RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels.

BACKGROUND: Removal of circulating plasma low-density lipoprotein cholesterol (LDL-C) by the liver relies on efficient endocytosis and intracellular vesicle trafficking. Increasing the availability of hepatic LDL receptors (LDLRs) remains a major clinical target for reducing LDL-C levels. Here, we describe a novel role for RNF130 (ring finger containing protein 130) in regulating plasma membrane availability of LDLR. METHODS: We performed a combination of gain-of-function and loss-of-function experiments to determine the effect of RNF130 on LDL-C and LDLR recycling. We overexpressed RNF130 and a nonfunctional mutant RNF130 in vivo and measured plasma LDL-C and hepatic LDLR protein levels. We performed in vitro ubiquitination assays and immunohistochemical staining to measure levels and cellular distribution of LDLR. We supplement these experiments with 3 separate in vivo models of RNF130 loss-of-function where we disrupted Rnf130 using either ASO (antisense oligonucleotides), germline deletion, or AAV CRISPR (adeno-associated virus clustered regularly interspaced short palindromic repeats) and measured hepatic LDLR and plasma LDL-C. RESULTS: We demonstrate that RNF130 is an E3 ubiquitin ligase that ubiquitinates LDLR resulting in redistribution of the receptor away from the plasma membrane. Overexpression of RNF130 decreases hepatic LDLR and increases plasma LDL-C levels. Further, in vitro ubiquitination assays demonstrate RNF130-dependent regulation of LDLR abundance at the plasma membrane. Finally, in vivo disruption of Rnf130 using ASO, germline deletion, or AAV CRISPR results in increased hepatic LDLR abundance and availability and decreased plasma LDL-C levels. CONCLUSIONS: Our studies identify RNF130 as a novel posttranslational regulator of LDL-C levels via modulation of LDLR availability, thus providing important insight into the complex regulation of hepatic LDLR protein levels.

Ovariectomy Activates Chronic Low-Grade Inflammation Mediated by Memory T Cells, Which Promotes Osteoporosis in Mice.

The loss of estrogen (E2 ) initiates a rapid phase of bone loss leading to osteoporosis in one-half of postmenopausal women, but the mechanism is not fully understood. Here, we show for the first time how loss of E2 activates low-grade inflammation to promote the acute phase of bone catabolic activity in ovariectomized (OVX) mice. E2 regulates the abundance of dendritic cells (DCs) that express IL-7 and IL-15 by inducing the Fas ligand (FasL) and apoptosis of the DC. In the absence of E2 , DCs become long-lived, leading to increased IL-7 and IL-15. We find that IL-7 and IL-15 together, but not alone, induced antigen-independent production of IL-17A and TNFα in a subset of memory T cells (TMEM ). OVX of mice with T-cell-specific ablation of IL15RA showed no IL-17A and TNFα expression, and no increase in bone resorption or bone loss, confirming the role of IL-15 in activating the TMEM and the need for inflammation. Our results provide a new mechanism by which E2 regulates the immune system, and how menopause leads to osteoporosis. The low-grade inflammation is likely to cause or contribute to other comorbidities observed postmenopause. © 2020 American Society for Bone and Mineral Research.

Genetic deficiency or pharmacological inhibition of miR-33 protects from kidney fibrosis.

Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can partially prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in 2 mouse models of kidney disease. These effects were not related to changes in circulating leukocytes because bone marrow transplants from miR-33-deficient animals did not have a similar impact on disease progression. Most important, targeted delivery of miR-33 peptide nucleic acid inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.

Doubled lifespan and patient-like pathologies in progeria mice fed high-fat diet.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating premature aging disease. Mouse models have been instrumental for understanding HGPS mechanisms and for testing therapies, which to date have had only marginal benefits in mice and patients. Barriers to developing effective therapies include the unknown etiology of progeria mice early death, seemingly unrelated to the reported atherosclerosis contributing to HGPS patient mortality, and mice not recapitulating the severity of human disease. Here, we show that progeria mice die from starvation and cachexia. Switching progeria mice approaching death from regular diet to high-fat diet (HFD) rescues early lethality and ameliorates morbidity. Critically, feeding the mice only HFD delays aging and nearly doubles lifespan, which is the greatest lifespan extension recorded in progeria mice. The extended lifespan allows for progeria mice to develop degenerative aging pathologies of a severity that emulates the human disease. We propose that starvation and cachexia greatly influence progeria phenotypes and that nutritional/nutraceutical strategies might help modulate disease progression. Importantly, progeria mice on HFD provide a more clinically relevant animal model to study mechanisms of HGPS pathology and to test therapies.

Therapeutic silencing of FSP27 reduces the progression of atherosclerosis in Ldlr-/- mice.

BACKGROUND AND AIMS: Obesity, hepatosteatosis, and hypertriglyceridemia are components of the metabolic syndrome and independent risk factors for cardiovascular disease. The lipid droplet-associated protein CIDEC (cell death-inducing DFFA-like effector C), known in mice as FSP27 (fat-specific protein 27), plays a key role in maintaining triacylglyceride (TAG) homeostasis in adipose tissue and liver, and controls circulating TAG levels in mice. Importantly, mutations and SNPs in CIDEC are associated with dyslipidemia and altered metabolic function in humans. Here we tested whether systemic silencing of Fsp27 using antisense oligonucleotides (ASOs) was atheroprotective in LDL receptor knock-out (Ldlr-/-) mice. METHODS: Atheroprone Ldlr-/- mice were fed a high-fat, high-cholesterol diet for 12 weeks while simultaneously dosed with saline, ASO-ctrl, or ASO-Fsp27. RESULTS: Data show that, compared to control treatments, silencing Fsp27 significantly reduced body weight gain and visceral adiposity, prevented diet-induced hypertriglyceridemia, and reduced atherosclerotic lesion size both in en face aortas and in the aortic root. CONCLUSIONS: Our findings suggest that therapeutic silencing of Fsp27 with ASOs may be beneficial in the prevention and management of atherogenic disease in patients with metabolic syndrome.

Genetic Ablation of miR-33 Increases Food Intake, Enhances Adipose Tissue Expansion, and Promotes Obesity and Insulin Resistance.

While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33-/- mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33-/- mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33-/- mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals.

Genetic Dissection of the Impact of miR-33a and miR-33b during the Progression of Atherosclerosis.

As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr-/- mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

Amelioration of diet-induced steatohepatitis in mice following combined therapy with ASO-Fsp27 and fenofibrate.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD progresses from benign steatosis to steatohepatitis (NASH) to cirrhosis and is linked to hepatocellular carcinoma. No targeted treatment is currently approved for NAFLD/NASH. We previously showed that fat-specific protein 27 (FSP27), a lipid droplet-associated protein that controls triglyceride turnover in the hepatocyte, is required for fasting- and diet-induced triglyceride accumulation in the liver. However, silencing Fsp27 with antisense oligonucleotides (ASOs) did not improve hepatosteatosis in genetic nor nutritional mouse models of obesity. Herein, we tested the therapeutic potential of ASO-Fsp27 when used in combination with the PPARα agonist fenofibrate. C57BL/6 mice were fed a high-trans-fat, high-cholesterol, high-fructose diet for eight weeks to establish NASH, then kept on diet for six additional weeks while dosed with ASOs and fenofibrate, alone or in combination. Data show that ASO-Fsp27 and fenofibrate synergize to promote resistance to diet-induced obesity and hypertriglyceridemia and to reverse hepatic steatosis, inflammation, oxidative stress, and fibrosis. This multifactorial improvement of liver disease noted when combining both drugs suggests that a course of treatment that includes both reduced FSP27 activity and activation of PPARα could provide therapeutic benefit to patients with NAFLD/NASH.

ABCG1 regulates pulmonary surfactant metabolism in mice and men.

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.

Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.

Obesity is a component of the metabolic syndrome, mechanistically linked to diabetes, fatty liver disease, and cardiovascular disease. Proteins that regulate the metabolic fate of intracellular lipid droplets are potential therapeutic candidates to treat obesity and its related consequences. CIDEC (cell death-inducing DFFA-like effector C), also known in mice as Fsp27 (fat-specific protein 27), is a lipid droplet-associated protein that prevents lipid mobilization and promotes intracellular lipid storage. The consequences of complete loss of FSP27 on hepatic metabolism and on insulin resistance are controversial, as both healthy and deleterious lipodystrophic phenotypes have been reported in Fsp27-/- mice. To test whether therapeutic silencing of Fsp27 might be useful to improve obesity, fatty liver, and glycemic control, we used antisense oligonucleotides (ASOs) in both nutritional (high-fat diet) and genetic (leptin-deficient ob/ob) mouse models of obesity, hyperglycemia, and hepatosteatosis. We show that partial silencing Fsp27 in either model results in the robust decrease in visceral fat, improved insulin sensitivity and whole-body glycemic control, and tissue-specific changes in transcripts controlling lipid oxidation and synthesis. These data suggest that partial reduction of FSP27 activity (e.g., using ASOs) might be exploited therapeutically in insulin-resistant obese or overweight patients.

Peroxisome proliferator-activated receptor-α accelerates α-chlorofatty acid catabolism.

α-Chlorofatty aldehydes are generated from myeloperoxidase-derived HOCl targeting plasmalogens, and are subsequently oxidized to α-chlorofatty acids (α-ClFAs). The catabolic pathway for α-ClFA is initiated by ω-oxidation. Here, we examine PPAR-α activation as a mechanism to increase α-ClFA catabolism. Pretreating both HepG2 cells and primary mouse hepatocytes with the PPAR-α agonist, pirinixic acid (Wy 14643), increased the production of α-chlorodicarboxylic acids (α-ClDCAs) in cells treated with exogenous α-ClFA. Additionally, α-ClDCA production in Wy 14643-pretreated wild-type mouse hepatocytes was accompanied by a reduction in cellular free α-ClFA. The dependence of PPAR-α-accelerated α-ClFA catabolism was further demonstrated by both impaired metabolism in mouse PPAR-α-/- hepatocytes and decreased clearance of plasma α-ClFA in PPAR-α-/- mice. Furthermore, Wy 14643 treatments decreased plasma 2-chlorohexadecanoic acid levels in wild-type mice. Additional studies showed that α-ClFA increases PPAR-α, PPAR-δ, and PPAR-γ activities, as well as mRNA expression of the PPAR-α target genes, CD36, CPT1a, Cyp4a10, and CIDEC. Collectively, these results indicate that PPAR-α accelerates important pathways for the clearance of α-ClFA, and α-ClFA may, in part, accelerate its catabolism by serving as a ligand for PPAR-α.

Truths and controversies concerning the role of miRNAs in atherosclerosis and lipid metabolism.

PURPOSE OF REVIEW: Better tools are sorely needed for both the prevention and treatment of cardiovascular diseases, which account for more than one-third of the deaths in Western countries. MicroRNAs typically regulate the expression of several mRNAs involved in the same biological process. Therapeutic manipulation of miRNAs could restore the expression of multiple players within the same physiologic pathway, and ideally offer better curative outcomes than conventional approaches that target only one single player within the pathway. This review summarizes available studies on the prospective value of targeting miRNAs to prevent dyslipidemia and atherogenesis. RECENT FINDINGS: Silencing the expression of miRNAs that target key genes involved in lipoprotein metabolism in vivo with antisense oligonucleotides results in the expected de-repression of target mRNAs in liver and atherosclerotic plaques. However, the consequences of long-term antimiRNA treatment on both circulating lipoproteins and athero-protection are yet to be established. SUMMARY: A number of studies have demonstrated the efficacy of miRNA mimics and inhibitors as novel therapeutic tools for treating dyslipidemia and cardiovascular diseases. Nevertheless, concerns over unanticipated side-effects related to de-repression of additional targets should not be overlooked for miRNA-based therapies.

miRNAs and High-Density Lipoprotein metabolism.

Altered lipoprotein metabolism plays a key role during atherogenesis. For over 50years, epidemiological data have fueled the proposal that HDL-cholesterol (HDL-c) in circulation is inversely correlated to cardiovascular risk. However, the atheroprotective role of HDL is currently the focus of much debate and remains an active field of research. The emerging picture from research in the past decade suggests that HDL function, rather than HDL-c content, is important in disease. Recent developments demonstrate that miRNAs play an important role in fine-tuning the expression of key genes involved in HDL biogenesis, lipidation, and clearance, as well as in determining the amounts of HDL-c in circulation. Thus, it has been proposed that miRNAs that affect HDL metabolism might be exploited therapeutically in patients. Whether HDL-based therapies, alone or in combination with LDL-based treatments (e.g. statins), provide superior outcomes in patients has been recently questioned by human genetics studies and clinical trials. The switch in focus from "HDL-cholesterol" to "HDL function" opens a new paradigm to understand the physiology and therapeutic potential of HDL, and to find novel modulators of cardiovascular risk. In this review we summarize the current knowledge on the regulation of HDL metabolism and function by miRNAs. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez.

Akt-mediated foxo1 inhibition is required for liver regeneration.

UNLABELLED: Understanding the hepatic regenerative process has clinical interest as the effectiveness of many treatments for chronic liver diseases is conditioned by efficient liver regeneration. Experimental evidence points to the need for a temporal coordination between cytokines, growth factors, and metabolic signaling pathways to enable successful liver regeneration. One intracellular mediator that acts as a signal integration node for these processes is the serine-threonine kinase Akt/protein kinase B (Akt). To investigate the contribution of Akt during hepatic regeneration, we performed partial hepatectomy in mice lacking Akt1, Akt2, or both isoforms. We found that absence of Akt1 or Akt2 does not influence liver regeneration after partial hepatectomy. However, hepatic-specific Akt1 and Akt2 null mice show impaired liver regeneration and increased mortality. The major abnormal cellular events observed in total Akt-deficient livers were a marked reduction in cell proliferation, cell hypertrophy, glycogenesis, and lipid droplet formation. Most importantly, liver-specific deletion of FoxO1, a transcription factor regulated by Akt, rescued the hepatic regenerative capability in Akt1-deficient and Akt2-deficient mice and normalized the cellular events associated with liver regeneration. CONCLUSION: The Akt-FoxO1 signaling pathway plays an essential role during liver regeneration.

miR-27b inhibits LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels in mice.

RATIONALE: Recently, there has been significant interest in the therapeutic administration of miRNA mimics and inhibitors to treat cardiovascular disease. In particular, miR-27b has emerged as a regulatory hub in cholesterol and lipid metabolism and potential therapeutic target for treating atherosclerosis. Despite this, the impact of miR-27b on lipid levels in vivo remains to be determined. As such, here we set out to further characterize the role of miR-27b in regulating cholesterol metabolism in vitro and to determine the effect of miR-27b overexpression and inhibition on circulating and hepatic lipids in mice. METHODS AND RESULTS: Our results identify miR-27b as an important regulator of LDLR activity in human and mouse hepatic cells through direct targeting of LDLR and LDLRAP1. In addition, we report that modulation of miR-27b expression affects ABCA1 protein levels and cellular cholesterol efflux to ApoA1 in human hepatic Huh7 cells. Overexpression of pre-miR-27b in the livers of wild-type mice using AAV8 vectors increased pre-miR-27b levels 50-fold and reduced hepatic ABCA1 and LDLR expression by 50% and 20%, respectively, without changing circulating and hepatic cholesterol and triglycerides. To determine the effect of endogenous miR-27b on circulating lipids, wild-type mice were fed a Western diet for one month and injected with 5 mg/kg of LNA control or LNA anti-miR-27b oligonucleotides. Following two weeks of treatment, the expression of ABCA1 and LDLR were increased by 10-20% in the liver, demonstrating effective inhibition of miR-27b function. Intriguingly, no differences in circulating and hepatic lipids were observed between treatment groups. CONCLUSIONS: The results presented here provide evidence that short-term modulation of miR-27b expression in wild-type mice regulates hepatic LDLR and ABCA1 expression but does not influence plasma and hepatic lipid levels.

CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.

UNLABELLED: The cell death-inducing DNA fragmentation factor alpha-like effector c (CIDEC; also known in rodents as FSP27 or fat-specific protein 27) is a lipid droplet-associated protein that promotes intracellular triglyceride (TAG) storage. CIDEC/Fsp27 is highly expressed in adipose tissue, but undetectable in normal liver. However, its hepatic expression rises during fasting or under genetic or diet-induced hepatosteatosis in both mice and patients. Herein, we demonstrate that CIDEC/Fsp27 is a direct transcriptional target of the nuclear receptor PPARα (peroxisome proliferator-activated receptor alpha) in both mouse and human hepatocytes, and that preventing Fsp27 induction accelerates PPARα-stimulated fatty acid oxidation. We show that adenoviral-mediated silencing of hepatic Fsp27 abolishes fasting-induced liver steatosis in the absence of changes in plasma lipids. Finally, we report that anti-Fsp27 short hairpin RNA and PPARα agonists synergize to ameliorate hepatosteatosis in mice fed a high fat diet. CONCLUSIONS: Together, our data highlight the physiological importance of CIDEC/Fsp27 in TAG homeostasis under both physiological and pathological liver steatosis. Our results also suggest that patients taking fibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient mobilization and catabolism of hepatic TAGs.

ABCG1 is required for pulmonary B-1 B cell and natural antibody homeostasis.

Many metabolic diseases, including atherosclerosis, type 2 diabetes, pulmonary alveolar proteinosis, and obesity, have a chronic inflammatory component involving both innate and adaptive immunity. Mice lacking the ATP-binding cassette transporter G1 (ABCG1) develop chronic inflammation in the lungs, which is associated with the lipid accumulation (cholesterol, cholesterol ester, and phospholipid) and cholesterol crystal deposition that are characteristic of atherosclerotic lesions and pulmonary alveolar proteinosis. In this article, we demonstrate that specific lipids, likely oxidized phospholipids and/or sterols, elicit a lung-specific immune response in Abcg1(-/-) mice. Loss of ABCG1 results in increased levels of specific oxysterols, phosphatidylcholines, and oxidized phospholipids, including 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine, in the lungs. Further, we identify a niche-specific increase in natural Ab (NAb)-secreting B-1 B cells in response to this lipid accumulation that is paralleled by increased titers of IgM, IgA, and IgG against oxidation-specific epitopes, such as those on oxidized low-density lipoprotein and malondialdehyde-modified low-density lipoprotein. Finally, we identify a cytokine/chemokine signature that is reflective of increased B cell activation, Ab secretion, and homing. Collectively, these data demonstrate that the accumulation of lipids in Abcg1(-/-) mice induces the specific expansion and localization of B-1 B cells, which secrete NAbs that may help to protect against the development of atherosclerosis. Indeed, despite chronic lipid accumulation and inflammation, hyperlipidemic mice lacking ABCG1 develop smaller atherosclerotic lesions compared with controls. These data also suggest that Abcg1(-/-) mice may represent a new model in which to study the protective functions of B-1 B cells/NAbs and suggest novel targets for pharmacologic intervention and treatment of disease.

Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice.

Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans.

Perilipin-5 is regulated by statins and controls triglyceride contents in the hepatocyte.

BACKGROUND & AIMS: Perilipin-5 (PLIN5) is a member of the perilipin family of lipid droplet (LD)-associated proteins. PLIN5 is expressed in oxidative tissues including the liver, and is critical during LD biogenesis. Studies showed that statins reduce hepatic triglyceride contents in some patients with non-alcoholic fatty liver disease and in rodent models of diet-induced hepatosteatosis. Whether statins alter triglyceride synthesis, storage, and/or utilization within the hepatocyte is unknown, though. Here we tested the hypothesis that statins alter the metabolism of LD in the hepatocyte during physiological conditions, such as fasting-induced steatosis. METHODS: Mice were gavaged with saline or atorvastatin, and the expression of LD-associated genes was determined in fed and fasted animals. The accumulation of triglycerides and LD was studied in mouse or human primary hepatocytes in response to statins, and following knock-down of SREBP2 or PLIN5. RESULTS: We show that statins decrease the levels of PLIN5, but not other LD-associated genes, in both mouse liver and mouse/human primary hepatocytes, which is paralleled by a significant reduction in both intracellular triglycerides and the number of LD. We identify an atypical negative sterol regulatory sequence in the proximal promoter of mouse/human PLIN5 that recruits the transcription factor SREBP2 and confers response to statins. Finally, we show that the statin-dependent reduction of hepatocyte triglyceride contents is mimicked by partial knock-down of PLIN5; conversely, ectopic overexpression of PLIN5 reverts the statin effect. CONCLUSIONS: PLIN5 is a physiological regulator of triglyceride metabolism in the liver, and likely contributes to the pleiotropic effects of statins.

Control of very low-density lipoprotein secretion by N-ethylmaleimide-sensitive factor and miR-33.

RATIONALE: Several reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist about the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG). OBJECTIVE: We test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion. METHODS AND RESULTS: Using therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3'UTR of NSF as miR-33 responsive elements and show that Nsf is specifically recruited to the RNA-induced silencing complex following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apolipoproteins and TAG in primary hepatocytes, compared with control cells. Importantly, Nsf rescues miR-33-dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG. CONCLUSIONS: Together, our data reveal key roles for the miR-33-NSF axis during hepatic secretion and suggest that caution should be taken with anti-miR-33-based therapies because they might raise proatherogenic VLDL-TAG levels.