Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in Livers of Mice.

The synthesis of cholesterol and fatty acids (FA) in the liver is independently regulated by SREBP-2 and SREBP-1c, respectively. Here, we genetically deleted Srebf-2 from hepatocytes and confirmed that SREBP-2 regulates all genes involved in cholesterol biosynthesis, the LDL receptor, and PCSK9; a secreted protein that degrades LDL receptors in the liver. Surprisingly, we found that elimination of Srebf-2 in hepatocytes of mice also markedly reduced SREBP-1c and the expression of all genes involved in FA and triglyceride synthesis that are normally regulated by SREBP-1c. The nuclear receptor LXR is necessary for Srebf-1c transcription. The deletion of Srebf-2 and subsequent lower sterol synthesis in hepatocytes eliminated the production of an endogenous sterol ligand required for LXR activity and SREBP-1c expression. These studies demonstrate that cholesterol and FA synthesis in hepatocytes are coupled and that flux through the cholesterol biosynthetic pathway is required for the maximal SREBP-1c expression and high rates of FA synthesis.

Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of hepatic low-density lipoprotein (LDL) receptors (LDLR), thereby, decreasing hepatocyte LDL-cholesterol (LDL-C) uptake. However, it is unknown whether PCSK9 has effects on atherogenesis that are independent of lipid changes. The present study investigated the effect of human (h) PCSK9 on plasma lipids, hepatic lipogenesis, and atherosclerotic lesion size and composition in transgenic mice expressing hPCSK9 (hPCSK9tg) on wild-type (WT), LDLR⁻/⁻, or apoE⁻/⁻ background. METHODS AND RESULTS: hPCSK9 expression significantly increased plasma cholesterol (+91%), triglycerides (+18%), and apoB (+57%) levels only in WT mice. The increase in plasma lipids was a consequence of both decreased hepatic LDLR and increased hepatic lipid production, mediated transcriptionally and post-transcriptionally by PCSK9 and dependent on both LDLR and apoE. Despite the lack of changes in plasma lipids in mice expressing hPCSK9 and lacking LDLR (the main target for PCSK9) or apoE (a canonical ligand for the LDLR), hPCSK9 expression increased aortic lesion size in the absence of apoE (268 655 ± 97 972 µm² in hPCSK9tg/apoE⁻/⁻ vs. 189 423 ± 65 700 µm(2) in apoE⁻/⁻) but not in the absence of LDLR. Additionally, hPCSK9 accumulated in the atheroma and increased lesion Ly6C(hi) monocytes (by 21%) in apoE⁻/⁻ mice, but not in LDLR⁻/⁻ mice. CONCLUSIONS: PCSK9 increases hepatic lipid and lipoprotein production via apoE- and LDLR-dependent mechanisms. However, hPCSK9 also accumulate in the artery wall and directly affects atherosclerosis lesion size and composition independently of such plasma lipid and lipoprotein changes. These effects of hPCSK9 are dependent on LDLR but are independent of apoE.

Local effects of human PCSK9 on the atherosclerotic lesion.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction.

On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a circulatory ligand that terminates the lifecycle of the low-density lipoprotein (LDL) receptor (LDLR) thus affecting plasma LDL-cholesterol (LDL-C) levels. Recent evidence shows that in addition to the straightforward mechanism of action, there are more complex interactions between PCSK9, LDLR and plasma lipoprotein levels, including: (a) the presence of both parallel and reciprocal regulation of surface LDLR and plasma PCSK9; (b) a correlation between PCSK9 and LDL-C levels dependent not only on the fact that PCSK9 removes hepatic LDLR, but also due to the fact that up to 40% of plasma PCSK9 is physically associated with LDL; and (c) an association between plasma PCSK9 production and the assembly and secretion of triglyceride-rich lipoproteins. The effect of PCSK9 on LDLR is being successfully utilized toward the development of anti-PCSK9 therapies to reduce plasma LDL-C levels. Current biochemical research has uncovered additional mechanisms of action and interacting partners for PCSK9, and this opens the way for a more thorough understanding of the regulation, metabolism, and effects of this interesting protein.

Elevated cholesteryl ester transfer protein (CETP) activity, a major determinant of the atherogenic dyslipidemia, and atherosclerotic cardiovascular disease in South Asians.

AIMS: Why South Asians are at increased risk of premature atherosclerotic cardiovascular diseases compared with other ethnic groups is not fully understood. Atherogenic dyslipoproteinemia - hypertriglyceridemia, elevated numbers of low-density lipoprotein (LDL) particles and low high-density lipoprotein cholesterol (HDL-C) - is more common in South Asians but the mechanisms responsible have not been explicated. Here we examined whether the circulating lipid transfer protein, cholesteryl ester transfer protein (CETP), plays a role in the pathogenesis of the atherogenic dyslipoproteinemia among South Asians. METHODS AND RESULTS: CETP activity was determined by exogenous substrate assay in the serum of healthy, metabolically well-characterized individuals of South Asian and European descent (N = 244 and 238, respectively). Serum and lipoprotein lipids and apolipoproteins were measured and lipoprotein particle number and size were quantified via nuclear magnetic resonance spectroscopy. All the elements of the atherogenic dyslipoproteinemia were more severe in South Asians and CETP activity was significantly greater by 30% in South Asians compared with Europeans, adjusted for age, sex, body mass index and waist circumference (p < 0.0001). CETP activity was directly associated with serum triglycerides and inversely with HDL-C in the whole population. CETP activity was also directly related to apoB and LDL particle number. Finally, increased CETP activity was associated with pro-atherogenic reductions in HDL and LDL particle size. CONCLUSIONS: We identified novel associations between elevated CETP activity and the triad of quantitative and qualitative lipoprotein abnormalities in the atherogenic dyslipidemia in South Asians, a major contributor of increased atherosclerotic cardiovascular diseases in South Asians.

Alirocumab: PCSK9 inhibitor for LDL cholesterol reduction.

The proof of concept that proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition affects cholesterol levels was first established after the demonstration that PCSK9 loss-of-function mutations result in a significant drop in circulating LDL cholesterol levels. Subsequent studies revealed that PCSK9 binds the epidermal growth factor precursor homology domain-A on the surface LDL Receptor (LDLR) and directs LDLR and PCSK9 for lysosomal degradation. Alirocumab (also known as SAR236553/REGN727) is a monoclonal antibody that binds circulating PCSK9 and blocks its interactions with surface LDLR. Alirocumab clinical trials with different doses on different administration schedules were shown to significantly reduce LDL cholesterol both as a mono-therapy and in combination with statins or ezetimibe. Although there is great potential for anti-PCSK9 therapies in the management of cholesterol metabolism, there is no clear evidence yet that blocking PCSK9 reduces cardiovascular disease outcome. This is being investigated in ongoing Phase III clinical trials with alirocumab.

Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms.

BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein (LDL) receptor (LDLR), and its deficiency in humans results in low plasma LDL cholesterol and protection against coronary heart disease. Recent evidence indicates that PCSK9 also modulates the metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins, another important coronary heart disease risk factor. Here, we studied the effects of physiological levels of PCSK9 on intestinal triglyceride-rich apoB lipoprotein production and elucidated for the first time the cellular and molecular mechanisms involved. METHODS AND RESULTS: Treatment of human enterocytes (CaCo-2 cells) with recombinant human PCSK9 (10 µg/mL for 24 hours) increased cellular and secreted apoB48 and apoB100 by 40% to 55% each (P<0.01 versus untreated cells), whereas short-term deletion of PCSK9 expression reversed this effect. PCSK9 stimulation of apoB was due to a 1.5-fold increase in apoB mRNA (P<0.01) and to enhanced apoB protein stability through both LDLR-dependent and LDLR-independent mechanisms. PCSK9 decreased LDLR protein (P<0.01) and increased cellular apoB stability via activation of microsomal triglyceride transfer protein. PCSK9 also increased levels of the lipid-generating enzymes FAS, SCD, and DGAT2 (P<0.05). In mice, human PCSK9 at physiological levels increased intestinal microsomal triglyceride transfer protein levels and activity regardless of LDLR expression. CONCLUSIONS: PCSK9 markedly increases intestinal triglyceride-rich apoB production through mechanisms mediated in part by transcriptional effects on apoB, microsomal triglyceride transfer protein, and lipogenic genes and in part by posttranscriptional effects on the LDLR and microsomal triglyceride transfer protein. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia.

PCSK9 and resistin at the crossroads of the atherogenic dyslipidemia.

The atherogenic dyslipidemia is a pathophysiological lipid triad, composed of high triglycerides and low-density lipoprotein and low high-density lipoprotein. The dyslipidemia is highly prevalent in individuals who are obese, insulin resistant and those with Type 2 diabetes and is the major contributing factor to the high atherosclerotic cardiovascular disease risk in these subjects. The primary initiating event in atherogenic dyslipidemia development is the hepatic overproduction of very-low-density lipoprotein (VLDL). The intracellular and extracellular protein triggers of hepatic VLDL production were not known until the recent identification of the causal roles of PCSK9 and resistin. Both PCSK9 and resistin act in large part by targeting and reducing the hepatic degradation of VLDL apoB through distinctly different mechanisms. In the current review, we discuss both the individual roles and the interaction of these proteins in driving atherogenic dyslipidemia, and thus, atherosclerotic cardiovascular disease progression in humans. We further explore the important therapeutic implications of these findings.

Discovery of a new role of human resistin in hepatocyte low-density lipoprotein receptor suppression mediated in part by proprotein convertase subtilisin/kexin type 9.

OBJECTIVES: In this study, our goal was to determine if human resistin plays a role in regulating the uptake of atherogenic low-density lipoproteins in human hepatocytes. BACKGROUND: Serum levels of resistin, an adipose tissue-derived adipokine, are increased in human obesity and are positively correlated with atherosclerotic cardiovascular diseases. However, the function of resistin in humans is enigmatic. METHODS: Human hepatocytes (HepG2 and primary) were treated (24 h) with the following: 1) purified human resistin at various concentrations, with and without lovastatin; and 2) obese human serum with elevated resistin levels or serum from which resistin was removed via antibody-immunoprecipitation. The effect of the treatments on cellular low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) messenger ribonucleic acid and protein levels were determined by using real-time polymerase chain reaction and Western blotting, respectively. RESULTS: Resistin, at physiological levels observed in human obesity, down-regulated hepatocyte LDLR expression substantially (by 40%). A key mechanism by which human resistin inhibited LDLR levels was by increased cellular expression of the recently identified protease, PCSK9, which enhances intracellular LDLR lysosomal degradation. The quantitatively important role of human resistin in LDLR expression was demonstrated by antibody-immunoprecipitation removal of resistin in human serum, which decreased serum stimulation of hepatocyte LDLRs markedly (by 80%). Furthermore, resistin diminished statin-mediated up-regulation of the LDLR by 60%, implicating resistin in the relative ineffectiveness of statins in selective target populations. CONCLUSIONS: These results reveal for the first time that resistin is a highly attractive therapeutic target in ameliorating elevated serum low-density lipoprotein and, thereby, atherosclerotic cardiovascular diseases in obese humans.

Adipocyte hypertrophy, fatty liver and metabolic risk factors in South Asians: the Molecular Study of Health and Risk in Ethnic Groups (mol-SHARE).

OBJECTIVE: We sought to determine if differences in the distribution and characteristics of adipose tissue between South Asians and white Caucasians account for differences in risk factors for cardiovascular disease. RESEARCH DESIGN AND METHODS: We recruited 108 healthy South Asians (36.8 years) and white Caucasians (34.2 years) within three BMI strata. Body composition, adipocyte size, abdominal fat area, and hepatic adiposity were assessed and related to fasting glucose, insulin, lipids and adiponectin. RESULTS: After adjustment for age, sex, and BMI, South Asians compared to white Caucasians had higher ln fasting insulin (mean difference (MD): 0.44; 95% CI: 0.20-0.69), lower HDL cholesterol (md: -0.13; 95% CI:-0.26 to -0.01), and lower adiponectin (md: -2.38; 95% CI: -3.59 to -1.17). South Asians also had more body fat (md: 2.69; 95% CI: 0.70 to 4.69), lower lean muscle mass (md: -3.25; 95%CI: -5.35 to -1.14), increased waist to hip ratio (md: 0.03; 95% CI: 0.01-0.05), less superficial subcutaneous abdominal adipose tissue (md: -2.94; 95% CI: -5.56 to-0.32), more deep/visceral to superficial adipose tissue ratio (md 0.34; 95% CI: 0.02 to 0.65), and more liver fat (md: 7.43%; 95% CI: 2.30 to 12.55%). Adipocyte area was increased in South Asians compared to white Caucasians (md: 64.26; 95% CI: 24.3 to 104.1) units(2). Adjustment for adipocyte area attenuated the ethnic differences in insulin (md: 0.22; 95% CI: -0.07 to 0.51), HDL (md: -0.01; 95% CI: -0.16 to 0.13) and adiponectin (md: -1.11; 95% CI: -2.61 to 0.39). Adjustment for differences in adipocyte area and fat distribution attenuated the ethnic difference in liver fat (md: 5.19; 95% CI: 0.31 to 10.06). CONCLUSION: South Asians have an increased adipocyte area compared to white Caucasians. This difference accounts for the ethnic differences in insulin, HDL cholesterol, adiponectin, and ectopic fat deposition in the liver.

Human resistin stimulates hepatic overproduction of atherogenic ApoB-containing lipoprotein particles by enhancing ApoB stability and impairing intracellular insulin signaling.

RATIONALE: Obese individuals are at high risk for developing atherosclerosis primarily attributable to elevated plasma concentrations of apolipoprotein (apo)B-containing particles, including very-low-density lipoprotein (VLDL). Plasma levels of the adipose tissue adipokine resistin are increased in human obesity, and resistin expression is positively correlated with coronary atherosclerosis and VLDL levels. OBJECTIVE: We sought to determine for the first time whether resistin directly stimulates human hepatocyte production of apoB-containing particles and to elucidate the mechanisms responsible. METHODS AND RESULTS: Treatment of human hepatocytes with resistin at levels observed in human obesity stimulated apoB secretion up to 10-fold, because of increased microsomal triglyceride transfer protein (MTP) activity and decreased expression/phosphorylation of proteins in the insulin signaling pathways (insulin receptor substrate-2, Akt, and extracellular signal-regulated kinase). Resistin also increased hepatocyte lipid content by stimulating de novo lipogenesis via the SREBP1 and SREBP2 pathways. Furthermore, obese serum with elevated resistin levels induced greater hepatocyte stimulation of apoB secretion than lean human serum, an effect that was ameliorated by antibody immunoprecipitation removal of serum resistin. CONCLUSIONS: Resistin has a direct deleterious impact on human hepatic lipid and lipoprotein regulation. Resistin greatly increased hepatocyte VLDL apoB and lipid secretion because of MTP activation and induction of hepatocyte insulin resistance. Conversely, antibody removal of serum resistin ameliorated human serum stimulation of apoB secretion. Increased hepatic cellular lipids mediated by resistin reflects the fatty liver/steatosis observed with elevated resistin in humans. Thus, human resistin is a novel therapeutic target for mitigating common hepatic pathophysiological processes associated with human obesity, dyslipidemia and atherosclerosis.

Identification of a novel human cellular HDL biosynthesis defect.

AIMS: Severe high-density lipoprotein cholesterol (HDL-C) deficiency is attributed to mutations in several genes and may contribute to the genetic basis of coronary artery disease. To identify the cellular basis of a novel HDL-deficiency phenotype, we screened 54 subjects of French Canadian ancestry with severe HDL deficiency. METHODS AND RESULTS: We excluded individuals with mutations in genes currently associated with low HDL (ABCA1, LCAT, APOA-I, and SMPD1). We identified two patients in which cellular phospholipid efflux in the HDL biosynthesis process is impaired, whereas cholesterol efflux is normal. Two-dimensional gel electrophoresis analysis further showed that the two patients with impaired phospholipid efflux were defective primarily in the larger alpha-HDL subpopulations. In fibroblasts from affected subjects, oxysterol stimulation resulted in increased ABCA1 protein expression and normalized their defective phospholipid efflux defect. CONCLUSION: Our results indicate for the first time in humans that phospholipid and cholesterol efflux are two separate and distinct processes in cellular HDL biosynthesis. They further show for the first time that normal cellular phospholipid efflux is necessary for the formation of larger alpha-HDL particles. The defect in phospholipid efflux is due to defective ABCA1 protein regulation and can be corrected by treatment with physiological oxysterols, a current therapeutic target of interest, that may, with further studies, be used to raise HDL levels in patients with severe HDL deficiencies.

Molecular mechanisms of hepatic steatosis and insulin resistance in the AGPAT2-deficient mouse model of congenital generalized lipodystrophy.

Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2(-/-) mice develop severe lipodystrophy affecting both white and brown adipose tissue, extreme insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased approximately 4-fold in Agpat2(-/-) mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2(-/-) mice, suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by approximately 50% in Agpat2(-/-) mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2(-/-) mice.

Key questions resulting from the JUPITER trial assessing cardiovascular disease intervention with rosuvastatin.

THIS PAPER PRESENTS AN ANALYSIS OF THE RECENTLY PUBLISHED JUSTIFICATION FOR THE USE OF STATINS IN PREVENTION (JUPITER: an intervention trial evaluating rosuvastatin) trial, which tested the statin rosuvastatin in apparently healthy individuals with no prior cardiovascular (CVD) disease and with normal plasma low density lipoprotein (LDL) cholesterol concentrations but with raised plasma high sensitivity C-reactive protein (hsCRP) levels. The rate of the combined primary CVD endpoint was significantly reduced in the treatment arm after a median of under 2 years. The JUPITER trial is distinct from previous studies examining statin use in primary prevention groups because the target group for drug therapy was apparently healthy men and women at low or intermediate risk for developing CVD. On the basis of JUPITER's findings, there are key questions that should be assessed on the therapeutic intervention of CVD regarding: the primary prevention groups that should be targeted for statin therapy, the utility of targets in addition to plasma LDL cholesterol levels, and the need to consider the metabolic state of individuals targeted for therapy (including the presence of obesity and inflammation). The conclusion from the current analysis is that the JUPITER results warrant further LDL cholesterol lowering than is currently targeted in primary prevention groups that have a pre-existing condition or lifestyle that elevates CVD risk but still do not have a high global CVD risk (as assessed with current algorithms). This group is not captured in current widely used CVD risk calculations, however, with the identification of useful biomarkers, such as hsCRP, this group can be better identified and targeted for intervention.