Hepatic enzyme induction patterns and phenothiazine side effects.

D-Glucaric acid excretion was followed in psychotic patients treated with phenothiazines for 12 days and in a control group of subjects who had no psychiatric disease. About half the psychiatric patients had a treatment-related rise in D-glucaric acid excretion compatible with enzyme induction. These patients had fewer and less severe neurologic side effects than those who did not have a significant rise in urinary D-glucaric acid levels. It is concluded that individual differences in metabolism of phenothiazines may in part account for the variability in clinical response to these drugs.

Response of mammary tumors to caloric restriction for different time periods during the promotion phase.

Rats treated with 7,12-dimethylbenz[a]anthracene were subjected to 25% caloric restriction for varying times during the promotion/progression phase of mammary tumorigenesis. Caloric restriction was inhibitory to a statistically significant extent when it was maintained for the entire four-month study or only during the first or last two months. Restriction for the first month with subsequent ad libitum feeding led to rapid weight gain with concomitant appearance of tumors. These data indicate that caloric restriction can be instituted late in the tumorigenic process with an acute response of tumor growth to caloric intake or body weight.

Inhibition of DMBA-induced mammary tumorigenesis by caloric restriction in rats fed high-fat diets.

Most previous studies on the inhibiting effect of caloric restriction during promotion of DMBA-induced mammary carcinogenesis have used low to moderate levels of dietary fat, i.e., about 4 to 14% by weight. The current study was designed to test whether a moderate degree of caloric restriction, 25%, would inhibit tumor growth in rats fed the equivalent of 20% dietary fat which approximates human consumption in affluent countries. Rats were fed diets ad libitum that contained 5, 15 or 20% corn oil. Groups of rats were pair-fed to the last 2 groups, but subjected to a 25% caloric restriction. These groups were fed 20 or 26.7% corn oil so that absolute fat intake in the paired groups was identical. Significant inhibition of tumor incidence, tumor weight, tumor burden, body fat deposition, and fasting serum insulin were observed in the 2 calorically restricted groups. We conclude that moderate caloric restriction is significantly more effective in inhibiting tumor growth than is the promoting effect of diets high in fat. Total body weight, body fat and serum insulin concentrations may be better correlates of risk of developing mammary tumors than is dietary fat.

Determination of degree of energy restriction necessary to reduce DMBA-induced mammary tumorigenesis in rats during the promotion phase.

This study was designed to determine the degree of energy restriction necessary to achieve significant inhibition of mammary tumor promotion in rats treated with 7,12-dimethylbenz[a]anthracene (DMBA). A control group of rats was fed a diet containing 5% corn oil ad libitum. Four other groups were pair-fed to the controls; these rats were subjected to energy restriction of 10, 20, 30, or 40%. Weight gains among the groups were proportional to energy intake. The differences in weight were due primarily to reductions in body fat stores. Tumor incidence was reduced slightly by 20% calorie restriction and significantly by 30 and 40% restriction. There were also reductions in number of tumors per tumor-bearing rat and in mean tumor weight. The groups subjected to 30 and 40% energy restriction had significantly reduced serum levels of insulin in the fasting state. These data suggest that body weight, body fat, and fasting serum insulin correlate with susceptibility to mammary tumor promotion and that insulin may be a growth factor for DMBA-induced tumors.

Reduction of enhanced mammary carcinogenesis in LA/N-cp (corpulent) rats by energy restriction.

Restriction of energy intake significantly reduces mammary tumorigenesis in normal rats exposed to carcinogens. Genetically obese LA/N-cp (corpulent) female rats were given 7,12-dimethylbenz[a]anthracene and fed purified diets ad libitum or restricted to 60% of the ad libitum caloric intake. Phenotypically lean littermates were also fed ad libitum. Obese animals developed large mammary tumors more rapidly than genetically normal rats so that 100% of the animals had tumors in less than 16 weeks. Only 21% of the lean animals developed tumors; the energy restricted obese animals had a tumor incidence of 27%. Although obese rats fed the restricted diet weighed significantly less than those fed ad libitum, percent body fat was not reduced, indicating that lean tissue was affected more. Obese animals were markedly hyperinsulinemic (1003 +/- 193 microunits/ml) and energy restriction reduced this to 328 +/- 41; the lean animals had insulin levels of 12 +/- 2. Tumor-bearing rats had higher insulin levels than rats without tumors. These data suggest that body fatness is not directly associated with risk of carcinogenesis. Lean body mass, adipose tissue mass, and their interaction with insulin in its capacity as a growth factor rather than body fatness per se may be determinants of tumor promotion.

ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.

The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a approximately 46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3beta-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.