Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice.

Obesity increases risk for liver toxicity by the anti-leukemic agent asparaginase, but the mechanism is unknown. Asparaginase activates the integrated stress response (ISR) via sensing amino acid depletion by the eukaryotic initiation factor 2 (eIF2) kinase GCN2. The goal of this work was to discern the impact of obesity, alone versus alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice. Following diet-induced obesity, biochemical analysis of livers revealed that asparaginase provoked hepatic steatosis that coincided with activation of another eIF2 kinase PKR-like endoplasmic reticulum kinase (PERK), a major ISR transducer to ER stress. Genetic loss of Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 failed to increase. Instead, mechanistic target of rapamycin complex 1 (mTORC1) signal transduction was unleashed, and this coincided with liver dysfunction reflected by a failure to maintain hydrogen sulfide production or apolipoprotein B100 (ApoB100) expression. In contrast, obese mice lacking hepatic activating transcription factor 4 (Atf4) showed an exaggerated ISR and greater loss of endogenous hydrogen sulfide but normal inhibition of mTORC1 and maintenance of ApoB100 during asparaginase exposure. In both genetic mouse models, expression and phosphorylation of Sestrin2, an ATF4 gene target, was increased by asparaginase, suggesting mTORC1 inhibition during asparaginase exposure is not driven via eIF2-ATF4-Sestrin2. In conclusion, obesity promotes a maladaptive ISR during asparaginase exposure. GCN2 functions to repress mTORC1 activity and maintain ApoB100 protein levels independently of Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway.

GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment.

The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone fibroblast growth factor 21 (FGF21) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF21 and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF21 and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis.

Loss of ß-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication.

Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles, where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets, cholesterol cannot be trafficked to replication organelles, and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral life cycle and host cell cholesterol homeostasis.

Over-expression of monoacylglycerol lipase (MGL) in small intestine alters endocannabinoid levels and whole body energy balance, resulting in obesity.

The function of small intestinal monoacylglycerol lipase (MGL) is unknown. Its expression in this tissue is surprising because one of the primary functions of the small intestine is to convert diet-derived MGs to triacylglycerol (TG), and not to degrade them. To elucidate the function of intestinal MGL, we generated transgenic mice that over-express MGL specifically in small intestine (iMGL mice). After only 3 weeks of high fat feeding, iMGL mice showed an obese phenotype; body weight gain and body fat mass were markedly higher in iMGL mice, along with increased hepatic and plasma TG levels compared to wild type littermates. The iMGL mice were hyperphagic and displayed reduced energy expenditure despite unchanged lean body mass, suggesting that the increased adiposity was due to both increased caloric intake and systemic effects resulting in a hypometabolic rate. The presence of the transgene resulted in lower levels of most MG species in intestinal mucosa, including the endocannabinoid 2-arachidonoyl glycerol (2-AG). The results therefore suggest a role for intestinal MGL, and intestinal 2-AG and perhaps other MG species, in whole body energy balance via regulation of food intake as well as metabolic rate.

Phosphatidate phosphatase activity plays key role in protection against fatty acid-induced toxicity in yeast.

The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity.

Diabetic dyslipidemia and exercise alter the plasma low-density lipoproteome in Yucatan pigs.

Although low-density lipoprotein (LDL) plays a predominant role in atherogenesis, the low-density lipoproteome has not been fully characterized. Moreover, alterations from a Western diet, diabetes, and physical inactivity on this proteome have yet to be determined. Accordingly, relative quantification was determined in LDL proteins from male Yucatan diabetic dyslipidemic (DD) swine in the early stages of atherosclerosis compared to healthy control (C) and non-diabetic hyperlipidemic (H) swine. Importantly, coronary vascular dysfunction was prevented by aerobic exercise training in these animals (DDX) without altering total LDL concentration. Using 2-DE, Western blot, label-free quantitative MS, and selected reaction monitoring, alterations in the abundance of apolipoproteins A-I, B, C-III, D, E, and J and noncovalently associated proteins were determined in LDL isolated using fast protein liquid chromatography. At least 28 unique proteins, many of which were novel, were identified with high confidence. An apolipoprotein E isoform demonstrated stronger correlation to disease (percent of coronary artery segments with intimal thickening) than some traditional risk factors (total cholesterol, LDL cholesterol, and LDL/HDL cholesterol). Taken together, this work identifies new possible biomarkers, potential therapeutic targets for atherosclerosis, and generates new hypotheses regarding the role of LDL in atherogenesis.

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells.

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.

Intestinal monoacylglycerol metabolism: developmental and nutritional regulation of monoacylglycerol lipase and monoacylglycerol acyltransferase.

Intestinal monoacylglycerol (MG) metabolism is well known to involve its anabolic reesterification to triacylglycerol (TG). We recently provided evidence for enterocyte MG hydrolysis and demonstrated expression of the monoacylglycerol lipase (MGL) gene in human intestinal Caco-2 cells and rodent small intestinal mucosa. Despite the large quantities of MG derived from dietary TG, the regulation of MG metabolism in the intestine has not been previously explored. In the present studies, we examined the mRNA expression, protein expression, and activities of the two known MG-metabolizing enzymes, MGL and MGAT2, in C57BL/6 mouse small intestine, as well as liver and adipose tissues, during development and under nutritional modifications. Results demonstrate that MG metabolism undergoes tissue-specific changes during development. Marked induction of small intestinal MGAT2 protein expression and activity were found during suckling. Moreover, while substantial levels of MGL protein and activity were detected in adult intestine, its regulation during ontogeny was complex, suggesting post-transcriptional regulation of expression. In addition, during the suckling period MG hydrolytic activity is likely to derive from carboxyl ester lipase rather than MGL. In contrast to intestinal MGL, liver MGL mRNA, protein and activity all increased 5-10-fold during development, suggesting that transcriptional regulation is the primary mechanism for hepatic MGL expression. Three weeks of high fat feeding (40% kcal) significantly induced MGL expression and activity in small intestine relative to low fat feeding (10% kcal), but little change was observed upon starvation, suggesting a role for MGL in dietary lipid assimilation following a high fat intake.

Overexpression of human diacylglycerol acyltransferase 1, acyl-coa:cholesterol acyltransferase 1, or acyl-CoA:cholesterol acyltransferase 2 stimulates secretion of apolipoprotein B-containing lipoproteins in McA-RH7777 cells.

The relative importance of each core lipid in the assembly and secretion of very low density lipoproteins (VLDL) has been of interest over the past decade. The isolation of genes encoding diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) provided the opportunity to investigate the effects of isolated increases in triglycerides (TG) or cholesteryl esters (CE) on apolipoprotein B (apoB) lipoprotein biogenesis. Overexpression of human DGAT1 in rat hepatoma McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of TG. These effects were associated with decreased intracellular degradation and increased secretion of newly synthesized apoB as VLDL. Similarly, overexpression of human ACAT1 or ACAT2 in McA-RH7777 cells resulted in increased synthesis, cellular accumulation, and secretion of CE. This led to decreased intracellular degradation and increased secretion of VLDL apoB. Overexpression of ACAT2 had a significantly greater impact upon assembly and secretion of VLDL from liver cells than did overexpression of ACAT1. The addition of oleic acid (OA) to media resulted in a further increase in VLDL secretion from cells expressing DGAT1, ACAT1, or ACAT2. VLDL secreted from DGAT1-expressing cells incubated in OA had a higher TG:CE ratio than VLDL secreted from ACAT1- and ACAT2-expressing cells treated with OA. These studies indicate that increasing DGAT1, ACAT1, or ACAT2 expression in McA-RH7777 cells stimulates the assembly and secretion of VLDL from liver cells and that the core composition of the secreted VLDL reflects the enzymatic activity that is elevated.

Porcine model of diabetic dyslipidemia: insulin and feed algorithms for mimicking diabetes mellitus in humans.

A weakness of many animal models of diabetes mellitus is the failure to use insulin therapy, which typically results in severe body wasting. Data collected from such studies must be interpreted cautiously to separate the effects of hyperglycemia from those of starvation. We provide several algorithms that were used by us in two long-term (20-week) experiments in which hyperglycemia (300 to 400 mg/dl), dyslipidemia (cholesterol [280 to 405 mg/dl] and triglycerides [55 to 106 mg/dl] concentrations), and positive energy balance were maintained in swine. Yucatan miniature swine groups included control, alloxan-induced diabetes mellitus, diabetes mellitus plus diet-induced dyslipidemia, and exercise-trained diabetic dyslipidemic pigs. The algorithms were developed for the porcine model because of several similarities to humans, including: cardiac anatomy and physiology, propensity for sedentary behavior, and metabolism of dietary carbohydrates and lipids. Acute toxic effects of alloxan (hypoglycemia, hyperglycemia, nephrotoxicosis) were minimized by preventive fluid loading and by use of algorithms in which insulin, food, and fluid therapy were administered. Long-term insulin and food maintenance algorithms elicited normal body weight gain in all three diabetic groups (lean experiment) and threefold greater body weight gain in pigs of an obesity experiment. Exercise-trained pigs of both experiments manifested significantly increased work performance and did not experience medical complications. We conclude that these algorithms can be used in swine, or similar algorithms can be developed for other animal species to maintain hyperglycemia and/or dyslipidemia, while avoiding diabetes-induced wasting. Importantly, animal models of diabetes mellitus that maintain positive energy balance and poor glycemic control provide a marked improvement over other models by more closely mimicking the human presentation of diabetes mellitus.

Increased calcium buffering in coronary smooth muscle cells from diabetic dyslipidemic pigs.

No studies exist concerning the ability of the plasma membrane Ca(2+) pump (PMCA), sarcoplasmic reticulum Ca(2+) pump (SERCA) and Na(+)-Ca(2+) exchanger (NCX) to regulate myoplasmic Ca(2+) (Ca(m)) in vascular smooth muscle cells from diabetic individuals with dyslipidemia. We tested the hypothesis that diabetic dyslipidemia would increase vascular smooth muscle cells to buffer Ca(m). Cells were isolated from the coronary artery of male Yucatan pigs treated for 20 weeks with: (1) a low fat diet (control group); (2) a high fat/cholesterol diet (F group); or (3) alloxan-induced diabetic pigs fed the high fat diet (DF group). The maximum Ca(m) response to a depolarizing 80 mM KCl (80 K) solution was evaluated in the absence and presence of thapsigargin (TSG; inhibits SERCA) and low Na (inhibits NCX). In response to 80 K alone, there was no difference in the Ca(m) response between groups. In the presence of TSG, the 80 K response decreased by 43% in the DF group; TSG did not affect the 80 K response in the control and F groups. When exposed to both TSG and low Na, the 80 K response also decreased by 55% in the DF group. This suggests increased Ca(m) buffering by the PMCA and/or mitochondria in the DF group when SERCA and NCX are inhibited. Compared to the control and F groups, low Na alone elicited a 50% lower Ca(m) amplitude in the DF group, which was reversed with TSG treatment; this suggests that SERCA activity is increased in DF pigs. Western blots also indicated a 7-fold increase in the approximately 115 kDa band density of an anti-SERCA2 antibody in DF compared to control pigs. This is the first report to demonstrate increased Ca(2+) buffering, specifically by SERCA, in vascular smooth muscle cells from diabetic individuals with dyslipidemia.

Increased atherosclerosis in diabetic dyslipidemic swine: protection by atorvastatin involves decreased VLDL triglycerides but minimal effects on the lipoprotein profile.

Male Yucatan swine were allocated to four groups (n = 5-6 pigs per group): low fat (3%) fed control, high fat/2% cholesterol (CH) fed (HF), high fat/CH fed with alloxan-induced diabetes (DF) and DF pigs that were treated with atorvastatin (80 mg/day; DF+A). Pigs were fed two meals per day and daily insulin injections were used in diabetic pigs to maintain plasma glucose between 250 and 350 mg/dl. Diabetic dyslipidemic (DF) pigs exhibited greater coronary atherosclerosis and increased collagen deposition in internal mammary artery compared with normoglycemic hyperlipidemic pigs. Although total and LDL CH concentrations did not differ, triglyceride (TG) were increased in DF pigs and FPLC analysis indicated that the LDL/HDL CH ratio was significantly increased in DF compared with HF pigs. The LDL fraction of DF pigs contained larger, lipid enriched particles resembling IDL. Consumption of the high fat/CH diet caused a moderate increase in the percentage of 14:0 fatty acids in plasma lipids and this was compensated by small-moderate declines in several unsaturated fatty acids. There was a significant increase in phospholipid arachidonic acid in DF compared with HF pigs. Atorvastatin protected diabetic pigs from atherosclerosis and decreased total and VLDL TG, but exerted minimal effects on the FPLC lipoprotein and plasma fatty acid profiles and plasma concentrations of total and LDL CH, vitamin A, vitamin E, and lysophosphatidylcholine. Across all groups the plasma CH concentration was positively correlated with hepatic CH concentration. These findings suggest that atorvastatin's protection against coronary artery atherosclerosis in diabetes may involve effects on plasma VLDL TG concentration. Lack of major effects on other lipid parameters, including the LDL/HDL ratio, suggests that atorvastatin may have yet other anti-atherogenic effects, possibly directly in the vessel wall.

Apolipoprotein B is synthesized in selected human non-hepatic cell lines but not processed into mature lipoprotein.

We studied apolipoprotein B100 (apoB) metabolism in a series of non-hepatic cell lines (HT29 colon adenocarcinoma, HeLa cervical epithelioid carcinoma, and 1321N1J astrocytoma human cell lines) and in the human hepatoma cell line HepG2. ApoB mRNA was detected by reverse transcription polymerase chain reaction in each non-hepatic cell line. ApoB was detected in HepG2 cells by immunoprecipitation, Western blotting, and immunocytochemistry using a polyclonal anti-human low-density lipoprotein (LDL) antibody, an anti-human apoB peptide antibody, and several monoclonal anti-apoB antibodies. ApoB was identified in the three non-hepatic cell lines by each method using the anti-apoB peptide and monoclonal antibodies, but not with the anti-LDL antibody. Immunocytochemistry indicated that epitopes of apoB were evident throughout the endoplasmic reticulum, and gel mobility of newly labeled apoB and immunoblot with anti-ubiquitin showed that apoB was highly ubiquinated in non-hepatic cells. The observations that apoB is synthesized in non-hepatic cell lines but never recognized by the anti-LDL antibody suggests that apoB is not processed into a nascent lipoprotein in these cells. Immunocytochemical localization of apoB epitopes at many locations throughout non-hepatic cells raises the exciting possibility that apoB can be used for other purposes in these cells.