Nirupa R. Matthan

Eicosapentaenoic Acid Prevents and Reverses Insulin Resistance in High-Fat Diet-Induced Obese Mice via Modulation of Adipose Tissue Inflammation

We investigated the effects of eicosapentaenoic acid (EPA) on prevention (P) and reversal (R) of high saturated-fat (HF) diet-induced obesity and glucose-insulin homeostasis. Male C57BL/6J mice were fed low-fat (LF; 10% energy from fat), HF (45% energy from fat), or a HF-EPA-P (45% energy from fat; 36 g/kg EPA) diet for 11 wk. A 4th group was initially fed HF for 6 wk followed by the HF-EPA-R diet for 5 wk. As expected, mice fed the HF diet developed obesity and glucose intolerance. In contrast, mice fed the HF-EPA-P diet maintained normal glucose tolerance despite weight gain compared with the LF group. Whereas the HF group developed hyperglycemia and hyperinsulinemia, both HF-EPA groups (P and R) exhibited normal glycemia and insulinemia. Further, plasma adiponectin concentration was lower in the HF group but was comparable in the LF and HF-EPA groups, suggesting a role of EPA in preventing and improving insulin resistance induced by HF feeding. Further analysis of adipose tissue adipokine levels and proteomic studies in cultured adipocytes indicated that dietary EPA supplementation of HF diets was associated with reduced adipose inflammation and lipogenesis and elevated markers of fatty acid oxidation. In C57BL/6J mice, EPA minimized saturated fat-induced insulin resistance and this is in part mediated by its effects on fatty acid oxidation and inflammation.

Extended-Release Niacin Alters the Metabolism of Plasma Apolipoprotein (Apo) A-I and ApoB-Containing Lipoproteins

Objectives—Extended-release niacin effectively lowers plasma TG levels and raises plasma high-density lipoprotein (HDL) cholesterol levels, but the mechanisms responsible for these effects are unclear. Methods and Results—We examined the effects of extended-release niacin (2 g/d) and extended-release niacin (2 g/d) plus lovastatin (40 mg/d), relative to placebo, on the kinetics of apolipoprotein (apo) A-I and apoA-II in HDL, apoB-100 in TG-rich lipoproteins (TRL), intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL), and apoB-48 in TRL in 5 men with combined hyperlipidemia. Niacin significantly increased HDL cholesterol and apoA-I concentrations, associated with a significant increase in apoA-I production rate (PR) and no change in fractional catabolic rate (FCR). Plasma TRL apoB-100 levels were significantly lowered by niacin, accompanied by a trend toward an increase in FCR and no change in PR. Niacin treatment significantly increased TRL apoB-48 FCR but had no effect on apoB-48 PR. No effects of niacin on concentrations or kinetic parameters of IDL and LDL apoB-100 and HDL apoA-II were noted. The addition of lovastatin to niacin promoted a lowering in LDL apoB-100 attributable to increased LDL apoB-100 FCR. Conclusion—Niacin treatment was associated with significant increases in HDL apoA-I concentrations and production, as well as enhanced clearance of TRL apoB-100 and apoB-48.

Dietary Hydrogenated Fat Increases High-Density Lipoprotein apoA-I Catabolism and Decreases Low-Density Lipoprotein apoB-100 Catabolism in Hypercholesterolemic Women

Objective—To determine mechanisms contributing to decreased high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein cholesterol (LDL-C) concentrations associated with hydrogenated fat intake, kinetic studies of apoA-I, apoB-100, and apoB-48 were conducted using stable isotopes. Methods and Results—Eight postmenopausal hypercholesterolemic women were provided in random order with 3 diets for 5-week periods. Two-thirds of the fat was soybean oil (unsaturated fat), stick margarine (hydrogenated fat), or butter (saturated fat). Total and LDL-C levels were highest after the saturated diet (P< 0.05; saturated versus unsaturated) whereas HDL-C levels were lowest after the hydrogenated diet (P< 0.05; hydrogenated versus saturated). Plasma apoA-I levels and pool size (PS) were lower, whereas apoA-I fractional catabolic rate (FCR) was higher after the hydrogenated relative to the saturated diet (P< 0.05). LDL apoB-100 levels and PS were significantly higher, whereas LDL apoB-100 FCR was lower with the saturated and hydrogenated relative to the unsaturated diet. There was no significant difference among diets in apoA-I or B-100 production rates or apoB-48 kinetic parameters. HDL-C concentrations were negatively associated with apoA-I FCR (r=−0.56, P=0.03) and LDL-C concentrations were negatively correlated with LDL apoB-100 FCR (r=−0.48, P=0.05). Conclusions—The mechanism for the adverse lipoprotein profile observed with hydrogenated fat intake is determined in part by increased apoA-I and decreased LDL apoB-100 catabolism.

Higher plasma docosahexaenoic acid is associated with reduced progression of coronary atherosclerosis in women with CAD

Fish intake, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and in some cases α-linolenic acid (ALA) have been associated with reduced risk of cardiovascular events and death. The association between n-3 fatty acids in plasma lipids and the progression of coronary artery atherosclerosis was assessed among women with established coronary artery disease (CAD). A prospective cohort study involved postmenopausal women (n = 228) participating in the Estrogen Replacement and Atherosclerosis Trial. Quantitative coronary angiography was performed at baseline and after 3.2 ± 0.6 (mean ± SD) years. Women with plasma phospholipid (PL) DHA levels above the median, compared with below, exhibited less atherosclerosis progression, as expressed by decline in minimum coronary artery diameter (−0.04 ± 0.02 and −0.10 ± 0.02 mm, respectively; P = 0.007) or increase in percentage stenosis (1.34 ± 0.76% and 3.75 ± 0.74%, respectively; P = 0.006), and had fewer new lesions [2.0% (0.5–3.5%) of measured segments (95% confidence interval) and 4.2% (2.8–5.6%), respectively; P = 0.009] after adjustments for cardiovascular risk factors. Similar results were observed for DHA in the triglycerides (TGs). EPA and ALA in plasma lipids were not significantly associated with atherosclerosis progression. Consistent with higher reported fish intake, higher levels of plasma TG and PL DHA are associated with less progression of coronary atherosclerosis in postmenopausal women with CAD.

Long-term fatty acid stability in human serum cholesteryl ester, triglyceride, and phospholipid fractions

Fatty acid profiles of biological specimens from epidemiological/clinical studies can serve as biomarkers to assess potential relationships between diet and chronic disease risk. However, data are limited regarding fatty acid stability in archived specimens following long-term storage, a variable that could affect result validity. Our objective was to determine the effect of prolonged storage at −80°C on the fatty acid profiles of serum cholesteryl ester (CE), triglyceride (TG), and phospholipid (PL) fractions. This was accomplished by determining the fatty acid profile of frozen, archived, previously unthawed serum samples from 22 subjects who participated in a controlled feeding trial. Initial analysis was performed after trial completion and the repeat analysis after 8–10 years of storage using GC. No significant differences were observed among the majority of fatty acids regardless of lipid fraction. Reliability coefficients were high for the fatty acid classes (saturated fatty acid : 0.70, MUFA : 0.90, PUFA : 0.80). When differences were identified, they were limited to low abundance fatty acids (≤1.5 mol%). These differences were quantitatively small and likely attributable to technical improvements in GC methodology rather than sample degradation. Thus, our data demonstrate that storage at −80°C up to 10 years does not significantly influence serum CE, TG, or PL fatty acid profiles.

Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans

Background: Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date. Methods: Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured. Results: Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations. Conclusions: Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a). Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.

Association of plasma n-6 and n-3 polyunsaturated fatty acids with synovitis in the knee: the MOST study.

In osteoarthritis (OA) the synovium is often inflamed and inflammatory cytokines contribute to cartilage damage. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have anti-inflammatory effects whereas omega-6 polyunsaturated fatty acids (n-6 PUFAs) have, on balance, proinflammatory effects. The goal of our study was to assess the association of fasting plasma phospholipid n-6 and n-3 PUFAs with synovitis as measured by synovial thickening on contrast enhanced (CE) knee MRI and cartilage damage among subjects in the Multicenter Osteoarthritis Study (MOST). MOST is a cohort study of individuals who have or are at high risk of knee OA. An unselected subset of participants who volunteered obtained CE 1.5T MRI of one knee. Synovitis was scored in six compartments and a summary score was created. This subset also had fasting plasma, analyzed by gas chromatography for phospholipid fatty acid content, and non-CE MRI, read for cartilage morphology according to the Whole-Organ Magnetic Resonance Imaging Score (WORMS) method. The association between synovitis and cartilage morphology and plasma PUFAs was assessed using logistic regression after controlling for the effects of age, sex, and BMI. 472 out of 535 subjects with CE MRI had complete data on synovitis, cartilage morphology and plasma phospholipids. Mean age was 60 years, mean BMI 30, and 50% were women. We found an inverse relation between total n-3 PUFAs and the specific n-3, docosahexaenoic acid with patellofemoral cartilage loss, but not tibiofemoral cartilage loss or synovitis. A positive association was observed between the n-6 PUFA, arachidonic acid, and synovitis. In conclusion, systemic levels of n-3 and n-6 PUFAs which are influenced by diet, may be related to selected structural findings in knees with or at risk of OA. Future studies manipulating the systemic levels of these fatty acids may be warranted to determine the effects on structural damage in knee OA.

Reduction in dietary omega-6 polyunsaturated fatty acids: Eicosapentaenoic acid plus docosahexaenoic acid ratio minimizes atherosclerotic lesion formation and inflammatory response in the LDL receptor null mouse ☆

Dietary very long chain omega (omega)-3 polyunsaturated fatty acids (PUFA) have been associated with reduced CVD risk, the mechanisms of which have yet to be fully elucidated. LDL receptor null mice (LDLr-/-) were used to assess the effect of different ratios of dietary omega-6 PUFA to eicosapentaenoic acid plus docosahexaenoic acid (omega-6:EPA+DHA) on atherogenesis and inflammatory response. Mice were fed high saturated fat diets without EPA and DHA (HSF omega-6), or with omega-6:EPA+DHA at ratios of 20:1 (HSF R=20:1), 4:1 (HSF R=4:1), and 1:1 (HSF R=1:1) for 32 weeks. Mice fed the lowest omega-6:EPA+DHA ratio diet had lower circulating concentrations of non-HDL cholesterol (25%, P<0.05) and interleukin-6 (IL-6) (44%, P<0.05) compared to mice fed the HSF omega-6 diet. Aortic and elicited peritoneal macrophage (Mphi) total cholesterol were 24% (P=0.07) and 25% (P<0.05) lower, respectively, in HSF R=1:1 compared to HSF omega-6 fed mice. MCP-1 mRNA levels and secretion were 37% (P<0.05) and 38% (P<0.05) lower, respectively, in elicited peritoneal Mphi isolated from HSF R=1:1 compared to HSF omega-6 fed mice. mRNA and protein levels of ATP-binding cassette A1, and mRNA levels of TNFalpha were significantly lower in elicited peritoneal Mphi isolated from HSF R=1:1 fed mice, whereas there was no significant effect of diets with different omega-6:EPA+DHA ratios on CD36, Mphi scavenger receptor 1, scavenger receptor B1 and IL-6 mRNA or protein levels. These data suggest that lower omega-6:EPA+DHA ratio diets lowered some measures of inflammation and Mphi cholesterol accumulation, which was associated with less aortic lesion formation in LDLr-/- mice.

Approaches to measuring cholesterol absorption in humans.

Under optimal conditions, plasma cholesterol homeostasis is maintained by a variety of mechanisms, balancing input and output, thereby preventing the net accumulation of cholesterol in circulation and tissues. Among these mechanisms, intestinal cholesterol absorption has recently re-emerged as a potentially important contributor to cholesterol homeostasis. However, its regulation has been difficult to study in humans because of technical limitations in methodologies. In this review the major methods available for measuring cholesterol absorption including those that utilize cholesterol balance, single dose isotopic feeding, dual isotope plasma ratio, continuous isotope feeding, intestinal perfusion, stable isotopes and serum plant sterols or cholestanol to cholesterol ratios are reviewed and contrasted. Emphasis is placed on the strengths, technical and interpretational limitations and their applicability for use in metabolic, small-scale outpatient, population and large-scale intervention studies.