Dietary cholesterol and the activity of stearoyl CoA desaturase in rats: evidence for an indirect regulatory effect.

The effect of cholesterol on stearoyl CoA desaturase (SCD) was investigated. Previous work had shown that the addition of cholesterol to the diet of rats produced higher liver SCD activity compared to non-cholesterol-fed controls. We have confirmed this result and investigated the mechanism responsible for this cholesterol-induced higher SCD activity. Rats were fed either a 10% corn oil (CO) or a 10% corn oil/1% cholesterol (CO/CH) diet for 1, 3, or 7 days. SCD mRNA abundance was 3.3, 1.9, and 2.4 times greater in livers from CO/CH-fed animals after 1, 3, and 7 days, respectively. Northern hybridization of RNA from kidney, intestinal mucosa, heart, adipose, and liver demonstrated that cholesterol feeding specifically altered liver SCD mRNA. Liver esterified cholesterol content increased 27-fold with cholesterol feeding. This esterified cholesterol increase was accompanied by a proportionately greater increase in oleic acid compared to other fatty acids. These studies indicate that cholesterol does influence the expression of SCD specifically in the liver and suggest that the product, oleic acid, is preferentially esterified to cholesterol in the liver. Preliminary liver nuclear run-on assays from rats fed CO or CO/CH diets for 1 and 3 days indicate that transcription regulation is not a factor.

Plasticity of brain alpha-adrenoceptors during the development of diet-induced obesity in the rat.

Male Sprague-Dawley rats, which are prone to develop diet-induced obesity (DIO) on a high energy (HE) diet can be separated from rats which are diet-resistant (DR) by several prospective tests. Using such tests, chow-fed DRl-prone rats have higher binding of 3H paraminoclonidine (PAC) to brain alpha2-adrenoceptors than do DIO-prone rats. These differences disappear after 3 months on a HE diet. To study the predictive value of these tests and possible associated changes in presynaptic membrane composition, brain alpha3(1-) (1nM 3H prazosin) and (alpha2-adrenoceptor (1nM) 3-H PAC) binding and synaptosomal fatty acid composition were assessed in 3-month-old male rats separated by weight gain into DR and DIO groups after 1 month on a HE diet. DIO had comparable total caloric intake but gained 30% and 43% more weight and were hyperinsulinemic compared to DR and chow-fed rats, respectively. After 1 month on a HE diet, DR rats still had 15%-53% higher 3H PAC binding than DIO and/or chow-fed rats in 14 of 16 brain areas assessed. A phenotype effect was present primarily in the amygdala where DR rats had higher 3H PAC binding than DIO rats. A diet effect was seen in some hypothalamic nuclei where both DR and DIO rats had higher 3H PAC binding than chow-fed rats. Conversely, DIO rats had 14%-21% higher 3H prazosin binding than DR rats in 3 brain areas. Changes in brain synaptosomal membranes' fatty acids reflected both phenotype and diet effects. Thus, while diet composition affects presynaptic membrane composition and alpha2-adrenoceptor binding in both DR and DIO rats, the predominance of plasticity of these parameters is limited to the brains of DR rats. This suggests that such plasticity may be an important determinant of the ability to resist the development of diet-induced obesity on a HE diet.

The effect of palmitoyl-CoA binding to albumin on the apparent kinetic behavior of carnitine palmitoyltransferase I.

Substrate saturation plots of carnitine palmitoyltransferase I activity from isolated rat liver mitochondria vs. palmitoyl-CoA concentration in the presence of bovine serum albumin have been reported to yield sigmoidal kinetics. Under identical assay conditions we have confirmed these observations as reflected by nonlinear Lineweaver-Burke plots (1/vi vs. 1/[S]) an average Hill coefficient of napp. = 1.98 +/- 0.09 (Mean +/- S.E. from four separate experiments). For these determinations the enzyme activity was plotted against the total [palmitoyl-CoA] in the presence of 0.13% bovine serum albumin. Utilizing the total [palmitoyl-CoA] to determine the kinetic properties of carnitine palmitoyltransferase I would be valid only if the relationship between total and free [palmitoyl-CoA] was linear, which is not the case as we have previously shown. When carnitine palmitoyltransferase I substrate saturation kinetics were reanalyzed using the previously determined free [palmitoyl-CoA]'s, the plots revealed a shift to standard hyperbolic kinetics. This observation was confirmed by an average Hill coefficient of napp. = 1.04 +/- 0.10 (Mean +/- S.E.) and linear Lineweaver-Burke plots. The double-reciprocal plots from these analyses yielded an average S0.5 of 2.55 +/- 0.82 microM (Mean +/- S.E.) palmitoyl-CoA and Vmax of 19.69 +/- 5.48 nmol/min per mg protein. These studies clearly demonstrate the importance of defining the free [palmitoyl-CoA] when analyzing the kinetics of carnitine palmitoyltransferase I in the presence of bovine serum albumin.

Improved survival of heterotopic cardiac allografts in rats with dietary n-3 polyunsaturated fatty acids.

A heterotopic cardiac transplant model, with male Fischer 344 rats as donors and Long Evans rats as recipients, was utilized to investigate the effect of dietary n-3 polyunsaturated fatty acids on acute rejection. Both donor and recipient rats were fed purified diets high in either n-3 polyunsaturated fatty acids (from concentrated n-3 ethyl esters [EE] or fish oil [FO]) or n-6 polyunsaturated fatty acids (from corn oil [CO]) for either 2-3 or 3-4 weeks before transplant. The recipient rats continued on their diets until rejection. The AIN-76A-based diets (with 30% of calories as fat) had adequate essential fatty acids and were balanced for sterols and antioxidants. Allograft survival was significantly increased by 45% when recipient rats were fed EE as compared to the control (CO diet fed to both donor and recipient), regardless of the diet fed to the donor. There was a slight but significant increase in allograft survival when only donor rats were fed the EE diet 2-3 weeks before transplant. With the FO diet (containing one third of the n-3 fatty acids in the EE diet), only the group fed FO to both donor and recipient (starting 2-3 weeks before transplant) showed a significant increase in allograft survival over the control. However, if the FO diets were fed for 3-4 weeks before transplant, increased survival was seen in groups fed FO to either the donor or recipient alone. In this case, allograft survival with FO feeding to both donor and recipient was not different from recipient treatment alone. In all the studies there was a significant and direct correlation between allograft survival and the donor heart phospholipid n-3/n-6 fatty acid ratio and the n-3 fatty acid content (at rejection). There was an indirect relationship with the n-6 fatty acid content. There was no detectable 20:3 (n-9) in the cardiac phospholipids, indicating the absence of essential fatty acid deficiency. Recipient diets were the strongest determinant of the fatty acid composition in the transplanted donor heart. The data indicate that providing dietary n-3 polyunsaturated fatty acids before and after cardiac transplant to recipient animals provides a significant protection against acute rejection.

The binding of palmitoyl-CoA to bovine serum albumin.

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.

Neuroendocrine and beta-adrenoceptor response to chronic ethanol and aggression in rats.

Male rats were administered either ethanol (6-8 g/kg/day) or dextrin-maltose, an isocaloric equivalent, for two weeks prior to a 24-hour resident-intruder test. After the first 20 minutes of the aggression test residents showed a greater increase in norepinephrine than intruders (216% vs. 97%), while intruders showed a greater increase in epinephrine (394% vs. 51%) and corticosterone (338% vs. 129%) than residents. Ethanol administration increased the initial epinephrine response of intruders almost two-fold compared to dextrin-maltose intruders. After 24 hours of aggression testing plasma norepinephrine was still elevated in residents (92%) and intruders (71%), however, only intruders continued to show an elevation in plasma corticosterone (98%) and epinephrine (107%). Using a cumulative dose-response technique, the dose of isoproterenol required to produce 50% of the maximal heart rate response (ED50) increased in intruders by 108% following aggression testing with ethanol intruders showing significantly smaller mean change. The increase in ED50 was related to drug type, behavior, and plasma corticosterone and epinephrine levels. Rats treated with ethanol had a greater beta-adrenoceptor density than control rats. However, no relationship was found between receptor density and the other measures in this study.

Effect of dietary fat and cholesterol supplements on glucagon receptor binding and adenylate cyclase activity of rat liver plasma membrane.

We have examined the effect on rat liver glucagon receptors and adenylate cyclase activity of a high saturated fat diet (butter fat), a high n-6 polyunsaturated fat diet (corn oil), and a high n-3 polyunsaturated fat diet (menhaden fish oil) with or without additional cholesterol. The number and affinity of the glucagon receptors were unaffected by diet. The glucagon-stimulated adenylate cyclase activity from fish oil-fed animals exhibited the greatest stimulation, followed by corn oil-fed animals. Butter fat-fed and all cholesterol-supplemented groups showed a depression in stimulation. The pattern of adenylate cyclase activity with fluoride stimulation was similar to that observed with glucagon. The effect of dietary fat on forskolin stimulation was similar to glucagon and fluoride for the groups without added cholesterol. However, the cholesterol-supplemented groups did not exhibit a decreased activity. It is suggested that the effect of dietary lipid on glucagon-stimulated adenylate cyclase is not due to changes in the glucagon receptor, but rather due to changes in signal transduction, the Gs-protein or the catalytic unit.

The effects of dietary sucrose on opiate receptor binding in genetically obese (ob/ob) and lean mice.

Weanling genetically obese (C57BL/6J-ob/ob) and lean (+/?) mice were given access to either a standard laboratory diet or the standard diet plus a 32% sucrose solution. At the end of a 4-week period, animals were sacrificed and opiate receptor binding determined. Both obese and lean mice given access to sucrose consumed approximately 30% more calories per day than animals given access to the standard diet alone. Obese animals consumed significantly more calories from the sucrose solution than the lean animals. Genetically obese animals weighed more than lean littermates throughout the course of the study. Differences in body weight due to sucrose supplementation in both genetically obese and lean mice were significant by day 10 and increased in magnitude until the termination of the study. Whereas there were no significant differences in specific opiate receptor binding (pmol 3H-naloxone bound/mg brain protein) between the genetically obese and lean animals, opiate receptor binding was significantly greater in genetically obese animals given access to sucrose than in obese animals which had access only to the standard diet. These data demonstrate that the sucrose-induced model of obesity functions in mice and that giving ob/ob mice access to sucrose in addition to a standard laboratory diet results in increased opiate receptor binding.

Dietary fat and the beta-adrenergic-mediated chronotropic response in the rat.

We have investigated the effect of dietary fat on isoproterenol-stimulated and propranolol-inhibited heart rate in the rat. In the first experiment, weanling male Sprague-Dawley rats were fed diets containing 10% butter (10B), 10% corn oil (10C) or 9% butter/1% corn oil (10M) for 4 wk. Heart rate was determined in response to increasing i.v. doses of isoproterenol. The percent stimulation at the highest dose of isoproterenol (100 microM) was found to be significantly higher (P less than 0.05) in the 10C-fed group than either the 10B- and 10M-fed groups. A similar study investigated the effect of propranolol inhibition of heart rate in low-fat (10C and 10B) as well as high-fat (25C) corn oil- and high-fat (25B) butter-fed animals. Heart rate was elevated with a constant i.v. infusion of isoproterenol and the inhibitory effect of propranolol was determined. The ED50 was found not to depend on the level of fat, but was found to be significantly lower in the corn oil-fed animals than in those red butter fat (P less than 0.05). Thus, animals appear to be more sensitive to these beta-adrenergic agonists/antagonists when fed a diet containing corn oil rather than butter fat as the lipid source.

Dietary fat ratios and liver plasma membrane lipid composition.

Male Sprague-Dawley weanling rats were fed isocaloric diets consisting of 10% (by wt) fat. The six groups differed in the ratio of corn oil and butter fat present in the diets such that: 10C, 10% corn oil (C); 8C2B, 8% C/2% butter fat (B); 6C4B, 6% C/4% B; 4C6B, 4% C/6% B; 2C8B, 2% C/8% B; and 10B, 10% B. Liver plasma membranes were analyzed for fatty acid composition and cholesterol/phospholipid molar ratio. The 18:2n-6 content was constant in the 10C and 8C2B diets and then decreased linearly through the 2C8B diet. The 20:4n-6 and 18:1n-9 contents were constant except in the 10B diet, in which a significant decrease and increase, respectively, were observed. The cholesterol/phospholipid molar ratio increased between the 10C and 6C4B diets and subsequently (4C6B and 10B diets) remained constant. This data indicates that changes in n-6 fatty acid content in the liver plasma membrane are directly related to dietary intake only for 18:2n-6. Arachidonic acid content in the membrane is maintained at a constant level until the linoleic acid content of the diet is reduced to 0.5% of calories. It also indicates that the cholesterol content of the membrane becomes saturated and does not increase with increasing concentrations of saturated fat in the diet.

Liver microsomal membrane fluidity and lipid characteristics in vitamin A-deficient rats.

Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.

Macrophage phagocytosis and membrane fluidity in mice: the effect of age and dietary protein.

Male C57BL/6NNia mice were used to investigate the effects of age and dietary protein intake on Fc and C3b receptor-mediated phagocytosis and on membrane fluidity. Six-month-old (adult) and 24-month-old (aged) mice were fed a 6% or 25% protein diet for 3, 5, or 6 weeks at which time thioglycollate elicited peritoneal macrophages were isolated. Both binding of IgG-coated sheep red blood cells to the macrophages and ingestion via the Fc-receptor were identical in all 4 groups after 3 and 6 weeks of feeding but were decreased at 5 weeks in the aged animals fed 6% protein. Phagocytosis via the C3b receptor was not depressed in either age group fed the low protein diet; it was, however, augmented significantly in the aged animals fed the 25% protein diet for 5 and 6 weeks. Membrane fluidity of the plasma membrane outer hemileaflet was monitored with an impermeant fluorescent probe. No changes were observed between adult and aged mice maintained up to 6 weeks on the diets.

Deuterated halothane--anesthetic potency, anticonvulsant activity, and effect on cerebellar cyclic guanosine 3',5'-monophosphate.

The effect of substituting deuterium for hydrogen in the halothane molecule on anesthetic potency, motor activity, and cerebellar cyclic guanosine 3',5'-monophosphate (cGMP) content was studied in mice. The concentration of halothane required to abolish the righting reflex in 50% of the mice (ED50RR) was chosen as index of anesthetic potency; cerebellar control of motor activity was evaluated by the incidence of isoniazid-induced convulsions. The ED50RR for deuterated (D)-halothane was similar to that of halothane (0.87 +/- 0.04 and 0.88 +/- 0.03 vol%, respectively). Both D-halothane and halothane (0.15-0.90 vol%) protected the mice against isoniazid-induced convulsions and decreased cerebellar cGMP content in a dose-dependent manner. D-halothane and halothane were equipotent on both parameters. Thus deuteration did not alter the anesthetic potency, the anticonvulsant activity, or the effect on cerebellar cGMP content of the anesthetic. Furthermore, the reactivity of the C-H bond is probably not critical for these actions of halothane.

Effect of protein restriction on macrophage phagocytic capacity in young mice.

C57BL/6J male mice were fed ad libitum either a 6% (low) or 25% (control) protein diet for 2 to 6 weeks. At periods of 2, 3, 5 or 6 weeks following initiation of the feeding protocol thioglycollate-elicited peritoneal macrophages were assayed for Fc receptor-mediated phagocytic capability with IgG-coated sheep red blood cells (SRBC). Binding via this receptor (as monitored at 2 degrees C) was not affected by the protein restriction throughout the study. Ingestion of prebound IgG-SRBC was initially suppressed (2 weeks on diet) in macrophages from animals fed low protein but rose to a level significantly higher than similarly treated control animals after 5 weeks on the diet. At 3 and 6 weeks macrophages from control and low protein-fed animals exhibited no differences in ingestion capability. Overall phagocytosis (no prebinding of IgG-SRBC) was similar in trend to the data observed for ingestion. The data indicate both an altered response to phagocytosis with the low protein diet and the importance of quantitating the relationship between the duration of the nutritional insult and the parameter being measured.

Transport and metabolism of pyridoxine in rat liver.

Evidence, obtained with in situ perfused rat liver, indicated that pyridoxine is taken up from the perfusate by a non-concentrative process, followed by metabolic trapping. These conclusions were reached on the basis of the fact that at low concentrations (0.125 microM), the 3H of [3H]pyridoxine accumulated against a concentration gradient, but high concentrations (333 microM) of pyridoxine or 4-deoxypyridoxine prevented this apparent concentrative uptake. Under no conditions did the tissue water:perfusate concentration ratio of [3H]pyridoxine exceed unity. The perfused liver rapidly converted the labeled pyridoxine to pyridoxine phosphate, pyridoxal phosphate and pyridoxamine phosphate and released a substantial amount of pyridoxal and some pyridoxal phosphate into the perfusate. Since muscle and erythrocytes failed to oxidize pyridoxine phosphate to pyridoxal phosphate, it is suggested that the liver plays a major role in oxidizing dietary pyridoxine and pyridoxamine as their phosphate esters to supply pyridoxal phosphate which then reaches to other organs chiefly as circulating pyridoxal.

Transport and metabolism of pyridoxine in the perfused small intestine and the hind limb of the rat.

The vascularly perfused small intestine and hind limb muscle of the rat were utilized to study the transport and metabolism of pyridoxine (PN), independent of other tissues, including erythrocytes. The transport of PN both into the mucosal tissue and transmurally into the perfusate was proportional to the dose over a 10,000-fold range of concentrations. The only labeled compound formed from [3H]-PN in the mucosa was PNP which accounted for 30.6% of the isotope found there. The data for the hind limb muscle suggest that transport occurs by passive diffusion followed by phosphorylative trapping. Over a 10,000-fold range of concentrations of PN in the perfusate, the percentage of 3H found in the muscle ranged from 10.4 to 15.7 for 30-minute experiments. As the dosage was increased the percentage of 3H in the muscle, present as PN increased and that in PNP decreased. In longer experiments, up to 75 minutes, with 20 nmole of [3H]-Pn, the PN in muscle decreased as phosphorylation occurred. There was no evidence of any conversion of PNP to PLP (pyridoxal phosphate) in the perfused hind limb.

Transport and metabolism of pyridoxamine and pyridoxamine phosphate in the small intestine of the rat.

The vascularly perfused small intestine of the rat was utilized to study the absorption and metabolism of pyridoxamine (PM) and pyridoxamine-5'-phosphate (PMP), independent of other tissues including erythrocytes. [3H]PM or [3H]PMP was administered intralumenally with or without the addition of unlabeled PM, or PMP or inorganic phosphate. The percentage absorption of PM was 17.1 to 19.7% in 10 minutes and was unaffected by dosages from 0.02 to 200 mumole. The isotope from [3H]PMP at a physiological level (0.02 mumole) was absorbed at the same rate as that from [3H]PM, and the distribution of the 3H remaining in the lumen and in the intestinal tissue and perfusate indicated that hydrolytic removal of the phosphate was occurring extensively in the gut. The spectrum of labeled compounds isolated from the lumen, the perfusate and the mucosa clearly indicated that PMP, unhydrolyzed, can be absorbed slowly and converted to a number of vitamin B-6 forms in the intestinal mucosa. However, the results support the view that under normal physiological conditions the majority of dietary PMP is hydrolyzed to PM which seems to be absorbed by passive diffusion and transported to other organs and tissues via the blood stream.