Hyperinsulinemia.

Patients with mild or early non-insulin-dependent diabetes mellitus often display a delay in insulin response followed by late hyperinsulinemia during oral glucose tolerance testing. Those patients with long-standing disease or elevations of fasting plasma glucose in excess of 140 mg/dl are generally hypoinsulinemic in response to an oral glucose tolerance test. Diabetic patients who do not have an acute response to intravenous glucose may have normal responses to intravenous tolbutamide or intravenous arginine, suggesting that delayed responsiveness to glucose is not due to decreased pancreatic insulin content. An association between hyperinsulinemia and hypertension has been suggested by recent studies from several laboratories. In a homogeneous population of men who suffered traumatic bilateral above-the-knee amputation in the Vietnam War with subsequent development of obesity, it was shown that there was strong correlation between hypertension and hyperinsulinemia during oral glucose tolerance testing despite only mild glucose intolerance. In addition, a subset of hypertensive women who were in their third trimester of pregnancy were markedly hyperinsulinemic during oral glucose tolerance testing in the absence of any abnormalities of glucose tolerance. Thus, the relationship between hyperinsulinemia and hypertension, and the possible reasons for this relationship, are fields of active investigation at present.

Early and long-term effects of acute caloric deprivation in obese diabetic patients.

PURPOSE: It is generally assumed that diet therapy can ameliorate the metabolic derangements experienced by obese type 2 diabetic patients, thereby leading to discontinuation of insulin or oral sulfonylurea drug therapy. We decided to retrospectively investigate which clinical and biochemical parameters affect therapeutic responses. PATIENTS AND METHODS: Sixty-four poorly controlled obese diabetic patients were hospitalized and placed on a precisely defined, hypocaloric diet. Known duration of diabetes, type of pharmacologic therapy, body weight, weight loss, fasting plasma glucose concentrations, C-peptide levels, hemoglobin A1C, and plasma lipid levels were assessed, as were nitrogen and electrolyte balances. RESULTS: Average weight loss was 13 pounds in a mean of 23 days. During hospitalization, the mean fasting plasma glucose value for the group fell from 221 +/- 10 to 122 +/- 5 mg/dl. In 45 patients (73 percent), the final fasting plasma glucose level was less than 125 mg/dl (mean: 102 +/- 2 mg/dl). Oral glucose tolerance even in those patients in whom fasting plasma glucose levels normalized was still grossly diabetic at the end of the hospital stay, deteriorating further after three days of liberalized caloric intake. In part this may have been due to decreased insulin secretory reserve as reflected by blunted plasma C-peptide response. Forty of 42 patients who entered the study taking insulin were able to discontinue the drug within one to seven days of hospitalization. After a mean follow-up period of 19 months, only 10 of 50 patients continued to maintain fasting euglycemia; five were on diet alone, and five were receiving oral hypoglycemic agents. Thirteen patients were receiving insulin therapy. CONCLUSION: Diet therapy in these patients resulted in short-term improvement of glycemic control and, in the majority, normalization of fasting plasma glucose levels. However, long-term outpatient follow-up revealed that relapse occurred in most patients.

Cardiovascular disease risk factors in combat veterans after traumatic leg amputations.

Traumatic leg amputation, but not arm amputation, in World War II combat veterans has been associated with subsequent increased ischemic heart disease mortality. In a pilot project we examined a group of 19 high-risk Vietnam War veterans with bilateral above-knee amputations in comparison with a control group with unilateral below-elbow amputations. Nine of the 19 above-knee amputees were hypertensive (p = 0.05) and obese by hydrostatic weighing (p less than 0.001). Obesity was strongly associated with hypertension, decreased glucose tolerance, and marked hyperinsulinemia. Cigarette smoking, blood lipid abnormalities, and decreased cardiovascular fitness were not implicated as significant risk factors. Long-term risks of amputation may be related to metabolic and hemodynamic sequelae of excessive maturity-onset weight gain in young men immobilized by loss of lower limbs.

Insulin as a potential factor influencing blood pressure in amputees.

War-injured, bilateral above-knee amputees are known to be at increased risk for cardiovascular mortality. To evaluate possible risk factors, we compared blood pressures and plasma glucose and insulin responses to orally administered glucose in 19 above-knee amputees from the Vietnam War (mean age, 36 +/- 1 years) with those of 12 age-matched unilateral below-elbow amputees. Body composition by densitometry and maximal oxygen consumption during arm or leg exercise were also determined. Nine of 19 leg amputees were hypertensive compared with one of 12 arm amputees. Their 3-hour average insulin responses were markedly increased (260 +/- 60 microU/ml) compared with those of normotensive leg (125 +/- 24 microU/ml) and arm amputees (101 +/- 20 microU/ml), and their mean body fat content (37.2%) also was elevated compared with that in both of these groups (23.2 and 22.6%, respectively). A unique finding was that both insulin response and body fat content were strongly and independently correlated with diastolic blood pressure (r = 0.55, p less than 0.01, and r = 0.62, p less than 0.01, respectively). We conclude that insulin may be a major factor in blood pressure regulation in the maturity-onset obesity that develops following traumatic leg amputation in young, healthy men.

Lecithin:cholesterol acyltransferase regulation. II. Effect of fluidity of egg phosphatidylcholine vesicles.

The regulation of human plasma lecithin:cholesterol acyltransferase (LCAT) by changes in bilayer fluidity of substrate egg phosphatidylcholine (egg PC) unilamellar vesicles was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity was decreased by adding up to 20% cholesterol or increased by adding up to 10% egg 2-lysophosphatidylcholine (lysoPC). The fluidizing effect of lysoPC was suppressed by the addition of cholesterol. LCAT activity with 10% cholesterol vesicles was decreased by adding 5% lysoPC, yet activity with 5% cholesterol vesicles was unaffected by adding 5% lysoPC. This difference may be explained by a balance between the known LCAT inhibitory effect of lysoPC and its ability to increase bilayer fluidity and thereby increase LCAT activity. LCAT esterification of up to 37% of vesicle cholesterol failed to alter the lysoPC/cholesterol balance sufficiently to influence activity in this system. The findings of our studies are in keeping with modulation of LCAT activity by bilayer fluidity, but fluidity changes caused by enzyme action are not sufficient to regulate that activity.

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles.

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.

Dependence of human plasma lecithin:cholesterol acyltransferase activity upon high-density lipoproteins.

The conclusion that HDL2 suppress lecithin:cholesterol acyltransferase activity has been re-examined using three assay methods. Removal by ultracentrifugation of lipoproteins of d 1.125 g/ml reduced activity to 53% (n = 15) of control plasma. Replacement of VLDL and LDL was without effect, but addition of HDL2 restored activity. Addition of HDL2 to d 1.125 g/ml infranate in quantities greater than had been present in original plasma had no further effect on activity, and addition of HDL2 to plasma had no effect on activity. It is concluded that HDL2 are required for full expression of plasma lecithin:cholesterol acyltransferase activity.

Lipoprotein products of lecithin: cholesterol acyltransferase and cholesteryl ester transfer.

High-density lipoprotein substrates and products of human plasma lecithin: cholesterol acyltransferase have been labelled with radioisotopic cholesteryl esters in order to facilitate identification. [3H]Cholesteryl esters were formed by endogenous HDL3/VHDL enzyme (d greater than 1.125 g/ml) following incubation with mixed vesicles of phosphatidylcholine, unesterified cholesterol and 3H-labelled unesterified cholesterol. Transfer of labelled esters to acceptor lipoproteins (VLDL+LDL, d less than 1.063 g/ml) was employed to distinguish a hypothetical transfer complex. Separation of labelled HDL3/VHDL was by gel-permeation chromatography. The results indicate that a subpopulation of labelled HDL3/VHDL cholesteryl esters (43-61% of total) were removed by VLDL/LDL during a 3 h transfer period and these derive from the smaller lipoproteins of the spectrum. HDL carrying non-transferable [3H]cholesteryl esters localize to the larger HDL3. Transfer rates were proportional to ratios of acceptor to donor lipoproteins. Net transfer of cholesteryl esters from the smaller HDL3 also occurred, but was smaller in magnitude (about 10.5% of total). Acyltransferase assays indicated that enzyme distribution is skewed to larger-sized HDL3, suggesting that the non-transferable components might be lecithin: cholesterol acyltransferase-containing parent complexes, while the smaller transfer products contain little acyltransferase. The results fit the hypothesis that a parent HDL3-lecithin: cholesterol acyltransferase complex generates a smaller-sized lipoprotein product which is active in cholesteryl ester transport.

Regulation of plasma lecithin:cholesterol acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.

Plasma lecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects. Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL(2), HDL(3), and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes. HDL(2) lipids and protein increased substantially, characterized primarily by enrichment with lecithin. HDL(3), which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others. Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity. The VHD cholesteryl esters and lysolecithin were also reduced. Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism. In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL(3) and VDH, but not in HDL(2), during incubation. Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteins is an unlikely explanation for activation. Since VHD and HDL(3), which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made. The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo. Rose, H. G., and J. Juliano. Regulation of plasma lecithin: cholesteryl acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.

Transfer and equilibration of LCAT-generated cholesteryl esters in human lipoproteins.

Non-enzymatic net transport of cholesteryl esters (CE) from HDL to VLDL in exchange for triglycerides has been reported in vitro. Likewise, in vivo observations have been adduced favoring the same process in human subjects and implicating LCAT product lipoproteins as carriers. Exchange of CE among lipoproteins is thought not to occur. In experiments in which LCAT-generated radioactive CE are formed from cholesterol dispersion in plasma, exchange to near equilibrium is observed. To better define this process, a labeling procedure employing lecithin: 3H-unesterified cholesterol single bilayer vesicles was devised. The order of lipoprotein labeling with isotopic CE is HDL greater than VLDL greater than LDL. After 19 hours of incubation, esters reached equilibrium. Control experiments with vesicles treated by purified LCAT showed that this result could not be explained by distribution of discrete ester-rich vesicle products or their binding to lipoproteins. Subfractionation of labeled lipoproteins on agarose columns confirmed completeness of equilibration, except for the HDL subfraction of smallest size, which is incompletely equilibrated. These results indicate that LCAT-generated CE are capable of equilibration among human lipoproteins.

Regulation of plasma lecithin:cholesterol acyltransferase. II. Activation during alimentary lipemia.

The effect of dietary fat on plasma lecithin:cholesterol acyltransferase (LCAT) activity has been investigated in 14 normal male subjects. After determination of postabsorptive lipid and LCAT levels, a high-fat liquid test meal (1 to 2 gm./kg. body weight) was fed, followed by lipid and LCAT determinations at 2.5 hour intervals. Plasma triglycerides were elevated by 2.5 hours, peaked at 5.0 hours, fell at 7.5 hours, and were normalized by 10 hours. LCAT was unchanged at 2.5 hours but was elevated by 5.0 hours, exhibiting a broad plateau through 10 hours. Most subjects manifested peak responses at 7.5 hours. The mean maximal increase in individual subjects was 37.2 +/- 13.3 (S.D.) percent. LCAT changes similarly followed the elevation and recession of chylomicrons (Sf greater than 400) and very-low-density lipoprotein triglycerides, both of which closely paralleled plasma triglycerides. Enzyme responses were proportional to percentage elevations of plasma triglycerides (r = 0.93, p less than 0.01) and related to quantity of fat in the test diet. Three subjects who ingested the test diet devoid of the fat component showed no significant change in enzyme activity. Enzyme progress curves revealed linearity for 3 hours for both postabsorptive and lipemic (7.5 hour) plasma from the same subjects, supporting the validity of the assay as a measure of enzyme rate. These studies demonstrate an increase in cholesterol esterifying activity temporally related to the clearance of alimentary particles, suggesting a physiologic role in the clearance process.

Regulation of plasma lecithin:cholesterol acyltransferase in man. I. Increased activity in primary hypertriglyceridemia.

Patients with primary hypertriglyceridemia have been reported to manifest increased in vivo turnover of plasma cholesteryl esters. To ascertain if this is due to plasma lecithin:cholesterol acyltransferase (LCAT) and to explore a possible link between triglyceride and cholesteryl ester turnover, we have measured LCAT in 15 patients with Type IV, 2 with Type V, 1 with Type III, and 9 with Type II B hyperlipoproteinemia. LCAT was significantly elevated (p less than 0.001) in hypertriglyceridemic subjects, regardless of lipoprotein pattern. In the Type IV group, but not in normal subjects, LCAT correlated significantly with measures of very low-density lipoprotein (VLDL) elevation, including plasma triglycerides and particularly VLDL-unesterified cholesterol, but not with body weight or substrate high-density lipoprotein (HDL) lipid levels. On repeated determinations in individual subjects, a relationship between triglyceride fluctuations and LCAT could be demonstrated in only one subject over an extreme range of triglyceride levels. Analysis of lipoprotein lipids revealed that the ester:free cholesterol ratio in VLDL was increased in hypertriglyceridemia, but was not correlated with enzyme level. In vitro removal of endogenous VLDL or addition of VLDL from lipemic plasmas to normal plasmas was without effect on enzyme activity. Regulation of enzyme activity does not appear to be a direct function of VLDL level.

Clofibrate-induced low density liporotein elevation. Therapeutic implications and treatment by colestipol resin.

Plasma lipid and lipoprotein responses to clofibrate were assessed in fifteen hypertriglyceridemic patients for the purpose of ascertaining low-density lipoprotein (LDL) changes. Subjects were grouped into either Type IV (11) or IIB (4) subgroups according to initial LDL level. Clofibrate was without effect on LDL in the IIB group, but consistent, often large, elevations were noted in Type IV cases (mean increase, 37.6%, P less than 0.001). In the IIB subgroup, addition of the bile-acid sequestrant, colestipol, lowered LDL (27.8%, P less 0.02) and total cholesterol (21.3%, P less 0.01) below pre-treatment values. In the Type IV subgroup, LDL fell to 19.5% above baseline (P great than 0.05). Significant LDL elevations induced by clofibrate in three of six subjects were restored to initial levels. In both groups, triglycerides and very-low density lipoproteins (VLDL) were not affected. The efficacy of colestipol in reducing LDL levels, expressed as either absolute or percentage reductions, increased as a function of increasing post-clofibrate LDL concentration (r = 0.84, P less than 0.001). In these subjects the level of LDL after treatment with clofibrate depended upon their LDL level prior to drug therapy, the effect of clofibrate on this level, and lipoprotein phenotype. Thus colestipol was most effective in IIB subjects, Type IV subjects with the lowest baseline VLDL and hence reciprocally highest LDL, and Type IV individuals who exhibited the largest LDL induction by clofibrate. The reported ineffectiveness of clofibrate on mortality and morbidity in patients with established coronary heart disease might be related to elevations and infrequent reductions of LDL. From the perspective of lipoprotein lowering, the combination with colestipol appears more favorable.