Changes in very low density lipoproteins with cholesterol loading in man

We have studied the effects of cholesterol loading in man, seeking changes in VLDL that may define a population of particles that resemble the atherogenic β-VLDL in experimental animals. Comparisons were made in 6 men during two diets, containing either 200 mg or 1700 mg cholesterol daily. Although the total plasma cholesterol did not rise significantly over 4 wk of cholesterol loading (mean ± SD 178 ± 41 to 194 ± 48 mg/dl), distinct changes in lipoprotein composition occurred; (1) HDL cholesterol rose significantly (34 ± 4 to 41 ± 5) and plasma apoprotein Al rose from 118 ± 10 to 129 ± 9 mg/dl, (2) Within VLDL, the proportion of apoproteins E:C rose from 0.18 to 0.32 (p < 0.005), though the apoprotein E concentration did not change, (3) Within VLDL, the ratio of cholesteryl esters:triglyceride rose, (4) Within VLDL (Sf 20–400) a population of particles that bound to heparin on heparin-sepharose columns increased threefold; since these particles were richer in apoprotein E and in cholesteryl ester than were VLDL not bound to heparin, we conclude that cholesterol loading leads to an increase in smaller VLDL particles, possibly partly catabolized VLDL or independently secreted IDL, that resemble findings in cholesterol fed animals, and (5) Transport kinetics of apoprotein B in VLDL studied in four subjects did not show a rise in production but this does not rule out increased secretion of a cholesteryl ester, apoprotein E enriched subpopulation of VLDL.     

Low density and high density lipoprotein kinetics and sterol balance in vegetarians

Vegetarians have lower than average plasma lipid levels. Possible mechanisms were studied by measuring the kinetics of LDL-B protein and HDL-AI protein in 7 young male vegetarians and 6 omnivorous control subjects; sterol balances were also determined in a further study with the 6 additional control subjects whose intake of cholesterol was low. Plasma total cholesterol, LDL cholesterol and LDL-B protein were significantly lower in the vegetarians and plasma AI protein concentrations also tended to be lower. The flux or production of LDL was significantly lower, 9.1 ± 2.3 mg/kg/d (mean ± SD) compared to 11.8 ± 0.6 mg/kg/d in the controls and appeared to explain the low levels of LDL. The fractional removal rate of HDL-AI protein was significantly greater in the vegetarians (0.043 ± 0.011 versus 0.027 ± 0.011 for the fractions of pool 1 cleared per hour) and appeared to account for the low plasma AI levels. In the second study, bile acid excretion was significantly lower in the vegetarians and estimated cholesterol synthesis was in the low normal range. Low rates of LDL-B protein formation have been reported in other subjects with low LDL cholesterol concentrations and in the vegetarians presumably reflect the composite of several dietary factors.     

Metabolism of apolipoproteins CII,CIII1, CIII2 and VLDL-B in human subjects consuming high carbohydrate diets

Simultaneous kinetics of apoproteins CII, CIII₁ and CIII₂ and VLDL-B were determined in six normal subjects initially while consuming a control diet and subsequently while consuming a high carbohydrate diet. Mean triglyceride levels in plasma and VLDL were significantly increased, from 110 to 173 mg/dl and 40 to 114 mg/dl respectively, by the high carbohydrate diet. Specific radioactivity of each C peptide was determined, following injection of ¹²⁵I-VLDL, in both VLDL and HDL from plasma samples taken over 48 hr using a technique involving analytical isoelectric focusing. Specific activity vs time curves for VLDL and HDL were almost superimposable on either diet, suggesting that CII, CIII₁ and CIII₂ were catabolized as a group. The high carbohydrate diet resulted in a significant increase in the mass of each C peptide which was brought about by an increase in production since no change in fractional removal rate (FRR) was observed. The pool size of VLDL apo B was also significantly increased and was a function of both increased production as well as decreased FRR. The proportion of CIII₂ mass relative to those of CII and CIII₁ was lower in both VLDL and HDL with the high carbohydrate diet. These observations indicate that the metabolism of the B and C apoproteins are independent of each other and do not respond in the same manner to changes in VLDL triglyceride and that increased triglyceride synthesis and secretion stimulates C apoprotein production.     

Increased catabolism of high density lipoprotein in alcoholic hepatitis

The kinetics of high density lipoprotein apoprotein A-I were determined in three subjects with severe alcoholic hepatitis. Lipoprotein was obtained from three healthy subjects, radioiodinated, and injected into the patients and the donors. The catabolic rate of A-I was two to four times higher in the patients compared to a group of 18 control subjects studied previously. In the patients only, about 20% of the radiolabeled apoproteins rapidly appeared in the lipoprotein-poor fraction of plasma, which also contained substantial amounts of unlabeled A-I. The findings show that the reduced concentration of A-I in alcoholic hepatitis (less than half-normal), is due not to diminished synthesis (synthesis was normal) but to rapid degradation. This may reflect disruption of abnormal high density lipoproteins due to lack of cofactors.     

Heterogeneity of very-low-density lipoprotein metabolism in hyperlipidemic subjects

Very-low-density lipoproteins (VLDL) are triglyceride-rich lipoporteins that have been shown, by physicochemical means, to comprise more than one group of particles. Because of the potential atherogenicity of catabolized VLDL, we used the technique of heparin-affinity chromatography to separate VLDL into two classes of particles, one of which appears to contain partly catabolized VLDL. This observation is based on the higher cholesterol/triglyceride and higher apoprotein E/apoprotein C ratios in VLDL particles that are bound to heparin, resembling in this way intermediate-density lipoproteins (IDL), which are certainly derived in the main through VLDL catabolism. Further studies showed separate metabolic characteristics for the unbound and heparin-bound VLDL particles. Radiolabeled whole VLDL or unbound particles were reinjected into seven hypertriglyceridemic subjects and the kinetics studied in serial samples of plasma over the next 18–48 hours. The specific radioactivity-time curves of apoprotein B in the unbound and bound particles showed that the bound particles were derived wholly or partly from the unbound particles and in turn, were the precursors of IDL. This confirmed that heparin-bound VLDL particles represented VLDL undergoing catabolism, although in one subject about one-half of the bound particles appeared to have an origin other than through VLDL catabolism. These studies show that VLDL metabolism is heterogeneous, that the kinetics of total VLDL must be interpreted accordingly, and that the technique of heparin-affinity chromatography can be used for more detailed studies of VLDL.     

Sucrose-induced changes in VLDL- and LDL-B apoprotein removal rates.

Dietary sucrose has been shown to increase triglyceride transport in very-low-density lipoproteins (VLDL), but it is not known whether the metabolism of the entire particle is affected. Measurements were therefore carried out on VLDL-B apoprotein flux and removal rate before and after the consumption of high sucrose diets (55% of calories). Autologous 125I-labeled VLDL were injected and the specific activity-time curves analyzed by two-pool kinetics. Two kinds of response to sucrose were seen. In two subjects, the pool of VLDL-B apoprotein decreased by 20% and 14% (despite increased fluxes) due to substantial increases in removal rates of 42% and 116%. In four subjects, pool size expanded by 154%, 426%, 50% and 105%, primarily as the result of decreased removal rates (decreases of 55%, 67%, 30%, and 43%). Changes in flux were inconstant, suggesting that accumulation of VLDL particles was related to delayed clearance rather than to increased formation. This may reflect the longer time required to remove the larger load of triglyceride from each particle. The catabolic rate of low-density lipoprotein (LDL) was also measured in five subjects: in four, the clearance of LDL increased with sucrose and was associated with decreases in LDL-B apoprotein and plasma cholesterol.     

Diminished plasma free fatty acid clearance in obese subjects

Obese subjects were compared with lean subjects to define the previously reported disturbance of plasma free fatty acid (FFA) kinetics in terms of altered net transport (lipolysis) or clearance (esterification). These measurements were made during prolonged constant infusions of 1 — ¹⁴C-palmitate toward the end of sustained glucose ingestion and again 6–8 hr after stopping glucose. Net transport of FFA was suppressed to equally low levels in obese and lean subjects, though at the expense of higher insulin concentrations in the obese. Whereas in the lean subjects the clearance of FFA was significantly stimulated with glucose, the obese subjects showed low clearance rates both during and after stopping glucose. When glucose was stopped, net transport rose more rapidly and to a greater extent in some obese than in the lean subjects. The increased influx of FFA led to a rise in the plasma triglyceride level only in the lean subjects. These studies suggest that clearance of plasma FFA, probably denoting esterification in tissues such as muscle and adipose tissue, is impared in obesity and cannot be readily stimulated with glucose and insulin. Lipolysis, measured as net transport of FFA, however, is suppressible with glucose and insulin in the obese, though this might be achieved only at insulin levels that are higher than those in lean subjects.     

Reduced triglyceride formation from long-chain polyenoic fatty acids in rat hepatocytes

The mechanism for the marked reduction in hepatic triglyceride secretion when rats are fed fish oils was explored in studies with isolated rat hepatocytes. Hepatocytes obtained from Sprague-Dawley rats fed either chow or fish oil or safflower oil were incubated in the presence of [3H]-glycerol to estimate triglyceride formation. In some experiments, various fatty acids, complexed to albumin, were added to the incubations. Similar experiments were carried out with hepatocytes from a genetic strain of hypertriglyceridemic, obese rats. In the absence of added fatty acid, hepatocytes from fish oil-fed rats produced and secreted substantially less triglyceride than cells from safflower oil-fed rats. However, the addition of 2 mmol/L Na oleate stimulated triglyceride formation similarly in both types of hepatocytes. When hepatocytes from chow fed rats were incubated with fatty acids of increasing chain length and unsaturation (oleate, linolenate, arachidonate, eicosapentaenoate, and docosahexaenoate), the latter two, which characterize the fish oil used, almost totally suppressed triglyceride formation. Coincubation with oleate partly reversed this effect. Hepatocytes from the hypertriglyceridemic rats synthesized significantly more triglyceride than hepatocytes from normal rats; however triglyceride formation was markedly reduced also in this strain of rat by feeding fish oil or by adding docosahexaenoate to hepatocytes in vitro. These studies confirm previous conclusions with perfused livers from fish oil-fed rats that showed diminished triglyceride production and secretion. These findings suggest that diversion of polyenoic acids from pathways of esterification is a major factor in the triglyceride lowering effect of fish oils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnover of apoproteins A-I and A-II of high density lipoprotein and the relationship to other lipoproteins in normal and hyperlipidemic individuals

The kinetics of the major apoproteins of high density lipoproteins (HDL), A-I(apoA-I) and A-II(apoA-II), were studied from the specific activity-time curves of these apoproteins, after reinjection of radioiodine-labeled HDL. In all 20 subjects, HDL apoprotein kinetics conformed to a two-pool model. The total fractional removal rates for the two apoproteins were similar, although the irreversible fractional removal rate appeared to be slightly greater for apoA-I. The mean transport for A-I and A-II was 12.2 mg/kg/day and 5.0 mg/kg/day, respectively. The mass of the apoprotein pools was strongly correlated with apoprotein production rate and also, to a lesser degree and inversely, with the irreversible fractional catabolic rate. Transport was directly correlated with body weight. Higher fractional catabolic rates, including the transfer rates between the two pools, were observed in five hypertriglyceridemic subjects; in contrast, five subjects with familial hypercholesterolemia tended to show lower fractional catabolic rates. These findings were supported by (1) a strongly positive correlation between the transport rates of HDL A-I and of very low density lipoprotein (VLDL) apoB, determined simultaneously in 10 subjects; and (2) a significant inverse correlation between the irreversible fractional removal rate of HDLA-I and the concentration of low density lipoprotein (LDL) apoB, measured in 15 subjects. These observations underline the metabolic interrelationships of the major lipoprotein classes. Two subjects with familial hyperalphalipoproteinemia showed enlarged pool sizes, but normal transport, with irreversible fractional removal rates that were in the lower range for the group of 20 subjects.     

Blunted norepinephrine responsiveness to changing energy states in obese subjects

We have previously reported in lean subjects a significant relationship between plasma norepinephrine metabolism and energy state. The present study has examined in six obese men the response in plasma norepinephrine flux to ten day periods of overeating (+ 1000 kcal/m2 above isocaloric requirements) or undereating (400 kcal/d). Despite significant gains or losses in body weight, norepinephrine flux, measured during constant infusions of 3H-l-norepinephrine, failed to change significantly. Measurements of glucose utilization during constant infusions of insulin, showed significant changes with changing energy state, falling with overeating and rising with undereating. Insulin sensitivity was not correlated with plasma norepinephrine metabolism.     

The mechanism of adaptive hyperlipogenesis: Insulin receptor binding and glucokinase activity in rat liver during fasting and refeeding

In the present study, rats were fasted for 3 days and subsequently refed for 1, 3, or 5 days. Measurements of insulin binding to its receptors on liver plasma membranes were carried out in conjunction with measurements of the activity of an insulin-regulated enzyme from liver cytosol, glucokinase. In response to the 3-day fast (chronic hypoinsulinemia), the insulin receptor number almost tripled, whereas the glucokinase activity was halved. The insulin receptor number slowly fell to control values during the 5 days of refeeding. In contrast, glucokinase activity rose to levels 2.5 times higher than control (5 times higher than the fasting values) after 1 day of refeeding. Although the activity fell off somewhat during refeeding it was still double control values after 5 days refeeding. It was concluded that in the fasted rat there was a dissociation between insulin receptor concentration and the activity of the insulin-regulated enzyme glucokinase. However, the fasting-induced increase in receptor concentration appeared to play a permissive role in the rapid overshoot of glucokinase activity observed in the early stages of refeeding. Such a scheme would explain the metabolic changes occurring in the fasted-refed rat.     

Responses of dog large coronary arteries to constrictor and dilator substances: implications for the cause and treatment of variant angina pectoris

Canine circumflex coronary artery ring segments were mounted in organ baths and contracted by ergonovine (ergometrine), serotonin, phenylephrine, norepinephrine (in the presence of propranolol), potassium (K⁺) and a thromboxane A₂ mimetic, U46619. Ergometrine was classified as a serotonin agonist since concentration-response curves were competitively inhibited by methysergide but not by alpha-adrenoceptor antagonists. Arteries precontracted to 80% of maximum were relaxed by verapamil, nifedipine and glyceryl trinitrate. However, nifedipine was 100-fold more potent against K⁺ than either serotonin- or phenylephrine-contracted vessels. Verapamil showed some selectivity for K⁺ and serotonin contractions compared with phenylephrine and was almost ineffective against U46619. Glyceryl trinitrate was without selectivity. Electrical field stimulation (2 and 20 Hz, 10-second) caused a weak contraction or relaxation or biphasic response in the isolated coronary ring. Contraction was amplified by propranolol (up to 1 μM) or metoprolol (50 ng/ml) and inhibited by alpha-adrenoceptor antagonists. In a blood-perfused left anterior descending coronary artery preparation, external diameter was measured by sonomicrometry under conditions of controlled flow and resistance. Intraarterial serotonin, U46619 and ergometrine infusions decreased the diameter by up to 18% without causing spasm (0 lumen diameter). Lowering the perfusion pressure from 90 to 60 mm Hg increased the decrease in diameter during serotonin infusions. Verapamil (50 to 150 ng/ml) and nifedipine (15 to 45 ng/ml) inhibited, to a similar degree, the decrease in coronary artery diameter in response to serotonin infusions. However, verapamil was more effective than nifedipine in depressing the sinoatrial node, abolishing reflex tachycardia and lengthening the atrioventricular conduction time. These studies suggest that verapamil and nifedipine are only selective antagonists of constrictor agents in the dog coronary artery, that both low perfusion pressure and beta-adrenoceptor blockade may amplify constrictor responses, and that nifedipine has fewer cardiotoxic effects than verapamil.     

Noradrenaline turnover during under- and over-eating in normal weight subjects

Adjustments to sympathetic nervous system activity may regulate constant body weight despite wide variations in energy intake. To test this six normal weight subjects were studied at three different energy intakes (low, weight maintaining and high). Noradrenaline turnover was measured on the tenth day of each diet. Both noradrenaline appearance rate and noradrenaline clearance increased significantly with increasing energy intake and were more sensitive indices than the plasma noradrenaline concentrations which rose, but not significantly. Fasting triiodothyronine (T₃) rose and reverse T₃ fell with increasing energy intake, while thyroxine (T₄) concentrations did not change. Systolic blood pressure also rose signficantly. Underfeeding resulted in reductions in noradrenaline appearance and clearance rates and in the T₃ level. These results demonstrate that sympathetic nervous system activity, as determined by noradrenaline turnover in plasma, varies in response to short-term changes in energy intake in normal weight subjects. These changes may partly explain why some individuals maintain body weight constant despite large differences in food intake. The present findings may also be relevant to the variability in susceptibility to become obese.     

The effects of meal feeding on the incorporation of D-[U-14C]-glucose into tissue lipids and glycogen as a function of increasing intravenous glucose dose.

The in vivo incorporation of D-[U-¹⁴C]-glucose into lipids and glycogen of adipose tissues, muscle tissues, and liver was measured 1 hr after the i.v. injection of increasing glucose doses (0.75, 1.5 and 2.5 g glucose/kg of body weight) in meal-fed and ad libitum-fed rats. In both perirenal and epididymal fat tissue, the levels of ¹⁴C-label in the total lipid extract was significantly higher in meal-fed than in nibbling rats at all glucose doses. As the glucose dose increased, the ¹⁴C-label in the lipids of both adipose tissues in meal-fed rats increased more than would be expected, assuming a linear dose dependency. In adipose tissues of nibbling rats, glucose dose dependency was linear. The ¹⁴C radioactivity in heart muscle lipids was significantly higher in meal-fed rats at all three glucose doses. In the diaphragm, this effect was seen only at the two higher doses; in liver, only at the highest dose; in skeletal muscle, there was no difference at any of the dose levels. The incorporation of ¹⁴C-label into tissue glycogen exhibited an entirely different pattern. Muscle glycogen synthesis tended to reach a plateau at the middle glucose dose in meal-fed rats, whereas it increased sharply with increasing glucose dose in nibbling rats. Indeed, muscle glycogen synthesis was much greater in nibbling rats than would be expected, assuming linear dose dependency. It was concluded that the two groups of rats responded quite differently to the increasing glucose load. The excess glucose tended to be incorporated into lipid in meal-fed rats and to muscle glycogen in nibbling rats.     

High density lipoprotein cholesterol and coronary artery occlusion.

The relationship between the levels of high density lipoprotein cholesterol (HDLC) and the extent of coronary artery occlusion (ascertained by arteriography) was studied in four hundred male patients. The group with HDLC levels over 50 mg/dl showed a significantly lower coronary artery occlusion score and lower plasma triglyceride levels than the patients with HDLC levels less than 35 mg/dl. The former group also imbibed more alcohol and had less smoking experience.     

Low dose heparin in the prevention of deepvein thromboses in patients with acute myocardial infarction

Patients with acute myocardial infarction of less than 48 hours duration were randomized into three groups. The “fully anticoagulated” group received heparin by intravenous infusion and warfarin sodium to maintain a whole blood clotting time of 30 to 90 minutes and a prothrombin index of 10% to 35%. The “low dose” heparin group received 500 units by intravenous infusion every 12 hours. The control group received no anticoagulants. The radioactive fibrinogen test was used to diagnose the presence of leg vein thromboses.The control group had an incidence of venous thrombosis of 29.7% compared with 13.9% in the low dose group and 11.3% in the fully anticoagulated group. Patients in the control group who had cardiac failure had a significantly higher incidence of venous thromboses (71.4%) when compared with patients not in failure (20.0%). In the two treatment groups no significant difference was observed in patients with and without cardiac failure.Patients with cardiac failure complicating an acute myocardial infarction have a high incidence of venous thromboses. Anticoagulants significantly reduce this incidence and low dose intravenous heparin is as efficacious as full anticoagulation.     

Increased high density lipoprotein cholesterol in obstructive pulmonary disease (predominant emphysematous type)

Since increased levels of high density lipoprotein-cholesterol (HDL-C) seem to be protective against coronary heart disease, there is increasing interest in mechanisms whereby HDL-C levels can be increased. Recent studies suggest that exercise can elevate HDL-C. Patients with chronic obstructive pulmonary disease (COPD), who are usually considered as leading sedentary lifestyles, have an increased work of breathing and their respiratory muscles may be considered to be under a chronic exercise load. Twenty-nine male patients with COPD were selected on the basis of their clinicalphysiologic data, which suggested a predominant emphysematous component of their disease. Clinical features and pulmonary function studies characterized the patients as having relatively pure emphysema (i.e., pink puffers): weight 98% ± 4% (SE) of ideal body weight; absence of cough and sputum; no evidence of cor pulmonale; overinflation in each patient by chest roentgenogram and either curvilinear lines or bullae in 25 of 29 patients; arterial oxygen tension, 62.7 ± 1.4 mm Hg; arterial carbon dioxide tension, 39.8 ± 1.3 mm Hg; severely reduced expiratory flow rate for the midportion of the forced expiratory curve, 0.27 ± 0.02 LPS; overinflation with a residual volume to total lung capacity ratio of 67% ± 1.5% (normal <35%); marked loss of elastic recoil with a transpulmonary pressure at total lung capacity of 14.9 ± 1.82 cm H20 (normal > 19) and a coefficient of retraction of 1.9 ± 0.27 cm H20/liter (normal range 2.5–8.5); minute ventilation (VE) of 10.8 ± .24 liters (normal 6.37 ± 0.9); oxygen consumption (Vo₂) of 251 ± 9 ml (normal 230 ± 30); ventilatory equivalent (V E/Vo₂), 44 ± 1.9 (normal 20–30). Levels of HDL-C were 72 ± 4 mg/dl vs. 54 ± 3 mg/dl for controls matched for age, obesity index, alcoholic intake, smoking history, and race. The HDL-C levels in the patient group were similar to those reported by other workers for long distance runners, and cross country skiers. We suggest these data provide another link between increased levels of HDL-C and exercise.     

In vitro exchanges of esterified cholesterol between serum lipoprotein fractions: studies of humans and rabbits.

Lipoproteins labeled with ³H in the free- and esterified-cholesterol moieties were isolated from the serum of rabbits that had been injected 14 hr before with ³H-mevalonic acid and from human serum that had been incubated with ³H-cholesterol for 6 hr at 37°C. Each of the labeled lipoprotein fractions of each species was subsequently incubated in vitro at 37°C with unlabeled lipoprotein fractions isolated from the same species in the presence of an inhibitor of lecithin:cholesterol acyltransferase (LCAT) activity. Incubations contained either the lipoprotein-free (the dialysed 1.21 g/ml infranate) fraction of serum or an equivalent volume of buffer. In incubations of both rabbit and human lipoproteins there was a significant bidirectional transfer of esterified ³H-cholesterol between each pair of lipoproteins so long as the 1.21 g/ml infranate was present, but in its absence there were no transfers. A minimum estimate of the rate at which esterified cholesterol exchanged between human lipoproteins was obtained; expressed as μmole/liter serum/hr, it was 30–166 between very low density lipoproteins (VLDL) and low density lipoproteins (LDL) 31–156 between VLDL and high density lipoproteins (HDL) and 97–140 between LDL and HDL. In subsequent experiments, labeled human VLDL was incubated with unlabeled human LDL in the presence of the 1.21 g/ml infranate isolated from the serum of different species. In terms of promoting a transfer of esterified ³H-cholesterol into the LDL, the rabbit 1.21 g/ml infranate proved to be more effective than that of the human, but the 1.21 g/ml infranate of rat serum was totally ineffective. It has been concluded that a process of esterified-cholesterol exchange between human serum lipoproteins may be rapid enough to have the potential of major physiologic importance.     

High density lipoprotein cholesterol and body composition of female runners

Twenty-two women participating in an endurance running program had determinations of body composition, of plasma total cholesterol (TC) and HDL cholesterol (HDLC) concentrations before and after increasing their running by 30 miles/wk. Mean total body weight, fat weight and relative body fat showed significant decreases, while mean lean weight significantly increased. Mean HDLC increased 5 mg/dl (p < 0.01). Although increases of HDLC correlated with increases in lean weight, exercise per se appears to increase HDLC of women.