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The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor α (LXRα). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor α (PPARα)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARα-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARα-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARα were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRα was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARα deficiency was abolished during re-feeding. However, although re-feeding the LXRα ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARα-deficient mice. We conclude that intact PPARα is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRα.

Peroxisome proliferator-activated receptor alpha (PPAR alpha)-null mice were used to investigate the nature of the relationship between the normal circadian rhythm of hepatic PPAR alpha expression and the expression of the lipogenic and cholesterogenic sterol regulatory element-binding protein (SREBP)-regulated genes, acetyl-CoA carboxylase, fatty acid synthase (FAS), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR). The expression of FAS and HMG-CoAR varied rhythmically over the diurnal cycle in the normal mice, with patterns that were the opposite of that of PPAR alpha. The diurnal variation of lipogenic and cholesterogenic gene expression was attenuated or abolished in the PPAR alpha-null mice. This resulted in decreased expression compared with normal mice, but only during the dark phase of the cycle, when food intake was high. The diurnal variation in hepatic fatty acid and cholesterol synthesis was also abolished in the PPAR alpha-null animals and the variations in the concentration of plasma triacylglycerol, nonesterified fatty acids, and cholesterol were all attenuated. The failure of HMG-CoAR expression to increase during the feeding period in the PPAR alpha-null mice was associated with a decrease in hepatic nonesterified cholesterol content and an increase in cholesteryl ester compared with normal mice. There was no defect in the downregulation of hepatic HMG-CoAR mRNA in response to dietary cholesterol in the PPAR alpha-null mice. Under these conditions, hepatic PPAR gamma expression increased in both the control and PPAR alpha-deficient mice. The results suggest that PPAR alpha-deficiency disturbs the normal circadian regulation of certain SREBP-sensitive genes in the liver, but does not affect their response to dietary cholesterol. -- Patel, D. D., B. L. Knight, D. Wiggins, S. M. Humphreys, and G. F. Gibbons. Disturbances in the normal regulation of SREBP-sensitive genes in PPAR alpha-deficient mice. J. Lipid Res. 2001. 42: 328--337.

Clones of HepG2 cells were selected that stably express the cDNA for hormone-sensitive lipase (HSL). When cells were cultured in the presence of labelled extracellular oleate, accumulation of labelled fatty acid as cellular triacylglycerol (TAG) was significantly lower in the transfectants compared with the wild-type cells. There was no change in the net rate of phospholipid (PL) synthesis. Culture of cells containing isotopically prelabelled TAG resulted in a greater net loss of TAG from the transfected cells than from the wild-type cells. The excess loss of labelled TAG was primarily due to an increased TAG fatty acid oxidation. Free fatty acid release into the medium was not increased in the transfectants, nor was the very low rate of lipoprotein lipid secretion. Also, there was no increased net trafficking of fatty acids from TAG into PLs. Changes in the 3H:14C ratio of TAG prelabelled with [3H]glycerol and [14C]oleate suggested that none of excess TAG fatty acid released in the transfected cells underwent intracellular re-esterification to TAG prior to oxidation. The results suggest that fatty acids mobilized by HSL are directed immediately into the oxidative pathway and are not available for biosynthetic processes. It appears likely, therefore, that intracellular TAG-derived fatty acids which enter the oxidative pathway exist in a different compartment from those that are directed towards synthesis.

Abstract —Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35 S methionine into newly synthesized apoB ( P P

Weanling Lewis rats were fed for 10 weeks on a low fat (2.5% by weight; LF) diet or on diets containing 20% by weight of hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), evening primrose oil (EPO), or menhaden (fish) oil (MO); all other components of the diets were identical. The chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions were isolated from the serum. The serum from MO-fed animals had lower LDL and HDL cholesterol concentrations than the serum from animals fed each of the other diets. The apolipoprotein A-1 concentration in the HDL fraction from animals fed the MO diet was also low. The serum from HCO-fed animals had a higher CM triacylglycerol concentration than serum from animals fed each of the other diets. The serum from OO-fed animals had a higher VLDL triacylglycerol concentration than serum from animals fed each of the other diets. The concentration of apolipoprotein B was also high in the VLDL fraction from OO-fed animals. The fatty acid composition of each lipoprotein fraction was affected by the nature of the lipid in the diet; the greatest effects were observed in the CM and VLDL fractions. Each lipoprotein fraction isolated from LF-fed rats inhibited mitogen-stimulated rat spleen lymphocyte proliferation in a concentration-dependent manner; LDL and HDL caused greater inhibition than CM and VLDL. Dietary lipid manipulation did not alter the inhibitory effects of any of the lipoprotein fractions upon lymphocyte proliferation, except that CM and HDL from MO-fed animals and HDL from OO-fed animals resulted in enhanced proliferation compared with either CM or HDL from animals fed the other diets. We conclude that the inhibition of lymphocyte proliferation caused by feeding rats certain dietary lipids (OO, EPO, MO) may be mediated by non-lipoprotein serum components.

Hepatocytes were prepared from rats fed a chow diet (control-fed) and from rats fed a similar diet in which the drinking water contained 10% (wt/vol) fructose (fructose-fed). Both types of hepatocyte preparations were cultured for < or = 48 hours in supplemented Waymouth's medium containing increasing concentrations of bovine insulin (0 to 780 nmol/L). During the first 24 hours of culture, hepatocytes from fructose-fed rats secreted more very-low-density lipoprotein (VLDL) triacylglycerol (TAG) than hepatocytes from control-fed rats. This difference persisted at all concentrations of insulin. There was no difference in the rate of secretion of apolipoprotein B (apo B). In both control-fed and fructose-fed animals, the inhibitory effect of insulin on the secretion of VLDL was greater on the second versus the first day of culture. Under these conditions, hepatocytes from fructose-fed groups were less sensitive to insulin inhibition as compared with those from the control-fed group. This was evidenced by the following: (1) the decreased inhibitory effect of insulin on the secretion of both total and newly synthesized VLDL TAG, (2) the attenuated inhibitory effect of insulin on the secretion of VLDL apo B, (3) the decreased potency of insulin in suppressing the secretion of VLDL TAG in TAG-depleted hepatocytes from fructose-fed as compared with control-fed animals, and (4) the larger proportion of newly synthesized TAG secreted as VLDL in hepatocytes from fructose-fed rats as compared with controls. This difference was exacerbated at higher concentrations of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular fatty acids entering the hepatocyte are either esterified to cytosolic TAG or oxidized to ketone bodies. Very little is esterified and secreted directly in association with VLDL. Thus, even when extracellular fatty acids are available, the major, direct source of VLDL TAG is the cytosolic pool. The recruitment of cytosolic TAG for VLDL assembly involves lipolysis followed by re-esterification. At least 70% of the secreted TAG is derived via this route. Fatty acids released at this lipolytic step are utilized exclusively for VLDL TAG synthesis and are not available for ketogenesis. Substantially more cytosolic TAG undergoes lipolysis than is required to meet the needs of VLDL assembly. The remaining fatty acids are re-esterified and re-cycled to the cell cytosol. From a physiological viewpoint, the presence of this indirect route for VLDL TAG recruitment would provide a means of regulation of VLDL secretion which is independent of the plasma fatty acid concentration. In this respect, several pathophysiological conditions are known in which there is a negative association between plasma fatty acid concentration and the rate of VLDL secretion. These are: (a) insulin-dependent diabetes, (b) starvation, (c) fat-feeding. Lipolysis of cytosolic TAG and transfer of fatty acids into the ER lumen may provide a regulatory focus for the control of hepatic VLDL output.

Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used anti-breast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a short-term treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMR-based metabolite analysis, we found that TMX-treated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly down-regulates FAS expression and activity as indicated by the accumulation of malonyl-CoA, a known inhibitor of mitochondrial beta-oxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonyl-CoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis.

Inclusion of the PPARα (peroxisome-proliferator-activated receptor α) activator WY 14,643 in the diet of normal mice stimulated the hepatic expression of not only genes of the fatty acid oxidation pathway, but also those of the de novo lipid synthetic pathways. Induction of fatty acid synthase mRNA by WY 14,643 was greater during the light phase of the diurnal cycle, when food intake was low and PPARα expression was high. Hepatic fatty acid pathway flux in vivo showed a similar pattern of increases. The abundance of mRNAs for genes involved in hepatic cholesterol synthesis was also increased by WY 14,643, but was associated with a decrease in cholesterogenic carbon flux. None of these changes were apparent in PPARα-null mice. Mice of both genotypes showed the expected decreases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels and cholesterol synthesis in response to an increase in dietary cholesterol. The increase in fatty acid synthesis due to WY 14,643 was not mediated by increased expression of SREBP-1c (sterol regulatory element binding protein-1c) mRNA, but by an increase in cleavage of the protein to the active form. An accompanying rise in stearoyl-CoA desaturase mRNA expression suggested that the increase in lipogenesis could have resulted from an alteration in membrane fatty acid composition that influenced SREBP activation.

Several missense mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor (PPAR)gamma have been described in subjects with dominantly inherited severe insulin resistance associated with partial lipodystrophy, hypertension, and dyslipidemia. These mutant receptors behave as dominant-negative inhibitors of PPARgamma signaling when studied in transfected cells. The extent to which such dominant-negative effects extend to signaling through other coexpressed PPAR isoforms has not been evaluated. To examine these issues further, we have created a PPARalpha mutant harboring twin substitutions, Leu459Ala and Glu462Ala, within the ligand binding domain (PPARalpha(mut)), examined its signaling properties, and compared the effects of dominant-negative PPARalpha and PPARgamma mutants on basal and ligand-induced gene transcription in adipocytes and hepatocytes. PPARalpha(mut) was transcriptionally inactive, repressed basal activity from a PPAR response element-containing promoter, inhibited the coactivator function of cotransfected PPAR-gamma coactivator 1alpha, and strongly inhibited the transcriptional response to cotransfected wild-type receptor. In contrast to PPARgamma, wild-type PPARalpha failed to recruit the transcriptional corepressors NCoR and SMRT. However, PPARalpha(mut) avidly recruited these corepressors in a ligand-dissociable manner. In hepatocytes and adipocytes, both PPARalpha(mut) and the corresponding PPARgamma mutant were capable of inhibiting the expression of genes primarily regulated by PPARalpha, -gamma, or -delta ligands, albeit with some differences in potency. Thus, dominant-negative forms of PPARalpha and PPARgamma are capable of interfering with PPAR signaling in a manner that is not wholly restricted to their cognate target genes. These findings may have implications for the pathogenesis of human syndromes resulting from mutations in this family of transcription factors.

Dietary supplementation with the peroxisome proliferator-activated receptor alpha (PPAR alpha) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPAR alpha-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor alpha (LXR alpha). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPAR alpha influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXR alpha.

Hamster hepatocytes, like human hepatocytes, secrete triacylglycerol (TAG) as very-low-density lipoprotein (VLDL) in association with apolipoprotein (apo) B100, whereas in the rat, TAG is secreted predominantly in association with apoB48. Nevertheless, in hepatocytes from both species, a minimum of between 60% and 70% [69. 1+/-1.4% (hamster), 60.6+/-2.5% (rat)] of the VLDL TAG was secreted following lipolysis and re-esterification of intracellular TAG. The fractional rates of hepatocellular TAG turnover (lipolysis and re-esterification) were similar in both species [1.83+/-0.28 pools/24 h (hamster), 1.39+/-0.23 pools/24 h (rat)]. Comparison of the relative changes in the 3H and 14C specific radioactivities of the VLDL and cellular TAG, pre-labelled with [3H]glycerol and [4C]oleate, suggested that fatty acids released by lipolysis either were recruited directly into a VLDL assembly pool or were recycled to the cellular pool following re-esterification. Recycling in the hamster was somewhat greater than in the rat (66.1+/-5.7% versus 53. 7+/-4.8% of TAG lipolysed respectively). Similarly, a larger proportion of newly synthesized TAG was retained within the cell, rather than secreted as VLDL, in the hamster compared with the rat (37.9+/-2.8% versus 20+/-3.8%, P

The effects of insulin (0-780 nM) on the secretion of very-low-density lipoprotein (VLDL) apolipoprotein B (apoB) and triacylglycerol (TAG) in hepatocytes from obese Zucker rats and from their lean littermates were studied over a total period of 48 h in chemically defined culture medium. Cells from the obese Zucker rats initially secreted more TAG than those from the lean animals. Cells from the former were resistant to the inhibitory effect of insulin on the secretion of TAG. These changes were accompanied by an increased rate of TAG synthesis. Although the hepatocytes from the obese animals initially secreted less apoB than those from the lean, apoB output from the former could not be suppressed by insulin. Prolonging the length of the culture period resulted in the acquisition of sensitivity to the inhibitory effect of insulin in the hepatocytes from the obese rats. This occurred more rapidly for the secretion of TAG than of apoB. Under these conditions, the initial difference in the rate of TAG synthesis in the hepatocytes from the obese and from the lean animals was also abolished.

1. 1. Urate synthesis was measured in hepatocytes from chickens after starvation or high-protein feeding. Adaptation occurred only on the high-protein diet. 2. 2. The theoretical balances of reactions from alanine ( 5 alanine + 3 O 2 = urate + 1.5 glucose + glycine ) and asparagine ( 3 asparagine + 2 O 2 = urate + ammonia + 0.5 glucose + glycine ) agree reasonably well with the experimental results. 3. 3. Enzymes directly involved in urate synthesis from these amino acids increase up to 12-fold on the high-protein diet; only amidophosphoribosyltransferase activity appears to be rate-limiting for urate synthesis. 4. 4. The processes of nitrogen disposal in chicken and rat are compared and discussed.

Umbilical blood venous-arterial differences for amino acids across the rat fetus show uptake of disproportionately large amounts of glutamine, alanine and lysine relative to their contribution to total body protein. Free amino acid concentrations in fetal liver tend to increase during development, but show a rapid adjustment to near-adult values within 24 h after birth.

Suspensions of rat hepatocytes are resistant to hypoxia for at least 24 hr at 4 degrees C. After storage for 24 hr with xylitol, sorbitol, or glycerol, their subsequent capacity for glucogenesis from these substrates is increased. Even storage under N2/CO2 as the gas phase has little deleterious effect.

The chelating agents (EGTA and EDTA) and inorganic phosphate (Pi) are the most variable components of experiments involving isolated liver mitochondria. In the absence of EGTA or EDTA, swelling induced by Pi leads to rapid loss of endogenous adenine nucleotides to adenosine. Chelating agents prevent swelling and loss of adenine nucleotides. Concentrations below about 0.1 mM are ineffective. The protective effects depend on the continuous presence of the chelating agent; they are lost on washing EGTA-containing suspensions with chelating-agent-free medium. We question the accepted view that chelating agents stabilize mitochondria by binding Ca2+ to prevent activation of phospholipase.

Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine.