Cristina M. Ramírez

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.

MicroRNA-758 Regulates Cholesterol Efflux Through Posttranscriptional Repression of ATP-Binding Cassette Transporter A1

Objective—The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of macrophage cholesterol efflux and protects cells from excess intracellular cholesterol accumulation; however, the mechanism involved in posttranscriptional regulation of ABCA1 is poorly understood. We previously showed that microRNA-33 (miR-33) is 1 regulator. Here, we investigated the potential contribution of other microRNAs (miRNAs) to posttranscriptional regulation of ABCA1 and macrophage cholesterol efflux. Methods and Results—We performed a bioinformatic analysis for identifying miRNA target prediction sites in ABCA1 gene and an unbiased genome-wide screen to identify miRNAs modulated by cholesterol excess in mouse peritoneal macrophages. Quantitative real-time reverse transcription–polymerase chain reaction confirmed that miR-758 is repressed in cholesterol-loaded macrophages. Under physiological conditions, high dietary fat excess in mice repressed miR-758 both in peritoneal macrophages and, to a lesser extent, in the liver. In mouse and human cells in vitro, miR-758 repressed the expression of ABCA1, and conversely, the inhibition of this miRNA by using anti-miR-758 increased ABCA1 expression. In mouse cells, miR-758 reduced cellular cholesterol efflux to apolipoprotein A1 (apoA1), and anti-miR-758 increased it. miR-758 directly targets the 3′-untranslated region of Abca1 as assessed by 3′-untranslated region luciferase reporter assays. Interestingly, miR-758 is highly expressed in the brain, where it also targets several genes involved in neurological functions, including Slc38a1, Ntm, Epha7, and Mytl1. Conclusion—We identified miR-758 as a novel miRNA that posttranscriptionally controls ABCA1 levels in different cells and regulates macrophage cellular cholesterol efflux to apoA1, opening new avenues to increase apoA1 and raise high-density lipoprotein levels.

MicroRNAs in Metabolic Disease

Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2-diabetes mellitus and atherosclerosis. Work over the last years has suggested that microRNAs (miRNAs) play an important role in regulating these physiological processes. The contribution of miRNAs in regulating metabolism is exemplified by miR-33, an intronic miRNA encoded in the Srebp genes. miR-33 controls cellular cholesterol export and fatty acid degradation, whereas its host genes stimulate cholesterol and fatty acid synthesis. Other miRNAs, such as miR-122, also play a critical role in regulating lipid homeostasis by controlling cholesterol synthesis and lipoprotein secretion in the liver. This review article summarizes the recent findings in the field, highlighting the contribution of miRNAs in regulating lipid and glucose metabolism. We will also discuss how the modulation of specific miRNAs may be a promising strategy to treat metabolic diseases.

Mir-33 regulates cell proliferation and cell cycle progression

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 G1 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.

A regulatory role for microRNA 33* in controlling lipid metabolism gene expression.

ABSTRACT hsa-miR-33a and hsa-miR-33b, intronic microRNAs (miRNAs) located within the sterol regulatory element-binding protein 2 and 1 genes (Srebp-2 and -1), respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes. Although the functional role of miR-33a and -b has been highly investigated, the role of their passenger strands, miR-33a* and -b*, remains unclear. Here, we demonstrate that miR-33a* and -b* accumulate to steady-state levels in human, mouse, and nonhuman primate tissues and share a similar lipid metabolism target gene network as their sister strands. Analogous to miR-33, miR-33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1), fatty acid metabolism (CROT and CPT1a), and insulin signaling (IRS2). Moreover, miR-33* also targets key transcriptional regulators of lipid metabolism, including SRC1, SRC3, NFYC, and RIP140. Importantly, inhibition of either miR-33 or miR-33* rescues target gene expression in cells overexpressing pre-miR-33. Consistent with this, overexpression of miR-33* reduces fatty acid oxidation in human hepatic cells. Altogether, these data support a regulatory role for the miRNA* species and suggest that miR-33 regulates lipid metabolism through both arms of the miR-33/miR-33* duplex.

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.

Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet–fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.

MiR-155 Has a Protective Role in the Development of Non-Alcoholic Hepatosteatosis in Mice

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155−/− mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155−/− livers, we identified and validated that Nr1h3 (LXRα) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155−/− mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes.

MicroRNA 33 Regulates Glucose Metabolism

ABSTRACT Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small noncoding RNAs (microRNAs [miRNAs]) in the posttranscriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a and miR33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression, leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression of PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.

MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL-cholesterol (LDL-C). While the transcriptional regulation of LDLR is well-characterized, the post-transcriptional mechanisms which govern LDLR expression are just beginning to emerge. Here, we developed a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen, we characterize miR-148a as a negative regulator of LDLR expression and activity, and define a novel SREBP1-mediated pathway by which miR-148a regulates LDL-C uptake. Importantly, inhibition of miR-148a increases hepatic LDLR expression and decreases plasma LDL-C in vivo. We also provide evidence that miR-148a regulates hepatic ABCA1 expression and circulating HDL-C levels. Collectively, these studies uncover miR-148a as an important regulator of hepatic LDL-C clearance through direct regulation of LDLR expression, and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate the elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.