Rajasekhar Ramakrishnan

Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles.

Hypertriglyceridemia is common in the general population, but its mechanism is largely unknown. In previous work human apo CIII transgenic (HuCIIITg) mice were found to have elevated triglyceride levels. In this report, the mechanism for the hypertriglyceridemia was studied. Two different HuCIIITg mouse lines were used: a low expressor line with serum triglycerides of approximately 280 mg/dl, and a high expressor line with serum triglycerides of approximately 1,000 mg/dl. Elevated triglycerides were mainly in VLDL. VLDL particles were 1.5 times more triglyceride-rich in high expressor mice than in controls. The total amount of apo CIII (human and mouse) per VLDL particle was 2 and 2.5 times the normal amount in low and high expressors, respectively. Mouse apo E was decreased by 35 and 77% in low and high expressor mice, respectively. Under electron microscopy, VLDL particles from low and high expressor mice were found to have a larger mean diameter, 55.2 +/- 16.6 and 58.2 +/- 17.8 nm, respectively, compared with 51.0 +/- 13.4 nm from control mice. In in vivo studies, radiolabeled VLDL fractional catabolic rate (FCR) was reduced in low and high expressor mice to 2.58 and 0.77 pools/h, respectively, compared with 7.67 pools/h in controls, with no significant differences in the VLDL production rates. In an attempt to explain the reduced VLDL FCR in transgenic mice, tissue lipoprotein lipase (LPL) activity was determined in control and high expressor mice and no differences were observed. Also, VLDLs obtained from control and high expressor mice were found to be equally good substrates for purified LPL. Thus excess apo CIII in HuCIIITg mice does not cause reduced VLDL FCR by suppressing the amount of extractable LPL in tissues or making HuCIIITg VLDL a bad substrate for LPL. Tissue uptake of VLDL was studied in hepatoma cell cultures, and VLDL from transgenic mice was found to be taken up much more slowly than control VLDL (P < 0.0001), indicating that HuCIIITg VLDL is not well recognized by lipoprotein receptors. Additional in vivo studies with Triton-treated mice showed increased VLDL triglyceride, but not apo B, production in the HuCIIITg mice compared with controls. Tissue culture studies with primary hepatocytes showed a modest increase in triglyceride, but not apo B or total protein, secretion in high expressor mice compared with controls. In summary, hypertriglyceridemia in HuCIIITg mice appears to result primarily from decreased tissue uptake of triglyceride-rich particles from the circulation, which is most likely due to increased apo CIII and decreased apo E on VLDL particles. the HuCIIITg mouse appears to be a suitable animal model of primary familial hypertriglyceridemia, and these studies suggest a possible mechanism for this common lipoprotein disorder.

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.

Effects of Reducing Dietary Saturated Fatty Acids on Plasma Lipids and Lipoproteins in Healthy Subjects: The Delta Study, Protocol 1

Few well-controlled diet studies have investigated the effects of reducing dietary saturated fatty acid (SFA) intake in premenopausal and postmenopausal women or in blacks. We conducted a multicenter, randomized, crossover-design trial of the effects of reducing dietary SFA on plasma lipids and lipoproteins in 103 healthy adults 22 to 67 years old. There were 46 men and 57 women, of whom 26 were black, 18 were postmenopausal women, and 16 were men > or =40 years old. All meals and snacks, except Saturday dinner, were prepared and served by the research centers. The study was designed to compare three diets: an average American diet (AAD), a Step 1 diet, and a low-SFA (Low-Sat) diet. Dietary cholesterol was constant. Diet composition was validated and monitored by a central laboratory. Each diet was consumed for 8 weeks, and blood samples were obtained during weeks 5 through 8. The compositions of the three diets were as follows: AAD, 34.3% kcal fat and 15.0% kcal SFA; Step 1, 28.6% kcal fat and 9.0% kcal SFA; and Low-Sat, 25.3% kcal fat and 6.1% kcal SFA. Each diet provided approximately 275 mg cholesterol/d. Compared with AAD, plasma total cholesterol in the whole group fell 5% on Step 1 and 9% on Low-Sat. LDL cholesterol was 7% lower on Step 1 and 11% lower on Low-Sat than on the AAD (both P<.01). Similar responses were seen in each subgroup. HDL cholesterol fell 7% on Step 1 and 11% on Low-Sat (both P<.01). Reductions in HDL cholesterol were seen in all subgroups except blacks and older men. Plasma triglyceride levels increased approximately 9% between AAD and Step 1 but did not increase further from Step 1 to Low-Sat. Changes in triglyceride levels were not significant in most subgroups. Surprisingly, plasma Lp(a) concentrations increased in a stepwise fashion as SFA was reduced. In a well-controlled feeding study, stepwise reductions in SFA resulted in parallel reductions in plasma total and LDL cholesterol levels. Diet effects were remarkably similar in several subgroups of men and women and in blacks. The reductions in total and LDL cholesterol achieved in these different subgroups indicate that diet can have a significant impact on risk for atherosclerotic cardiovascular disease in the total population.

Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol.

Plasma levels of HDL apo A-I are reduced in individuals with low HDL cholesterol (HDL-C) concentrations as a result of increased fractional catabolic rates (FCRs). To determine the basis for the high apo A-I FCRs, seven subjects with low HDL-C levels (31.0 +/- 4.3 mg/dl) were compared with three subjects with high HDL-C levels (72.0 +/- 4.5 mg/dl). Each subject received autologous HDL that was labeled directly by the iodine-monochloride method (whole-labeled) and autologous HDL that was labeled by exchange with homologous radiolabeled apo A-I (exchange-labeled). Blood was obtained for 2 wk, specific activities determined, and FCRs (d-1 +/- SD) estimated. In every subject, whether in the low or high HDL-C group, the exchange-labeled FCR was greater than the whole-labeled FCR. The exchange-labeled FCR was higher in the low HDL-C group (0.339 +/- 0.043) versus the high HDL-C group (0.234 +/- 0.047; P < 0.009). The whole-labeled FCR was also greater in the low HDL-C group (0.239 +/- 0.023) versus the high HDL-C group (0.161 +/- 0.064; P < 0.02). In addition, in both low and high HDL groups ultracentrifugation resulted in more radioactivity in d > 1.210 (as percentage of total plasma counts per minute) with the exchange-labeled tracer than with the whole-labeled tracer (12.55 +/- 4.95% vs. 1.02 +/- 0.38%; P < 0.003). With both HDL tracers, more radioactivity was found in d > 1.210 in the low versus the high HDL-C groups. When apo A-I catabolism was studied by perfusing isolated rabbit kidneys with whole-labeled HDL, there was twice as much accumulation (cpm/g cortex) of HDL apo A-I isolated from subjects with low HDL-C than from subjects with high HDL-C (P < 0.0025). Finally, HDL that had been isolated from subjects with high levels of HDL-C was triglyceride enriched and exposed to partially purified lipases before perfusion through kidneys. Threefold more apo A-I from modified HDL accumulated in the cortex compared with the unmodified preparation (P < 0.007). The results of these in vivo and ex vivo studies indicate that individuals with low HDL-C levels have more loosely bound, easily exchanged apo A-I and that this exchangeable apo A-I is more readily cleared by the kidney.

Lipoprotein(a): An Elusive Cardiovascular Risk Factor

Lipoprotein (a) [Lp(a)], is present only in humans, Old World nonhuman primates, and the European hedgehog. Lp(a) has many properties in common with low-density lipoprotein (LDL) but contains a unique protein, apo(a), which is structurally different from other apolipoproteins. The size of the apo(a) gene is highly variable, resulting in the protein molecular weight ranging from 300 to 800 kDa; this large variation may be caused by neutral evolution in the absence of any selection advantage. Apo(a) influences to a major extent metabolic and physicochemical properties of Lp(a), and the size polymorphism of the apo(a) gene contributes to the pronounced heterogeneity of Lp(a). There is an inverse relationship between apo(a) size and Lp(a) levels; however, this pattern is complex. For a given apo(a) size, there is a considerable variation in Lp(a) levels across individuals, underscoring the importance to assess allele-specific Lp(a) levels. Further, Lp(a) levels differ between populations, and blacks have generally higher levels than Asians and whites, adjusting for apo(a) sizes. In addition to the apo(a) size polymorphism, an upstream pentanucleotide repeat (TTTTA(n)) affects Lp(a) levels. Several meta-analyses have provided support for an association between Lp(a) and coronary artery disease, and the levels of Lp(a) carried in particles with smaller size apo(a) isoforms are associated with cardiovascular disease or with preclinical vascular changes. Further, there is an interaction between Lp(a) and other risk factors for cardiovascular disease. The physiological role of Lp(a) is unknown, although a majority of studies implicate Lp(a) as a risk factor.

Reduction of Plasma Cholesterol Levels in Normal Men on an American Heart Association Step 1 Diet or a Step 1 Diet with Added Monounsaturated Fat

The design of diets to achieve optimal changes in plasma lipid levels is controversial. In a randomized, double-blind trial involving 36 healthy young men, we evaluated the effects on plasma lipid levels of both an American Heart Association Step 1 diet (in which 30 percent of the total calories were consumed as fat: 10 percent saturated, 10 percent monounsaturated, and 10 percent polyunsaturated fats, with 250 mg of cholesterol per day) and a monounsaturated fat-enriched Step 1 diet (with 38 percent of the calories consumed as fat: 10 percent saturated, 18 percent monounsaturated, and 10 percent polyunsaturated fats, with 250 mg of cholesterol per day). The effects of these diets were then compared with those of an average American diet, in which 38 percent of the total calories were consumed as fat: 18 percent saturated, 10 percent monounsaturated, and 10 percent polyunsaturated fats, with 500 mg of cholesterol per day. The men consumed the average American diet for 10 weeks before random assignment to one of the two Step 1 diets or to continuation of the average diet for an additional 10 weeks. Caloric intake was adjusted to maintain a constant body weight. As compared with the mean (+/- SD) change in the plasma total cholesterol level in the group that followed the average American diet throughout the study (-0.05 +/- 0.36 mmol per liter), there were statistically significant reductions (P less than 0.025) in the plasma total cholesterol level in the group on the Step 1 diet (-0.37 +/- 0.27 mmol per liter) and in the group on the monounsaturated fat-enriched Step 1 diet (-0.46 +/- 0.36 mmol per liter). There were parallel reductions in the plasma low-density lipoprotein cholesterol levels in these two groups. Neither the plasma triglyceride levels nor the high-density lipoprotein cholesterol concentrations changed significantly with any diet. We conclude that enrichment of the Step 1 diet with monounsaturated fat does not alter the beneficial effects of the Step 1 diet on plasma lipid concentrations.

Effects of the PPARγ agonist pioglitazone on lipoprotein metabolism in patients with type 2 diabetes mellitus

Elevated plasma levels of VLDL triglycerides (TGs) are characteristic of patients with type 2 diabetes mellitus (T2DM) and are associated with increased production rates (PRs) of VLDL TGs and apoB. Lipoprotein lipase–mediated (LPL-mediated) lipolysis of VLDL TGs may also be reduced in T2DM if the level of LPL is decreased and/or the level of plasma apoC-III, an inhibitor of LPL-mediated lipolysis, is increased. We studied the effects of pioglitazone (Pio), a PPARγ agonist that improves insulin sensitivity, on lipoprotein metabolism in patients with T2DM. Pio treatment reduced TG levels by increasing the fractional clearance rate (FCR) of VLDL TGs from the circulation, without changing direct removal of VLDL particles. This indicated increased lipolysis of VLDL TGs during Pio treatment, a mechanism supported by our finding of increased plasma LPL mass and decreased levels of plasma apoC-III. Lower apoC-III levels were due to reduced apoC-III PRs. We saw no effects of Pio on the PR of either VLDL TG or VLDL apoB. Thus, Pio, a PPARγ agonist, reduced VLDL TG levels by increasing LPL mass and inhibiting apoC-III PR. These 2 changes were associated with an increased FCR of VLDL TGs, almost certainly due to increased LPL-mediated lipolysis.

Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E.

The mechanism of apolipoprotein (apo) CIII-induced hypertriglyceridemia remains uncertain. We crossed apoCIII transgenic and apoE gene knockout (apoE0) mice, and observed severe hypertriglyceridemia with plasma triglyceride levels of 4,521+/-6, 394 mg/dl vs. 423+/-106 mg/dl in apoE0 mice, P < 0.00001 for log(triglycerides [TG]). Cholesterols were 1,181+/-487 mg/dl vs. 658+/-151 mg/dl, P < 0.0001. Lipoprotein fractionation showed a marked increase in triglyceride-enriched chylomicrons+VLDL. This increase was limited to the lowest density (chylomicrons and Sf 100-400) subfractions. Intermediate density lipoproteins (IDL)+LDL increased moderately, and HDL decreased. There was no significant increase in triglyceride production in apoCIII transgenic/apoE0 mice. The clearance of VLDL triglycerides, however, was significantly decreased. Lipoprotein lipase in postheparin plasma was elevated, but activation studies suggested LPL inhibition by both apoCIII transgenic and apoCIII transgenic/apoE0 plasma. ApoCIII overexpression also produced a marked decrease in VLDL glycosaminoglycan binding which was independent of apoE. The predominant mechanism of apoCIII-induced hypertriglyceridemia appears to be decreased lipolysis at the cell surface. The altered lipoprotein profile that was produced also allowed us to address the question of the direct atherogenicity of chylomicrons and large VLDL. Quantitative arteriosclerosis studies showed identical results in both apoCIII transgenic/apoE0 and apoE0 mice, supporting the view that very large triglyceride-enriched particles are not directly atherogenic.

Suppression of apolipoprotein B production during treatment of cholesteryl ester storage disease with lovastatin. Implications for regulation of apolipoprotein B synthesis.

Cholesteryl ester storage disease (CESD) is characterized by the deficient activity of lysosomal cholesteryl ester (CE) hydrolase, accumulation of LDL-derived CE in lysosomes, and hyperlipidemia. We studied the kinetics of VLDL and LDL apolipoprotein B (apoB), using 125I-VLDL and 131I-LDL, in a 9-yr-old female with CESD and elevated total cholesterol (TC) (271.0 +/- 4.4 mg/dl), triglyceride (TG) (150.0 +/- 7.8 mg/dl), and LDL cholesterol (184.7 +/- 3.4 mg/dl). These studies demonstrated a markedly elevated production rate (PR) of apoB, primarily in LDL, with normal fractional catabolism of apoB in VLDL and LDL. Urine mevalonate levels were elevated, indicative of increased synthesis of endogenous cholesterol. Treatment with lovastatin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, resulted in significant reductions in TC (196.8 +/- 7.9 mg/dl), TG (100.8 +/- 20.6 mg/dl), and LDL cholesterol (102.0 +/- 10.9 mg/dl). Therapy reduced VLDL apoB PR (5.2 vs. 12.2 mg/kg per d pretreatment) and LDL apoB PR (12.7 vs. 24.2 mg/kg per d pretreatment). Urine mevalonate levels also decreased during therapy. These results indicate that, in CESD, the inability to release free cholesterol from lysosomal CE resulted in elevated synthesis of endogenous cholesterol and increased production of apoB-containing lipoproteins. Lovastatin reduced both the rate of cholesterol synthesis and the secretion of apoB-containing lipoproteins.

Autonomic nervous system influences on QT interval in normal subjects

OBJECTIVES We sought to determine whether the relationship between heart rate (HR) and QT interval (QT) differs as HR increases in response to exercise, atropine and isoproterenol. BACKGROUND Autonomic nervous system influences on repolarization are poorly understood and may complicate the interpretation of QT measurements. METHODS Twenty-five normal subjects sequentially underwent graded-intensity bicycle exercise, atropine injection and isoproterenol infusion. Serial 12-lead electrocardiograms were recorded at steady state during each condition and analyzed using interactive computer software. The HR-QT data were modeled linearly and the slopes (quantifying QT adaptation to HR) as well as the QT intervals at 100 beats/min for each intervention were compared by repeated-measures analysis of variance. RESULTS As HR increased, QT was longer for isoproterenol in comparison to exercise or atropine, which were similar. The HR-QT slope (ms/beats/min) was less steep for isoproterenol (-0.83 +/- 0.53) than for atropine (-1.45 +/- 0.21) or exercise (-1.37 +/- 0.23) (p < 0.0001). In comparison to men, women had more negative HR-QT slopes during all interventions. At 100 beats/min, the QT was 364 ms during isoproterenol, which was significantly longer than that during exercise (330 ms) or atropine (339 ms) (p < 0.0001). Isoproterenol produced a dose-dependent increase in U-wave amplitude that was not observed during exercise or atropine. CONCLUSIONS In comparison to exercise and atropine, isoproterenol is associated with much less QT shortening for a given increase in HR and, therefore, greater absolute QT intervals. Our findings demonstrate that autonomic conditions directly affect the ventricular myocardium of healthy subjects, causing differences in QT that are independent of HR.