Alpha and omega of carotenoid cleavage.

In early 1900s, based on indirect evidence, Steenbock and Morton independently predicted that beta-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore showed the in vivo formation of vitamin A from beta-carotene. But it was not until Jim Olson and DeWitt Goodman independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A from beta-carotene in cell-free extracts of liver and intestine, that this vital pathway of beta-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of beta-carotene to retinal by many investigators, there were some careful independent studies by Glover et al., Ganguly et al., Hansen and Meret and Krinsky et al. showing the eccentric cleavage of beta-carotene resulting in the formation of apocarotenoids both in vivo and in vitro. In an attempt to resolve this controversial issue, we revisited this problem in 1989 and showed beyond doubt the formation of retinal as the sole enzymatic product of a cytosolic enzyme from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product. This was confirmed in 1996 by Nagao using the pig intestinal extract. Yeum et al. confirmed in 2000 that retinal is the sole product of beta-carotene cleavage in the presence of alpha-tocopherol, and that the observed formation of apocarotenoids occurs only in the absence of an antioxidant like alpha-tocopherol. In the same year, Barua and Olson also concluded from their in vivo studies in rats that central cleavage is by far the major pathway for the formation of vitamin A from beta-carotene. Beta, beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves beta-carotene into two molecules of retinal. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver. It is a 66 kDa sulfhydryl protein, requires molecular oxygen and is activated by ferrous ions. It is highly specific for 15:15' ethylenic bond of carotenoids although it has fairly broad specificity towards a number of carotenoids with at least one intact beta-ionone ring. The dioxygenase was recently cloned from Drosophila melanogaster and from the chicken intestine. The recombinant protein was found to form retinal as the sole cleavage product of beta-carotene. No apo-carotenoids were formed. Therefore, it is unequivocally proven that the major, if not the sole, pathway of beta-carotene cleavage to vitamin A is by oxidative cleavage of the central ethylenic bond of beta-carotene to yield two molecules of retinal. Most recently, human dioxygenase has also been cloned. Thus, the wisdom, vision and epoch-making mission of Jim Olson in the science of beta-carotene metabolism have been accomplished. I have no doubt that the impact of his original discovery of the dioxygenase and its importance in vitamin A nutriture should be forthcoming in the near future.

Plasma sialic-acid index of apolipoprotein J (SIJ): a new alcohol intake marker.

Although plasma carbohydrate-deficient transferrin (CDT) is considered a viable biochemical marker for chronic alcohol consumption, it is valid only when an individual's daily alcohol consumption exceeds 60 g. In addition, it is less sensitive in women drinkers than in men drinkers. We have established that chronic alcohol consumption impairs the hepatic sialylation of a number of glycoproteins by specifically down-regulating Gal-beta-1,4GlcNAc alpha2,6-sialyltransferase mRNA. Significantly, we found that chronic ethanol consumption markedly inhibits hepatic sialylation of apolipoprotein J (Apo J), a 70-kDa N-glycosylated protein of plasma HDL. Because the sialic-acid index of Apo J (SIJ; moles of sialic acid per mole of Apo J protein) is approximately seven times more than that for transferrin (28 vs. 4), we have evaluated whether plasma SIJ would be an even more sensitive marker for chronic ethanol consumption than CDT in both rats and human subjects. The method involves immunoaffinity purification of plasma HDL-Apo J, followed by its sialic acid determination. We have found that chronic ethanol feeding resulted in loss of sialic acid residues of plasma HDL-Apo J in rats. This loss of sialic acid was positively correlated with both amount and duration of ethanol treatment. In human subjects, an intake of about 60 g of alcohol for 30 days led to almost 50% (P <.01) depletion of sialic acid from plasma HDL-Apo J. Further, we established that there was a positive correlation of alteration in SIJ with alcohol consumption, detoxification, abstinence, and relapse in human alcohol-dependent patients (sensitivity, 90%-92%). In addition, plasma SIJ was decreased by 50%-57% (P <.01) in both male and female alcohol-dependent subjects. We suggest that plasma SIJ can be used as a viable marker for early detection of chronic alcohol consumption in human beings.

Chronic ethanol exposure in rats affects rabs-dependent hepatic trafficking of apolipoprotein E and transferrin.

Because of the important roles of rabs in protein trafficking, we tested whether chronic ethanol exposure affected the trafficking of newly synthesized apolipoprotein E (apoE) or transferrin (O-glycosylated and N-glycosylated proteins, respectively) attached to acylated or prenylated rabs. The in vivo 30-min incorporation ratios of [3H]palmitate:[35S]methionine or [3H]mevalonate:[35S]methionine (relative ratios of rabs acylation or prenylation to total protein or to immunoisolated apoE or transferrin) were measured in various hepatic subcellular organelles of 8 week-ethanol-fed (E) and pair-fed control (C) Wistar-Furth rats. With respect to total protein trafficking, ethanol increased rabs acylation ratio by 136% (P <.01), 69% (P <.05), and 64% (P <.01) in the endoplasmic reticulum (ER), Golgi light fraction (GLF), and Golgi heavy fraction (GHF), respectively, and decreased this ratio by 76% (P <.01) in carrier vesicle fraction 2 (CV2). With respect to apoE trafficking, ethanol increased rabs acylation ratio by 121% in GHF and decreased this ratio by 27% in CV2. Rabs prenylation ratio increased by 21% and 53% in GHF and CV2, respectively, and decreased by 42% in GLF. With respect to transferrin trafficking, ethanol increased rabs acylation ratio by 53% (P <.01) in GHF, with no significant effect in ER, whereas rabs prenylation ratio increased by 26% (P <.05) in ER, with no significant effect in GHF. Therefore, we conclude that ethanol-induced impaired trafficking of newly synthesized O- and N-glycosylated proteins occurs primarily in ER and Golgi and is due to altered lipidation of rabs, possibly rabs 1, 2, or 6 or combinations of these three rabs.

High-density lipoproteins from human alcoholics exhibit impaired reverse cholesterol transport function.

We have previously shown that chronic alcohol consumption leads to inhibition of sialylation of apolipoprotein E (apo E) that results in its impaired binding to high-density lipoprotein (HDL) molecule. Because apo E plays a major role in reverse cholesterol transport (RCT), we speculated that ethanol-mediated formation of HDL molecules without apo E may affect the RCT process. Therefore, we have investigated whether the RCT function of HDL is affected in chronic alcoholics with or without liver disease compared with nondrinkers. HDL was isolated from fasting plasma of normal subjects, n = 9 (nondrinkers), chronic alcoholics, n = 8 (ALC), and chronic alcoholics with liver disease, n = 6 (ALD). A portion of HDL sample from each subject was evaluated for its cholesterol efflux capacity from [3H]cholesterol oleate preloaded mouse macrophages. The remaining portion of each HDL sample was labeled with [3H]cholesterol oleate and evaluated for its ability to deliver cholesterol to the liver using HepG2 cells in culture. Cholesterol efflux capacity of HDLs was decreased by 83% (P < .0002) in alcoholics without liver disease and by 84% (P < .0006) in alcoholics with liver disease compared with the HDLs from nondrinkers. The capacities of HDLs to deliver cholesterol to the liver were decreased by 54% (P < .005) in alcoholics without liver disease and by 64% (P < .005) in alcoholics with liver disease compared with the HDLs from nondrinkers. The fact that further complications by liver disease in alcoholic subjects did not significantly exacerbate the extent of impairment in RCT function of HDL suggest that alcohol per se is responsible for its deleterious effects on RCT. Significantly, plasma HDL apo E concentration relative to that of apo A1 (apo E/apo A1 ratio) was also decreased by 31% to 32% (P < .0005) in alcoholics without or with liver disease compared with nondrinkers. It is therefore concluded that chronic alcohol consumption adversely affects the RCT function of HDL by altering its association with apo E due to ethanol-induced desialylation of apo E.

Effects of chronic alcohol treatment on the synthesis, sialylation, and disposition of nascent apolipoprotein E by peritoneal macrophages of rats.

BACKGROUND: Plasma apolipoprotein (apo) E, a sialoprotein, plays an important role in reverse cholesterol transport. Previously, we showed that chronic alcohol consumption impairs glycosylation of apo E in rat liver. Peritoneal macrophages are another significant apo E synthesis site. OBJECTIVE: The main purpose of this study was to determine the effects of chronic alcohol feeding of rats on the synthesis, sialylation, and sialic acid content of macrophage apo E and its ability to bind to the HDL(3) molecule in vitro. DESIGN: Rats were fed an alcoholic diet or an isoenergetic control diet for 8 wk, after which peritoneal macrophages isolated from them were cultured and analyzed for apo E metabolism. RESULTS: Macrophages from alcohol-fed rats accumulated 33.3% more (P < 0.05) cholesterol than did those from control rats when incubated with acetylated LDL. These macrophages showed a 51-57% lower relative sialylation rate of apo E (P < 0.001) but no significant difference in relative protein synthetic rate. The sialic acid content of the intracellular and secreted forms of apo E was reduced by 41.8% (P < 0.001) and 50.3% (P < 0.001), respectively, with chronic alcohol treatment. Secretion of newly synthesized apo E was impaired by 53.7% (P < 0.001) and 26. 1% (P < 0.001) in the absence and presence of HDL in the medium, respectively. Macrophages of alcohol-treated rats secreted apo E with 47.6-67.2% lower (P < 0.001) HDL(3) binding ability; binding ability was restored completely by resialylation of the desialylated apo E. CONCLUSION: In rats, an alcohol-mediated decrease in sialylation rate resulting in loss of sialic acid residues in apo E impairs the ability of apo E to bind to HDL and consequently in defective reverse cholesterol transport.

Effect of dietary omega-3 fatty acids and chronic ethanol consumption on reverse cholesterol transport in rats.

We previously showed that chronic ethanol feeding leads to a decrease of apolipoprotein E (apoE) in high-density lipoprotein (HDL), whereas supplementing this diet with 2.8% of total dietary calories as omega3-fatty acids (omega3FAs) restores HDL-apoE to the control values. Since HDL containing apoE plays a major role in reverse cholesterol transport (RCT), we measured the effects chronic ethanol intake and omega3-FAs on RCT in the present study. Four groups of rats, control normal fat (CN), alcohol-normal fat (AN), control omega3FA fat (CF), and alcohol-omega3FA fat (AF), were fed their respective diets for 8 weeks, after which hepatocytes and HDLs from each group were evaluated for RCT capacity (cholesterol efflux from macrophages and uptake by liver cells). Compared with the control diet (CN), chronic ethanol (AN) feeding inhibited the cholesterol efflux capacity of HDL by 21% (P < .01), whereas omega3FA feeding (2.8% of total dietary calories) stimulated this capacity by 79% (P < .01) and 25% (P < .01) in CF and AF rats, respectively. With respect to cholesterol uptake by the liver, there were no significant 3-way or 4-way interactions between the 4 factors, HDL-alcohol, HDL-fish oil, hepatocyte-alcohol, and hepatocyte-fish oil. The main effects for HDL-alcohol, HDL-fish oil, and hepatocyte-alcohol were all highly significant (P = .0001, .0001, and .007, respectively). There was a significant HDL-alcohol and HDL-fish oil interaction (P = .0001). Hepatocyte-alcohol was not a factor in any 2-way interactions. Our study indicates no evidence of an interaction between the effects of omega3FAs and the effects of alcohol on hepatocytes in terms of RCT function. Thus, feeding as little as 2.8% of the total dietary calories as omega3FA not only restored the impaired RCT function of HDL caused by chronic ethanol intake, but also enhanced by severalfold the ability of HDL to promote RCT even in normal animals.

Alcohol and molecular regulation of protein glycosylation and function.

Chronic alcohol exposure leads to the appearance of carbohydrate-deficient transferrin (CDT), a N-glycosylated protein and sialic acid-deficient apolipoprotein E (apoE), an O-glycosylated protein. We show that chronic ethanol treatment destabilizes sialyltransferase (ST) mRNA resulting in a concomitant decreased steady-state level of ST mRNA. As a result, alcohol markedly decreases the hepatic synthetic rate of ST. This leads to impaired sialylation of transferrin and apoE. Consequently, apoE content in plasma high-density lipoproteins (HDL) is decreased. ApoE plays a significant role in the delivery of HDL cholesterol to the liver via apo B/E receptor, a process called reverse cholesterol transport (RCT). Desialylation of apoE results in its decreased association with HDL. Thus, the dissociation constant of HDL for binding to sialo-apoE is 90 +/- 35 nM, whereas that for desialo-apoE is 1010 +/- 250 nM. More importantly, the uptake of labeled cholesterol by human HepG2 cells is decreased by 30-40% from reconstituted HDL particles (rHDL)-containing desialo-apoE compared to rHDL with sialo-apoE. We conclude that chronic alcohol exposure down-regulates the expression of sialyltransferase genes resulting in impaired sialylation of apoE. This leads to its decreased binding to plasma HDL and thereby, impairs the RCT function of HDL.

Desialylation of human apolipoprotein E decreases its binding to human high-density lipoprotein and its ability to deliver esterified cholesterol to the liver.

Apolipoprotein E (apoE) plays a significant role in the delivery of high-density lipoprotein (HDL) cholesterol to the liver via the apoB/E receptor. The roles of the apoE sialylation status in its association with HDL and in the reverse cholesterol transport (RCT) function of HDL have not been well defined. Furthermore, long-term ethanol treatment impairs apoE sialylation and leads to its decreased content in HDL. Therefore, we investigated the association of either sialo apoE (SapoE) or desialo apoE (DSapoE) with HDL and its effect on the RCT function of HDL. The dextran sulfate precipitation method showed that [125I]DSapoE binding to HDL was 27.3% (P < .02) to 35.5% (P < .001) lower versus [125I]SapoE. Scatchard analysis of the specific binding data showed that [125I]SapoE had 11.2 times more affinity for HDL than [125I]DSapoE based on size-exclusion chromatography (Kd = 89.7 v 1,010 nmol/L). Similarly, [1251]HDL had 4.5 times more affinity for SapoE compared with DSapoE based on solid-phase binding (Kd = 21.9 v 104.4 nmol/L). Furthermore, esterified cholesterol uptake from reconstituted HDL particles (rHDLs) by HepG2 cells increased over basal uptake up to 153% when rHDLs contained SapoE, versus only 37% with DSapoE. Enzymatic resialylation of DSapoE completely restored its HDL-binding and RCT properties, identical to those of SapoE. It is therefore concluded that desialylation of apoE decreases its binding to plasma HDL, leading to an impaired RCT function.

Chronic ethanol consumption leads to destabilization of rat liver beta-galactoside alpha2,6-sialyltransferase mRNA.

Chronic ethanol consumption in rats is accompanied by decreased levels of Gal beta1,4GlcNAc alpha2,6-sialyltransferase (2,6-ST) activity in the liver. Our previous studies have shown that there is a concomitant decrease in the levels of 2,6-ST mRNA. In this study, the alteration in the regulation of 2,6-ST expression by chronic ethanol consumption was assessed by Northern hybridization, nuclear run-on experiments, and 2,6-ST mRNA stability studies. 2,6-ST downregulation was found at 4 weeks of feeding an ethanol diet (36% of calories from ethanol) and remained up to 8 weeks. The decrease in 2,6-ST mRNA levels was found to be dose-dependent, with lower dose of ethanol (12% and 24% of total dietary calories from ethanol) being ineffective and the effects being manifested only when 36% of the dietary calories were from ethanol. The effects of chronic ethanol feeding could be completely reversed within 1 week after ethanol consumption was stopped, when 2,6-ST mRNA levels were restored to normal. The downregulation was not sensitive to actinomycin D, indicating that the regulation was not affected at the transcriptional level but at the posttranscriptional level. This was confirmed by nuclear run-on experiments showing that the rate of 2,6-ST mRNA transcription was unaffected by ethanol. Finally, mRNA stability experiments showed that the half-life of 2,6-ST mRNA was reduced 50% in ethanol-fed rat livers compared with control rat livers. Taken together, the results show that 2,6-ST mRNA is regulated at the posttranscriptional level and chronic ethanol intake downregulates 2,6-ST expression by destabilizing its mRNA.

Chronic ethanol increases ganglioside sialidase activity in rat leukocytes, erythrocytes, and brain synaptosomes.

In view of the chronic alcohol-mediated pathological changes in various sialic acid-deficient glycoconjugates and the potential importance of sialidase in the generation of these glycoconjugates in the blood compartment and in the brain, we have investigated the effects of chronic ethanol feeding for 8 weeks on ganglioside sialidase activities in rat blood and brain. Ganglioside sialidase activity in erythrocytes (whether expressed as units/mg of protein or units/ml of blood) was 1.37- to 1.40-fold higher (p < 0.01) in the ethanol-fed group than in the control group. On the other hand, the same ethanol treatment increased sialidase activity in the leukocyte soluble fraction by 2.50- to 2.60-fold (p < 0.01) and by 1.61- to 1.63-fold (p < 0.01) in the leukocyte particulate fraction, compared with the control group. More importantly, most of the blood compartment sialidase activity was localized in the leukocytes particulate fraction (80 to 86% of total blood activity). Similarly, chronic ethanol treatment increased brain synaptosomal sialidase activity (whether expressed as units/gram of brain or units/mg of protein) 2.16- to 2.43-fold (p < 0.01). In contrast, brain lysosomal sialidase was not significantly altered by ethanol treatment, even though the major proportion of the brain sialidase activity was localized in the lysosomes. The proportion of synaptosomal sialidase activity as the percentage of total brain sialidase activity increased markedly from 13% in the control group to 24% in the ethanol group. Thus, chronic ethanol-mediated increases in sialidase activity in the leukocytes and brain synaptosomes could account for alterations in the ganglioside status of the animal and consequent adverse effects of chronic ethanol on behavioral and pathological changes.

Diuretics and beta-blockers do not have adverse effects at 1 year on plasma lipid and lipoprotein profiles in men with hypertension. Department of Veterans Affairs Cooperative Study Group on Antihypertensive Agents.

BACKGROUND: Concern based on the reported short-term adverse effects of antihypertensive agents on plasma lipid and lipoprotein profiles (PLPPs) has complicated the therapy for hypertension. OBJECTIVE: To compare the long-term (1-year) effects of 6 different antihypertensive drugs and placebo on PLPPs in a multicenter, randomized, double-blind, parallel-group clinical trial in 15 US Veterans Affairs medical centers. PATIENTS AND METHODS: A total of 1292 ambulatory men, 21 years or older, with diastolic blood pressures (DBPs) ranging from 95 to 109 mm Hg taking placebo were randomized to receive placebo or 1 of 6 antihypertensive drugs: hydrochlorothiazide, atenolol, captopril, clonidine, diltiazem, or prazosin. After drug titration, patients with a DBP of less than 90 mm Hg were followed up for 1 year. Plasma lipids and lipoprotein profiles were determined at baseline, after initial titration, and at 1 year. RESULTS: After 8 weeks on a regimen of hydrochlorothiazide, increases of 3.3 mg/dL (0.09 mmol/L) in total cholesterol and 2.7 mg/dL in apolipoprotein B were significantly different (P< or =.05) from decreases of 9.3 mg/dL in total cholesterol and 5.4 mg/dL in ApoB levels while receiving prazosin but not from placebo. Patients achieving positive DBP control using hydrochlorothiazide (responders) showed no adverse changes in PLPPs, whereas nonresponders exhibited increases in triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels. Plasma lipids and lipoprotein profiles did not change significantly among treatment groups after 1 year except for minor decreases in high-density lipoprotein 2 levels using hydrochlorothiazide, clonidine, and atenolol. CONCLUSIONS: None of these 6 antihypertensive drugs has any long-term adverse effects on PLPPs and, therefore, may be safely prescribed. Previously reported short-term adverse effects from using hydrochlorothiazide are limited to nonresponders.

Purification and characterization of cellular carotenoid-binding protein from mammalian liver.

A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. beta-[14C]Carotene copurified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites; a high-affinity site with an apparent Kd of 56 x 10(-9) M and a low-affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high-affinity site was 1 mol/mol whereas that for the low-affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambda max with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa size has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.

Taurocholate stimulates the absorption and biotransformation of beta-carotene in intact and lymph duct-cannulated ferrets.

We have determined the influence of dietary taurocholate and beta-carotene on the absorption and biotransformation of newly administered beta-14C]carotene. Male ferrets were fed the control or beta-carotene diet (0.05% beta carotene wt/wt) with and without taurocholate (1% wt/wt) for four weeks, and then the absorption and biotransformation of newly administered beta-[14C]carotene was measured after eight hours in intact or thoracic lymph duct-cannulated animals. Percent recover of beta-[14C]carotene in the liver was increased 3.6-fold (p < 0.05) in the taurocholate-fed ferrets regardless of whether they were fed the control or beta-carotene diet. Percent recovery of labeled vitamin A in the liver was also increased by the same magnitude (p < 0.05). These results were confirmed in thoracic lymph duct-cannulated ferrets. The recoveries of beta-carotene label in the lymph were comparable to the corresponding values in livers of intact animals. The recovery of beta-carotene label in the liver was 50% (p M 0.05) higher in beta-carotene-fed than in control animals. Taurocholate stimulates intestinal absorption of newly administered beta [14C]carotene and its metabolic conversion to 14C-labeled vitamin A (retinol + retinyl ester) 3.6-fold. Beta-Carotene absorption is as efficient in thoracic lymph duct-cannulated ferrets as in intact animals. Prior beta-carotene feeding also stimulates the absorption of newly administered beta-carotene by 50%.

Purification and partial characterization of a cellular carotenoid-binding protein from ferret liver.

A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]beta-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites, a high affinity site with an apparent Kd of 56 x 10(-9) M and a low affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambdamax with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.

Cholesterogenesis, lipogenesis, cholesterol degradation to bile acids, and histopathology of the liver in LA/N-cp obese rats.

Various lipid parameters were determined in lean control and LA/NIH-corpulent (LA/N-cp) rats, a normotensive strain showing metabolic characteristics associated with human Type IV hyperlipidemia. Hepatic and plasma total cholesterol, high density lipoproteins (HDL) cholesterol and triglycerides were significantly higher in the obese group than in the control group. Depending upon whether the data were expressed as per gram tissue or per organ, the rates of de novo fatty acid synthesis in the liver and adipose tissue were higher by 61% to 127% (P < .05) and 79% to 355% (P < .05), respectively, in the obese group compared with the lean control group. Similarly, hepatic rate of cholesterol synthesis was higher by 46% to 107% (P < .05) in the obese animals compared with the lean ones. In vivo hepatic rate of HDL2 cholesterol degradation to bile acids was lower in the obese group by 48% to 63% (P < .05). This was confirmed in the perfused liver in spite of the fact that cholesterol uptake from HDL2 was 3- to 4-fold higher in the obese group. These changes in lipid parameters of the obese animals were neither caused by hyperphagia because they were pair-fed with the control group nor caused by increased rate of food consumption because they were meal-fed. At the same time, all these lipid parameters were 17% to 20% higher in ad libitum-fed obese than in pair-fed obese group. Histopathological evaluation of the livers in the obese and control groups also showed prominent lipid droplets in the cytoplasm of the obese liver but not in the lean control liver. Thus, the possible causes of obesity in the LA/N-cp obese rats are higher synthetic rates of lipids coupled with lower rate of degradation of cholesterol to bile acids.

Chronic ethanol downregulates Gal-beta-1,4GlcNAc alpha 2,6-sialyltransferase and Gal-beta-1,3GlcNAc alpha 2,3-sialyltransferase mRNAs in rat liver.

We have previously demonstrated that chronic ethanol specifically decreases the hepatic level and rate of synthesis of 2,6-sialyltransferase (2,6-ST). To understand its mechanism of action, effects of 8 weeks of chronic ethanol feeding on the expression of sialyltransferase (ST) genes in rat liver and kidneys were determined by Northern-blot analysis of ST mRNAs. It was found that, compared with the pair-fed control rats, the percentage decreases in ST mRNAs in the ethanol-fed group were as follows: liver-Gal-beta-1,4GlcNAc alpha 2,6-ST (2,6-ST): 59% (p < 0.001); liver-Gal-beta-1,3GlcNAc alpha 2,3-ST (2,3-ST): 32% (p < 0.01); and kidneys-2,6-ST: 5% (NS). In contrast, glyceral-dehyde-3-phosphate dehydrogenase mRNA in both liver and kidneys was unaffected by the same ethanol treatment. Taken together, these results demonstrate that chronic ethanol downregulates the expression of 2,6-ST and 2,3-ST genes in rat liver.

Chronic ethanol induced impairment of hepatic glycosylation machinery in rat is independent of dietary carbohydrate.

In recent years, a number of studies have been reported that have clearly established that hepatic glycosylation machinery is affected by chronic ethanol treatment in rats. We have previously reported that chronic ethanol treatment in rats resulted in decreased glycosylation of transferrin and apolipoprotein E with concomitant decreases in enzymatic activities of Golgi galactosyltransferases and sialyltransferases. In all these studies investigators have invariably used the well-accepted dietary formulation of alcohol diet as proposed by Lieber and DeCarli. However, questions were raised whether the lower carbohydrate content in Lieber's alcohol diet may be responsible for observed effects of ethanol on hepatic glycosylation machinery. Therefore, to verify whether or not the crucial effects of chronic ethanol treatment on hepatic glycosylation machinery as observed in our studies, were truly caused by ethanol, we have extended our studies on protein glycosylation with the inclusion of a third dietary group that was compensated for carbohydrate content. In this investigation, rats were fed with three dietary regimen corresponding to control, ethanol, and carbohydrate compensated ethanol group and studies were done on (i) labeled leucine, galactose and N-acetylmannosamine incorporation into transferrin and apolipoprotein E, and (ii) hepatic galactosyltransferase and sialyltransferase activities in Golgi rich fraction in rat. Our results clearly showed that regardless of the carbohydrate content, marked decreases in the incorporation of labeled sugars into transferrin and the enzymatic activities of galactosyltransferase and sialyltransferase occurred in rats administered chronic ethanol. Thus, it is reasonable to conclude that it is not the carbohydrate content of the diet but ethanol per se, when administered chronically, greatly impairs the glycosylation machinery of rat liver and that the magnitudes of these effects are selectively specific with regard to the type of sugar or the glycosylation enzyme.

Comparison of plasma lipid and lipoprotein profiles in hypertensive black versus white men. Department of Veterans Affairs Cooperative Study Group on Antihypertensive Agents.

An abnormal plasma lipid and lipoprotein profile is an independent and strong predictor of mortality and morbidity from coronary artery disease (CAD). We report on plasma lipid and lipoprotein profiles with respect to race, age, obesity, blood pressure (BP), smoking, and drinking history in 1,292 male veterans with a diastolic BP of 95 to 109 mm Hg while off antihypertensive medications. Blacks had 24% (p <0.001) lower triglycerides than whites. In contrast, the following parameters were higher in blacks than in whites by the indicated percentages: high-density lipoprotein (HDL) cholesterol, 16% (p <0.001); HDL2 cholesterol, 36% (p <0.001); apolipoprotein (Apo) A1, 8% (p <0.001); HDL/low-density lipoprotein (LDL), 18% (p = 0.018); HDL2/LDL, 36% (p = 0.031); HDL2/HDL3, 21% (p <0.001); and Apo A1/Apo B, 15% (p <0.001). Triglycerides were unchanged up to age 60, but were lower by 24% (p <0.001) in those aged > or = 70. Apo A1 levels were higher (p <0.001), whereas LDL cholesterol was lower (p <0.008) in moderate alcohol consumers versus abstainers. Triglycerides were higher (p <0.001), whereas HDL, HDL2 cholesterol, and Apo A1 were lower (p <0.001) with increasing obesity. Moderate alcohol consumption had a strong favorable effect on HDL, HDL2, and HDL3 cholesterol among subjects of normal weight, but this effect was diminished in obese subjects. Total and LDL cholesterol were higher by 6.4% (p = 0.001) and 9.4% (p <0.003), respectively, whereas HDL cholesterol remained unchanged in those with diastolic BP of 105 to 109 mm Hg versus those with diastolic BP of 95 to 99 mm Hg. We conclude that hypertensive black men have lipid and lipoprotein profiles indicative of less CAD risk than white men. Chronic moderate alcohol consumption correlates with a favorable plasma lipid and lipoprotein profile in normal, but not obese, men. Obesity is associated with an adverse plasma lipid and lipoprotein profile. Thus, race, alcohol intake, and obesity may be important modifiers of CAD in untreated hypertensive men.

The effects of dietary taurocholate, fat, protein, and carbohydrate on the distribution and fate of dietary beta-carotene in ferrets.

Dietary beta-carotene has been shown to have cancer chemopreventive action on the basis of epidemiologic evidence and studies in animals. Because the anticarcinogenic property of beta-carotene may be exerted per se, it is desirable to achieve the maximum absorption and accumulation of intact beta-carotene in various parts of the body. Therefore the effects of dietary taurocholate, fat, protein, and carbohydrate on the absorption, accumulation, and fate of dietary beta-carotene (3.730 nmol/g diet) in selected tissues of ferrets were explored. Taurocholate (0.2-1.0% wt/wt) and fat (6-23% wt/wt) caused two- to threefold (p < 0.05) increases in the absorption and accumulation of beta-carotene in the liver, lungs, and adipose tissue in a dose-dependent manner. In contrast, neither dietary protein (10-40% wt/wt) nor carbohydrate (25-55% wt/wt) affected the absorption and accumulation of beta-carotene in various tissues. Significantly, taurocholate, 23% fat, or 40% protein also markedly increased the amounts of hepatic retinol and retinyl esters derived from dietary beta-carotene. These results indicate that dietary taurocholate, fat, and high protein have a marked influence on the exposure of beta-carotene to intestinal carotene cleavage enzyme or its activity. Thus an ideal combination of dietary components (wt/wt) in ferrets for the maximal absorption and accumulation of beta-carotene in different tissues is 0.5% taurocholate and 13.4% fat, whereas 1% taurocholate, 23% fat, or 40% protein stimulates its conversion to vitamin A.