John J. Mulligan

Oral Nicotine Induces an Atherogenic Lipoprotein Profile

Abstract Male squirrel monkeys were used to evaluate the effect of chronic oral nicotine intake on lipoprotein composition and metabolism. Eighteen yearling monkeys were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine primates given liquid diet supplemented with nicotine at 6 mg/kg body wt/day. Animals were weighed biweekly, plasma lipid, glucose, and lipoprotein parameters were measured monthly, and detailed lipoprotein composition, along with postheparin plasma lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activity, was assessed after 24 months of treatment. Although nicotine had no effect on plasma triglyceride or high density lipoproteins (HDL), the alkaloid caused a significant increase in plasma glucose, cholesterol, and low density lipoprotein (LDL) cholesterol plus protein while simultaneously reducing the HDL cholesterol/plasma cholesterol ratio and animal body weight. Levels of LDL precursors, very low density (VLDL) and intermediate density (IDL) lipoproteins, were also lower in nicotine-treated primates while total postheparin lipase (LPL + HTGL) activity was significantly elevated. Our data indicate that long-term consumption of oral nicotine induces an atherogenic lipoprotein profile (+LDL, +HDL/total cholesterol ratio) by enhancing lipolytic conversion of VLDL to LDL. These results have important health implications for humans who use smokeless tobacco products or chew nicotine gum for prolonged periods.

Effect of drinking pattern on plasma lipoproteins and body weight

The effect of drinking pattern on plasma lipoproteins and body weight was examined in three groups of squirrel monkeys: (1) controls fed isocaloric liquid diet; (2) regular drinkers given liquid diet containing ethanol (EtOH) substituted isocalorically for carbohydrate at 12% of calories daily; and (3) binge drinkers fed 6% EtOH calories daily for a four-day period followed by three days of 20% EtOH to mimic a weekend bout drinking cycle. The number of calories offered per day was the same for all groups, and the average weekly EtOH consumption (12% calories) was identical for the two alcohol treatments. The entire study lasted six months. There were no significant differences in plasma cholesterol, triglyceride or liver function tests. Regular drinkers had the highest high density lipoprotein2/high density lipoprotein3 (HDL2/HDL3) protein and apolipoprotein A-I/B ratios of any group and exhibited a significant elevation in the molar plasma lecithin:cholesterol acyltransferase (LCAT) rate (nmol/min/ml). Binge drinking produced a selective increase in low density lipoprotein (LDL) cholesterol and apolipoprotein B, and a depression in the fractional LCAT rate (% esterified/min). During the course of the study, controls ate 92% of their diet while the alcohol groups each consumed 95% of the liquid diet. Despite this difference, body weight and Quetelet index (weight/height2) decreased progressively in the order controls greater than regular drinkers greater than binge drinkers. Results from our study indicate that moderate, regular daily consumption of EtOH at 12% of calories causes a modest reduction in body weight and produces a coronary protective lipoprotein profile (increases HDL2/HDL3, increases apolipoprotein A-I/B, low LDL cholesterol). By contrast, when this same average weekly dose is concentrated in a binge cycle, unfavorable alterations in lipoprotein composition (increases LDL cholesterol, increases apolipoprotein B) and metabolism (decreases LCAT activity) occur along with weight loss and depletion of body fat. These studies point to the value of the squirrel monkey model in evaluating both favorable and pathophysiological effects of chronic EtOH intake.

Ethanol Induced Alterations in Low and High Density Lipoproteins

Abstract Male squirrel monkeys fed ethanol (ETOH) at variable doses were used to determine whether alcohol modifies levels of plasma low density lipoproteins (LDL) in addition to increasing high density lipoproteins (HDL). Because we earlier showed that high alcohol consumption enhances lipoprotein cholesterol synthesis, experiments were also performed to further assess whether ETOH alters lipoprotein clearance and plasma transfer processes in vivo- Monkeys were divided into three groups: Controls fed isocaloric liquid diet; and Low and High ETOH animals fed liquid diet with vodka substituted isocalorically for carbohydrate at 12 and 24% of calories, respectively. High ETOH primates had significantly more LDL lipid and protein while serum glutamate oxaloacetate transaminase was similar for the three groups. Although removal of 3H LDL cholesteryl ester (CE) from the plasma compartment was not affected by dietary ETOH, transfer of LDL CE to HDL was impaired in the High ETOH group suggesting a mechanism for the enlarged circulating pool of LDL. Transfer of 14C HDL CE to lower density lipoproteins was similar for the three groups. However, ETOH at both doses delayed clearance of radiolabeled HDL CE from circulation. Thus besides enhancing synthesis of lipoproteins, ETOH at a moderately high dose (24% of calories) influences lipoprotein levels in primates by modifying lipid transfer processes (LDL) as well as by altering clearance (HDL) without adversely affecting liver function.

Alcohol delays clearance of lipoproteins from the circulation

Long-term (18-month) consumption of high-dose ethanol ([EtOH] 24% of total calories) by squirrel monkeys results in marked elevations in plasma antiatherogenic high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-1, and atherogenic low-density lipoprotein (LDL) cholesterol and apo B. In an effort to determine whether alterations in lipoprotein turnover could explain the above findings, 131I-HDL apo A-1 and 125I-LDL apo B were injected into EtOH and control animals, following which in-vivo catabolic and production rates were determined. For both lipoproteins, synthetic rates were unaltered, while fractional catabolic rates (FCR) were significantly reduced in EtOH monkeys. Results from this study implicate EtOH-induced changes in hepatic metabolism as the basis for delayed lipoprotein clearance and hence elevated plasma apolipoprotein levels.

Oral nicotine impairs clearance of plasma low density lipoproteins.

Abstract The effect of chronic oral nicotine intake on plasma low density lipoprotein (LDL) clearance, lipid transfer protein, and lecithimcholesterol acyltransferase (LCAT) was examined in male atherosclerosis susceptible squirrel monkeys. Eighteen yearling primates were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine monkeys given liquid diet supplemented with nicotine at 6 mg/kg body wt/day for a two-year period. Averaged over 24 months of treatment, animals in the Nicotine group had significantly higher levels of plasma and LDL cholesterol compared to Controls while plasma LCAT activity was similar for both groups. Following simultaneous injection of 3H LDL and 14C high density lipoprotein (HDL) cholesteryl ester (CE), removal of the latter was not altered by oral nicotine while plasma clearance of 3H LDL was dramatically delayed in Nicotine monkeys. Transfer of 14C HDL CE to very low density lipoprotein (VLDL)-LDL particles was greatly accelerated in the Nicotine group vs Controls while the reciprocal movement of 3H LDL CE to HDL was only higher in experimental animals at two time points following injection of the isotopes. Results from this study provide evidence that one major detrimental effect of long-term oral nicotine use is an increase in the circulating pool of atherogenic LDL which is due to: 1) accelerated transfer of lipid from HDL; and 2) impaired clearance of LDL from the plasma compartment. Diminished removal of LDL is of particular importance because an extended residence time of these particles in circulation would increase the likelihood of their deposition in the arterial wall.

Alcohol Produces Dose-Dependent Antiatherogenic and Atherogenic Plasma Lipoprotein Responses

Abstract A comprehensive assessment of lipoprotein compositional/metabolic response to incremental caloric ethanol (EtOH) doses ranging from low to moderate to high was undertaken using male squirrel monkeys. Control monkeys were maintained on a chemically defined, isocaloric liquid diet, while experimental primates were fed increasing doses of alcohol (6, 12, 18, 24, 30, and 36% of energy) substituted isocalorically for carbohydrate at 3-month intervals. Liver function tests and plasma triglyceride were normal for all animals. Plasma cholesterol showed a transient increase at the 12% caloric dose that was attributed solely to an increase in high density lipoprotein (HDL). A more pronounced increase in plasma sterol, beginning at 24% and continuing to 36% EtOH, was the result of increments in both HDL and low density lipoprotein (LDL) cholesterol, although the contribution by the latter was substantial primarily at the 36% dose. Plasma apolipoprotein elevations (HDL apolipoprotein A-1, LDL apolipoprotein B) generally accompanied the lipoprotein lipid increases, although the first atherogenic response for LDL became manifest as a significant increase in apolipoprotein B at 18% EtOH calories. Postheparin plasma lipoprotein lipase was not affected by dietary alcohol, whereas hepatic triglyceride lipase activity showed significant increases at higher (24 and 36%) EtOH doses. Plasma lecithin-cholesterol acyltransferase activity was normal at the 6 and 12% EtOH doses, but exhibited a significant reduction beginning at 18% and continuing to 36% EtOH. Alterations in these key lipoprotein regulatory enzymes may represent the underlying metabolic basis for the observed changes in lipoprotein levels and our earlier findings of HDL2/HDL3 subtraction modifications. Results from our study indicate that in squirrel monkeys, moderate (12%) EtOH caloric intake favors an antiatherogenic lipoprotein profile (↑HDL, normal LDL levels, and lecithin-cholesterol acyltransferase activity), whereas higher doses (24–36%) produce both coronary-protective (↑HDL) and atherogenic (↑LDL) responses. Moreover, the 18% EtOH level represents an important transition dose which signals early adverse alterations in lipoprotein composition (↑apolipoprotein B) and metabolism (↓lecithin-cholesterol acyltransferase).

Ethanol enhances de novo synthesis of high density lipoprotein cholesterol.

Abstract Male squirrel monkeys fed ethanol at variable doses were used to assess whether alcohol enhances de novo synthesis of high density lipoprotein (HDL) cholesterol in vivo. Monkeys were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys fed liquid diet with vodka substituted isocalorical-ly for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. High Ethanol primates had significantly higher levels of HDL nonesterified cholesterol than Control and Low Ethanol animals while serum glutamate oxaloacetate transaminase was similar for the three treatments. There were no significant differences between the groups in HDL cholesteryl ester mass or specific activity following intravenous injection of labeled mevalonolactone. By contrast, High Ethanol monkeys had significantly greater HDL nonesterified cholesterol specific activity with approximately 60% of the radioactivity distributed in the HDL3 sub-fraction. This report provides the first experimental evidence that ethanol at 24% of calories induces elevations in HDL cholesterol in primates through enhanced de novo synthesis without adverse effects on liver function.

Effect of ethanol on lipoprotein synthesis and fecal sterol excretion

Abstract The effect of variable doses of ethanol on plasma lipoprotein composition, lipoprotein synthesis and fecal sterol excretion was examined in male, atherosclerosis susceptible squirrel monkeys. Primates were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys given liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. Circulating high density lipoprotein (HDL) free cholesterol and phospholipid, very low density-low density lipoprotein (VLDL-LDL) total cholesterol, and total plasma cholesterol and triglyceride were significantly elevated in High Ethanol primates compared to the other treatments. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups. There were no significant differences in serum glutamate oxalo-acetate transaminase. High Ethanol primates also had significantly greater HDL free cholesterol specific activity following intravenous injection of 3 H mevalonolactone compared to the other groups while radioactive VLDL-LDL free cholesterol was elevated in both High and Low Ethanol animals. Although, total fecal bile acid mass was significantly greater in both alcohol treatment groups compared to Controls, fecal neutral sterol specific activity was only higher in monkeys fed the high ethanol diet. This study provides evidence that ethanol at 24% of calories: 1) raises HDL cholesterol levels by enhancing lipoprotein synthesis; 2) increases the fecal output of newly synthesized cholesterol without causing liver dysfunction; and 3) maintains a constant relative distribution of cholesterol among lipoprotein classes.

Cigarette smoking impairs hepatic uptake of high density lipoproteins

The effect of chronic inhalation of cigarette smoke on hepatic uptake of high density lipoproteins (HDL) in White Carneau pigeons was examined. Four treatment groups included: 1) Shelf Control birds fed a chow diet and retained in their cages; 2) Sham pigeons fed a cholesterol-saturated fat diet and exposed to fresh air by a smoking machine; 3) Low nicotine-low carbon monoxide (LoLo) animals also fed the cholesterol diet and exposed to low concentrations of these cigarette smoke products; and 4) High nicotine-high carbon monoxide (HiHi) birds fed the cholesterol diet and subjected to high concentrations of these components. Livers from both smoke exposed groups contained significantly more triglyceride than those from Sham animals while livers from HiHi birds alone had elevated concentrations of protein. Liver slices from LoLo and HiHi pigeons incorporated significantly less HDL 3H free and esterified cholesterol and HDL 14C apoprotein from media during in vitro incubation than livers from Sham birds. Impaired hepatic uptake of HDL suggests a permanent alteration in liver function resulting from chronic exposure to tobacco smoke and may represent one mechanism by which cigarette smoking attenuates HDLs anti-atherogenic properties.

Alcohol dose and low density lipoprotein heterogeneity in squirrel monkeys (Saimiri sciureus)

The present study was designed to determine whether normolipidemic male squirrel monkeys (Saimiri sciureus) exhibit low density lipoprotein (LDL) heterogeneity similar to that observed in humans and if present, whether LDL subfractions are altered by consumption of low vs. high dose ethanol (EtOH). Primates were divided into three groups designated control, low, and high EtOH and fed isocaloric liquid diets containing 0%, 12% and 24% of calories as EtOH, respectively, for 6 months. The 12% EtOH caloric level resulted in a modest, non-significant increase in high density lipoprotein (HDL) cholesterol and no change in LDL cholesterol or plasma apolipoprotein B (apo B), while the 24% dose produced significant elevations in plasma, LDL and HDL cholesterol and apo B. Using a single-spin density gradient ultracentrifugation procedure developed for humans, three distinct LDL subclasses designated LDL1a (d = 1.031 g/ml), LDL1b (d = 1.038 g/ml) and LDL 2 (d = 1.046 g/ml) were isolated from all three treatment groups. Monkey LDL subfractions were nearly identical to very light, light and heavy LDL subspecies isolated from human plasma in terms of their: (1) isopycnic densities following ultracentrifugation; (2) co-migration as single bands with beta-electrophoretic mobility in cellulose acetate and agarose electrophoretic gels; (3) size-dependent migration pattern in polyacrylamide gradient electrophoretic gels; (4) co-migration as a single band corresponding to apo B-100, following SDS polyacrylamide gel electrophoresis; and (5) decrease in total cholesterol/protein ratios with increasing LDL subclass density. Although there were no treatment differences in LDL particle size, within each treatment group, mean particle size for each LDL subfraction was significantly different from every other subfraction. Low (12%) dose alcohol had no effect on LDL subfraction mass relative to controls while high alcohol consumption resulted in marked increases in all lipid (except triglyceride) and protein of the larger, buoyant LDL subspecies (LDL1a and LDL1b). Moreover, the best correlation between plasma apo B and LDL subfraction total mass was demonstrated with LDL1b (r = 0.735). Since neither the lipid nor the protein concentration of the small, dense, purportedly more atherogenic, LDL2 changed with the 24% EtOH dose, we propose that the LDL subfraction alterations associated with high alcohol intake in squirrel monkeys (increased LDL1a, increased LDL1b, LDL2 no effect) may represent a compensatory response to modulate the overall atherogenic lipoprotein profile associated with elevations in total LDL cholesterol and plasma apolipoprotein B.