Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR.

Mipomersen is a 20mer antisense oligonucleotide (ASO) that inhibits apolipoprotein B (apoB) synthesis; its low-density lipoprotein (LDL)-lowering effects should therefore result from reduced secretion of very-low-density lipoprotein (VLDL). We enrolled 17 healthy volunteers who received placebo injections weekly for 3 weeks followed by mipomersen weekly for 7 to 9 weeks. Stable isotopes were used after each treatment to determine fractional catabolic rates and production rates of apoB in VLDL, IDL (intermediate-density lipoprotein), and LDL, and of triglycerides in VLDL. Mipomersen significantly reduced apoB in VLDL, IDL, and LDL, which was associated with increases in fractional catabolic rates of VLDL and LDL apoB and reductions in production rates of IDL and LDL apoB. Unexpectedly, the production rates of VLDL apoB and VLDL triglycerides were unaffected. Small interfering RNA-mediated knockdown of apoB expression in human liver cells demonstrated preservation of apoB secretion across a range of apoB synthesis. Titrated ASO knockdown of apoB mRNA in chow-fed mice preserved both apoB and triglyceride secretion. In contrast, titrated ASO knockdown of apoB mRNA in high-fat-fed mice resulted in stepwise reductions in both apoB and triglyceride secretion. Mipomersen lowered all apoB lipoproteins without reducing the production rate of either VLDL apoB or triglyceride. Our human data are consistent with long-standing models of posttranscriptional and posttranslational regulation of apoB secretion and are supported by in vitro and in vivo experiments. Targeting apoB synthesis may lower levels of apoB lipoproteins without necessarily reducing VLDL secretion, thereby lowering the risk of steatosis associated with this therapeutic strategy.

OBJECTIVE:Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA)-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP.APPROACH AND RESULTS:Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II, and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, ANA treatment increased plasma HDL-C (63.0%; P<0.001) and apoA-I levels (29.5%; P<0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate.CONCLUSIONS:ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein.

Background/Objective: In a previous report of HIV-infected patients with fat redistribution, we found that recombinant human growth hormone (rhGH) therapy reduced visceral adipose tissue (VAT) but increased insulin resistance, and that the addition of rosiglitazone reversed the negative effects of rhGH on insulin sensitivity. In this study, we sought to determine the effects of rhGH and rosiglitazone therapy on an array of inflammatory and fibrinolytic markers.Methods: 72 patients with HIV-associated abdominal obesity and insulin resistance were randomized to treatment with rhGH, rosiglitazone, the combination of rhGH and rosiglitazone, or placebo for 12 weeks. Subjects with plasma and serum samples available at weeks 0 (n = 63) and 12 (n = 46-48) were assessed for adiponectin, C-reactive protein, homocysteine, interleukin-1, interleukin-6, tumor necrosis factor alpha, interferon gamma, fibrinogen, plasminogen activator inhibitor-1 antigen, and tissue plasminogen activator antigen.Results: Treatment with both rosiglitazone alone and the combination of rosiglitazone and rhGH for 12 weeks resulted in significant increases in adiponectin levels from baseline. Adiponectin levels did not change significantly in the rhGH arm alone. There were no significant changes in the other biomarkers among the different treatment groups.Discussion: In this study of HIV-infected patients with altered fat distribution, treatment with rosiglitazone had beneficial effects on adiponectin concentrations, an effect that was also seen with a combination of rosiglitazone and rhGH. RhGH administration alone, however, did not demonstrate any significant impact on adiponectin levels despite reductions in VAT.

Background: CETP inhibition with anacetrapib reduces triglyceride (TG) and cholesteryl ester transfer among lipoproteins and increases very low density lipoprotein (VLDL) apolipoprotein B (apoB) clearance. This study reports the effects of anacetrapib on VLDL-TG, apoC-II, apoC-III and apoE metabolism. Methods: Mildly hypercholesterolemic subjects were randomized to background placebo (N=10) or atorvastatin 20 mg (N=29) for 4 weeks, followed by addition of anacetrapib 100 mg for another 8-weeks. Subjects underwent stable isotope kinetic studies to determine fractional catabolic rates (FCR) and production rates (PR) of VLDL-TG, apoC-II, C-III and E. Results: Anacetrapib reduced the circulating VLDL-TG pool by 14% (p=.006) in subjects on atorvastatin due to an increase in FCR (29%; p=.002) without changing plasma TG. VLDL-TG pool size did not change in the anacetrapib monotherapy group; although FCR increased by 40% (p=.11), these changes were offset by a 39% increase in PR (p=.01), which was not observed in the atorvastatin plus anacetrapib group. Anacetrapib increased plasma apoE (with a trend towards increased PR; p=.10) and increased plasma apoC-II (with a trend towards decreased FCR; p=.08). Anacetrapib also increased plasma apoC-III and tended to increase PR (p=.07) and decrease FCR (p=.08); these changes could be due an increased association of apoCIII with HDL particles. Conclusion: Anacetrapib increases VLDL-TG FCR, possibly through effects mediated by apoE and apoC-II. In subjects on atorvastatin background, this was associated with reduced circulating VLDL-TG. In anacetrapib monotherapy subjects, there were no changes in VLDL-TG due to competing mechanisms of FCR and PR.

BACKGROUND:Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib.METHODS:We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR).RESULTS:Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR.CONCLUSION:These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance.TRIAL REGISTRATION:ClinicalTrials.gov NCT00990808.FUNDING:Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Adults with type 2 diabetes (T2D) have an increased risk of fractures [1]. This increased risk, despite normal bone mineral density (BMD) [2, 3], has led to investigation of deficits in diabetic skeletal properties. Skeletal dynamics are reduced in T2D [4, 5], possibly as a result of inflammation [6]. Inflammation plays an important role in T2D, as obesity activates the transcription-factor-nuclear-factorjB (NF-jB), which increases the risk for T2D [7]. Inflammation also compromises skeletal remodeling by reducing bone formation and increasing bone resorption [8], possibly via activation of receptoractivator of nuclear-factor-j-B ligand (RANKL) [9]. RANKL and NF-jB may act as hormones, exerting effects at sites distant from where they are produced. We analyzed stored samples from the TINSAL-T2D trial [10], where it was found that salsalate, a prodrug of salicylate which reduces NF-jB activity [11, 12], decreased HbA1c levels [10]. We hypothesized that reducing inflammation in T2D would rebalance the bone remodeling process.

Background: Alirocumab is a monoclonal antibody that inhibits binding of PCSK9 to LDL receptors, decreasing their lysosomal degradation and increasing their numbers on cell surfaces. In Phase 2/3 studies, alirocumab 150 mg lowered LDL-cholesterol (C) ~61%, apoB 53%, and fasting triglycerides (TG) 16%. The mechanisms that increased LDL removal or the effects on postprandial (PP) TG levels after alirocumab therapy have not been reported. Method: 18 (10F, 8 M), healthy volunteers completed a Phase 1, placebo-controlled, single-blind, single-sequence study. Subjects received 2 placebo doses followed by 5 alirocumab doses, 150 mg SC every 2 wks. At the end of each treatment period, we measured fasting lipids and lipoproteins and subjects received a high fat meal with PP TG and apoB48 levels measured over 8hrs. Stable isotope studies were performed 3-5 days later to measure apoB turnover in very low density (VLDL), intermediate density (IDL) and LDL. Results: In 18 subjects, Alirocumab significantly reduced plasma total-C by 35% (172±32 to 111±24 mg/dL), LDL-C by 60% (104±23 to 44±21 mg/dL) and, apoB by 45% (90±21 to 45±12 mg/dL). Plasma TG levels decreased by 13% (99±44 to 86±40mg/dL). LDL-C, LDL-TG, and LDL-apoB isolated from plasma by ultracentrifugation fell by 50±15%, 5±2%, and 41±16%, respectively, on alirocumab. Preliminary Kinetic results on N=10, show that the reductions in LDL-apoB were due to an increase in the fractional clearance rate (FCR) of LDL-apoB from 0.50±0.18 on placebo to 1.02±0.35 pools/day on alirocumab (p<0.001). IDL-C, IDL-TG and IDL-apoB were also reduced significantly, with a trend toward an increase in IDL-apoB FCR on alirocumab (placebo: 9.2±4 vs alirocumab: 10.8±3 pools/day; p=0.06). Alirocumab had no effects VLDL-C, VLDL-TG, or VLDL-apoB, nor did alirocumab change VLDL FCR or PR. The area under the curve for PP TG and PP apoB48 did not change from placebo to alirocumab. No serious adverse event or treatment discontinuation occurred. Results for all 18 subjects will be available for presentation. Summary: Alirocumab treatment significantly reduced levels of IDL and LDL-apoB by increasing the FCRs of these lipoproteins, particularly LDL. Alirocumab did not alter the metabolism of VLDL-apoB or PP TG-rich lipoproteins.

Background: Proprotein convertase subtilisin/kexin type (PCSK9) inhibitors are promising new drugs for the treatment of hypercholesterolemia. They inhibit the binding of PCSK9 to the low density lipoprotein (LDL) receptor that, in turn, decreases lysosomal degradation of LDL receptors and increases their numbers on the cell surface. In Phase 2/3 studies, alirocumab significantly lowered plasma levels of LDL-cholesterol (C) and apolipoprotein B (apoB). The mechanism underlying the LDL-C lowering effects of PCSK9 inhibition has not been reported. Method: We enrolled 10 healthy volunteers (4 male, 6 female), into a Phase 1, placebo-controlled, single-blind, single-sequence study to examine the effects of alirocumab, 150 mg administered subcutaneously every two weeks, on lipid and lipoproteins levels and the metabolism of apoB in very low density (VLDL), intermediate density (IDL) and LDL. Subjects received 2 doses of placebo followed by 5 doses of alirocumab. At the end of each treatment period, fasting lipids and lipoprotein levels were measured, and stable isotope studies of the apoB turnover in VLDL, IDL and LDL were performed. Results: Alirocumab significantly reduced plasma levels of total-C by 37% (178.4±34 to 112.7±29 mg/dL), LDL-C by 59% (110.2±25 to 45.5±26 mg/dL), and apoB by 51% (93.6±25 to 45.5±13 mg/dL) compared to placebo. Plasma triglycerides (TG) and HDL-C did not change. Levels of LDL-C, LDL-TG, and LDL-apoB fell by 55.8±10%, 33.9±13%, and 56.0±11%, respectively (all p<0.0001), on alirocumab. The reductions in LDL apoB were explained by a dramatic increase in the fractional clearance rate (FCR) of LDL apoB from 0.50±0.18 on placebo to 1.02±0.35 pools/day on alirocumab (p<0.001) and a trend toward lower LDL apoB production rates on alirocumab (15.1±4.6 vs 12.9±3.3 mg/kg/day; p=0.10). Levels of IDL-C, IDL-TG and IDL-apoB were also reduced significantly and there was a trend toward an increase in IDL apoB FCR on alirocumab (placebo: 9.2±4 vs alirocumab: 10.8±3 pools/day; p=0.06). Additional kinetic parameters will be presented at the meeting. Summary: Alirocumab treatment significantly reduced the levels of IDL and LDL, and these changes were due to increases in the FCRs of these lipoproteins, particularly LDL.

Objectives: Mipomersen (MIPO), a second generation antisense oligonucleotide, targets apoB mRNA, thereby inhibiting apolipoprotein B (apoB) synthesis. In humans, MIPO reduces plasma levels of low density lipoprotein-cholesterol (LDL-C), and plasma triglycerides (TG). We hypothesized that these changes are due to reduced assembly and secretion of very low density lipoproteins (VLDL) and lower production of LDL. Methods: Healthy volunteers (HVs) (9M, 8F), mean age 43.5 ± 14.2 yr, completed a single-blind, fixed-sequence, phase I study. They received sc-placebo injections once weekly for 3-wks followed by 200mg sc-MIPO injections once weekly for 7-9 wks. Stable isotope turnover studies were performed after each treatment. Blood samples were collected over 48-hrs to determine fractional catabolic rates (FCRs) and production rates (PRs) of apoB in VLDL, IDL, and LDL, and of TG in VLDL. Rates of de novo lipogenesis (DNL) were also measured. Results: MIPO treatment resulted in significant reductions in plasma LDL-C (45%), TG (29%), and apoB (40%). VLDL, IDL, and LDL apoB levels fell by 29%, 25%, and 42%, respectively. These changes were associated with increases in FCRs of VLDL apoB (42%) and LDL apoB (30%), and by reductions in PRs of IDL apoB (15%) and LDL apoB (27%). The PR of VLDL apoB was unaffected. The FCR of VLDL-TG increased 46% without change in PR. DNL did not change. Conclusion: In summary, 7 wks of MIPO significantly reduced levels of all apoB-lipoproteins in HVs by increasing the FCRs of VLDL and LDL apoB. The absence of a reduction in VLDL apoB secretion is consistent with many studies in isolated hepatocytes demonstrating both intracellular degradation and secretion of newly synthesized apoB. Thus, if MIPO submaximally inhibited apoB synthesis in this study, the liver could have compensated by increasing the efficiency of VLDL assembly and secretion. The basis of increases in VLDL and LDL FCRs requires further investigation.

Background: Recombinant human growth hormone (rhGH) reduces visceral adipose tissue (VAT) volume in HIV-infected patients but can worsen glucose homeostasis and lipoatrophy. We aimed to determine if adding rosiglitazone to rhGH would abrogate the adverse effects of rhGH on insulin sensitivity (SI) and subcutaneous adipose tissue (SAT) volume.Methodology/Principal Findings: Randomized, double-blind, placebo-controlled, multicenter trial using a 2x2 factorial design in which HIV-infected subjects with abdominal obesity and insulin resistance were randomized to rhGH 3 mg daily, rosiglitazone 4 mg twice daily, combination rhGH + rosiglitazone, or double placebo (control) for 12 weeks. The primary endpoint was change in SI by frequently sampled intravenous glucose tolerance test from entry to week 12. Body composition was assessed by whole body magnetic resonance imaging (MRI) and dual Xray absorptiometry (DEXA). Seventy-seven subjects were randomized of whom 72 initiated study drugs. Change in SI from entry to week 12 differed across the 4 arms by 1-way ANCOVA (P = 0.02); by pair-wise comparisons, only rhGH (decreasing SI; P = 0.03) differed significantly from control. Changes from entry to week 12 in fasting glucose and glucose area under the curve on 2-hour oral glucose tolerance test differed across arms (1-way ANCOVA P = 0.004), increasing in the rhGH arm relative to control. VAT decreased significantly in the rhGH arms (-17.5% in rhGH/rosiglitazone and -22.7% in rhGH) but not in the rosiglitazone alone (-2.5%) or control arms (-1.9%). SAT did not change significantly in any arm. DEXA results were consistent with the MRI data. There was no significant rhGH x rosiglitazone interaction for any body composition parameter.Conclusions/Significance: The addition of rosiglitazone abrogated the adverse effects of rhGH on insulin sensitivity and glucose tolerance while not significantly modifying the lowering effect of rhGH on VAT.

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 PPAR gamma 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 PPAR gamma 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.

Many patients with type 2 diabetes fail to achieve or maintain the American Diabetes Association's recommended treatment goal of glycosylated hemoglobin levels. This multicenter, double-blind trial enrolled patients with type 2 diabetes who had inadequate glycemic control [glycosylated hemoglobin A(1C) (A1C), >7% and <12%) with diet and exercise alone to compare the benefits of initial therapy with glyburide/metformin tablets vs. metformin or glyburide monotherapy. Patients (n = 486) were randomized to receive glyburide/metformin tablets (1.25/250 mg), metformin (500 mg), or glyburide (2.5 mg). Changes in A1C, fasting plasma glucose, fructosamine, serum lipids, body weight, and 2-h postprandial glucose after a standardized meal were assessed after 16 wk of treatment. Glyburide/metformin tablets caused a superior mean reduction in A1C from baseline (-2.27%) vs. metformin (-1.53%) and glyburide (-1.90%) monotherapy (P = 0.0003). Glyburide/metformin also significantly reduced fasting plasma glucose and 2-h postprandial glucose values compared with either monotherapy. The final mean doses of glyburide/metformin (3.7/735 mg) were lower than those of metformin (1796 mg) and glyburide (7.6 mg). First-line treatment with glyburide/metformin tablets provided superior glycemic control over component monotherapy, allowing more patients to achieve American Diabetes Association treatment goals with lower component doses in drug-naive patients with type 2 diabetes.