MicroRNA-33a/b in Lipid Metabolism

– Novel “Thrifty” Models –

Abstract

MicroRNAs (miRNAs; miRs) are small non-protein-coding RNAs that negatively regulate gene expression. They bind to the 3’ UTR of specific mRNAs and either inhibit translation or promote mRNA degradation. There is emerging evidence linking miR-33a/b to lipid homoeostasis, targeting ABCA1, SREBF1, etc and it would appear that they have acted as “thrifty genes” during evolution to maintain cholesterol levels both at the cellular and whole body level. As we are now living in a period of “satiation”, miR-33a/b no longer seem to be useful and could be potential therapeutic targets for lipid disorders and/or atherosclerosis. In this review, we describe the current understanding of the function of miR-33a/b in lipid homeostasis, focusing on the “thrifty” aspect. (Circ J 2015; 79: 278–284)

MicroRNAs (miRNAs; miRs) are endogenous, small (approximately 20–22 nucleotides in length), nonprotein-coding RNAs. miRNAs bind to the 3’ untranslated region (UTR) of specific mRNAs according to the complementarity of their sequences and they either inhibit translation or promote mRNA degradation.^1,2 miRNAs were initially discovered in Caenorhabditis elegans^3,4 and were later found to be evolutionarily conserved.^5,6 More than 60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs, and so far, approximately 2,500 miRNAs have been identified in humans.^6,7

miRNAs are usually transcribed as longer primary miRNAs (Pri-miRNAs) by RNA polymerase II (Pol II) and then processed by the Drosha (RNase III)/DGCR8 complex to pre-mature miRNAs (Pre-miRNAs) in the nucleus. Pre-miRNAs are exported to the cytoplasm through exportin 5 and then processed by another ribonuclease enzyme, Dicer, to form mature miRNAs, which typically comprise 20–22 nucleotides. Moreover, it is known that miR-451 does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic center to generate an intermediate 3’ end, which is further trimmed.⁸ Mature miRNAs are assembled into an RNA-inducing silencing complex and post-transcriptionally inhibit mRNA expression by binding to the 3’ UTR of their target mRNAs.⁹

In addition to their existence in tissues, recent studies have indicated that miRNAs also exist in serum, plasma, urine, and other body fluids in highly stable forms that are secure from endogenous RNase activity.¹⁰ Altered levels of circulating miRNAs have been found in acute myocardial infarction,¹¹ acute coronary syndrome,¹² stable coronary artery disease (CAD),¹³ heart failure,¹⁴ essential hypertension,¹⁵ and stroke.¹⁶

miRNAs have many functions in physiological and pathological states, and some miRNAs have been shown to have a significant effect on lipid homeostasis.^9,17,18 Dyslipidemia and related metabolic disorders continue to rise at an alarming rate worldwide and are associated with increased cardiovascular disease risk. A high plasma level of low-density lipoprotein cholesterol (LDL-C) is a major risk factor for CAD. Therapy with statins [inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR)], which inhibit cholesterol biosynthesis, effectively reduces the levels of both LDL-C and modified forms of LDL in human plasma,¹⁹ and significantly reduces the risk of CAD, as evidenced by primary and secondary clinical intervention studies.^20,21 However, patients who are treated with high doses of statins, regardless of their treated LDL-C level, are still at considerable risk for cardiovascular disease.^22,23 Thus, we still need other therapeutic options to treat the residual risk.^24–26 Elucidation of the function of miRNAs may provide avenues to developing novel treatments of dyslipidemia.

In particular, we have intensively investigated the functions of miR-33a/b, in vivo, using genetically modified mice.^27–30 In this review, we summarize the functions of miR-33a/b and therapeutic strategies to suppress these functions.

In Vivo Regulation of HDL-C by miR-33a/b

Recent studies have indicated that the miR-33s, located in the intron of sterol-regulatory element-binding proteins (SREBPs), control cholesterol homeostasis.^27,31–34 In humans, miR-33a and -33b are encoded in the introns of SREBF2 and SREBF1, respectively,^32,35 whereas in rodents there is a deletion in part of the miR-33b encoding lesion and miR-33b cannot be expressed (Figure 1). Furthermore, miR-33a and -33b share the same seed sequence and differ in only 2 nucleotides. SREBF1 encodes SREBP-1a and -1c, which mainly regulate lipogenic genes, such as fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), and acetyl-CoA carboxylase 1 (ACC1). SREBF2 encodes SREBP-2, which mainly regulates cholesterol-regulating genes, such as HMGCR, and low-density lipoprotein receptor (LDLR).^36–38 miR-33a and -33b are considered to be cotranscribed and regulate lipid homeostasis with their host genes.

Figure 1.

Mice have a single copy of microRNA (miR)-33, but humans have 2 copies (miR-33a and miR-33b). SREBP, sterol-regulatory element-binding protein.

Several groups, including ours, have reported that miR-33a targets ATP-binding cassette transporter A1 (Abca1) in vivo, using either antisense technology or by generating miR-33a knockout mice.^27,31–33 ABCA1 mediates the efflux of cholesterol to lipid-poor apolipoprotein A-I (apoA-I) and forms nascent HDL. Therefore, ABCA1 is an essential molecule for HDL biogenesis and reverse cholesterol transport (RCT) (Figure 2). ABCA1 mRNA and protein half-lives are very short (1–2 h), suggesting that de novo transcription and translation are important for controlling its expression in response to environmental stimuli.³⁹ It was shown that mice treated with LNA antisense oligonucleotides or anti-miR-33a lentivirus exhibited increased ABCA1 expression in the liver and ABCA1 and ABCG1 expression in macrophages (Abcg1 is another target of miR-33a in rodents). More importantly, in anti-miR-33a-treated mice, plasma HDL levels increased to 35–50%, without affecting other lipoproteins.^31–33 miR-33a knockout mice also showed a significant increase in the expression of ABCA1 in the liver and macrophages, and a 25–40% increase in serum HDL²⁷ (Figures 3A,B). Moreover, we analyzed the liver and measured the serum lipid profile of miR-33b knock-in (KI) mice, which have miR-33b in the same intron as humans.³⁰ In contrast to the results with miR-33a-deficient mice, HDL-C levels in these mice were reduced by almost 35%, even in miR-33b KI hetero mice compared with the control mice (Figures 3C,D).

Figure 2.

High-density lipoprotein cholesterol (HDL-C) and reverse cholesterol transport. The liver is the site of HDL biogenesis, where ABCA1-mediated lipidation of lipid-poor apolipoprotein A-I (apoA-I) generates nascent HDL. Free cholesterol on nascent HDL particles is esterified to cholesteryl esters (CE) by lecithin-cholesterol acyltransferase (LCAT). In turn, ABCG1 mediates cholesterol transfer to mature HDL. CETP mediates CE transfer from HDL to apolipoprotein B-containing lipoproteins (VLDL/LDL) in exchange for triglycerides (TG), promoting plasma cholesterol clearance by the uptake of VLDL/LDL lipoproteins through the LDLR pathway. Hepatic SR-BI mediates the removal of FC and CE from HDL, through the selective uptake pathway, and excess cholesterol is excreted from the liver into the bile. Both the ABCB11 and ATP8B1 transporters promote hepatic clearance, and have been shown to be the target genes of miR-33a.⁴⁰ LDLR, low-density lipoprotein receptor.

Figure 3.

Western blot analysis and lipid profiles of wild-type, miR-33a-deficient mice, and miR-33b KI mice. (A) Western blot analysis of hepatic ABCA1 and SREBP-2 in 16-week-old male wild-type and miR-33a-deficient mice. GAPDH was used as a loading control. (B) Representative HPLC analysis of serum cholesterol from male wild-type and miR-33a-deficient mice. (C) Western blot analysis of hepatic ABCA1 and SREBP-1 in 8-week-old male wild-type and miR-33b KI^+/+ mice. β-actin was used as the loading control. (D) Representative HPLC analysis of serum cholesterol from male wild-type and miR-33b KI^+/+ mice. HPLC, high-performance liquid chromatography; KI, knock-in; miR, microRNA; SREBP, sterol-regulatory element-binding protein. (Cited and modified from references 27 and 30).

Effect of miR-33 Inhibition on Atherosclerosis

In mammals, somatic cells do not catabolize cholesterol; thus, the removal of excess cellular cholesterol by HDL-C is central to the maintenance of sterol homeostasis. A major component of the atheroprotective function of HDL-C is the removal of cholesterol from lipid-loaded macrophages in the vessel wall, and its delivery to the liver for excretion, thereby playing a key role in RCT. Anti-miR-33 therapy contributes to enhancing of this pathway not only by increasing HDL-C through ABCA1 upregulation but also by increasing bile secretion through upregulation of ABCB11 and ATP8B1, which are the other targets of miR-33s.⁴⁰ It has already been proven that antisense inhibition of miR-33a results in regression of atherosclerotic plaque in LDLR-deficient mice by promoting RCT.⁴¹ Moreover, miR-33a-deficiency reduced the progression of atherosclerosis in apoE-deficient mice (Figure 4).²⁸ In that study, miR-33a-deficient mice not only had higher and functional HDL-C but also macrophages, which have a higher cholesterol efflux capacity, resulting in lower lipid accumulation in atherosclerotic areas, proven by bone marrow transplantation experiments. Other possible beneficial properties of anti-miR-33a therapy include an antiinflammatory response via upregulation of ABCA1. ABCA1 modulates cell-surface cholesterol levels, inhibits its partitioning into lipid rafts, and decreases the responsiveness of inflammatory signals from innate immune receptors. Furthermore, ABCA1 has been reported to directly act as an antiinflammatory receptor, independent of its lipid transport activities.⁴²

Figure 4.

miR-33a-deficiency reduced atherosclerosis. (A) Representative images of the en face analysis of the total aorta in miR-33a^+/+ Apoe^−/− and miR-33a^−/− Apoe^−/− male mice. (B) Quantification of the atherosclerotic lesion area in the en face analysis of the total aorta in male mice. Values are mean±SE (n=15–16 each). ***P<0.001. ApoE, apolipoprotein E; miR, microRNA; SE, standard error. (Cited and modified from reference 28.)

Different results have been recently reported by 2 groups regarding the effect of antisense inhibition of miR-33a on atherosclerosis progression in LDLR-deficient mice. Marquart et al reported that anti-miR-33a therapy did not alter the progression of atherosclerosis in LDLR-deficient mice,⁴³ whereas Rotllan et al reported that silencing of miR-33a inhibited the progression of atherosclerosis in LDLR-deficient mice.⁴⁴ There are some discrepancies between these studies, which include differences in the antisense technology used and a considerable difference in the animals’ diet (1.25% vs. 0.15% cholesterol).

Obesity and Hepatic Steatosis in miR-33a-Deficient Mice

Although inhibition of the miR-33 s shows several beneficial effects, such as raising HDL-C and preventing atherosclerosis, we noticed that obesity and hepatic steatosis in the miR-33a-deficient mice at the age of 50 weeks or when fed a high-fat diet (HFD) for 12 weeks (Figure 5). In order to determine the cause of the phenotypic changes observed in miR-33a^−/− mice fed a HFD or in older miR-33a^−/− mice, we analyzed the gene expression profiles by microarray analysis using the livers of miR-33a^+/+ and miR-33a^−/− mice fed NC at the age of 16 weeks when their weights were the same. Most strikingly, the genes classified in the fatty acid metabolism pathway showed the greatest change. We validated their expression levels in the liver by quantitative RT-PCR. Significant differences were observed in the expression levels of several lipogenic genes, including Srebf1, Pparg, and their downstream genes. We also measured de novo hepatic fatty acid synthesis rates, and they had significantly increased in the miR-33a^−/− mice compared with those in the miR-33a^+/+ mice. Srebf1 proved to be a good target of miR-33a, and miR-33a^−/− mice had enhanced expression of SREBP-1 in the liver. To elucidate the role of SREBP-1 in the phenotypic changes in miR-33^−/− mice fed a HFD, we generated miR-33a^−/− mice that had SREBP-1 expression levels similar to wild-type mice. Protein levels of SREBP-1 were the same in miR-33a^−/− Srebf1^+/− and miR-33a^+/+ Srebf1^+/+ mice. Generation of miR-33a^−/− Srebf1^+/− mice clearly showed that enhanced expression of SREBP-1 caused fatty liver and obesity in miR-33a^−/− mice.

Figure 5.

miR-33a^−/− mice become obese and develop hepatic steatosis. (A) Representative image of miR-33a^+/+ and miR-33a^−/− mice that were fed a high-fat diet (HFD). Lower images show the livers of the mice. Scale bar=1.0 cm. (B) Representative CT images of miR-33a^+/+ and miR-33a^−/− mice fed a HFD. Pink color indicates abdominal fat in 2 different slices of each mice. (C) Representative microscopic images of the livers of miR-33a^+/+ and miR-33a^−/− mice fed or not fed a HFD. Fatty change is shown by the increase in large fat vacuoles, which are optically “empty”. Scale bars=200 μm. (D) Representative microscopic images of the adipose tissue (epididymal fat) of miR-33a^+/+ and miR-33a^−/− mice fed or not fed a HFD. Scale bars=200 μm. miR, microRNA. (Cited and modified from reference 29.)

These results indicate a previously unrecognized association between SREBP-1 and SREBP-2 through miR-33a (Figure 6). Until now, it has only been shown that interactions between SREBP-1 and SREBP-2 were mediated by changes in sterol levels. It is known that in cholesterol-rich dietary conditions, SREBP-2 is downregulated at the cleavage level and SREBP-1c is transcriptionally activated through activation of liver X receptors (LXRs) by the binding of oxysterols.^45–48 However, in sterol-depleted conditions, SREBP-2 is cleaved in the Golgi and the active N-terminal region translocates to the nucleus. Reduction in oxysterol levels inactivates LXRs, resulting in a decrease in SREBP-1c mRNA levels. Therefore, suppression of SREBP-1 by miR-33a indicates the existence of a more direct and fine regulatory mechanism between SREBPs.

Figure 6.

Schematic overview of the function of miR-33a in the regulation of SREBP-1 expression levels. Context-dependent change in the role of miR-33a in lipid metabolism. LDLR, low-density lipoprotein receptor; LXR, liver X receptor; miR, microRNA; SREBP, sterol-regulatory element-binding protein. (Cited from reference 29.)

Novel “Thrifty” Models of miR-33a/b

From our findings, we speculate that miR-33a and -33b have evolved to keep cholesterol levels in 2 different modes. At the cellular level, they target ABCA1 and reduce cholesterol efflux to keep intracellular cholesterol levels under cholesterol-depleted conditions. In cholesterol-rich conditions, SREBP-2 and miR-33a levels decrease, resulting in the desuppression of ABCA1.

At the whole body level, miR-33a maintains cholesterol levels at the cost of fatty acid synthesis. Our data showed that miR-33a targeted the 3’ UTR of Srebf1 and upregulation of miR-33a by cholesterol depletion, considerably affecting the reduction of SREBP-1 expression.²⁹ Therefore, based on our finding that miR-33a regulates SREBP-1, miR-33a in the intron of SREBF2 may amplify the reduction in SREBP-1 levels in sterol-depleted conditions (Figure 6). SREBP-1c is known to activate transcription genes involved in fatty acid and triglyceride synthesis, such as genes encoding ACC1, FASN, and ELOVL6 and SCD. Therefore, it is possible that in sterol-depleted conditions, acetyl-CoA is the preferred substrate for cholesterol production, and not for fatty acid production, through upregulation of miR-33a. On the other hand, in cholesterol-rich conditions, the miR-33a level decreases³¹ and its negative regulation of SREBP-1 may be reduced. Thus, in this situation, acetyl-CoA is the preferred substrate for fatty acid production (Figure 6).

We have found that miR-33b targets similar genes to miR-33a.³⁰ It is known that fasting-refeeding causes strong transcriptional activation of SREBF1. Therefore, we speculate that miR-33b is also involved in the maintenance of cholesterol levels at both the cellular and whole body level, even during famine (Figure 7). It is true that activation of miR-33a/b decreases HDL-C levels; however, low HDL-C is not a life-threatening risk at that time most people/animals are dying of infectious diseases. At present, it is the time of “satiation”; thus, miR-33a/b no longer seems to be useful and could potentially be a therapeutic target for lipid disorders and/or atherosclerosis.

Figure 7.

Schematic overview of the function of miR-33a and miR-33b in the regulation of cellular cholesterol levels. miR-33a and miR-33b may act as ‘thrifty’ genes to maintain intracellular cholesterol levels. HDL-C, high-density lipoprotein cholesterol; miR, microRNA; SREBP, sterol-regulatory element-binding protein.

Future Direction of miR-Based Therapeutic Strategies

miRNAs are important regulators of physiological and pathological states. Recent studies provide considerable evidence about the effect of miR-33 s on lipid metabolism, particularly in the preclinical stage.⁴⁹ Because one miRNA can have many targets and affect the expression levels of many genes, further investigations are required to understand the complexity of miRNA biology. In the case of the miR-33s, complete inhibition leads to the development of obesity and liver steatosis;²⁹ therefore, spatiotemporal regulation may be required.

Conclusions

miR-33a/b are involved in lipid homoeostasis by targeting ABCA1, SREBF1, etc. and as such, appear to have acted as “thrifty genes” during evolution to maintain cholesterol levels at both the cellular and whole body level. Fine regulation of miR-33a/b could be a promising new approach to preventing or treating cardiovascular diseases in the future.

Acknowledgments

This work was supported in part by grants from the Japan Society for the Promotion of Science; by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. Kimura, T. Kita, K.H., T.H., and K.O.); by a Grant-in-Aid for Scientific Research on Innovative Areas “Crosstalk between transcriptional control and energy pathways, mediated by hub metabolites” (3307) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.O.); by Otsuka Pharmaceutical Co, Ltd. Kyoto University SRP Program (to T. Kimura, T. Kita, M.Y., T.H., and K.O.); by grants from Banyu Life Science Foundation International, Takeda Memorial Foundation, Suzuken Memorial Foundation, Sakakibara Memorial Foundation, Japan Foundation of Applied Enzymology, SENSHIN Medical Research Foundation, Kowa Life Science Foundation, and ONO Medical Research Foundation (to T.H.); and by grants from ONO Medical Research Foundation, the Cell Science Research Foundation, Daiichi-Sankyo Foundation of Life Science, and Takeda Memorial Foundation (to K.O.).

Disclosures

Conflict of Interest: All authors report none.

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