Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA.

The plasma cholesteryl ester transfer protein (CETP) catalyzes the transfer of cholesteryl esters from high density lipoproteins (HDL) to triglyceride-rich lipoproteins and plays a major role in the catabolism of HDL. Lipoprotein lipase (LPL) is the rate-limiting enzyme for hydrolysis of circulating triglyceride and is involved in HDL formation. We show that tissues containing LPL are major sources of CETP mRNA in several mammalian species, including some with low cholesteryl ester transfer activity in plasma. In hamsters, adipose tissue and heart were found to be the richest sources of both CETP and LPL mRNA; in situ hybridization studies showed that the same cell types (i.e. adipocytes or myocytes) contained CETP and LPL mRNA in these tissues. Isolated adipocytes synthesized active CETP. Dietary studies revealed a complex pattern of response of CETP mRNA levels in different tissues, which showed partial similarity to the changes in LPL mRNA abundance. However, high cholesterol diets resulted in increased CETP mRNA abundance in adipose tissue, heart, and skeletal muscle, without equivalent changes in LPL mRNA. Plasma HDL cholesteryl ester levels showed strong inverse correlations with CETP mRNA abundance in adipose tissue. The results suggest a conserved function of CETP in adipose tissue and heart, such as a co-ordinate action with LPL to enhance HDL turnover. Although there is considerable overlap in the tissue- and cell-specific pattern of CETP and LPL gene expression, dietary studies revealed only limited parallelism in response at the mRNA level. The increase in CETP mRNA in peripheral tissues in response to increased dietary cholesterol suggests that local induction of CETP synthesis may help to recycle cholesterol deposited in these tissues during lipolysis of dietary lipoproteins.

The effect of acarbose on the food intake, weight gain, and adiposity of LA/N-cp rats.

1. Groups of lean and obese LA/N-cp rats were administered the intestinal glucosidase inhibitor acarbose at 150 or 300 mg/kg diet from 7 until 17 weeks of age and the effects of the drug on food intake patterns and adiposity determined. 2. Dose related effects on body weights, adiposity and feed efficiency ratio were observed (control greater than 150 mg greater than 300 mg drug/kg diet) following treatment in both phenotypes, with the greatest differences observed in the obese phenotype. 3. Acarbose at both dosages was associated with phenotype-specific alterations in food intake amount and feeding pattern, resulting in an attenuation of age-associated increases in food intake. The feed efficiency ratio decreased in both phenotypes, and approached normally fed lean controls in obese rats administered the greater dosage. 4. These results indicate that patterns of food intake and weight gain differ markedly between lean and obese rats of this strain, and acarbose brings about a dose-related attenuation of developing food intake patterns in both phenotypes and which are associated with decreases in weight gain and adiposity. Thus, this drug may have therapeutic potential as an adjunct agent in the treatment of obesity and/or other disorders of carbohydrate intolerance.

The effects of the intestinal glucosidase inhibitor acarbose on cholesterogenesis in corpulent rats.

1. Groups of lean and obese LA/N-cp and obese Type II diabetic SHR/N-cp rats were fed semisynthetic diets with or without the alpha-glucosidase inhibitor acarbose (ACB, 100 mg/kg diet, p.o.) from 8 until 15 weeks of age, and measures of fasting serum total cholesterol (TC), insulin (INS), and hepatic HMG-CoA synthase activity determined at the end of the study. 2. ACB was without marked effect on mean food intake in either strain or either phenotype, and resulted in less weight gain and decreased adipose mass in obese LA/N-cp rats. INS was greater in the obese than the lean phenotype of both strains, and ACB resulted in greater reductions in INS in obese LA/N-cp than in obese LA/N-cp rats. 3. Serum TC concentrations were greater in the obese than in the lean phenotype of both strains, and ACB resulted in decreases in TC in both strains and in lower beta:alpha lipoprotein cholesterol ratios in obese LA/N-cp rats. Liver HMG Co-A synthase activity was greater in lean than obese rats and ACB resulted in normalization of enzyme activity in obese LA/N-cp but not SHR/N-cp rats. 4. These results confirm the hypercholesterolemia which occurs in the obese phenotype of the corpulent rat strains, and indicates that ACB may bring about significant reductions in body weight and fatness, TC, and in improved beta:alpha lipoprotein ratios and HMG-CoA synthase activity in obesity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acyl-CoA: cholesterol O-acyltransferase inhibition on cholesterol absorption and plasma lipoprotein composition in hamsters.

1. The ACAT inhibitors, CL 277082 and SA 58-035 were administered for 7 days to hamsters fed diets containing 0.5% cholesterol. 2. Both agents inhibited cholesterol absorption, decreased hepatic. VLDL and IDL cholesterol esters, plasma HDL and HDL apoE and A-I. 3. In addition, CL 277082 treatment produced significant decreases in plasma cholesterol, VLDL apoB and plasma IDL. 4. The cholesteryl esters in VLDL and LDL but not HDL were more polyunsaturated in CL 277082 treated animals. 5. These results support the hypothesis that ACAT inhibition in the cholesterol fed hamster results in an inhibition of dietary cholesterol absorption, thus limiting the cholesterol supply required for the hepatic production of triglyceride-rich lipoproteins.

Effects of phenotype, sex, and diet on plasma lipids in LA/N-cp rats.

The LA/N-corpulent (cp) rat is a recently developed congenic strain which exhibits obesity. The effects of phenotype and sex on serum and lipoprotein lipid content were examined in LA/N-cp rats fed either a control or an atherogenic diet high in saturated fat and protein. Obese rats were pair-fed to equivalent lean animals. Results from this study indicate that sex, phenotype, and diet exert significant effects on plasma and lipoprotein cholesterol content. Plasma cholesterol levels were higher in obese compared with lean rats, females than in males, and rats consuming the atherogenic diet compared with the control diet. Plasma and lipoprotein triglyceride levels were significantly increased only in obese compared with lean animals. The increased plasma cholesterol and triglyceride was observed primarily in the chylomicron and very low density lipoprotein fractions. Increased levels of plasma cholesterol were not a result of increased dietary cholesterol absorption or increased liver cholesterol biosynthesis. These data suggest that LA/N-cp rats can serve as a unique rodent model for the study of the interrelationships between hyperlipidemia, obesity, and coronary heart disease.

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity.

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.

Toxicity of palmitoyl glycerol to mice: hypothermia and reversal of the toxicity.

Adult and weanling mice kept at low ambient temperatures show an increased sensitivity to the toxicity of dietary rac-1(3)-palmitoyl glycerol. When fed the palmitoyl glycerol, mice less than 6 wk old show a pronounced hypothermia that is prevented by adding safflower oil to the diet. A more moderate degree of hypothermia is seen with older animals. Once body temperature fell below 28 degrees C, replacing the toxic monoacylglycerol with safflower oil and/or raising the environmental temperature to 34 degrees C did not reverse the ultimate fatality caused by palmitoyl glycerol ingestion. If hypothermia was between 28 and 32 degrees C, high mortality was not reversed by feeding the unsaturated fat or raising the environmental temperature to 34 degrees C. However, a combination of both treatments reduced the mortality. Irrespective of body temperature, the hypothermia was eliminated by the warm ambient temperature, but mortality was high. Thus, although hypothermia is a sign of the toxicity of rac-1(3)-palmitoyl glycerol, it is not the immediate cause of death.

Characterization of HMG CoA synthase activity of rat liver and CHO-K1 cells.

This report describes the characterization and partial purification of rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase activity. A preliminary characterization of Chinese hamster ovary (CHO) cell HMG CoA synthase activity is also presented. Ion-exchange chromatography of ammonium sulfate precipitates of rat liver cytosol indicate the existence of two isoenzymes of HMG CoA synthase. These isoenzymes are physically, catalytically, and immunologically distinct. One of these isoenzymes, peak 1, resembles mitochondrial HMG-CoA synthase activity as evidenced by similarities in elution upon ion-exchange chromatography, inhibition by MgCl2, and cross reactivity with an antibody prepared against the mitochondrial enzyme. As peak 1 activity is unstable, further purification studies were performed on peak 2 activity. Peak 2 can be further resolved into two activities (peaks 2A and 2B) by gel filtration. In contrast, CHO-K1 cells (a permanent fibroblast line) possess only peak 2 type HMG CoA synthase activity.

The effect of mevinolin on cytosolic acetoacetyl coenzyme a thiolase activity in Chinese hamster ovary fibroblasts.

The effects of mevinolin on cytosolic acetoacetyl CoA thiolase activity were studied in wild type Chinese hamster ovary fibroblasts and in CHO cells adapted to growth in high levels of mevinolin. Acetoacetyl CoA thiolase, HMG CoA synthase and HMG CoA reductase activities were elevated in the mevinolin resistant line, KH 2.0. Thiolase activity was also increased when wild type cells were incubated for 5 days with 1 micron mevinolin. These results are consistent with the hypothesis that the regulation of the first three enzymes in the cholesterol biosynthetic pathway is mediated at least in part via a common mechanism.

A somatic cell mutant with a kinetically altered 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A somatic cell mutant has been isolated which is resistant to killing and growth inhibition by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The resistance phenotype is dominant with respect to the wild-type cell and can largely be ascribed to a 6-7-fold lowering of the KM for HMG-CoA. We thus conclude that mevinolin resistance can be utilized to obtain a genetic marker for the structural gene encoding HMH-CoA reductase.

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts.

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.

Analysis of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant auxotrophic for mevalonate.

Regulation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and synthesis by 25-hydroxycholesterol is aberrant in a somatic cell mutant of the Chinese hamster ovary K1 cell auxotrophic for mevalonate by virtue of a defect in HMG-CoA synthase activity. Normal regulation of HMG-CoA reductase activity and synthesis in this mutant by 25-hydroxycholesterol can be restored by simultaneous incubation with a small (0.4 mM) mevalonate supplement. Normal regulation of HMG-CoA reductase is also observed in a revertant of the mutant cell with normal HMG-CoA synthase activity.

Isolation and characterization of a mammalian cell mutant defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 cell auxotrophic for mevalonic acid has been isolated by means of the bromodeoxyuridine-visible light technique. This mutant can incorporate labeled mevalonate but not labeled acetate into cholesterol and, thus, is apparently defective in mevalonate biosynthesis. The mutant is recessive with respect to the parental cell phenotype. Assessment of the in vitro activities of the enzymes responsible for mevalonate biosynthesis under varying growth conditions indicates that the mutant, Mev-1, is defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

Effect of fluoride on the absorption of dietary fat in rats.

The inhibition of lipid absorption by sodium fluoride in adult male rats was investigated following intraduodenal infusion of labeled lipid and cannulation of the intestinal lymphatics. A striking delay in both the appearance and total amount of lymph radioactivity was observed when 26 mM fluoride was included in the infusate. Effective inhibition of lipid absorption was observed over a 15-fold wide range of fluoride concentration (3.3-56 mM). The inhibitory effect of fluoride was not due to an inhibition of lipolysis since the absorption of free palmitic acid was inhibited to the same extent as that of tripalmitoyl glycerol. Studies on the uptake and distribution of infused lipid in the upper small intestine an experiments examining the extent of fluoride-induced absorptive inhibition of different fatty acids suggest that the effect of fluoride may be on the mucosal reesterification of dietary lipid.

Interstitial pneumonitis induced by ingestion of palmitoyl glycerol.

Weanling male mice fed rac-1(3)-palmitoyl glycerol at levels of 30 mmoles/100 g of diet or higher develop, within a few days, a severe pulmonary inflammation characterized by marked infiltration of the interstitium by macrophages and few polymorphonuclear leukocytes. This results in severe vascular stasis, alveolar collapse and death of the animal. Adult mice and weanling rats also show the syndrome, but only at higher levels of palmitoyl glycerol. Neither the position of palmitate on the glycerol nor the level of myo-inositol in the diet affects the toxicity of palmitoyl glycerol. Supplementation of the diet with small amounts of linoleate or oleate prevents the toxicity although oleate is less effective than linoleate. There are no differences between mice fed linoleate and those that were not in: the rate of absorption of palmitoyl glycerol, oxidative phosphorylation by liver or heart mitochondria, excretion of carbon dioxide and tissue distribution of radioactivity following gavage of rac-1(3)-[1-14C]palmitoyl glycerol.

Toxicity of rac-1(3)-palmitoyl glycerol in weanling mice.

Reinvestigation of the previously reported toxicity of saturated fat on weanling mice has shown rac-1(3)-palmitoyl glycerol to be a more potent toxic agent than free palmitic acid when fed as the sole source of dietary fat. As shown before, protection against this toxicity can be afforded by the addition of 2 to 4% safflower oil. We have now shown that if the rac-1(3)-palmitoyl glycerol is acetylated the toxicity is much less. The protective effect of diacetyl rac-1(3)-palmitoyl glycerol cannot be totally ascribed either to the presence of acetate itself or to the blocking of the free hydroxyls of palmitoyl glycerol by acylation. In vivo absorption studies coupled with in vitro experiments with pancreatic lipase suggest that the major protective effects result from increased lipolysis of the acylated palmitoyl glycerol, causing conversion to the less toxic free palmitic acid.