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.
Synopsis
Although short‐term anti‐miR‐33 therapy was reported to increase circulating HDL‐cholesterol and reduce atherosclerosis, long‐term adverse effects are here shown for the first time in mice fed a high‐fat diet to result in hypertriglyceridemia and moderate hepatic steatosis.
The effect of long‐term inhibition of miR‐33 was determined in mice fed a chow diet and high‐fat diet.
Chronic therapeutic silencing of miR‐33 increased circulating triglycerides and lipid accumulation in the livers of mice fed a high‐fat diet.
miR‐33 inhibition raised the expression of genes involved in fatty acid synthesis and lipid metabolism.
Further studies are warranted to understand the complex gene regulatory network controlled by miR‐33.
Although short‐term anti‐miR‐33 therapy was reported to increase circulating HDL‐cholesterol and reduce atherosclerosis, long‐term adverse effects are here shown for the 1st time in mice fed a high‐fat‐diet to result in hypertriglyerimedia and moderate hepatic steatosis.
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. (Hepatology 2016;63:1660‐1674)
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.
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. (Hepatology 2015;61:1227–1238)
Abstract only 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 to 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. Animals were fed a high-fat, high-cholesterol diet for 12 weeks while simultaneously dosed with saline, ASO-ctrl, or ASO-Fsp27. Data show that, compared to control treatments, silencing Fsp 27 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. 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.
Abstract only The cell death-inducing DFFA-like effector c (CIDEC; known as FSP27 or fat-specific protein 27 in rodents) is a lipid droplet (LD)-associated protein that is mainly expressed in white ...adipose tissue, where it promotes triglyceride storage. Fsp27 levels are undetectable in the livers of fed mice, but rise during fasting or under hepatosteatosis in both obese mice and patients. The Peroxisome Proliferator Activated Receptor α (PPARα) is a nuclear receptor that controls the fatty acid oxidative pathway. Hepatic PPARα signaling is activated physiologically during fasting, and pharmacologically by fibrates, a class of hypotriglyceridemic drugs. Here we show that CIDEC/Fsp27 is induced in primary hepatocytes and in the liver by synthetic PPARα agonists, and promoter reporter and ChIP assays identified CIDEC/Fsp27 as a conserved, direct PPARα target. Importantly, adenoviral-mediated shRNA silencing of hepatic Fsp27 resulted in a strong reduction of fasting-induced hepatosteatosis. Likewise, in vivo shRNA against Fsp27 resulted in decreased hepatic triglyceride contents in mice fed a high-fat diet, and in mice dosed with a PPARα agonist. Further studies in mouse primary hepatocytes showed that loss of Fsp27 activity enhanced PPARα-stimulated fatty acid oxidation. Collectively, our results highlight the physiological role of CIDEC/Fsp27 in hepatic triglyceride homeostasis. Additionally, these data suggest that patients taking fibrates likely have elevated levels of hepatic CIDEC, which may limit the efficient catabolism of hepatic triglycerides.
The sterol regulatory element binding protein 2 (SREBP-2) and the liver X receptor (LXR) control antagonistic transcriptional programs that stimulate cellular cholesterol uptake and synthesis, and ...cholesterol efflux, respectively. The clinical importance of SREBP-2 is revealed in patients with hypercholesterolemia treated with statins, which reduce low-density lipoprotein (LDL) cholesterol levels by increasing hepatic expression of SREBP-2 and its target, the LDL receptor. Here we show that miR-33 is encoded within SREBP-2 and that both mRNAs are coexpressed. We also identify sequences in the 3′ UTR of ABCA1 and ABCG1, sterol transporter genes both previously shown to be regulated by LXR, as targets for miR-33—mediated silencing. Our data show that LXR-dependent cholesterol efflux to both ApoAI and serum is ameliorated by miR-33 overexpression and, conversely, stimulated by miR-33 silencing. Finally, we show that ABCA1 mRNA and protein and plasma HDL levels decline after hepatic overexpression of miR-33, whereas they increase after hepatic miR-33 silencing. These results suggest novel ways to manage hypercholesterolemic patients.
Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. microRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene ...expression, acting predominantly at posttranscriptional level. Recent work from our group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the Srebp genes, regulate cholesterol and fatty acid metabolism in concert with their host genes. Here, we show that hsa-miR-33 family members modulate the expression of genes involved in cell cycle regulation and cell proliferation. MiR-33 inhibits the expression of the cyclin-dependent kinase 6 (CDK6) and cyclin D1 (CCND1), thereby reducing cell proliferation and cell cycle progression. Overexpression of miR-33 induces a significant G
1
cell cycle arrest in Huh7 and A549 cell lines. Most importantly, inhibition of miR-33 expression using 2'fluoro/methoxyethyl-modified (2'F/MOE-modified) phosphorothioate backbone antisense oligonucleotides improves liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these results suggest that Srebp/miR-33 locus may cooperate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.
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.
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
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.
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
using ASO, germline deletion, or AAV CRISPR results in increased hepatic LDLR abundance and availability and decreased plasma LDL-C levels.
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.
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