M. Nazeem Nanjee

Weight and Metabolic Outcomes 12 Years after Gastric Bypass

BACKGROUND Few long‐term or controlled studies of bariatric surgery have been conducted to date. We report the 12‐year follow‐up results of an observational, prospective study of Roux‐en‐Y gastric bypass that was conducted in the United States. METHODS A total of 1156 patients with severe obesity comprised three groups: 418 patients who sought and underwent Roux‐en‐Y gastric bypass (surgery group), 417 patients who sought but did not undergo surgery (primarily for insurance reasons) (nonsurgery group 1), and 321 patients who did not seek surgery (nonsurgery group 2). We performed clinical examinations at baseline and at 2 years, 6 years, and 12 years to ascertain the presence of type 2 diabetes, hypertension, and dyslipidemia. RESULTS The follow‐up rate exceeded 90% at 12 years. The adjusted mean change from baseline in body weight in the surgery group was ‐45.0 kg (95% confidence interval [CI], ‐47.2 to ‐42.9; mean percent change, ‐35.0) at 2 years, ‐36.3 kg (95% CI, ‐39.0 to ‐33.5; mean percent change, ‐28.0) at 6 years, and ‐35.0 kg (95% CI, ‐38.4 to ‐31.7; mean percent change, ‐26.9) at 12 years; the mean change at 12 years in nonsurgery group 1 was ‐2.9 kg (95% CI, ‐6.9 to 1.0; mean percent change, ‐2.0), and the mean change at 12 years in nonsurgery group 2 was 0 kg (95% CI, ‐3.5 to 3.5; mean percent change, ‐0.9). Among the patients in the surgery group who had type 2 diabetes at baseline, type 2 diabetes remitted in 66 of 88 patients (75%) at 2 years, in 54 of 87 patients (62%) at 6 years, and in 43 of 84 patients (51%) at 12 years. The odds ratio for the incidence of type 2 diabetes at 12 years was 0.08 (95% CI, 0.03 to 0.24) for the surgery group versus nonsurgery group 1 and 0.09 (95% CI, 0.03 to 0.29) for the surgery group versus nonsurgery group 2 (P<0.001 for both comparisons). The surgery group had higher remission rates and lower incidence rates of hypertension and dyslipidemia than did nonsurgery group 1 (P<0.05 for all comparisons). CONCLUSIONS This study showed long‐term durability of weight loss and effective remission and prevention of type 2 diabetes, hypertension, and dyslipidemia after Roux‐en‐Y gastric bypass. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others.)

Secretion of adipokines by human adipose tissue in vivo: partitioning between capillary and lymphatic transport

Peptides secreted by adipose tissue (adipokines) may enter blood via capillaries or lymph. The relative importance of these pathways for a given adipokine might influence its biological effects. Because this has not been studied in any species, we measured the concentrations of seven adipokines and eight nonsecreted proteins in afferent peripheral lymph and venous plasma from 12 healthy men. Data for nonsecreted proteins were used to derive indices of microvascular permeability, which in conjunction with the molecular radii of the adipokines were used to estimate the amounts leaving the tissue via capillaries. Transport rates via lymph were estimated from the lymph adipokine concentrations and lymph flow rates and total transport (secretion) as the sum of this and capillary transport. Concentrations of nonsecreted proteins were always lower in lymph than in plasma. With the exception of adiponectin, adipokine concentrations were always higher in lymph (P < 0.01). Leptin and MCP-1 were secreted at the highest rates (means: 43 μg/h or 2.7 nmol/h and 32 μg/h or 2.4 nmol/h, respectively). IL-6 and MCP-1 secretion rates varied greatly between subjects. The proportion of an adipokine transported via lymph was directly related to its molecular radius (r(s) = +0.94, P = 0.025, n = 6), increasing from 14 to 100% as the radius increased from 1.18 (IL-8) to 3.24 nm (TNFα). We conclude that the lymph/capillary partitioning of adipokines is a function of molecular size, which may affect both their regional and systemic effects in vivo. This finding may have implications for the physiology of peptides secreted by other tissues.

Effects of Intravenous Apolipoprotein A-I/Phosphatidylcholine Discs on LCAT, PLTP, and CETP in Plasma and Peripheral Lymph in Humans

Objective—We have previously shown that intravenous apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) discs increase plasma pre-&bgr; HDL concentration and stimulate reverse cholesterol transport (RCT) in humans. We have now investigated the associated changes in the following 3 HDL components that play key roles in RCT: lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP). Methods and Results—apoA-I/PC discs (40 mg/kg over 4 hours) were infused into 8 healthy men. Samples of blood and prenodal peripheral lymph were collected for 24 to 48 hours. At 12 hours, plasma LCAT concentration had increased by 0.40±0.90 mg/L (+7.8%; mean±SD; P <0.05), plasma cholesterol esterification rate by 29.0±9.0 nmol/mL per h (+69.5%; P <0.01), plasma CETP concentration by 0.5±0.2 mg/L (+29.7%; P <0.01), and plasma PLTP activity by 1.45±0.67 &mgr;mol/mL per h (+23.9%; P <0.01). In contrast, plasma PLTP concentration had decreased by 4.4±2.7 mg/L (−44.8%; P <0.01). The changes in PLTP were accompanied by alterations in the relative proportions of large lipoproteins containing inactive PLTP and small particles containing PLTP of high specific activity. No changes were detected in peripheral lymph. Conclusions—Nascent HDL secretion may induce changes in PLTP, LCAT, and CETP that promote RCT by catalyzing pre-&bgr; HDL production, cholesterol esterification in HDLs, and cholesteryl ester transfer from HDLs to other lipoproteins.

Expression of Human Apolipoprotein A-I/C-III/A-IV Gene Cluster in Mice Induces Hyperlipidemia but Reduces Atherogenesis

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257±9, 7.1±0.5, and 1.0±0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean±SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB48–containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

An in-silico model of lipoprotein metabolism and kinetics for the evaluation of targets and biomarkers in the reverse cholesterol transport pathway.

High-density lipoprotein (HDL) is believed to play an important role in lowering cardiovascular disease (CVD) risk by mediating the process of reverse cholesterol transport (RCT). Via RCT, excess cholesterol from peripheral tissues is carried back to the liver and hence should lead to the reduction of atherosclerotic plaques. The recent failures of HDL-cholesterol (HDL-C) raising therapies have initiated a re-examination of the link between CVD risk and the rate of RCT, and have brought into question whether all target modulations that raise HDL-C would be atheroprotective. To help address these issues, a novel in-silico model has been built to incorporate modern concepts of HDL biology, including: the geometric structure of HDL linking the core radius with the number of ApoA-I molecules on it, and the regeneration of lipid-poor ApoA-I from spherical HDL due to remodeling processes. The ODE model has been calibrated using data from the literature and validated by simulating additional experiments not used in the calibration. Using a virtual population, we show that the model provides possible explanations for a number of well-known relationships in cholesterol metabolism, including the epidemiological relationship between HDL-C and CVD risk and the correlations between some HDL-related lipoprotein markers. In particular, the model has been used to explore two HDL-C raising target modulations, Cholesteryl Ester Transfer Protein (CETP) inhibition and ATP-binding cassette transporter member 1 (ABCA1) up-regulation. It predicts that while CETP inhibition would not result in an increased RCT rate, ABCA1 up-regulation should increase both HDL-C and RCT rate. Furthermore, the model predicts the two target modulations result in distinct changes in the lipoprotein measures. Finally, the model also allows for an evaluation of two candidate biomarkers for in-vivo whole-body ABCA1 activity: the absolute concentration and the % lipid-poor ApoA-I. These findings illustrate the potential utility of the model in drug development.

Vitamin D dependent effects of APOA5 polymorphisms on HDL cholesterol

OBJECTIVES Vitamin D and serum lipid levels are risk factors for cardiovascular disease. We sought to determine if vitamin D (25OHD) interacts at established lipid loci potentially explaining additional variance in lipids. METHODS 1060 individuals from Utah families were used to screen 14 loci for SNPs potentially interacting with dietary 25OHD on lipid levels. Identified putative interactions were evaluated for (1) greater effect size in subsamples with winter measures, (2) replication in an independent sample, and (3) lack of gene-environment interaction for other correlated dietary factors. Maximum likelihood models were used to evaluate interactions. The replicate sample consisted of 2890 individuals from the Family Heart Study. Putative 25OHD receptor binding site modifying SNPs were identified and allele-specific, 25OHD-dependent APOA5 promoter activity examined using luciferase expression assays. An additional sample with serum 25OHD measures was analyzed. RESULTS An rs3135506-25OHD interaction influencing HDL-C was identified. The rs3135506 minor allele was more strongly associated with low HDL-C in individuals with low winter dietary 25OHD in initial and replicate samples (p=0.0003 Utah, p=0.002 Family Heart); correlated dietary factors did not explain the interaction. SNP rs10750097 was identified as a putative causative polymorphism, was associated with 25OHD-dependent changes in APOA5 promoter activity in HEP3B and HEK293 cells (p<0.01), and showed similar interactions to rs3135506 in family cohorts. Linear interactions were not significant in samples with serum 25OHD measures; however, genotype-specific differences were seen at deficient 25OHD levels. CONCLUSIONS A 25OHD receptor binding site modifying APOA5 promoter polymorphism is associated with lower HDL-C in 25OHD deficient individuals.

Altered composition of triglyceride-rich lipoproteins and coronary artery disease in a large case-control study.

BACKGROUND Traditional beta-quantification of plasma lipoproteins by ultracentrifugation separates triglyceride-rich lipoproteins (TGRL) from higher density lipoproteins. The cholesterol in the TGRL fraction is referred to as measured very low-density lipoprotein cholesterol (VLDL-C) recognizing that other TGRL may be present. The measured VLDL-C to total plasma triglyceride (VLDL-C/TG) has long been considered an index of average TGRL composition with abnormally high VLDL-C/TG ratios (>or=0.30 with TG>150mg/dL) indicative of atherogenic remnant accumulation (type III hyperlipidemia). However, virtually no reports are available which examine potential associations between CAD and VLDL-C/TG at the lower end of the spectrum. METHODS AND RESULTS We performed ultracentrifugation in 1170 cases with premature-onset, familial CAD and 1759 population-based controls and examined the VLDL-C/TG ratio as an index of TGRL composition. As expected, we found very high CAD risk associated with severe type III hyperlipidemia (OR 10.5, p=0.02). Unexpectedly, however, we found a robust, graded, and independent association between CAD risk and lower than average VLDL-C/TG ratios (p<0.0001 as ordered categories or as a continuous variable). Among those in the lowest VLDL-C/TG category (a ratio <0.12), CAD risk was clearly increased (OR 4.5, 95% CI 2.9-6.9) and remained significantly elevated in various subgroups including those with triglycerides below 200mg/dl, in males and females separately, as well as among those with no traditional CAD risk factors (OR 5.8, 95% CI 1.5-22). Significant compositional differences by case status were confirmed in a subset whose samples were re-spun with measurement of lipids and apolipoprotein B (apo B) in each subfraction. CONCLUSIONS We found a strong, graded, independent, and robust association between CAD and lower VLDL-C/TG ratios. We consider this a novel, hypothesis-generating observation which will hopefully generate additional future studies to provide confirmation and further insight into potential mechanisms.

Lipoprotein remodeling generates lipid-poor apolipoprotein A-I particles in human interstitial fluid

Although much is known about the remodeling of high density lipoproteins (HDLs) in blood, there is no information on that in interstitial fluid, where it might have a major impact on the transport of cholesterol from cells. We incubated plasma and afferent (prenodal) peripheral lymph from 10 healthy men at 37°C in vitro and followed the changes in HDL subclasses by nondenaturing two-dimensional crossed immunoelectrophoresis and size-exclusion chromatography. In plasma, there was always initially a net conversion of small pre-β-HDLs to cholesteryl ester (CE)-rich α-HDLs. By contrast, in lymph, there was only net production of pre-β-HDLs from α-HDLs. Endogenous cholesterol esterification rate, cholesteryl ester transfer protein (CETP) concentration, CE transfer activity, phospholipid transfer protein (PLTP) concentration, and phospholipid transfer activity in lymph averaged 5.0, 10.4, 8.2, 25.0, and 82.0% of those in plasma, respectively (all P < 0.02). Lymph PLTP concentration, but not phospholipid transfer activity, was positively correlated with that in plasma (r = +0.63, P = 0.05). Mean PLTP-specific activity was 3.5-fold greater in lymph, reflecting a greater proportion of the high-activity form of PLTP. These findings suggest that cholesterol esterification rate and PLTP specific activity are differentially regulated in the two matrices in accordance with the requirements of reverse cholesterol transport, generating lipid-poor pre-β-HDLs in the extracellular matrix for cholesterol uptake from neighboring cells and converting pre-β-HDLs to α-HDLs in plasma for the delivery of cell-derived CEs to the liver.

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (kp-if) or its mean residence time in interstitial fluid (MRTif). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. kp-if averaged 0.75%/h (range, 0.33–1.32), and MRTif averaged 29.1 h (14.1–40.0). Neither parameter was correlated with the distribution volume (57–105 ml/kg) or the fractional elimination rate (1.44–2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.