Edwin L. Bierman

Hyperlipidemia in Coronary Heart Disease II. GENETIC ANALYSIS OF LIPID LEVELS IN 176 FAMILIES AND DELINEATION OF A NEW INHERITED DISORDER, COMBINED HYPERLIPIDEMIA

To assess the genetics of hyperlipidemia in coronary heart disease, family studies were carried out in 2520 relatives and spouses of 176 survivors of myocardial infarction, including 149 hyperlipidemic and 27 normolipidemic individuals. The distribution of fasting plasma cholesterol and triglyceride values in relatives, together with segregation analyses, suggested the presence of five distinct lipid disorders. Three of these-familial hypercholesterolemia, familial hypertriglyceridemia, and familial combined hyperlipidemia-appeared to represent dominant expression of three different autosomal genes, occurring in about 20% of survivors below 60 yr of age and 7% of all older survivors. Two other disorders-polygenic hypercholesterolemia and sporadic hypertriglyceridemia-each affected about 6% of survivors in both age groups. The most common genetic form of hyperlipidemia identified in this study has hitherto been poorly defined and has been designated as familial combined hyperlipidemia. Affected family members characteristically had elevated levels of both cholesterol and triglyceride. However, increased cholesterol or increased triglyceride levels alone were also frequently observed. The combined disorder was shown to be genetically distinct from familial hypercholesterolemia and familial hypertriglyceridemia for the following reasons: (a) the distribution pattern of cholesterol and triglyceride levels in relatives of probands was unique; (b) children of individuals with combined hyperlipidemia did not express hypercholesterolemia in contrast to the finding of hypercholesterolemic children from families with familial hypercholesterolemia; and (c) analysis of informative matings suggested that the different lipid phenotypes owed their origin to variable expression of a single autosomal dominant gene and not to segregation of two separate genes, such as one elevating the level of cholesterol and the other elevating the level of triglyceride. Heterozygosity for one of the three lipid-elevating genes identified in this study may have a frequency in the general population of about 1%, constituting a major problem in early diagnosis and preventive therapy.

The Significance of Basal Insulin Levels in the Evaluation of the Insulin Response to Glucose in Diabetic and Nondiabetic Subjects

The level of insulin after an overnight fast (basal) in 37 obese and nonobese male subjects with normal and abnormal carbohydrate tolerance was directly related to the increase in insulin concentration during a 3 hr 100 g oral glucose tolerance test. Obesity, but not diabetes, was associated with an elevation of this basal insulin level. Thus obesity predicted with the magnitude of the insulin response to glucose ingestion. When the individual insulin values were expressed as per cent change from the basal level, this effect of obesity was excluded. The insulin levels of all subjects with normal carbohydrate tolerance promptly rose 5-7-fold, and reached peak values 1 hr after oral glucose. In contrast, the diabetic response (as per cent increase) was markedly reduced during the 1st hr, and maximal (but still subnormal) insulin levels were not attained until 2 hr. In all subjects the insulin response (quantitated by calculation of the area circumscribed by a plot of the per cent change in insulin with time) showed a significant inverse correlation with the glucose response. Thus increasing degrees of carbohydrate intolerance were associated with decreasing insulin responses. Elevated levels of insulin, in both the basal state and in response to glucose, were related to obesity.

Hyperlipidemia in Coronary Heart Disease I. LIPID LEVELS IN 500 SURVIVORS OF MYOCARDIAL INFARCTION

Plasma cholesterol and triglyceride levels were measured after an overnight fast in 500 consecutively studied 3-mo survivors of myocardial infarction. Virtually all patients under 60 yr of age (95% ascertainment) and a randomly chosen group of older survivors admitted to 13 Seattle hospitals during an 11 mo period were included. A comparison of their lipid values with those of 950 controls demonstrated that 31% had hyperlipidemia. These lipid abnormalities were most commonly found in males under 40 yr of age (60% frequency) and in females under 50 yr of age (60% frequency). Elevation in triglyceride levels with (7.8%) or without (15.6%) an associated elevation in cholesterol levels was three times more common in survivors than a high cholesterol level alone (7.6%). These results raise the possibility that hypertriglyceridemia may be as an important a risk factor for coronary atherosclerosis as hypercholesterolemia. The identification of hyperlipidemic survivors of myocardial infarction provided a unique source of probands for family studies designed to disclose the genetic origin of hyperlipidemia in coronary heart disease.

Lipoprotein Uptake and Metabolism by Rat Aortic Smooth Muscle Cells in Tissue Culture

Aortic smooth muscle cells from the rat were successfully grown in tissue culture and shown to have characteristic morphology. 125I-labeled homologous very low density lipoproteins and high density lipoproteins were taken up by these smooth muscle cells during incubation for 48 hours at the stationary phase. Despite multiple washings, a large proportion of the lipoprotein radioactivity associated with the cells was apparently surface bound and trypsin releasable. With both lipoprotein fractions, lipid and protein uptake by the cells measured after trypsinization was related to time and to the amount of lipoprotein protein added to the medium. Compared with protein, there was a disproportionately greater entry of lipid radioactivity into the cells. Light and electron microscope autoradiography localized the label intracellularly over the cell cytoplasm, cell boundaries, and, in some cells, over lysosomes. On the basis of either protein uptake or whole particle uptake, approximately four times as much high density lipoprotein as very low density lipoprotein was taken up by the smooth muscle cells. To assess metabolism and degradation of high density lipoproteins, aortic smooth muscle cells were incubated in fresh unlabeled medium for 48 hours after exposure to 125I-labeled high density lipoproteins. A large proportion of radioactivity released was trichloroacetic acid precipitable, suggesting some release of whole lipoprotein protein; however, these lipoproteins appeared to be modified when they were tested with anti-high density lipoprotein antiserum. Also, water-soluble radioactivity (presumably protein breakdown products) was released in amounts that averaged 3% of the protein label in the cells. These results indicate that although aortic smooth muscle cells growing in tissue culture can rapidly take up lipids and lipoproteins, catabolism of lipoprotein protein is slow. Correlative biochemical and ultrastructural analysis suggests the possibility of regurgitation of noncatabolized lipoprotein protein by reverse endocytosis.

Evidence for a Common, Saturable, Triglyceride Removal Mechanism for Chylomicrons and Very Low Density Lipoproteins in Man

Hypertriglyceridemic subjects were fed diets in which dietary fat calories were held constant, but carbohydrate calories were varied. Three subjects with fasting chylomicronemia (Type V) were given less carbohydrate and four subjects without fasting chylomicronemia (Type IV) were fed diets with more calories as carbohydrate. The restricted carbohydrate intake led to disappearance of chylomicronemia in those subjects who had chylomicronemia on a normal diet (Type V to IV). In those subjects without chylomicronemia, chylomicronemia appeared in response to increased carbohydrate intake (Type IV to V). Thus chylomicron concentrations in plasma were altered even though fat intake and presumably chylomicron input into plasma was kept constant. These findings provide evidence for saturation of chylomicron removal mechanisms by alteration of endogenous triglyceride-rich lipoprotein concentrations. They suggest that chylomicrons compete with very low density lipoproteins for similar removal mechanisms. The relationship between endogenous triglyceride concentration and the lipolytic activity in plasma following heparin was then evaluated with the use of long-term heparin infusions to release and maintain lipolytic activity in the circulation. 10 subjects were placed on fatfree diets to remove circulating dietary fat. The plasma lipolytic rate during the heparin infusion was measured consecutively on different days in individuals whose triglyceride concentrations were varied by either increasing or decreasing calories. The lipolytic rate was curvilinearly related to the plasma triglyceride concentrations. This curvilinear relationship followed Michaelis-Menton saturation kinetics over a wide range of triglyceride concentrations on fat-free, high-carbohydrate diets, in multiple studies in a group of individuals. These studies suggest that endogenous and exogenous triglyceride compete for a common, saturable, plasma triglyceride removal system related to lipoprotein lipase.

Effect of insulin on the proliferation of cultured primate arterial smooth muscle cells.

Smooth muscle cells were grown from thoracic aortas of 1-year-old monkeys (Macaca nemistrina). The effect of insulin on the proliferation of these cells was studied by comparing the growth of cells in culture medium to which insulin had been added with that of cells in basal (1% monkey serum) medium and in growth-promoting 5% monkey serum. Insulin in concentrations of 10, 100, 1,000, and 10,000 μunits/ml resulted in successively greater stimulation of growth which was highly significant (P < 0.001) by analysis of variance. There was a significant linear relationship between the logarithm of the insulin dose and cell growth. However, the highest concentration of insulin produced only 50% of the effect of 5% monkey serum. Serum from which insulin had been removed stimulated growth less well (P < 0.05) than did untreated serum at the same concentration (5%) but had significant (P < 0.05) stimulating properties compared with whole serum at a lower concentration. Cells that were older in culture life (eight or nine passages) did not show a growth response to insulin and had an attenuated response to 5% serum. The effect of insulin (100 μunits/ml) was inhibited by dibutyryl cyclic adenosine monophosphate (db-cAMP) (5 × 10−5M), although there was a latent period of 3 days before inhibition occurred; db-cAMP had no effect on cell counts in the absence of insulin. The electron microscopic appearance of the cells was unaltered by insulin.

An Abnormality of Nonesterified Fatty Acid Metabolism in Diabetes Mellitus

The nonesterified fatty acid (NEFA) fraction of plasma, with its exceedingly rapid turnover,2 appears to be a major link in the transport of fatty acids from depots to liver and peripheral tissues.4 In normal subjects, plasma NEFA concentration falls sharply after an injection of insulin, paralleling the fall of blood glucose. On the other hand, feeding of glucose or injection of glucagon causes the two components to vary in opposite directions: Blood glucose rises while NEFA falls.6 Apparently the common denominator responsible for reduction of NEFA in these two situations is an increased utilization of glucose promoted by insulin or by an input of extra glucose in the presence of adequate endogenous insulin. Diabetic subjects show an abnormally prolonged elevation of blood sugar after a glucose load and a reduced capacity to burn carbohydrate or synthesize fat. The present studies indicate that control of NEFA concentration is likewise abnormal in diabetes. Disturbances of fat metabolism have long been recognized in diabetes, and indeed continue to cause the majority of deaths. Before insulin was available, ketosis, arising from incomplete combustion of fatty acids, was the main hazard. The defects of lipid metabolism in diabetes are now more chronic, but no less lethal. About half of the diabetics in one recent series died from coronary occlusion, and among the entire group, the great majority showed arteriosclerosis. As further evidence of faulty fat metabolism, xanthomata and fatty liver are frequently associated with diabetes, and obesity may be the forerunner of the disease. Hyperlipemia, mainly an increased concentration of the triglyceride plus phospholipid fractions in plasma of fasting subjects, occurs frequently in diabetics, especially those in poor control. In one series, studied before the availability of insulin, blood fat was found to be increased almost as consistently as blood glucose

Myocardial infarction in the familial forms of hypertriglyceridemia

Among 74 hypertriglyceridemic patients who were referred for study because of hypertriglyceridemia, family investigations detected 19 with familial hypertriglyceridemia and 24 with familial combined hyperlipidemia. The frequency of myocardial infarction among adult living hyperlipidemic relatives of patients with familial combined hyperlipidemia was 17.5% (10/57). Five of these relatives had their infarct between the ages of 40 and 50 yr of age, and five before the age of 40 yr. The frequency of myocardial infarction in living hyperlipedemic relatives with familial hypertriglyceridemia was 4.7% (2/43) and was similar to the frequency of myocardial infarction among normolipidemic relatives (4.5%) or among spouse controls (5.2%). Mortality data due to myocardial infarction among relatives of index patients failed to contribute meaningful information.

Regulation of High Density Lipoprotein Receptor Activity in Cultured Human Skin Fibroblasts and Human Arterial Smooth Muscle Cells

Cultured human skin fibroblasts and human arterial smooth muscle cells possess high-affinity binding sites specific for high density lipoproteins (HDL). Results from the present study demonstrate that binding of HDL to these sites is up-regulated in response to cholesterol loading of cells. When fibroblasts or smooth muscle cells were preincubated with nonlipoprotein cholesterol, cellular binding of 125I-HDL3 was enhanced severalfold. This enhancement was sustained in the presence of cholesterol but was readily reversed when cells were exposed to cholesterol-free medium. The stimulatory effect of cholesterol treatment was prevented by cycloheximide, suggesting the involvement of protein synthesis. Kinetic analysis of HDL3 binding showed that prior exposure to cholesterol led to an induction of high-affinity binding sites on the cell surface. In the up-regulated state, the apparent dissociation constant (Kd) of these sites was approximately 2 micrograms protein/ml. Competition studies indicated that the HDL binding sites recognized either HDL3 or HDL2 but interacted weakly with low density lipoprotein (LDL). Exposure of cells to lipoprotein cholesterol in the form of LDL also enhanced HDL binding by a process related to delivery of sterol into cells via the LDL receptor pathway. Enhancement of HDL binding to fibroblasts by either nonlipoprotein cholesterol or LDL was associated with an increased cell cholesterol content, a suppressed rate of cholesterol synthesis, decreased LDL receptor activity, and an enhanced rate of cholesterol ester formation. A comparison of HDL3 binding with the effects of HDL3 on cholesterol transport from cells revealed similar saturation profiles, implying a link between the two processes. Thus, cultured human fibroblasts and human arterial smooth muscle cells appear to possess specific receptors for HDL that may function to facilitate cholesterol removal from cells.

Improved Glucose Tolerance with High Carbohydrate Feeding in Mild Diabetes

Abstract To evaluate the effect of increased dietary carbohydrate in diabetes mellitus, glucose and immunoreactive insulin levels were measured in normal persons and subjects with mild diabetes mai...