two lipid transfer proteins, liver fatty acid-binding protein (L-FABP) and MTP, which cooperatively shunt fatty acids into de novo synthesized glycerolipids and the transfer of lipids into VLDL, respectively, act together to maintain hepatic lipid homeostasis. FAO rat hepatoma cells express L-FABP and MTP and demonstrate the ability to assemble and secrete VLDL. In contrast, L35 cells, derived as a single cell clone from FAO cells, do not express L-FABP or MTP nor do they assemble and secrete VLDL. We used these hepatoma cells to elucidate how a conserved DR1 promoter element present in the promoters of L-FABP and MTP affects transcription, expression, and VLDL production. In FAO cells, the DR1 elements of both L-FABP and MTP promoters are occupied by peroxisome proliferator-activated receptor alpha-retinoid X receptor alpha (RXRalpha), with which PGC-1beta activates transcription. In contrast, in L35 cells the DR1 elements of both L-FABP and MTP promoters are occupied by chicken ovalbumin upstream promoter transcription factor II, and transcription is diminished. The combined findings indicate that peroxisome proliferator-activated receptor alpha-RXRalpha and PGC-1beta coordinately up-regulate L-FABP and MTP expression, by competing with chicken ovalbumin upstream promoter transcription factor II for the DR1 sites in the proximal promoters of each gene. Additional studies show that ablation of L-FABP prevents hepatic steatosis caused by treating mice with an MTP inhibitor. Our findings show that reducing both L-FABP and MTP is an effective means to reduce VLDL secretion without causing hepatic steatosis.

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia.

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.

Bile acids decrease hepatic paraoxonase 1 expression and plasma high-density lipoprotein levels via FXR-mediated signaling of FGFR4.

OBJECTIVE: The purpose of this research was to determine how dietary bile acids repress hepatic expression of paraoxonase 1 (PON1). METHODS AND RESULTS: C57BL/6 mice and C3H/HeJ mice, having different susceptibilities to atherosclerosis, were fed a chow diet and an atherogenic diet containing taurocholate. Compared with the more atherosclerosis-susceptible C57BL/6 mice, C3H/HeJ mice display resistance to dietary bile acid repression of hepatic PON1 mRNA and decreased high-density lipoprotein cholesterol. Whereas knockout of toll receptor 4 did not affect response to taurocholate, deletion of either FXR or FGFR4 blocked taurocholate repression of PON1 and CYP7A1. FGF19, an activator of FGFR4 expressed in human ileum, decreased expression of both PON1 and CYP7A1 expression by human hepatoma cells. In all of the mice studied, dietary taurocholate increased ileal expression of FGF15, a FXR-inducible murine homologue of human FGF19. CONCLUSIONS: Hepatic PON1 and CYP7A1 mRNA expression is repressed by bile acids via FXR-mediated induction of FGF15. Thus, the inability of C3H/HeJ mice to display taurocholate repression of PON1 and CYP7A1 mRNAs was not because of a lack of induction of FGF15 but rather signaling events distal to FGF15-FGFR4 association.

Facilitated replacement of Kupffer cells expressing a paraoxonase-1 transgene is essential for ameliorating atherosclerosis in mice.

Resident macrophages (i.e., Kupffer cells) are derived from hematopoietic stem cells (HSCs) and are primarily responsible for the removal from plasma of oxidized forms of low-density lipoprotein (LDL). The therapeutic potential of Kupffer cell expression of a transgene encoding paraoxonase-1 (PON1), whose plasma activity correlates with the protection from atherosclerosis, was examined in mice rendered atherosclerosis-susceptible through genetic deletion of the LDL receptor. Mice having their bone marrow engrafted with HSCs expressing the PON1 transgene (PON1-Tg) driven by a macrophage-specific promoter were injected i.v. with saline (vehicle only) or with gadolinium chloride (GdCl(3)), an agent that rapidly causes Kupffer cell apoptosis. One month later, GdCl(3)-facilitated Kupffer cell apoptosis increased the hepatic expression of transgenic PON1 mRNA by 9-fold. After 12 weeks of being fed a cholesterol-enriched atherogenic diet, mice injected with GdCl(3) exhibited 50% reductions in both aortic sinus atherosclerotic lesions (P < 0.0097) and surface lesions of the abdominal aorta (P < 0.006). In contrast, mice receiving HSCs expressing the PON1-Tg but not treated with GdCl(3) showed no protection from atherosclerosis. In addition, mice engrafted with HSCs not expressing the PON1-Tg but injected with GdCl(3) also showed no protection from atherosclerosis. These findings, showing that GdCl(3)-enhanced hepatic expression of the PON1-Tg is essential for reducing atherosclerosis, indicate that Kupffer cells play an important role in atherogenesis. GdCl(3)-facilated replacement of Kupffer cells may enhance the efficacy of other HSC-based gene therapies.

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha, beta/delta, and gamma.

PPARalpha, beta/delta, and gamma regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor-ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARalpha, beta, and gamma agonists on foam-cell formation and atherosclerosis in male LDL receptor-deficient (LDLR(-/-)) mice. Like the PPARgamma agonist, a PPARalpha-specific agonist strongly inhibited atherosclerosis, whereas a PPARbeta-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARalpha and PPARgamma agonists, but not the PPARbeta agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARalpha and PPARgamma agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARalpha required LXRs, activation of PPARgamma reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

Mice lacking thioredoxin-interacting protein provide evidence linking cellular redox state to appropriate response to nutritional signals.

Thioredoxin-interacting protein (Txnip) is a ubiquitous protein that binds with high affinity to thioredoxin and inhibits its ability to reduce sulfhydryl groups via NADPH oxidation. HcB-19 mice contain a nonsense mutation in Txnip that eliminates its expression. Unlike normal animals, HcB-19 mice have approximately 3-fold increase in insulin levels when fasted. The C-peptide/insulin ratio is normal, suggesting that the hyperinsulinemia is due to increased insulin secretion. Fasted HcB-19 mice are hypoglycemic, hypertriglyceridemic, and have higher than normal levels of ketone bodies. Ablation of pancreatic beta-cells with streptozotocin completely blocks the fasting-induced hypoglycemia/hypertriglyceridemia, suggesting that these abnormalities are due to excess insulin secretion. This is supported by increased hepatic mRNA levels of the insulin-inducible, lipogenic transcription factor sterol-responsive element-binding protein-1c and two of its targets, acetyl-CoA carboxylase and fatty acid synthase. During a prolonged fast, the hyperinsulinemia up-regulates lipogenesis but fails to down-regulate hepatic phosphoenolpyruvate carboxykinase mRNA expression. Hepatic ratios of reduced:oxidized glutathione, established regulators of gluconeogenic/glycolytic/lipogenic enzymes, were elevated 30% in HcB-19 mice, suggesting a loss of Txnip-enhanced sulfhydryl reduction. The altered hepatic enzymatic profiles of HcB-19 mice divert phosphoenolpyruvate to glyceroneogenesis and lipogenesis rather than gluconeogenesis. Our findings implicate Txnip-modulated sulfhydryl redox as a central regulator of insulin secretion in beta-cells and regulation of many of the branch-points of gluconeogenesis/glycolysis/lipogenesis.

ARP-1/COUP-TF II determines hepatoma phenotype by acting as both a transcriptional repressor of microsomal triglyceride transfer protein and an inducer of CYP7A1.

L35 and FAO cells were derived as single cell isolates from H35 cells. Whereas L35 cells do not express microsomal triglyceride transfer protein (MTP), which regulates lipoprotein secretion, they express CYP7A1, which regulates bile acid synthesis from cholesterol. FAO cells display the opposite phenotype (i.e. expression of MTP but not CYP7A1). We examined the molecular basis of the transcriptional inactivation of the MTP gene in L35 cells. Nested deletion and mutagenesis studies show that a conserved DR1 element within the 135-bp proximal MTP promoter is responsible for differential expression by L35 and FAO cells. Yeast one-hybrid screening identified apolipoprotein A1 regulatory protein-1/chicken ovalbumin upstream promoter transcription factor II (ARP-1/COUP-TFII) and retinoid X receptor (RXRalpha) as the protein factors that can bind to the conserved DR1 element. Nuclear extracts from L35 cells contained 2-fold more ARP-1/COUP-TFII and 50% less RXRalpha than those from FAO cells. Immunologic studies show that in L35 cells, ARP-1/COUP-TFII is bound to the DR1 element, whereas in FAO cells, a complex containing RXRalpha is bound to the DR1 element. Co-transfection studies show that ARP-1/COUP-TFII repressed MTP promoter activity by approximately 70% in FAO hepatoma cells, whereas RXRalpha and its ligand 9-cis-retinoic acid increased MTP promoter activity by 6-fold in L35 cells. The combined data suggest that in the context of the MTP promoter, ARP-1/COUP-TFII (repressor) and a complex containing RXRalpha (inducer) compete for the DR1 element. Analysis of the CYP7A1 promoter revealed that it is approximately 5-fold more active in L35 cells than in FAO cells. Co-transfection of an ARP-1/COUP-TFII expression vector showed that it enhances CYP7A1 promoter activity by 6-fold in FAO cells. These combined findings indicate that ARP-1/COUP-TFII acts as both a transcriptional repressor (of MTP) and as a transcription activator (of CYP7A1). This dual function of ARP-1/COUP-TFII may play an important role in determining the metabolic phenotype of individual liver cells.

Blocking microsomal triglyceride transfer protein interferes with apoB secretion without causing retention or stress in the ER.

Microsomal triglyceride transfer protein (MTP) is an intraluminal protein in the endoplasmic reticulum (ER) that is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins. In this study, we examine how the livers of mice respond to two distinct methods of blocking MTP function: Cre-mediated disruption of the gene for MTP and chemical inhibition of MTP activity. Blocking MTP significantly reduced plasma levels of triglycerides, cholesterol, and apoB-containing lipoproteins in both wild-type C57BL/6 and LDL receptor-deficient mice. While treating LDL receptor-deficient mice with an MTP inhibitor for 7 days lowered plasma lipids to control levels, liver triglyceride levels were increased by only 4-fold. Plasma levels of apoB-100 and apoB-48 fell by >90% and 65%, respectively, but neither apoB isoform accumulated in hepatic microsomes. Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. Levels of microsomal luminal chaperone proteins [e.g., protein disulfide isomerase, glucose-regulated protein 78 (GRP78), and GRP94] and cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) were unaffected by MTP inhibition. These findings show that the liver responds rapidly to inhibition of MTP by degrading apoB and preventing its accumulation in the ER. The rapid degradation of secretion-incompetent apoB in the ER may block the induction of proteins associated with unfolded protein and heat shock responses.

Microsomal triglyceride transfer protein is essential for hepatic secretion of apoB-100 and apoB-48 but not triglyceride.

Despite a complete lack of microsomal triglyceride transfer protein (MTP), L35 rat hepatoma cells secrete triglyceride-containing lipoproteins, albeit at a rate 25% of that of parental FAO hepatoma cells, which express high levels of MTP. The inability to express MTP was associated with a complete block in the secretion of both apolipoprotein (apo)B-100 and apoB-48. Stable expression of a MTP transgene restored the secretion of both apoB-100 and apoB-48 in L35 cells, indicating that MTP is essential for the secretion of both forms of apoB. Treatment with the MTP inhibitor BMS-200150 reduced the secretion of triglyceride by 70% in FAO cells, whereas the inhibitor did not affect the secretion of triglycerides by L35 cells. Thus, in the presence of the MTP inhibitor, both cell types secreted triglycerides at similar rates. Essentially, all of the triglycerides secreted by L35 cells were associated with HDL containing apoA-IV and apoE but devoid of apoB-100 or apoB-48. These results suggest that these triglyceride-containing lipoproteins are assembled and secreted via a pathway that is independent of both apoB and MTP. Our findings support the concept that apoB and MTP co-evolved and provided a means to augment the secretion of triglyceride through the formation of lipoproteins containing large hydrophobic cores enriched with triglycerides.

Regulation of cholesterol-7alpha-hydroxylase: BAREly missing a SHP.

Cholesterol-7alpha-hydroxylase (CYP7A1) regulates the pathway through which cholesterol is converted into bile acids. The unique detergent properties of bile acids are essential for the digestion and intestinal absorption of hydrophobic nutrients. Bile acids have potent toxic properties (e.g., membrane disruption) and there are a plethora of mechanisms to limit their accumulation in blood and tissues. The discovery of farnesoid X receptor (FXR), the nuclear receptor activated specifically by bile acids, has opened new insights into these mechanisms. Bile acid activation of FXR has been shown to repress the expression of CYP7A1 via increasing the expression of small heterodimer partner (SHP), a non-DNA binding protein. The increased abundance of SHP causes it to associate with liver receptor homolog (LRH)-1, an obligate factor required for transcription of CYP7A1. Recent studies show there is an "FXR/SHP-independent" mechanism that also represses CYP7A1 expression. This "FXR/SHP-independent" pathway involves the interaction of bile acids with liver macrophages (i.e., Kupffer cells), which induces the expression, and secretion of cytokines. These inflammatory cytokines, which include tumor necrosis factor alpha and interleukin-1beta, act upon liver parenchymal cells causing a rapid repression of the CYP7A1 gene.

Expression of cholesterol-7alpha-hydroxylase in murine macrophages prevents cholesterol loading by acetyl-LDL.

Unlike macrophages, the hepatic parenchymal cells express cholesterol-7 alpha-hydroxylase (CYP7A1) which regulates the conversion of cholesterol into bile acids, the major quantitative pathway maintaining cholesterol homeostasis. We examined if CYP7A1 expression in RAW 264.7 macrophages could prevent the accumulation of cholesterol when they were incubated with acetyl-LDL. A single cell clone (designated as 7 alphaRAW cells) that stably expresses rat CYP7A1 displayed similar rates of growth as non-transfected RAW cells. The CYP7A1 enzymatic activity expressed by microsomes obtained from 7 alphaRAW cells was similar to that expressed by microsomes obtained from mouse liver. Incubating non-transfected RAW with increasing amounts of acetyl-LDL caused a parallel accumulation of cholesterol, whereas 7 alphaRAW cells displayed a complete resistance to cholesterol accumulation. 7 alphaRAW cells displayed increased expression of both ABCA1 mRNA (3.1-fold, P < 0.001) and ABCG1 mRNA (2.2-fold, P < 0.01), whereas the expression of scavenger receptor class A mRNA was unchanged. 7 alphaRAW cells also displayed small but significant increases in the rate of efflux of [(3)H]cholesterol to both delipidated apolipoprotein A1 and to HDL.Thus, CYP7A1 expression in RAW cultured macrophages prevented the accumulation of cholesterol from acetyl-LDL via both increased metabolism and efflux of cholesterol.

Transgenic expression of cholesterol-7-alpha-hydroxylase prevents atherosclerosis in C57BL/6J mice.

C57BL/6J mice are susceptible to atherosclerosis when fed a diet consisting of fat, cholesterol, and taurocholate. The susceptibility to diet-induced atherosclerosis is linked to a reduction in plasma high density lipoprotein (HDL). Diet-induced reduction of plasma HDL shows a physiological and a genetic correlation with repression of cholesterol-7-alpha-hydroxylase, the liver-specific enzyme that regulates the conversion of cholesterol into bile acids. To examine the hypothesis that the repression of cholesterol-7-alpha-hydroxylase is responsible for initiating the metabolic alterations leading to the formation of atherosclerosis and gallstones, we determined whether constitutive transgenic expression of cholesterol-7-alpha-hydroxylase in C57BL/6J mice would confer resistance to these 2 common human diseases. When fed the atherogenic diet, nontransgenic littermates, but not cholesterol-7-alpha-hydroxylase transgenic mice, accumulated cholesterol and cholesterol esters in their livers and plasma. Although the atherogenic diet caused a marked decrease in plasma HDL cholesterol in nontransgenic mice, HDL levels in transgenic mice remained relatively unchanged. Moreover, the ability of cholesterol-7-alpha-hydroxylase transgenic mice to maintain cholesterol and lipoprotein homeostasis completely prevented the formation of atherosclerosis and gallstones. These data establish the integral role that cholesterol-7-alpha-hydroxylase has in maintaining hepatic cholesterol homeostasis and, thus, in the susceptibility to the formation of gallstones and atherosclerosis.