Loss of hepatic miR-33 improves metabolic and liver function without altering body weight or atherosclerosis

Nathan L. Pricea,b,1, Xinbo Zhanga,b,1, Pablo Fernández-Tussya,b,1, Abhishek K. Singha,b, Sean A. Burnapc, Noemi Rotllana,b, Leigh Goedekea,b, Jonathan Suna,b, Alberto Canfrán-Duquea,b, Binod Aryala,b, Manuel Mayrc, Yajaira Suáreza,b,d, and Carlos Fernández-Hernandoa,b,d,2

aVascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT 06520; bIntegrative Cell Signaling and Neurobiology of Metabolism Program, Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520; cKing’s College London British Heart Foundation Centre, School of Cardiovascular Medicine and Sciences, King’s College London, WC2R 2LS London, United Kingdom; and dDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06520

Edited by Rudolf Zechner, University of Graz, Graz, Austria, and approved December 20, 2020 (received for review April 6, 2020) miR-33 is an intronic microRNA within the encoding the miR-33 is an intronic miRNA encoded within the Sterol reg- SREBP2 transcription factor. Like its host gene, miR-33 has been ulatory element-binding protein 2 (Srebf2) gene (2–4). The shown to be an important regulator of lipid metabolism. Inhibition protein encoded by this gene, SREBP2, is the primary tran- of miR-33 has been shown to promote cholesterol efflux in mac- scription factor responsible for regulating cellular cholesterol rophages by targeting the cholesterol transporter ABCA1, thus re- levels by promoting cholesterol uptake and synthesis in response ducing atherosclerotic plaque burden. Inhibition of miR-33 has also to low intracellular sterol levels. miR-33 is transcribed along with been shown to improve high-density lipoprotein (HDL) biogenesis its host gene, and has also been found to be an important reg- in the liver and increase circulating HDL-C levels in both rodents ulator of cholesterol metabolism, by targeting ATP Binding and nonhuman primates. However, evaluating the extent to Cassette Subfamily A Member 1 (Abca1), a cholesterol trans- which these changes in HDL metabolism contribute to atherogen- porter critical for HDL-C biogenesis. miR-33 has also been esis has been hindered by the obesity and metabolic dysfunction

shown to target numerous other mRNAs, including key factors in PHYSIOLOGY observed in whole-body miR-33–knockout mice. To determine the other functions related to atherogenesis. In , which impact of hepatic miR-33 deficiency on obesity, metabolic function, make up the bulk of atherosclerotic plaques, miR-33 has been and atherosclerosis, we have generated a conditional knockout shown to regulate a number of important functions related to mouse model that lacks miR-33 only in the liver. Characterization plaque development, including fatty acid oxidation (FAO), mi- of this model demonstrates that loss of miR-33 in the liver does not tochondrial function, polarization, efferocytosis, and cholesterol lead to increased body weight or adiposity. Hepatic miR-33 defi- efflux (2–9). Cholesterol efflux is the first step in the RCT ciency actually improves regulation of glucose homeostasis and im- pathway, the process by which macrophages are able to remove pedes the development of fibrosis and . We further cholesterol from the plaque for transport to the liver via circu- demonstrate that hepatic miR-33 deficiency increases circulating lating HDL-C. In addition to this, within the liver, miR-33 has HDL-C levels and reverse cholesterol transport capacity in mice fed been shown to further regulate RCT by targeting factors involved a chow diet, but these changes are not sufficient to reduce athero- sclerotic plaque size under hyperlipidemic conditions. By elucidating Significance the role of miR-33 in the liver and the impact of hepatic miR-33 deficiency on obesity and atherosclerosis, this work will help inform ongoing efforts to develop novel targeted therapies against Prior work has suggested that the ability of miR-33, a micro- cardiometabolic diseases. RNA involved in regulation of lipid metabolism, to regulate liver function underlies its effects on obesity and/or athero- sclerosis. In this work, we selectively remove miR-33 from the miRNA | metabolism | atherosclerosis | obesity | fibrosis liver and demonstrate that, unlike mice globally deficient for miR-33, mice lacking miR-33 in the liver are not predisposed to ncreased circulating levels of low-density lipoprotein choles- diet-induced obesity and are actually protected from hepatic Iterol (LDL-C) is the primary risk factor for the development of insulin resistance and fibrosis. While loss of liver miR-33 in- atherosclerotic plaques. While the use of statins to lower LDL-C creases circulating high-density lipoprotein and increases re- levels has proven an effective treatment for patients at risk for verse cholesterol transport in mice on a chow diet, it is not developing cardiovascular disease (CVD), heart disease remains sufficient to decrease atherosclerotic plaque burden under the leading cause of death in developed countries. More re- hyperlipidemic conditions where hepatic miR-33 is already re- cently, researchers have sought to develop treatments able to pressed. This information will help improve efforts to develop regulate other factors related to atherosclerosis, including re- novel therapies against cardiometabolic diseases. verse cholesterol transport (RCT), inflammation, and plaque stability. Among the novel approaches to treat CVD, micro- Author contributions: N.L.P., X.Z., P.F.-T., Y.S., and C.F.-H. designed research; N.L.P., X.Z., RNAs (miRNAs) have shown a great deal of potential due to P.F.-T., A.K.S., S.A.B., N.R., L.G., J.S., A.C.-D., B.A., and M.M. performed research; N.L.P., their ability to target many different mRNAs involved in pro- X.Z., P.F.-T., M.M., Y.S., and C.F.-H. analyzed data; and N.L.P. and C.F.-H. wrote the paper. cesses related to plaque development. For example, inhibition of The authors declare no competing interest. miR-148 has been shown to both decrease circulating LDL-C This article is a PNAS Direct Submission. levels and increase circulating high-density lipoprotein choles- Published under the PNAS license. terol (HDL-C) levels through targeting of multiple different 1N.L.P., X.Z., and P.F.-T. contributed equally to this work. mRNAs (1). However, the promiscuous nature of miRNAs also 2To whom correspondence may be addressed. Email: [email protected]. increases the potential for unintended effects, necessitating an This article contains supporting information online at https://www.pnas.org/lookup/suppl/ in-depth exploration of the impact of altering miRNA expres- doi:10.1073/pnas.2006478118/-/DCSupplemental. sion/activity in different organs and tissues. Published January 25, 2021.

PNAS 2021 Vol. 118 No. 5 e2006478118 https://doi.org/10.1073/pnas.2006478118 | 1of12 Downloaded by guest on September 29, 2021 in HDL-C biogenesis (Abca1) and the final step of the RCT mice was very low, residual expression that was observed is likely process, bile acid secretion and synthesis, which facilitates re- due to expression of miR-33 in other less abundant cell types moval of cholesterol from the body (10, 11). found within the liver. Kupffer cells were found to express an These findings demonstrated that miR-33 is in a unique position appreciable amount of miR-33, but this was lower than the ex- to regulate many different factors related to atherosclerotic plaque pression in primary hepatocytes (SI Appendix, Fig. S1A). Dele- formation and could provide an important therapeutic target for tion of miR-33 in hepatocytes did not alter hepatic Srebf2 mRNA the treatment of CVD. Consistent with this, initial work with both levels or SREBP2-regulated (SI Appendix, Fig. rodents and nonhuman primates was able to show that inhibition S1B). Consistent with the known role of miR-33 in suppressing of miR-33 could increase circulating HDL levels (2–4, 12–14). the posttranscriptional expression of ABCA1 and ABCG1, LKO Moreover, miR-33 inhibition or genetic ablation was shown to mice expressed higher levels of both transporters in the liver reduce atherosclerotic plaque size in mouse models of athero- compared to WT mice (Fig. 1C). As expected by the derepres- sclerosis (15–17). However, long-term treatment with miR-33 in- sion of ABCA1, we also observed an increase in circulating levels hibitors was found to increase circulating triglycerides (TAGs) and of total cholesterol and HDL-C in both male and female LKO promote hepatic steatosis (18, 19). Moreover, genetic ablation of mice (Fig. 1 D and E), while circulating TAG levels were not miR-33 resulted in increased susceptibility to obesity and meta- altered (Fig. 1F). FPLC fractionation further demonstrates an bolic dysfunction (20, 21). We have demonstrated that trans- increase in the HDL peak of LKO mice (Fig. 1G). plantation of bone marrow from miR-33–deficient animals into We next sought to directly assess whether these changes were − − the Ldl receptor-knockout (Ldlr / ) mouse model of atheroscle- sufficient to improve RCT capacity in vivo. To accomplish this, rosis was sufficient to promote RCT and reduce we isolated peritoneal macrophages from wild-type (WT) mice plaque burden. Moreover, specific disruption of the interaction and preloaded them with radioactively labeled cholesterol. between miR-33 and Abca1 largely mimicked the effects of miR- These cells were then injected intraperitoneally (IP) into LKO 33 deficiency on atherosclerosis without impacting body weight or and control mice. By measuring the amount of radioactively la- metabolic function. beled cholesterol present in the plasma and feces of these mice, While these findings demonstrate the importance of macro- we were able to show that LKO mice have an increased ability to phage miR-33 for promoting plaque formation, prior work has take up excess cholesterol into the plasma from peripheral cells been unable to address how the regulation of HDL biogenesis and remove it from the body through the fecal bile acids and bile acid metabolism by miR-33 in the liver impacts ath- (Fig. 1H). In an attempt to determine protein factors that could erosclerosis due to the metabolic alterations observed in whole- be related to the increase in circulating HDL-C and RCT ob- body knockout mice. Additionally, it is not clear whether loss of served above in LKO animals, a quantitative mass spectrometry miR-33 in the liver may contribute to these metabolic pheno- (MS)-based proteomic approach was taken. Plasma from WT types. In this work, we have developed a conditional miR-33 and LKO mice was analyzed to quantify global plasma proteome knockout model and used this to selectively remove miR-33 from alterations. Of interest, lecithin:cholesterol acyltransferase (LCAT), the liver (LKO). Through characterization of this model, we an enzyme responsible for the esterification and incorporation of demonstrate that liver-specific loss of miR-33 increases circu- cholesterol within HDL (23), was significantly enriched within the lating HDL-C and improves in vivo RCT. Moreover, we dem- plasma of LKO mice (Fig. 1I). The higher levels of circulating onstrate that hepatic miR-33 deficiency does not result in the LCAT observed in LKO mice was likely due to increased plasma predisposition to diet-induced obesity and metabolic dysfunction HDL-C observed in these animals, since the 3′UTR of Lcat does observed in whole-body knockout animals. Conversely, we find not have a predicted binding site for miR-33 (SI Appendix,Fig.S2A) that, after long-term high-fat diet (HFD) feeding, LKO mice and the hepatic LCAT mRNA and protein expression was similar in have an improved ability to regulate metabolic homeostasis and both groups of mice (SI Appendix,Fig.S1B and C). Together, these reduced expression of factors related to hepatic fibrosis and in- findings demonstrate that LKO mice have an improved circulating flammation. Our data further indicate that LKO mice are pro- lipid profile, which may contribute to the reduced atherosclerotic tected from CCl4-induced liver fibrosis, which may be in part plaque size observed in studies using miR-33 inhibitors. mediated by up-regulation of Ski, a negative regulator of TGFβ signaling (22). However, despite the beneficial alterations ob- Hepatic miR-33 Deficiency Does Not Promote Obesity, and Actually served in LKO mice, we did not observe any differences in ath- Improves Metabolic Function after HFD Feeding. As global miR- erosclerotic plaque size. These findings support the conclusion 33–knockout mice are predisposed to the development of obesity that the proatherogenic effects of miR-33 are primarily due to and metabolic dysfunction, we sought to determine the extent to direct effects on macrophages within the atherosclerotic plaque. which hepatic miR-33 contributes to this phenotype. However, This work also indicates that adverse metabolic effects observed we did not observe any differences in body weight (Fig. 2A)or in global miR-33–knockout mice are unlikely to be due to loss of body composition (Fig. 2B) in male or female LKO mice after miR-33 in the liver, which aids in the development of new ap- HFD feeding. Moreover, we observed that insulin sensitivity is proaches for treating atherosclerosis and obesity. actually improved in LKO mice, as the phosphorylation of AKT (pAKTs473) and the ratio of pAKTs473/total were increased in the Results livers of these mice following injection of insulin (Fig. 3A). Interest- Loss of Hepatic miR-33 Increases Circulating HDL-C and RCT Capacity. ingly, we also observed an increase in pAKTs473 and pAKTs473/total To determine the specific impact of hepatic miR-33 on obesity, in the skeletal muscle of LKO mice (SI Appendix,Fig.S3A), metabolic function, and atherogenesis, we generated a condi- suggesting that the improved metabolic regulation by the liver in tional miR-33 knockout model (miR-33loxP/loxP). In these mice, these mice could lead to a secondary improvement in insulin the intronic region of the Srebf2 gene encoding miR-33 is flanked sensitivity in other tissues. However, this response is not global, as by loxP sites, so Cre recombinase activity leads to the excision of visceral white adipose tissue did not show a similar response (SI this region, causing miR-33 deficiency (Fig. 1 A, Left). By Appendix, Fig. S3B). Consistent with this, we find that male LKO crossing these mice to well-established Albumin-Cre strain, we mice have an improved ability to regulate circulating blood glu- have generated liver-specific miR-33–knockout mice (LKO). cose levels (Fig. 3B), and female LKO mice show a very similar Excision of the floxed region could be visualized by PCR am- trend that does not quite reach statistical significance (SI Appen- plification followed by agarose gel electrophoresis (Fig. 1 A, dix,Fig.S4A). This is in stark contrast to global miR-33–deficient Right), and loss of miR-33 in the liver was confirmed by qPCR animals that show a profound impairment in regulation of glucose analysis (Fig. 1B). While the amount of miR-33 detected in LKO homeostasis after HFD feeding (20, 21).

2of12 | PNAS Price et al. https://doi.org/10.1073/pnas.2006478118 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis Downloaded by guest on September 29, 2021 ABMale Female 2.0 * 2.0 * miR-33lox/ox miR-33lox/ox (WT) AlbCRE 1.5 1.5 (LKO) miR-33 1.0 1.0 excision product 0.5 0.5

0.0 0.0

miR-33 (relative expression) WT LKO miR-33 (relative expression) WT LKO

CEWT LKO D F Male Female Male Female Male Female ABCA1 NS 200 * 200 **80 * 80 150 NS ABCG1 200 Vinculin 150 150 60 * 60 100 150 1.8 * 2.0 * 100 1.5 1.4 100 100 40 40 50 1.0 TAG (mg/dL) TAG TAG (mg/dL) TAG HDL-C (mg/dL)

HDL-C (mg/dL) 50 1.0 0.5 Total cholesterol (mg/dL) Total protein levels) protein levels) 0.0 cholesterol (mg/dL) Total 50 50 20

ABCA1 (Relative 20 0 0.6 ABCG1 (Relative 0

WT WT WT WT WT WT WT LKO LKO WT LKO LKO LKO LKO LKO LKO Plasma G 60 WT 40 WT HIFecal

HDL ) LKO 3 20 WT LKO LKO HDL 30 * ) * 3

30 0.01 PHYSIOLOGY 40 Male Female 15 LCAT 20 IGFALS 20 SOD1 10 0.1 20 p-Value IDL/LDL 10 10 5 IDL/LDL Cholesterol (mg/dL) Cholesterol VLDL (mg/dL) Cholesterol VLDL H]-Bile acids (cpm X10 H]-Cholesterol (cpm X10 3 3 0 0 0 [ 0 [ 1 24 28 32 36 40 44 48 52 56 60 64 68 24 28 32 36 40 44 48 52 56 60 64 68 -2 -1 0 1 2 WT WT LKO Fraction number Fraction number LKO Log 2 Fold Change (LKO / WT) Reverse cholesterol transport

Fig. 1. Loss of miR-33 in the liver increases circulating HDL levels and improves RCT capacity. (A) Schematic diagram depicting the generation of conditional miR-33–knockout mice. The construct is composed of a short flippase recombination enzyme (Flp)-recognition target (FRT), reporter, and a Cre recombinase recognition target (loxP). The first loxP site is followed by the miR-33 coding region, then the first FRT site, the neomycin selection cassette driving the neomycin resistance gene, a second FRT site, and the second loxP site (Top). Mice with the floxed allele but lacking the neo cassette were generated by crossing with flp recombinase-deleter mice (Middle). Subsequently, these mice were bred with mice expressing Cre recombinase to produce tissue-specific miR-33–knockout mice (Bottom). PCR amplification of the excised genomic region containing miR-33 in the liver of LKO mice (Right). (B) qPCR analysis of miR- 33 expression in the liver of miR-33loxP/loxP (WT) and miR-33loxP/loxP/AlbuminCre (LKO) animals (n = 3). (C) Representative Western blots and densitometric analysis of ABCA1, ABCG1, and housekeeping standard Vinculin in liver lysates from male WT and LKO mice. (D–F) Levels of total cholesterol (D), HDL-C (E), and TAGs (F) in plasma of WT and LKO mice (male, n = 15 to 16; female, n = 7 to 8). (G) Cholesterol content of FPLC-fractionated lipoproteins from pooled plasma of n = 5to8WT and LKO mice. (H) The [3H]-cholesterol in plasma (Left) and in fecal bile acids (Right) from male WT and LKO mice injected IP with [3H]- cholesterol–labeled BMDMs (n = 9 to 10). (I) Plasma proteome analysis from male WT and LKO mice. The graph indicates the significantly increased proteins found in WT and LKO plasma (n = 5). Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals).

To better understand the metabolic changes that occur in LKO revealed a trend toward improved regulation of glucose ho- mice, a pyruvate tolerance test (PTT) was performed to assess meostasis, but this was not found to be statistically significant hepatic gluconeogenesis, but this did not reveal any differences (Fig. 3E). Importantly, circulating insulin levels were found to be between LKO and control animals (SI Appendix, Fig. S4B). reduced in chow diet-fed LKO mice, which likely contributed to Additionally, despite the improved induction of pAKT that we the less substantial response to glucose administration (Fig. 3F). observe, no differences were found in the ability of LKO mice to Insulin levels were increased after 3 mo of HFD feeding in male modulate blood glucose levels in response to a bolus of insulin mice, and the difference between WT and LKO mice was no [i.e., insulin tolerance test (ITT); Fig. 3C]. To determine whether longer observed (Fig. 3G). In female mice, the increase in insulin the metabolic differences between LKO and control mice were levels in response to HFD was less pronounced and tended to be diminished due to the severe insulin resistance that develops even lower in LKO mice compared to controls. This may in part after prolonged HFD feeding (3 mo), we performed additional account for the more moderate effects observed in regulation of characterization of metabolic function in mice fed a chow diet glucose homeostasis (SI Appendix, Fig. S4C). and after a shorter period (1 mo) of HFD feeding. Chow-fed Metabolic characterization of mice fed an HFD for only 1 mo mice were more responsive to insulin during an ITT (Fig. 3D). also shows that LKO animals have improved regulation of glu- Glucose tolerance tests (GTTs) in chow-fed animals also cose homeostasis during a GTT (Fig. 3H). Additionally, although

Price et al. PNAS | 3of12 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering https://doi.org/10.1073/pnas.2006478118 body weight or atherosclerosis Downloaded by guest on September 29, 2021 LKO A Male B WT demonstrate that neither the macrophages nor adipose tissue 60 WT Male contribute substantially to the obesity and metabolic dysfunc- LKO 30 tion observed in mice lacking miR-33 globally. To assess what factors contribute to the improved metabolic 40 20 function we observe in LKO mice, we performed RNA se- quencing (RNA-seq) analysis in hepatic tissue from LKO and control animals on chow or HFD. Principal component analysis

20 Mass (g) 10 Body Weight (g) Body Weight of this dataset reveals clear separation based on genotype (Fig. 4A). In HFD-fed animals, there were 735 whose 0 0 expression was significantly altered in LKO mice, among which 1 8 Fat Lean 15 22 29 36 43 50 57 64 71 78 85 92 99 106 mass mass 278 genes were up-regulated and 457 down-regulated (Fig. 4B). Days on HFD We then used Ingenuity Pathway Analysis to assess the relative WT LKO 50 Female functions of these dysregulated genes and were able to identify a WT Female number of canonical pathways that were overrepresented in this LKO 30 40 dataset (Fig. 4C). The top pathway in this analysis was the he- 30 patic fibrosis and stellate cell activation pathway, which included 20 numerous genes involved in promoting hepatic fibrosis that 20 showed reduced expression in LKO mice compared to controls

Mass (g) 10 (Fig. 4D). Further analysis revealed inflammatory response as Body Weight (g) Body Weight 10 one of the top diseases and functions associated with loss of miR- 0 0 33 in the liver of HFD-fed mice, which was predicted to be de- 1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 Fat Lean creased (SI Appendix, Table S1). Consistent with this, leukocyte 106 Days on HFD mass mass extravasation (Fig. 4E) and other pathways related to inflam- mation (Fig. 4C) were also down-regulated in the liver of these Fig. 2. Hepatic miR-33 deficiency does not predispose mice to diet-induced loxP/loxP loxP/loxP animals. Upstream regulator analysis identified decreased ac- obesity. (A) Body weight trajectories in miR-33 (WT)andmiR-33 / β Cre = = tivity of a number of (TNF, IL1 ), as well as the pro- Albumin (LKO) mice fed an HFD (male, n 14 to 19; female, n 21 to 24). β (B) Body composition analysis of fat mass and lean mass in HFD-fed WT and fibrotic factor TGF 1. These key factors are responsible for LKO mice (male, n = 10 to 13; female, n = 20 to 21). Data represent the regulation of numerous other upstream regulators, and therefore mean ± SEM. likely to be responsible for many of the transcriptional changes observed (SI Appendix, Fig. S7). As expression of miR-33 in stellate cells was not found to be substantially impacted by Al- the differences are less pronounced than those observed in chow bumin-Cre–specific excision (SI Appendix, Fig. S1 A and B), it is diet-fed mice, regulation of glucose levels in response to insulin likely that the changes in genes related to stellate cell activation was also significantly improved (Fig. 3I). While the metabolic and inflammation were secondary to alterations in hepatocyte phenotype in these animals is not dramatic, our original results function, which have been shown to have an important impact on and the additional data we have provided support the conclusion stellate cell activation and fibrosis (25). As hepatic fibrosis and that LKO mice have improved insulin sensitivity. In relatively inflammation can promote chronic liver disease and impair liver healthy control mice fed a chow diet, this difference is largely function, these changes may directly contribute to the improved offset by increasing circulating insulin levels, but HFD feeding metabolic function we observe in these animals. leads to an impaired ability to regulate glucose homeostasis. As such, the improved insulin sensitivity of LKO mice results in a Hepatic miR-33 Deficiency Protects against Acute and HFD-Induced significant improvement in regulation of glucose homeostasis Liver Fibrosis. To directly evaluate whether miR-33 deficiency after HFD feeding. can protect against HFD-induced liver fibrosis, we performed To examine other potential explanations for the obesity phe- picrosirius red staining in histologic liver sections. This analysis notype associated with global loss of miR-33, we also assessed demonstrated that collagen accumulation was significantly re- the impact of adipocyte- and macrophage-specific miR-33 defi- duced in the livers of LKO mice compared to controls after HFD ciency. Our characterization of miR-33loxP/loxP mice crossed tob feeding (Fig. 5A). Consistent with this, Western blot analysis Adiponectin-Cre animals (ATKO) did not reveal any differ- revealed a significant reduction in the protein levels of markers ences in body weight or body composition in male or female of fibrosis (αSMA) and inflammation (pJNK; Fig. 5B). F4/80 mice fed chow or HFD (SI Appendix,Fig.S5A–C). ATKO mice staining also revealed a decrease in macrophage accumulation also did not show the differences in fat tolerance tests or GTTs within the livers of LKO mice (Fig. 5C), and circulating levels of that were observed in whole-body miR-33–knockout mice (SI alanine aminotransferase (ALT), a common biomarker of liver Appendix,Fig.S5D–F), and no differences were observed in damage, were reduced (Fig. 5D). The reduction of hepatic circulating HDL-C levels under either chow- or HFD-fed macrophage content in LKO mice was independent of circulating conditions (SI Appendix,Fig.S5G). Additionally, miR-33loxP/loxP leukocyte distribution, which was similar in WT and LKO mice mice were crossed with LysM-Cre animals to generate mice fed an HFD for 1 mo (SI Appendix, Fig. S9). To further assess deficient for miR-33 specifically in macrophages and neutro- the extent to which miR-33 deficiency protects against hepatic phils (MKO). As our prior work has demonstrated that bone fibrosis, we treated LKO and control animals with carbon tet- marrow transplant from miR-33–deficient animals was suffi- rachloride (CCl4), a commonly used model of acute liver fibrosis. cient to reduce atherosclerotic plaque formation, these animals While the extent of fibrosis induced using this model was much were characterized primarily in the context of hyperlipidemia. greater than that observed in our HFD-fed animals, the pro- Similar to what was observed after HFD feeding, whole-body tection elicited from hepatic miR-33 deficiency was similarly miR-33–knockout mice showed a pronounced body-weight and pronounced. Histologic assessment of hepatic sections demon- insulin-resistance phenotype under these conditions (24). MKO strated that LKO mice had a significant reduction in the accu- mice were not found to have any differences in body weight mulation of both necrotic regions (Fig. 5E) and collagen fibers with either chow or Western diet (WD; SI Appendix,Fig.S6A). after CCl4 treatment (Fig. 5F). Western blot analysis also Circulating levels of HDL-C were also not different in these revealed a reduction in αSMA and pJNK, as well as Col3a1, in mice (SI Appendix,Fig.S6B). Together, these findings LKO mice treated with CCl4 compared to controls (Fig. 5G).

4of12 | PNAS Price et al. https://doi.org/10.1073/pnas.2006478118 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis Downloaded by guest on September 29, 2021 Insulin (5 min) A WTLKO WT LKO B C 500 250 pAKT DFH )m3( 50 * DFH )m3( 20 AKT 400 200 Vinculin * * 40 15 8 150 WT LKO 300 * 30 10 6 200 100 4 20 WT 5

WT Glucose (mg/dL) Glucose (mg/dL) 100 50 LKO LKO 2 pAKT/AKT Area under curve (X100) 0 Area under curve (X100) 10 0 0 (fold change) 0 0 0 15 30 60 15 30 60 120 WT 120 WT Untreated Insulin (5 min) Time after injection (min) LKO Time after injection (min) LKO

D 200 CD E 400 CD F * 20 * 50 1.50 1.25 150 300 40 CD 15 1.00 30 100 10 200 0.75 20 0.50 WT WT 50 5 100 Insulin (ng/ml) Glucose (mg/dL) LKO Glucose (mg/dL) LKO 10 0.25 Area under curve (X100) 0 Area under curve (X100) 0 0 0 0.00 0 0 15 30 60 15 30 60 WT LKO 120 120 WT LKO Time after injection (min) WT LKO Time after injection (min)

600 DFH )m1( DFH )m1( G HFD (3m) H 60 I 200 20 5 * * * PHYSIOLOGY * * 4 150 15 400 40 * 3 100 10 2 200 20 WT 50 5 Insulin (ng/ml) WT

1 Glucose (mg/dL) LKO Glucose (mg/dL) LKO

* Area under curve (X100) 0 0 0 Area under curve (X100) 0 0 0 0 WT LKO 15 30 60 15 30 60 120 120 WT Time after injection (min) WT LKO Time after injection (min) LKO

Fig. 3. Loss of miR-33 in the liver improves metabolic function. Characterization of metabolic function in male miR-33loxP/loxP (WT) and miR-33loxP/loxP/ AlbuminCre (LKO) mice fed chow diet or subjected to short-term (1 mo) or long-term (3 mo) HFD feeding. (A) Representative Western blots of total AKT (tAKT), phosphorylated AKT (pAKTs473), and housekeeping standard Vinculin in liver lysates from WT and LKO mice injected with insulin 5 min prior to euthanasia (Top). Densitometric analysis of the ratio of pAKTs473/total (Bottom; basal, n = 2; insulin, n = 5). Data represent the mean ± SEM. GTTs (B) and ITTs (C)inWT and LKO mice fed an HFD for 3 mo with areas under the curve (Right; n = 9 to 13). ITT (D) and GTT (E) in chow diet-fed WT and LKO mice with areas under the curve (Right; GTT, n = 9 to 10; ITT, n = 5). (F) Plasma insulin levels in chow diet-fed WT and LKO mice (n = 7 to 8). (G) Plasma insulin levels in WT and LKO mice fed an HFD for 3 mo (n = 4 to 5). GTT (H) and ITT (I)inWT and LKO mice fed an HFD for 1 mo with areas under the curve (Right;n= 5). Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals).

Finally, in keeping with our findings in HFD-fed mice, the ac- found to be significantly enriched in this dataset (SI Appendix, cumulation of F4/80-positive macrophages in response to CCl4 Fig. S10). In chow diet-fed animals, loss of miR-33 led to sig- treatment was reduced in LKO mice (Fig. 5H). Together, these nificant alterations in the expression of 698 genes, 30 of which findings demonstrate that loss of miR-33 protects against the were identified as strong potential targets of miR-33 based on development of hepatic fibrosis, which may account in part for the TargetScan prediction algorithm (Fig. 6A). Among these the improved metabolic function of LKO mice on HFD. predicted targets, a higher proportion of genes (24 of 30) were up-regulated than would be predicted based on the rest of the Identification of Differentially Expressed miR-33 Target Genes. We dataset (Fig. 6B). Two of these predicted targets, Dusp1 and Tfrc, next used RNA-seq data to assess what known and predicted were identified as likely upstream regulatory factors in this targets of miR-33 may be involved in mediating these effects. As dataset. Two of the top transcriptional regulators in this analysis previous reports have demonstrated that miR-33 is repressed in were Tlr4 and Tgfb1, and these targets, as well as other known the liver along with SREBP2 under conditions where hepatic (Abca1) and predicted (Ski and Hipk2) miR-33 targets, have lipid levels are elevated (4, 26), we assessed changes in predicted been linked to TLR4 and TGFβ signaling. Together, these five miR-33 targets in liver samples from mice under chow diet potential miR-33 targets are predicted to directly regulate a conditions to identify target genes that may be altered in these number of other genes identified as being dysregulated in LKO conditions and contribute to the protection from HFD-induced mice under chow diet-fed conditions, and many of these were fibrosis and metabolic dysfunction that we observe. Principal also identified as important upstream regulators, demonstrating component analysis demonstrated a clear separation between the ability of miR-33 to directly mediate many of the transcrip- control and LKO, and genes from a number of pathways were tional changes observed under chow diet conditions (Fig. 6C).

Price et al. PNAS | 5of12 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering https://doi.org/10.1073/pnas.2006478118 body weight or atherosclerosis Downloaded by guest on September 29, 2021 AC

B

DE

Fig. 4. Liver-specific miR-33 deficiency reduces the expression of genes related to hepatic fibrosis and inflammation. RNA-seq from livers of male miR- 33loxP/loxP (WT) and miR-33loxP/loxP/AlbuminCre (LKO) mice fed an HFD. (A) Principal component analysis of RNA-seq data from WT and LKO mice. (B) Heat map depicting differentially expressed genes between WT and LKO mice. (C) Top 15 canonical pathways overrepresented among differentially expressed genes in RNA-seq analysis of WT and LKO mice. Orange bars indicate pathways in which genes are consistently up-regulated, blue indicates pathways in which genes are consistently down-regulated, and gray indicates pathways in which some genes are significantly altered in both directions. (D and E) Heat maps depicting differentially expressed genes involved in hepatic fibrosis and hepatic stellate cell activation (D) and leukocyte extravasation (E) pathways.

For comparison, under HFD-fed conditions, 28 predicted miR- treatment with CCl4, while expression of the proapoptotic factor 33 targets were found to be significantly altered, but only 11 of Bcl2-associated protein X (Bax), which is induced by fibrosis and these were up-regulated, and, among these, only ABCA1 was TGFβ, was reduced (Fig. 6D). identified as a potential upstream regulator (SI Appendix, Fig. Consistent with the repression of miR-33 and SREBP2 under S11). Of particular relevance, SKI has been demonstrated to be hyperlipidemic conditions (26), most of the miR-33 targets that an important negative regulator of TGFβ signaling. Since TGFβ were identified as being up-regulated in the liver of LKO mice signaling is considered the primary pathway responsible for in- under chow diet conditions were not found to be altered after duction of fibrotic response, impairment in this signaling path- prolonged HFD feeding, although ABCA1 was still elevated way due to derepression of Ski could play a direct role in both at the mRNA and protein level (SI Appendix, Fig. S12). As mediating the antifibrotic response observed in LKO mice. such, many of the changes that we observe after HFD feeding are Consistent with this hypothesis, expression of Ski was also found likely secondary to differences in metabolic regulation and liver to be elevated in LKO mice compared to controls following function. Enhanced FAO has previously been demonstrated to

6of12 | PNAS Price et al. https://doi.org/10.1073/pnas.2006478118 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis Downloaded by guest on September 29, 2021 ABCDSirius Red WT LKO F4/80 SMA * 2.5 * 100 * 0.9 pJNK 0.8 Vinculin 2.0 WT WT 0.7 0.6 1.5 * * 1.5 0.5 50 0.4 1.0 1.0 0.3 ALT (u/L) 0.2 0.5 levels 0.5 0.1 F4/80 (% positive area) LKO LKO 0.0 0

0.0 Relative protein

Sirius Red (% positive area) WT LKO WT LKO 0.0 WT LKO WT LKO WT LKO SMA pJNK E H&E F Sirius Red CCl4 Vehicle CCl4 Vehicle * 17.5 * a) 2.00 15.0 1.50

12.5 WT WT NC 10.0 1.00 7.5 5.0 0.50 2.5 H&E (% necrotic area) LKO LKO 0.0 0.00 WT LKO WT LKO Sirius Red (% positive are WT LKO WT LKO Vehicle CCl4 Vehicle CCl4

GHF4/80 1.00 * CCl4 1.5 * 1.5 Vehicle CCl4 1.5 * * PHYSIOLOGY WT LKO 0.75 SMA

Non-specific 1.0 1.0 1.0 WT 0.50 Col3a1

0.5 0.5 0.5 pJNK 0.25 F4/80 (% positive area) LKO Vinculin (Relative protein levels) SMA 0.00 0 0 pJNK (Relative protein levels) 0 WT LKO Col3a1 (Relative protein levels) WT LKO WT LKO WT LKO WT LKO Vehicle CCl4

Fig. 5. Loss of miR-33 in liver protects against HFD- and CCl4-induced hepatic fibrosis. Characterization of factors related to fibrosis, inflammation, and liver function in male miR-33loxP/loxP (WT) and miR-33loxP/loxP/AlbuminCre (LKO) mice. (A) Representative images and quantification of Sirius red staining in liver sections from WT and LKO mice fed an HFD (n = 9 to 10). (B) Representative Western blots and densitometric analysis of αSMA, pJNK, and housekeeping standard Vinculin in WT and LKO mice fed an HFD (n = 5). (C) Representative images and quantification of F4/80 staining in liver sections from WT and LKO mice fed an HFD (n = 9 to 10). Arrows indicate F4/80-positive cells. (D) Serum ALT levels in WT and LKO mice fed an HFD (n = 8 to 10). (E) Representative

images of H&E staining in liver sections from WT and LKO mice treated with vehicle or CCl4 for 6 wk. (Insets) Increased-magnification views of the presence or lack of necrotic areas, which are also quantified (Right; vehicle, n = 3; CCl4, n = 8). (F) Representative images and quantification of Sirius red staining in liver sections from WT and LKO mice treated with vehicle or CCl4 for 6 wk (vehicle, n = 2; CCl4, n = 8). (G) Representative Western blots and densitometric analysis of αSMA, Col3a1, pJNK, and housekeeping standard Vinculin in WT and LKO mice treated with CCl4 for 6 wk (n = 7 to 8). (H) Representative images and quantification of F4/80 staining in liver sections from WT and LKO mice treated with vehicle or CCl4 for 6 wk (vehicle, n = 3; CCl4, n = 8). Arrows indicate F4/80- positive cells. Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals).

protect against fibrosis (27), and we observed a dramatic increase biogenesis and RCT in the liver impact atherosclerosis. As these in the capacity for FAO in LKO mice on both chow and HFD phenotypes are not observed in our LKO mice, we next sought to (Fig. 6E). miR-33 has been shown to regulate a number of fac- determine whether these animals were protected from the devel- tors involved in FAO in the liver as well as other tissues. Con- opment of atherosclerotic plaques. To do this, we injected LKO sistent with this, we observe increased protein expression of and control mice with an adenovirus encoding a gain-of-function CPT1a in the livers of our LKO mice (Fig. 6F). Despite this, we mutant of the proprotein convertase subtilisin/kexin type 9 do not observe any differences in hepatic TAGs between LKO (PCSK9) enzyme (AAV8-PCSK9). This approach has previously and control animals on HFD, and only a trend toward reduced been demonstrated to cause degradation of the LDL receptor TAGs in chow diet-fed mice (SI Appendix, Fig. S13). Overall, our (LDLR), thereby promoting hypercholesterolemia and athero- findings demonstrate that hepatic miR-33 targets a number of sclerotic plaque formation in mice fed a WD (40% fat Kcal, factors related to the regulation of lipid metabolism, fibrosis, and 1.25% cholesterol). Similar to what we observed in mice without inflammatory response that together lead to improved metabolic hypercholesterolemia, LKO mice in this study did not show any regulation by the liver. differences in body weight even after WD feeding (Fig. 7A). However, following AAV8-PCSK9 injection and WD feeding, Loss of miR-33 in the Liver Is Not Sufficient to Reduce Atherosclerotic we no longer observed any differences in circulating levels of Plaque Size. In our previous work, the obesity and metabolic total cholesterol or HDL-C between control and LKO mice dysfunction observed in whole-body miR-33–knockout mice (Fig. 7 B and C). TAGs were once again unaltered (Fig. 7D). prevented any meaningful assessment of how changes in HDL These effects are consistent with what we previously observed in

Price et al. PNAS | 7of12 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering https://doi.org/10.1073/pnas.2006478118 body weight or atherosclerosis Downloaded by guest on September 29, 2021 A BC

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Fig. 6. RNA-seq in chow diet-fed mice identifies potential targets of miR-33 that are up-regulated in LKO mice and could mediate effects on gene expression and functional changes. RNA-seq from livers of male miR-33loxP/loxP (WT) and miR-33loxP/loxP/AlbuminCre (LKO) mice fed a chow diet. (A) Heat map depicting differentially expressed genes between WT and LKO mice. (B) Venn diagrams depicting the overlap between miR-33 target genes and total up-regulated transcripts. (C) Network depicting known and predicted miR-33 targets that are up-regulated in LKO mice and predicted to contribute to changes in other

dysregulated genes. (D) qPCR analysis of mRNA expression of Bax, Tgfb1, and Ski in WT and LKO mice treated with CCl4 for 6 wk (n = 8). (E) Analysis of FAO in the livers of male WT and LKO mice on a chow diet (Left) or HFD (Right; n = 5 to 7). (F) Representative Western blots and densitometric analysis of CPT1a, CROT, and housekeeping standard Vinculin in male WT and LKO mice fed an HFD. Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals).

miR-33–knockout mice in an Ldlr-knockout background and are did not show any protection from the formation of atherosclerotic likely due to the fact that transcription of Srepf2, and therefore plaques. miR-33, is repressed under hyperlipidemic conditions (4, 26). Similarly, we did not observe any differences in total cholesterol, Discussion HDL-C, or TAGs in LKO mice after HFD feeding (SI Appendix, While miRNAs represent an entirely new level of regulation that Fig. S14). Assessment of circulating leukocytes by Hemavet could provide novel therapeutic approaches for the treatment of (Fig. 7E) and fluorescence-activated cell sorting (FACS; Fig. 7F) human diseases, the promiscuous nature of these miRNAs and did not reveal differences in most immune cells between LKO our limited understanding of the mechanisms by which they mice and control animals, but CD4+ cells were found to be el- function has raised concerns over the potential utility of miRNA- based therapies. miR-33 is an excellent example of both the evated in LKO mice. As LKO mice do not show the pronounced potential utility and pitfalls of this type of approach. Work by obesity and hyperlipidemia phenotype observed in mice deficient numerous groups has demonstrated that loss or inhibition of for miR-33 globally, this model provides an excellent opportunity miR-33 can promote macrophage cholesterol efflux, increase to assess the impact of hepatic miR-33 on atherogenesis. circulating HDL-C, and limit the development of atherosclerotic Analysis of the atherosclerotic plaque formation in LKO and plaques in mouse models. Recent work has also demonstrated control animals also did not demonstrate any significant differ- that loss or inhibition of miR-33 could also impede the devel- ences in the size of plaques within the aortic root (Fig. 7G). Lipid opment of fibrosis in both the heart and kidney (28, 29). How- accumulation, as assessed by Oil Red O staining of the aorta en ever, global loss of miR-33 leads to the development of obesity, − − face (Fig. 7H) and in histological sections of the aortic root dyslipidemia, and insulin resistance (20, 21). In Ldlr / mice, (Fig. 7I), was also unaltered. In vivo foam cell formation was also these detrimental whole-body effects were found to offset the unchanged in LKO mice, consistent with the similar levels of beneficial changes observed in plaque macrophages (24). We circulating lipids (Fig. 7J). Overall, this work demonstrates that have shown that pH low insertion peptides can be used to deliver loss of miR-33 in the liver does not lead to increased body weight miR-33 inhibitors specifically to the kidney and other low-pH and actually improves measures of liver function and whole-body microenvironments (28), and other recent work by our group has metabolic regulation. Despite this, and the increased circulating demonstrated that this approach may also be effective for de- HDL-C and RCT observed in chow-diet conditions, LKO mice livering miR-33 inhibitors to macrophages within the tumor

8of12 | PNAS Price et al. https://doi.org/10.1073/pnas.2006478118 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis Downloaded by guest on September 29, 2021 A B C NS D 50 130 NS WT 1750 NS 500 LKO 1500 110 400 40 1250 90 1000 70 300 750 30 50 200 500 30 TAG (mg/mL) TAG 100 250 HDL-C (mg/mL) Body Weight (mg/dl) Body Weight 10 20 cholesterol (mg/dL) Total 0 0 0 0 14 28 42 58 72 86 WT LKO WT LKO WT LKO 100 E Days on WD 1400 F 40 1000 WT WT LKO LKO 600 30 10 L

µ 8 20

K/ * 6 4 10

2 % of parental cells 0 0 WBC NE LY MO EO BA Platelet Ly6Chi Ly6clow NE BC CD4+ CD8+

NS PHYSIOLOGY G 0.45 H WT LKO 14 NS 0.40 )

2 12 0.35

WT 10 0.30 0.25 8 0.20 6 0.15 4 0.10 Plaque area (mm LKO 0.05 2

0.00 Area % Oil-Red O Positive 0 WT LKO WT LKO NS NS I J Thiglycollate (i.p.) 50 500 3m 4d

WT 40 WD 400

30 300

20 200

10 100 LKO

0 ORO+ area per cell (a.u.) 0 % Oil-Red O Positive Area % Oil-Red O Positive WT LKO WT LKO WT LKO

Fig. 7. Hepatic miR-33 deficiency does not impact atherosclerotic plaque development. Characterization of male miR-33loxP/loxP (WT) and miR-33loxP/loxP/ AlbuminCre (LKO) mice treated with AAV8-PCSK9 and fed a WD for 16 wk. (A) Body weight trajectories of WT and LKO mice fed a WD (n = 12 to 13). (B–D) Levels of total cholesterol (B), HDL-C (C), and TAGs (D) in plasma of WT and LKO mice following AAV8-PCSK9 treatment and WD feeding (n = 15 to 20). (E) Peripheral blood counts from WT and LKO mice following AAV8-PCSK9 treatment and WD feeding using a Hemavet hematology analyzer (n = 6 to 7). (F) Flow cytometry analysis of circulating leukocytes from WT and LKO mice following AAV8-PCSK9 treatment and WD feeding. Data are expressed as per- centages of live cells (n = 5). (G) Representative images (enlarged, Right) and quantification of H&E-stained atherosclerotic plaques within the aortic root of WT and LKO mice following AAV8-PCSK9 treatment and WD feeding (n = 20 to 25). (H) Representative images and quantification of Oil Red O-stained en face preps of WT and LKO mice following AAV8-PCSK9 treatment and WD feeding (n = 10 to 11). (I) Representative images and quantification of Oil Red O-stained atherosclerotic plaques within the aortic root of WT and LKO mice following AAV8-PCSK9 treatment and WD feeding (n = 5 to 6). (J) Representative images and quantification of Oil Red O staining in peritoneal macrophages from WT and LKO mice treated with AAV8-PCSK9 and fed a WD for 12 wk (n = 5). Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals).

Price et al. PNAS | 9of12 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering https://doi.org/10.1073/pnas.2006478118 body weight or atherosclerosis Downloaded by guest on September 29, 2021 microenvironment (30). While this type of approach may prove Further assessment demonstrated that LKO mice were protected very important for limiting unintended consequences of miRNA- against both HFD- and CCl4-induced hepatic fibrosis. Mechanis- based therapies, it will still be necessary to explore in detail the tically, we have identified known targets of miR-33 (ABCA1, specific role of miRNAs in different tissues to properly assess the CPT1) related to lipid metabolism, as well as miR-33 targets more potential benefits and hazards of manipulating these miRNAs in directly involved in fibrotic response (SKI, HIPK2), that may be human patients. involved in mediating these effects. These findings indicate that, To this end, we generated a conditional miR-33 knockout rather than being detrimental to hepatic function, inhibition of mouse model (miR-33loxP/loxP) and used this to selectively elim- miR-33 in the liver may actually have beneficial effects. This inate miR-33 from key metabolic tissues. Our initial efforts have suggests that targeted miR-33 inhibition in the liver could provide focused on the liver, as alterations in hepatic lipid accumulation a viable therapeutic approach both for metabolic disorders asso- and biosynthesis have been suggested to be responsible for some ciated with impaired liver function and more acute cases of liver of the negative effects associated with inhibition or loss of miR- damage. This is important, as miR-33 inhibitors have been shown 33 (19, 20). Our recent work demonstrated that the ability of the to effectively target miR-33 in the liver. Moreover, approaches liver to regulate lipid synthesis in response to changes in nutrient such as triantennary N-acetylgalactosamine–conjugated antisense status was not impacted by miR-33, and that changes in food oligonucleotides have been demonstrated to be effective at tar- consumption were primarily responsible for driving the obesity geted delivery specifically to the liver (31). and metabolic dysfunction observed in these animals (21). We have previously demonstrated that loss of miR-33 in However, as the liver is one of the primary tissues responsible for macrophages and other hematopoietic cells is sufficient to re- sending signals to the brain to coordinate feeding behavior and duce atherosclerotic plaque burden. These effects appear to be other metabolic functions, we crossed our miR-33loxP/loxP mice primarily due to targeting of ABCA1, as selective disruption of to the well-established Albumin-Cre mouse line to generate the interaction between ABCA1 and miR-33 was found to animals that lack miR-33 selectively in the liver. Additionally, we largely mimic these effects. However, the increased feeding and used Adiponectin-Cre and LysM-Cre to selectively remove miR- corresponding predisposition to obesity and metabolic dysfunc- 33 from the adipose tissue and macrophages, respectively. tion of miR-33–deficient mice has prevented any meaningful Initial characterization of these animals clearly demonstrated assessment of whether the enhanced biogenesis of HDL in the that loss of miR-33 in the liver, adipose tissue, and macrophages liver of miR-33–deficient animals also contributes to the bene- was not responsible for promoting the obesity and metabolic ficial effects on delaying atherogenesis. As LKO mice were not dysfunction observed in mice with global miR-33 deficiency. found to have altered body weight or metabolic function, we Indeed, male LKO mice actually had significantly enhanced in- sought to address this question in these animals. Consistent with sulin sensitivity and an improved ability to regulate glucose ho- previous work, we found that loss of miR-33 in the liver resulted meostasis after HFD feeding. Consistent with this, we observe an in increased circulating HDL-C levels and enhanced RCT in increase in FAO in the liver of LKO mice. While the results of young mice on a chow diet. We further reveal, through a pro- our PTT indicate that the capacity for hepatic gluconeogenesis is teomic approach, a potential secondary mechanism whereby similar between LKO and control animals, the response to in- miR-33 can directly regulate HDL function through the regula- sulin, as measured by AKT phosphorylation, is improved in LKO tion of LCAT, which may also contribute to the increased RCT animals. This indicates that the ability to suppress gluconeogenesis capacity of LKO mice. As LCAT levels were not found to be in response to insulin is likely enhanced, which could mediate the altered in the liver, and LCAT in circulation is primarily bound improved metabolic function that is observed. Additionally, we to circulating HDL-C, it is likely that the increased LCAT we also observe enhanced AKT phosphorylation in the skeletal observe in the plasma of LKO animals is a reflection of the in- muscle of LKO animals, demonstrating that the improved hepatic creased HDL-C levels observed in these animals. However, dif- function observed in LKO mice can also improve insulin sensitivity ferences in HDL-C were not observed under hyperlipidemic in other important metabolic organs, consistent with the liver’s conditions. It is therefore not surprising that these mice did not role as a master regulator of lipid and glucose metabolism. These show any protection from atherosclerotic plaque development. peripheral effects may also contribute to some extent to the im- While it is certainly possible that enhanced HDL biogenesis and proved metabolic function that is observed. other effects of miR-33 in the liver could contribute to the re- A similar trend toward improved glucose homeostasis was duced plaque burden observed with inhibition of miR-33, our observed in female LKO mice, although this did not reach statis- findings clearly demonstrate that these changes are not sufficient tical significance. It is possible that hormonal factors or other to drive this effect. differences between the sexes may have contributed to the These findings indicate that the ability of miR-33 to alter somewhat greater variability in females, but we have no direct cholesterol efflux and other functions within the plaque macro- evidence to suggest that this is the case. Alternatively, it is possible phages is the primary mechanism by which miR-33 impacts that the window for observing metabolic alterations in the male atherosclerotic plaque formation. It is important to note that mice was larger. Our data show that the ability to restore blood mice have only one isoform of miR-33, while humans have two glucose levels was impaired to a greater degree by HFD in male isoforms, miR-33a and miR-33b. Like the mouse isoform, miR- mice than in their female counterparts, consistent with previous 33a is encoded within the Srebf2 gene and would be expected to comparisons between sexes, suggesting this may have contributed be repressed under hyperlipidemic conditions. However, miR- to the more significant increase observed in male mice. Similarly, 33b is encoded within the Srebf1 gene, which is regulated in an the levels of circulating insulin after HFD were higher in male independent manner (26). This suggests that, in humans, hepatic mice. Female animals show a trend toward reduced insulin levels miR-33b may play a more important role in regulation of ath- in the LKO mice, similar to what is observed in chow diet-fed erosclerosis than we observe in mice. In the future, it will be animals, which may also contribute to the slightly less substantial important to determine how miR-33 impacts the function of changes in regulation of glucose homeostasis. As our metabolic other key metabolic tissues and how this contributes to the phenotype was more consistent in male animals and our breeding obesity and metabolic dysfunction observed in whole-body miR- strategy provided more male mice for analysis, some of the follow- 33–deficient mice. In future work, it will also be important to up analyses and experiments on fibrosis and atherosclerosis were determine the specific tissues and cell types that are responsible performed only in male mice. for mediating the obesity and feeding phenotypes observed in RNA-seq revealed a reduction in the expression of genes and whole-body knockout animals as well as the mechanisms through pathways related to inflammation and fibrosis after HFD feeding. which miR-33 promotes these effects.

10 of 12 | PNAS Price et al. https://doi.org/10.1073/pnas.2006478118 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis Downloaded by guest on September 29, 2021 Materials and Methods performed following overnight fasting (16 h) by IP injection of 1.5 g/kg pyruvate. Blood glucose measurements were taken 0, 15, 30, 60, and The plasma proteomics, RNA-seq analysis, and the isolation, cellular purifi- 120 min after injection of pyruvate. Plasma insulin levels were measured cation, and statistical methods are described in SI Appendix, Materials and with Mercodia Mouse Insulin ELISA (no. 10-1247-01) according to manufac- Methods. turer instructions. Fat tolerance test was performed as previously described (32). Briefly, mice were fasted for 4 h beginning at 7:00 AM, followed by oral Animals. Generation of conditional miR-33–knockout mice (miR-33loxP/loxP) gavage of 10 μL olive oil per gram body weight. Blood samples were col- was accomplished with the assistance of Cyagen Biosciences. The success of lected from the tail vein 0, 1, 2, 4, and 6 h after administration. this approach has been verified by Southern blotting and confirmed by PCR- based genotyping using specific primers. To generate liver-specific miR-33– Western Blot Analysis. Tissues were homogenized by manual disruption and knockout mice, miR-33loxP/loxP mice were crossed with transgenic mice the Bullet Blender Homogenizer in ice-cold buffer containing 50 mM Tris·HCl, expressing Cre recombinase under the control of tissue-specific promoters: pH 7.5, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 0.1 mM Albumin promoter (JAX stock 003574), AdipoQ promoter (JAX stock EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM 010803), and Lyz2 promoter (JAX stock 004781). For diet-induced obesity orthovanadate, 1 mg/mL protease inhibitor mixture (Roche), and 0.25 experiments, mice were fed a standard chow diet for 8 to 10 wk, after which mg/mL 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Roche). they were either switched to an HFD containing 60% calories from fat Lysates were sonicated and rotated at 4 °C for 1 h before the insoluble (D12492; Research Diets) for 8 to 20 wk or maintained on chow diet. For × atherosclerosis experiments, proprotein convertase subtilisin/kexin type 9 material was removed by centrifugation at 12,000 g for 10 min. After (PCSK9) adenoassociated virus (AAV8-PCSK9) was injected IP (1.0 × 1011 VC) normalizing for equal protein concentration, cell lysates were resuspended to promote the degradation of LDLR and increase circulating cholesterol in SDS sample buffer before separation by SDS polyacrylamide gel electro- levels. Accelerated atherosclerosis was induced by feeding mice for 12 to 16 phoresis. Following transfer of the proteins onto nitrocellulose membranes, wk with a WD containing 1.25% cholesterol (D12108; Research Diets). Body the membranes were probed with the following antibodies: ABCA1 (Abcam – weights were measured throughout HFD or WD feeding studies, and anal- no. 18180; 1:1,000), ABCG1 (Novus no. 400 132; 1:1,000), AKT (Cell Signaling ysis of body composition was performed by Echo MRI (Echo Medical System). no. 4691; 1:500), phosphorylated-AKT(S473) (Cell Signaling no. 9271; 1:500), α Mice used in all experiments were sex- and age-matched and kept in indi- SMA (Sigma A5228; 1:2,000), COLIII (Sigma ab7778; 1:1,000), CROT (Novus vidually ventilated cages in a pathogen-free facility. All of the experiments no. 3144; 1:1,000); CPT1A (Abnova H00001374-P01; 1:1,000), Vinculin (Sigma were approved by the institutional animal care use committee of Yale V9131; 1:2,000), and LCAT (Abcam no. 109417; 1:1,000). Protein bands were University School of Medicine. visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnol- ogy), and densitometry was performed using ImageJ software. RNA Isolation and Real-Time qPCR. Total RNA and miRNAs from liver tissue were isolated using the miRNeasy miRNA Isolation Kit according to the Liver Histology. Mouse livers were perfused with phosphate-buffered saline (PBS) solution and fixed in 4% paraformaldehyde (PFA) overnight. After manufacturer’s instructions. For mRNA expression analysis, cDNA was syn- PHYSIOLOGY thesized using iScript RT Supermix (Bio-Rad) following the manufacturer’s PFA, livers were washed three times in PBS and transferred to 70% EtOH. protocol. Real-time qPCR analysis was performed in duplicate using SsoFast Then, livers were submitted for standard paraffin processing though the EvaGreen Supermix (BioRad) on an iCycler Real-Time Detection System Yale Pathology Tissue Services core. Sections were stained with H&E, Sirius (Eppendorf). The mRNA levels were normalized to 18S. To quantify miR-33 red, and F4/80 to evaluate liver morphology and global injury associated levels, RNAs were reverse-transcribed using the TaqMan MicroRNA Reverse with liver inflammation and fibrosis. For each quantification, pictures from Transcription Kit and quantified with the miRNA-specific TaqMan miRNA eight randomly selected fields were taken with an EVOS microscope. assay (Life Technologies/Invitrogen). The amount of the indicated miRNA Quantification of stained areas was performed with ImageJ software. was normalized to the amount of U6 RNA. CCl4-Induced Liver Fibrosis. CCl4 (Sigma-Aldrich no. 289116; NH2COCH2)was Lipoprotein Profile and Lipid Measurements. Mice were fasted for 12 to 16 h administered by IP injection at a dose of 0.6 mL/kg once per week for a total μ overnight before blood samples were collected by retroorbital venous plexus of 6 wk. CCl4 was diluted in corn oil at a final volume of 50 L. Untreated mice μ puncture, and plasma was separated by centrifugation. HDL-C was isolated received weekly doses of 50 L of corn oil. After 6 wk, mice were euthanized, by precipitation of non–HDL-C, and both HDL-C fractions and total plasma and liver and blood were collected for analysis. For CCl4 administration, age- = = were stored at −80 °C. Total plasma cholesterol and TAGs were enzymati- matched WT (n 8) and LKO (n 8) mice were used. For the control untreated = = cally measured (Wako Pure Chemicals) according to the manufacturer’sin- groups, WT (n 3) and LKO (n 3) were included in the study. structions. The lipid distribution in plasma lipoprotein fractions was assessed by fast-performed liquid chromatography (LC) gel filtration with two ALT Measurements. ALT activity was determined in serum with the ALT Ac- Superose 6 HR 10/30 columns (Pharmacia Biotech). tivity Assay Kit (Sigma-Aldrich MAK052) following the manufacturer’s recommendations. In Vivo Macrophage-Specific RCT. Bone marrow-derived macrophages (BMDMs) from WT mice were differentiated in vitro. After 7 d of differentiation, cells FAO. FAO was assayed as previously described (33). In brief, livers were re- were cultured in 75-cm tissue culture flasks at 5 × 106 cells per flask and grown moved from control and LKO mice and homogenized in five volumes of to 90% confluence in Roswell Park Memorial Institute (RPMI) 1640 medium chilled STE buffer (pH 7.4, 0.25 M sucrose, 10 mM Tris·HCl, and 1 mM EDTA). supplemented with 10% fetal bovine serum. Mouse macrophages were incu- The homogenate was immediately centrifuged, and the pellet was resus- bated for 48 h in the presence of 5 μCi/mL of [1α,2α(n)-3H]-cholesterol pended and incubated with a reaction mixture containing 0.5 mmol/L pal- (GE Healthcare), 100 μg/mL of acetylated LDL, and 10% lipoprotein-depleted mitate (conjugated to 7% BSA/[14C]-palmitate at 0.4 μCi/mL) for 30 min. serum. These cells were washed, equilibrated, detached by gently pipetting, After this incubation period, the resuspended pellet-containing reaction and resuspended in 0.9% (wt/vol) saline and pooled before being injected IP mixture was transferred to an Eppendorf tube, the cap of which housed a into control or LKO mice. Equal plasma counts per minute were injected in Whatman filter paper disk that had been presoaked with 1 mol/L sodium 14 both groups of mice. Mice were then individually housed, and stools were hydroxide. The CO2 trapped in the reaction mixture media was then re- collected over the next 2 d. Plasma counts per minute were determined at 48 h leased by acidification of media using 1 mol/L perchloric acid and gentle by liquid scintillation counting. At that time, mice were euthanized. Fecal lipids agitation of the tubes at 37 °C for 1 h. Radioactivity that had become were extracted with isopropyl alcohol/hexane (2:3; vol/vol). The lipid layer was adsorbed onto the filter disk was then quantified by liquid scintillation collected and evaporated and [3H]-cholesterol radioactivity measured by liquid counting in a β-counter. scintillation counting. The [3H]-tracer detected in fecal bile acids was deter- mined in the remaining aqueous portion of fecal material extracts. Circulating Leukocyte Analysis. Blood was collected by retroorbital puncture in heparinized microhematocrit capillary tubes. Measurement of total circu- Metabolic Characterization. GTTs were performed following overnight fasting lating numbers of blood leukocytes was performed using a Hemavet system. (16 h) by IP injection of glucose at a dose of 2 g/kg for chow diet and 1-mo For further FACS analysis, erythrocytes were lysed with ACK lysis buffer HFD-fed mice or 1 g/kg for 3-mo HFD-fed mice. Blood glucose was measured (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.01 mM at 0, 15, 30, 60, and 120 min post injection. ITTs were performed following a EDTA, pH 7.4). White blood cells were resuspended in 3% fetal bovine serum − 6-h fast by IP injection of 0.75 U/kg insulin. Blood glucose measurements (FBS) in PBS, blocked with 2 μgmL 1 of FcgRII/III, then stained with a mixture were taken 0, 15, 30, 60, and 120 min after injection of insulin. PTTs were of antibodies. Monocytes were identified as CD115hi and subsets as Ly6-Chi

Price et al. PNAS | 11 of 12 Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering https://doi.org/10.1073/pnas.2006478118 body weight or atherosclerosis Downloaded by guest on September 29, 2021 and Ly6-Clo; neutrophils were identified as CD11bhiLy6Ghi; B cells were lesion area was quantified as percent of Oil Red O staining area in total identified as CD19hiB220hi; T cells were identified as CD4hi or CD8hi. The aorta area. following antibodies were used (all from BioLegend): FITC-Ly6-C (HK1.4), PE- CD115 (AFS98), APC- Ly6-G (1A8), PB-CD11b (M1/70), APC-CD19 (6D5), PE/ Cell Culture. For in vivo foam cell formation experiments, mice were fed a WD Cy7-B220 (RA3-6B2), APC/Cy7-CD4 (RM4-5), and BV421-CD8a (53-6.7). All for 12 wk, and cells were harvested by peritoneal lavage 4 d after IP injection antibodies were used at 1:300 dilutions. of thioglycollate (3% wt/vol). Cells were plated in RPMI medium 1640 sup- plemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin. Histology, Immunohistochemistry, and Morphometric Analyses of Atherosclerotic After 2 h, nonadherent cells were removed and remaining cells were stained Plaques. Mouse hearts were perfused with PBS and put in 10 mL 4% para- for 30 min with 0.3% Oil Red O solution in 60% isopropanol. The mean area formaldehyde for 4 h. After incubation in paraformaldehyde, hearts were of Oil Red O-stained region per cell was quantified from six or more fields of washed with PBS, left with PBS for 1 h, and put in 30% sucrose overnight. containing at least 200 total cells using the ImageJ software from the NIH. Finally, hearts were embedded in OCT compound and frozen. Serial sections were cut at 6-μm thickness using a cryostat. Every fourth slide from the serial Data Availability. The genomic data were deposited in the Gene Expression sections was stained with hematoxylin and eosin, and each consecutive slide Omnibus (accession no. GSE164517) (34). All study data are included in the was stained with Oil Red O for quantification of the lesion area and lipid ac- article and SI Appendix. cumulation, respectively. Aortic lesion size of each animal was obtained by averaging the lesion areas in at least nine sections from the same mouse. ACKNOWLEDGMENTS. This work was supported by grants from the NIH (R35HL135820 to C.F.-H.; R01HL105945, R01HL135012 to Y.S.; and 1K01DK120794 to N.L.P.), the American Heart Association (16EIA27550005 to C.F.-H.), Yale Liver En Face Oil Red O Staining. Oil Red O stock solution (35 mL; 0.2% wt/vol in Center (P30 DK34989), and the Programa Postdoctoral de Perfecionamiento de methanol) was mixed with 10 mL of 1 M NaOH and filtered. Aortas opened up Personal del Govierno Vasco (Spain) (to P.F.-T.). M.M. is a British Heart longitudinally were briefly rinsed with 78% methanol, stained with 0.16% Oil Foundation (BHF) Chair Holder (CH/16/3/32406) with BHF programme grant Red O solution for 50 min, and then destained in 78% methanol for 5 min. The support (RG/16/14/32397).

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