Role of miR-181c in Diet-induced obesity through regulation of lipid synthesis in liver.

We recently identified a nuclear-encoded miRNA (miR-181c) in cardiomyocytes that can translocate into mitochondria to regulate mitochondrial gene mt-COX1 and influence obesity-induced cardiac dysfunction through the mitochondrial pathway. Because liver plays a pivotal role during obesity, we hypothesized that miR-181c might contribute to the pathophysiological complications associated with obesity. Therefore, we used miR-181c/d-/- mice to study the role of miR-181c in hepatocyte lipogenesis during diet-induced obesity. The mice were fed a high-fat (HF) diet for 26 weeks, during which indirect calorimetric measurements were made. Quantitative PCR (qPCR) was used to examine the expression of genes involved in lipid synthesis. We found that miR-181c/d-/- mice were not protected against all metabolic consequences of HF exposure. After 26 weeks, the miR-181c/d-/- mice had a significantly higher body fat percentage than did wild-type (WT) mice. Glucose tolerance tests showed hyperinsulinemia and hyperglycemia, indicative of insulin insensitivity in the miR-181c/d-/- mice. miR-181c/d-/- mice fed the HF diet had higher serum and liver triglyceride levels than did WT mice fed the same diet. qPCR data showed that several genes regulated by isocitrate dehydrogenase 1 (IDH1) were more upregulated in miR-181c/d-/- liver than in WT liver. Furthermore, miR-181c delivered in vivo via adeno-associated virus attenuated the lipogenesis by downregulating these same lipid synthesis genes in the liver. In hepatocytes, miR-181c regulates lipid biosynthesis by targeting IDH1. Taken together, the data indicate that overexpression of miR-181c can be beneficial for various lipid metabolism disorders.

Introduction

Lipogenesis during obesity is a major pathological condition, and there are many efforts to mitigate its harmful consequences [1]. Recently, altering microRNA (miRs) expression levels has been postulated as one way to reduce lipid production in the liver [2]. However, more studies are needed to delineate the relevant miRs involved in this process, and their mechanism of regulation.

We have previously studied one particular miR, miR-181c, which regulates mitochondrial gene expression and alters mitochondrial function [3-5]. Overexpression of miR-181c in rats causes oxidative stress, which leads to cardiac dysfunction [3], similar to the reported upregulation of miR-181c in human cardiac dysfunction [6]. Recently, we showed that myocardial lipid accumulation from a high-fat (HF) diet upregulates miR-181c expression in the heart [7]. Additionally, mitochondria activate reactive oxygen species (ROS) production in a type 2 diabetes (T2D) diet-induced obesity (DIO) model, indicating a significant role for mitochondrial dysfunction in glucose homeostasis and insulin resistance [8-10]. As mitochondrial function changes, multiple cellular and physiological functions are affected, contributing to the development of common T2D-related complications.

One miRNA can have different targets in different organs [11,12]. miR-181c is expressed mainly in the mitochondrial fraction of heart, and targets a mitochondrial gene (cytochrome c oxidase subunit I, mt-COX1) [3-5]. However, it is possible that miR-181c does not localize to the mitochondria in other organs. In fact, it has been shown that miR-181c targets genes in a gastric cancer cell line, suggesting that it mainly localizes to the cytosolic compartment in the gastrointestinal tract [13]. Similarly, miR-181c was found to interact with tumor necrosis factor α in hematopoietic progenitor cells, further suggesting non-mitochondrial localization [14]. It has been demonstrated that miR-181c plays an important role in the priming phase of liver regeneration [15] and that overexpression inhibits Hepatitis C virus replication by directly targeting the 3′-untranslated region (UTR) of homeobox A1 [16].

Normal homeostasis requires a balance between lipid synthesis and lipid oxidation to prevent lipid deposition. In obesity and T2D, lipids such as triglycerides and total cholesterol are elevated [17-20]. The role of miRNAs in lipid metabolism is well documented [21]. The liver-specific miRNA miR-122 has been shown to regulate lipid synthesis by targeting multiple mRNAs that are responsible for lipid biosynthesis, such as fatty acid synthase and acetyl-CoA carboxylases 1 and 2 [22-24]. Sterol regulatory element binding transcription factor 1 (SREBF1) regulates cholesterol homeostasis by downregulating ATP-binding cassette transporters [25]. miR-33 has been shown to alter cholesterol and HDL generation by targeting SREBF1 mRNA [26]. Mitochondrial function directly influences lipid biosynthesis [27-29].

The aim of this study was to identify the role of miR-181c in liver lipid metabolism in the context of obesity. We took advantage of our global miR-181c/d-/- (c/d KO) mouse model, which allowed us to investigate liver lipid metabolism in the absence of miR-181c during DIO. Based on previous finding that the hearts of c/d KO mice are protected against ischemia-reperfusion injury [5] and diet-induced obesity associated cardiac dysfunction [7], we hypothesized that c/d KO mice would also be protected from HF-induced metabolic stress in the liver.

Material and methods

Animals

Male wild-type (WT) C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) and previously described c/d KO mice [5] were used where indicated. Mice were provided ad libitum access to standard laboratory chow (CH; 2018 Teklad, Envigo, Frederick, MD) or purified HF diet (60% fat, D12492, Research Diets, New Brunswick, NJ) and tap water. The mice were divided into four experimental groups: WT-CH, WT-HF, c/d KO-CH, and c/d KO-HF (n = 6–7 per group). In a subset of mice on the HF diet, a glucose tolerance test was performed at 29 weeks of age. A separate cohort of WT mice was used for the miR-181c overexpression study as described below. All mice were housed in standard polycarbonate cages in a humidity- and temperature-controlled vivarium on a 12 h:12 h light:dark cycle with light onset at 6 am. All procedures were approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University School of Medicine.

Indirect calorimetry

Energy expenditure, respiratory exchange ratio, locomotor activity, and food intake of WT and c/d KO mice were determined by taking indirect calorimetric measurements in an open-flow indirect calorimeter (Oxymax, Columbus Instruments) at 0, 10, and 20 weeks of HF exposure. Data were collected for 3 days to confirm acclimation to the calorimetry chambers (stable body weights and food intake). Data from the third day were analyzed. Rates of oxygen consumption (VO2, ml·kg−1·h−1) and carbon dioxide production (, ml·kg−1·h−1) were measured for each chamber every 16 min throughout the study. The respiratory exchange ratio (RER = VCO2/VO2), calculated by Oxymax software (v. 4.02), was used to estimate relative oxidation of carbohydrate (RER = 1.0) versus fat (RER approaching 0.7), not accounting for protein oxidation.

Intraperitoneal glucose tolerance test

Prior to the intraperitoneal glucose tolerance test (IPGTT) the mice were fasted overnight (food was removed at 6 pm). Mice were moved into the testing room 1.5 h before glucose injection for habituation (8 am). The IPGTT was performed according to methods previously described [30]. A baseline blood sample was taken (9:30 am) via a small nick of the tail. Then, the mice were injected intraperitoneally with 1.5 mg/g glucose (20% glucose in sterile water solution; 10 am). Additional blood samples (20 μl) were taken 15, 30, 45, 60, and 120 min after glucose injection. Glucose levels in the blood were determined immediately with a handheld glucose analyzer (Freestyle; TheraSense, Alameda, CA, USA). Blood was collected into heparinized capillary tubes and stored in microcentrifuge tubes on ice. After the blood was centrifuged at 4°C, plasma was collected and stored at -80°C. Plasma insulin concentration was determined with a commercially available ultrasensitive mouse insulin ELISA kit (Crystal Chem, Downers Grove, IL, USA).

Tissue collection

Animals were deeply anesthetized with isoflurane, and blood was collected via cardiac puncture. The heart and liver were rapidly dissected, flash-frozen in liquid nitrogen, and stored at -80°C for further analysis. Blood was centrifuged at 4°C and plasma was collected and stored at -80°C.

Hormone and triglyceride analyses

Plasma leptin levels were determined with a commercially available mouse leptin ELISA kit (EMD Millipore, St. Charles, MO, USA). The intra-assay variation of the kit was 1.06–1.76%, and the inter-assay variation was 3.01–4.59%. Plasma and liver triglyceride levels were determined with a commercially available colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI, USA). For liver triglyceride levels, samples were further diluted (1:5) and used for triglyceride analysis according to the manufacturer’s instructions. Intra- and inter-assay variations of the kit were 1.34% and 3.17%, respectively.

Mitochondrial isolation protocol

Mitochondria were freshly isolated from hearts and livers by differential centrifugation [4]. Briefly, after incubation in RNAlater, the tissues were dissected and placed in Buffer A (in mM: 180 KCl, 2 EGTA, 5 MOPS, 0.2% BSA; pH: 7.25). The tissues were then digested with trypsin (0.0001 g/0.1 g tissue) in 0.7 ml of ice-cold Buffer B (in mM: 225 mannitol, 75 sucrose, 5 MOPS, 0.5 EGTA, 2 taurine; pH: 7.25) and finally homogenized in Buffer B containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). To further separate the heart mitochondria from other cellular components and tissue debris, a series of differential centrifugations was carried out in a Microfuge 22R centrifuge (Beckman Coulter, Fullerton, CA) at 4°C. The crude pellet was then lysed with QIAzol (Qiagen, Valencia, CA).

qRT-PCR

Total RNA was isolated from hearts or liver tissue with an miRNeasy kit (Qiagen) for the miRNA-enriched fraction or an RNEasy kit (Qiagen) for a larger RNA fraction. For the mitochondrial miRNA-enriched fraction, we used a modified protocol [4]. In both cases, an RNase-free DNase kit (Qiagen) was used to eliminate genomic DNA contamination. To characterize the integrity of the isolated RNA, we performed spectrophotometric evaluation with a microspectrophotometer (Nanodrop, Thermo Scientific, Wilmington, DE). Only RNA samples for which the absorbance at 260 nm (A260) was greater than 0.15 were used for further experiments. The ratio of the readings at 260 nm and 280 nm (A260/A280) was used to evaluate the purity of the isolated RNA. For further and more accurate purity and integrity estimation of the isolated RNA, we profiled the samples in the Bioanalyzer 2100 (Agilent Technologies, CA). Only RNA with A260/A280 ~2.00 and RNA integrity number > 8 was used for the experiments. RNA from heart and liver were reverse transcribed with the miScript Reverse Transcription Kit (Qiagen). HiFlex and HiSpec buffers were used for mRNA and miRNA, respectively. PCR for miRNA and mRNA was carried out with an miScript SYBR green PCR kit (Qiagen) using a QuantStudio 5 (Thermo Scientific). qRT-PCR was carried out with primers for miR-181c, SNORD61, and 5S rRNA (Qiagen). All reactions were repeated in triplicate. Primer sequences for all other genes are reported in .

Western blot analysis

Liver, heart, and isolated mitochondrial pellets were lysed with RIPA buffer (Cat. #9806S, Cell signaling Technology), and protein content was measured by Bradford assay. Cell homogenate protein was separated by 1D gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk for 2 h at room temperature before being incubated overnight at 4°C with antibodies to isocitrate dehydrogenase 1 (IDH1; 1:1000), isocitrate dehydrogenase 2 (IDH2; 1:1000; Cell Signaling, Danvers, MA), or α-tubulin (Abcam, Cambridge, MA) in Tris-buffered saline (pH 7.4) containing 1% TWEEN-20 (TBS-T) and 1% bovine serum albumin (BSA). Membranes were incubated with the appropriate horseradish peroxidase–conjugated IgG secondary antibody in TBS-T with 1% BSA for 1 h at room temperature. Immunoreactive protein was visualized with an enhanced chemiluminescence analysis kit (GE HealthCare, Piscataway, NJ). The signals emitted for chemiluminescence were detected with the iBright Imaging System (ThermoFisher Scientific).

Body composition analysis

After the mice were sacrificed, their body composition was determined by whole-body nuclear magnetic resonance imaging (EchoMRI, Echo Medical Systems, Waco, TX).

Oil-Red-O staining

Liver samples were immediately frozen after collection. Samples were embedded in O.C.T for sectioning, and tissue sections were mounted on glass slides. The mounted sections were stained with Oil-Red-O.

miR-181c overexpression in WT mice

To overexpress miR-181c in WT mice, we packaged an miR-181c expression construct into an adeno-associated virus (serotype 8; AAV-8). Control scrambled miRNA (“Scr”; Cat# AA08-CmiR0001-MR14-100, GeneCopoeia Inc., Rockville, MD, USA) and overexpression miR-181c (“miR-181c OE”; Cat# AA08-MmiR3275-MR14-200, GeneCopoeia Inc.) were packaged into a hepatocyte-specific serotype, AAV-8 construct. C57BL/6J mice were fed a HF diet for 4 weeks before being injected with virus construct (1011 viral particles in PBS; n = 8 mice per construct) and remained on the HF diet until sacrifice 6 weeks later. A total of 50 μl of scrambled or miR-181c oligonucleotide was injected retro-orbitally. Mice were tested in an IPGTT 5 weeks after virus injection and sacrificed 1 week later for collection of blood and tissues as described above.

Statistical analysis

Data were analyzed by 1-way or 2-way analysis of variance (ANOVA), and Bonferroni or Tukey corrected post hoc tests were used where appropriate. All analyses were carried out in Prism 9 (GraphPad Software Inc., CA). A p value <0.05 was considered statistically significant. Results are presented as mean ± standard error of the mean (mean ± SEM).

Results

Metabolic profile of mice lacking miR-181c/d

As shown previously, c/d KO mice were born in the expected Mendelian ratios and exhibited no body weight differences at birth compared to WT littermates [5]. Prior to HF exposure, c/d KO mice had higher oxygen consumption (VO2; Fig 1A) than did WT mice (p = 0.01), but no differences in CO2 production (Fig 1B). Thus, c/d KO mice had a lower RER, indicating a preferential mobilization of fat, rather than carbohydrates, for energy needs (p<0.0001; Fig 1C). In addition, c/d KO mice had increased energy expenditure compared to that of WT mice (p = 0.01; Fig 1D) on chow diet. However, after 10 and 20 weeks of HF access, no significant group differences remained in oxygen consumption, CO2 production, RER, or energy expenditure between the WT and c/d KO mice (Fig 1A–1D). There were significant group x time interactions for VO2 (F (2, 18) = 59.78, p<0.0001; Fig 1A), RER (F (2, 18) = 468.6, p<0.0001; Fig 1C), and energy expenditure (F (2, 18) = 78.54, p<0.0001; Fig 1D).

Fig 1

Metabolic profiling of miR-181c/d-/- (c/d KO) mice.

(A) Oxygen consumption (VO2), (B) carbon dioxide production (VCO2), (C) respiratory exchange rate (RER), and (D) energy expenditure (EE) were measured before and during the 26 weeks of high-fat (HF) diet. Data are expressed as mean ± SEM (n = 4/genotype). *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 2-way ANOVA. *p<0.05, ****p<0.0001 by two-sample t-test.

Lack of miR-181c leads to the accumulation of white adipose tissue and impaired glucose tolerance after HF exposure

While on chow diet (6–8 week of age), c/d KO and WT mice exhibited no differences in body weight (Fig 2A). When placed on the HF diet, both WT and c/d KO mice increased in body weight compared to their respective chow controls (F (3, 48) = 5.315, p = 0.003; Fig 2A). After 26 weeks of chow or HF exposure, there was a significant genotype diet effect on body fat (F (3, 37) = 30.83, p<0.0001; Fig 2B). Bonferroni post hoc analysis showed that c/d KO-HF mice had a higher body fat percentage than did WT-CH, c/d KO-CH, and WT-HF mice. In addition, there were significant effects of genotype and diet with respect to lean mass (F (3, 40) = 28.44, p<0.0001; Fig 2C). c/d KO-CH mice had lower lean body mass than did WT-CH mice, but not WT-HF mice. When exposed to a HF diet for 26 weeks, c/d KO-HF mice had lower lean mass as a percent of total carcass weight compared to all groups.

Fig 2

Effect of high-fat (HF) diet on miR-181c/d-/- (c/d KO) mice.

(A) Body weight (n = 4/genotype/diet), (B) percent fat mass (n = 8-13/genotype/diet), and (C) percent lean mass (n = 8-13/genotype/diet) were measured before and during the 26 weeks of HF diet. Data are expressed as mean ± SEM. *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 1-way ANOVA. *p<0.05, ***p<0.001, ****p<0.0001 by two-sample t-test.

Under baseline conditions when all mice were 6–8 weeks old and on a normal chow diet, light cycle postprandial blood glucose levels did not differ between c/d KO (149.0±4.4 mg/dl) and WT mice (147.4±7.4 mg/dl). After 26 weeks of a HF diet, fasting glucose levels did not differ between WT and c/d KO mice (Fig 3A). However, fasting plasma insulin levels were significantly higher in the c/d KO mice than in the WT mice (p = 0.03; Fig 3B). When challenged with a glucose tolerance test, there was an overall time by group interaction effect on plasma glucose levels (F (5, 72) = 24.21, p<0.0001; Fig 3A). Bonferroni post hoc correction analysis showed that at 30, 45, 60, and 120 min after glucose injection, circulating blood glucose levels were higher in c/d KO mice than in WT mice. In addition, c/d KO mice had an elevated area under the curve (AUC; p = 0.004) compared to that of WT mice. There was a main effect of group (F (1, 71) = 18.47, p<0.0001) and time (F (5, 71) = 2.714, p = 0.0266) for plasma insulin levels (Fig 3B) but no statistically significant differences in the AUC between WT and c/d KO mice.

Fig 3

Metabolic consequences of high-fat (HF) diet on miR-181c/d-/- (c/d KO) mice.

(A) Blood glucose and area under the curve during an intraperitoneal glucose tolerance test (IPGTT) and (B) plasma insulin levels and area under the curve during an IPGTT. Data are expressed as mean ± SEM and are from 6–7 mice/genotype/diet. *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 2-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-sample t-test.

Lack of miR-181c leads to impaired lipid biosynthesis in the liver after HF exposure

After 26 weeks of HF exposure, lipid accumulation in the liver of c/d KO mice was elevated compared to that in WT mice as measured by Oil-Red-O staining (Fig 4A). Plasma leptin level is considered a key biomarker for obesity and metabolic diseases [31,32]. Measurements showed no differences in plasma leptin between c/d KO and WT mice on normal chow diets (Fig 4B). However, after exposure to HF, both WT and c/d KO mice had elevated plasma leptin levels (F (3, 19) = 53.43, p<0.0001). There was a main effect of diet (F (3, 18) = 19.79, p<0.0001) on plasma triglyceride levels (Fig 4C). Post hoc analysis showed that plasma triglyceride levels of c/d KO-HF mice were elevated compared to those of WT-CH mice. Post hoc analysis also revealed that within the HF groups, liver triglyceride levels were significantly higher in the c/d KO than in the WT mice and respective chow-fed controls.

Fig 4

Effect of high-fat (HF) diet on the liver of c/d KO mice.

(A) Liver Oil-Red-O staining for lipid droplets. Analysis of plasma leptin levels (B), plasma triglyceride levels (C), and liver triglyceride content (D) in WT and c/d KO groups fed normal chow or a HF diet. Data are expressed as mean ± SEM (n = 5–7 mice/genotype/diet). *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 1-way ANOVA. *p<0.05, **p<0.01, ****p<0.0001 by two-sample t-test.

Effect of HF diet on liver miR-181c expression

Recent data showed that a HF diet upregulates miR-181c in the heart [7]. To determine the regulation of miR-181c in the liver during diet-induced metabolic stress, we measured the expression of miR-181c in WT mice after exposure to 26 weeks of HF. Interestingly, miR-181c was decreased in the liver of WT mice after exposure to HF (p = 0.02; Fig 5A). In the heart, miR-181c mainly translocates to the mitochondrial fraction [3-5]. Whole tissue analysis in WT mice showed that expression of miR-181c was markedly higher in the liver than in heart tissue (Fig 5B). Next, to detect miR-181c in liver mitochondria, we isolated mitochondrial miRNA-enriched total RNA from both the heart and liver mitochondrial fraction. We detected significantly higher levels of miR-181c expression in the mouse heart mitochondria than in liver mitochondria (Fig 5C). These data suggest that unlike heart, miR-181c is mainly expressed in the cytosolic fraction of liver.

Fig 5

Cytosolic localization of miR-181c in the liver.

Quantitative polymerase chain reaction (qRT-PCR) analysis of miR-181c expression in total RNA from the liver of normal chow (CH)-fed and high-fat (HF)-fed WT mice (A), in total RNA from the heart and liver tissue (B), and in the mitochondrial pellets of WT heart and liver tissue (C). Data are expressed as mean ± SEM (n = 3-7/genotype/diet). *p<0.05 by t-test analysis after intergroup differences were found by 2-way ANOVA. *p<0.05 by two-sample t-test.

miR-181c targets IDH1 in the cytoplasm to inhibit lipid biosynthesis in the liver

Previously, it was shown that the “seed” sequence for miR-181 can directly bind to the 3′-UTR of the IDH1 gene at one putative site (UGAAUGU) [2]. In addition, overexpression of cytosolic-specific IDH (IDHc) resulted in elevated liver triglycerides and adipose tissue accumulation [33]. Because miR-181c is expressed mainly in the cytosolic fraction of the liver (Fig 5B and 5C) and IDHc is involved in lipid biosynthesis in the liver, we measured IDH protein expression in the liver of chow-fed WT and c/d KO mice. The expression of IDH1 is in the cytoplasm, whereas IDH2 is expressed in the mitochondrial fraction. Therefore, we compared the protein expression of both IDH1 and IDH2 in c/d KO mouse and WT liver. Loss of miR-181c led to the upregulation of IDH1 protein in liver (p = 0.015 versus WT mice; Fig 6A). However, IDH2 protein expression did not differ between c/d KO and WT mice (Fig 6B). These data suggest that miR-181c targets IDH1 mRNA in the cytoplasmic fraction of hepatocytes.

Fig 6

In the absence of miR-181c, lipogenesis is activated by IDH1 upregulation.

Western blot analysis of protein lysate from liver IDH1 (A) and IDH2 (B). (C) Quantitative polymerase chain reaction (qRT-PCR) was used to assess liver mRNA expression of genes involved in lipogenesis. Sterol regulatory element binding transcription factor 1 (SREBP1), fatty acid synthase (FASN), and ATP citrate lyase (ACLY) were more highly expressed in WT than in c/d KO mice. Data are expressed as mean ± SEM (n = 5-7/genotype/diet). *p<0.05 by t-test analysis after intergroup differences were found by 2-way ANOVA. *p<0.05 by two-sample t-test.

Finally, to determine potential mechanisms by which the IDH1 pathway might contribute to impaired metabolic functioning in the liver of mice on a HF diet, we measured liver mRNA expression of genes involved in lipogenesis in chow-fed WT and c/d KO mice (Fig 6C). We found that expression of mRNA for genes involved in de novo lipid synthesis (ATP citrate lyase [ACLY]; p = 0.03, fatty acid synthase [FASN]; p = 0.03) and fatty acid synthesis (SREBP1; p = 0.03) were increased in the livers of c/d KO mice compared to those of WT controls.

Liver-specific miR-181c overexpression

Next, to determine if overexpression of miR-181c can protect against the liver-specific metabolic consequences of HF exposure, we used an AAV-8 package system. First, the dose of viral particles, retro-orbital vein delivery, and optimal time points were optimized (Fig 7A). After 4 weeks of HF diet, the mice were randomly selected for either AAV-8 scramble or AAV-8-miR-181c injection. Both groups of mice then maintained a HF diet for an additional 6 weeks. Using this treatment regimen of in vivo miR-181c delivery, we successfully overexpressed miR-181c in the liver by 2.5-fold (p = 0.03; Fig 7B). Importantly, miR-181c was not overexpressed in heart (Fig 7C), lung (Fig 7D), spleen (Fig 7E), or kidney (Fig 7F) after AAV-8-miR-181c injections.

Fig 7

Liver-specific miR-181c delivery and validation.

(A) In vivo miR-181c overexpression protocol. Quantitative polymerase chain reaction (qRT-PCR) analysis of miR-181c expression in total RNA isolated from liver (B), heart (C), lung (D), spleen (E), and kidney (F). OE, overexpression; Scr, scrambled oligonucleotide. Data are expressed as mean ± SEM; n = 8 for panels B-F. * p<0.05 by t-test analysis. *p<0.05 by two-sample t-test.

Groups injected with AAV-8-miR-181c and AAV-8 scramble did not differ in body weight after 10 weeks of HF diet (Fig 8A). Similarly, after 10 weeks of the HF diet, there were no significant differences in baseline (0 min) blood glucose (Fig 8B) or insulin levels (Fig 8C) between groups injected with AAV-8-miR-181c and AAV-8 scramble. When the mice were challenged with a glucose tolerance test, we found an overall time by group interaction effect on plasma glucose level (Fig 8B). No circulating blood glucose or insulin level differences were present between AAV-8-miR-181c and AAV-8 scramble groups.

Fig 8

Effect of miR-181c overexpression in liver on insulin secretion.

(A) Body weight. (B) Blood glucose and area under the curve during an IPGTT. (C) Plasma insulin levels and area under the curve during an IPGTT. OE, overexpression; Scr, scrambled oligonucleotide. Data are expressed as mean ± SEM; n = 8 per group. *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 2-way ANOVA.

Overexpression of miR-181c mitigates HF-induced lipogenesis

Liver-specific overexpression of miR-181c had no effect on IDH1 mRNA level (Fig 9A) but significantly downregulated IDH1 protein expression (Fig 9B). In contrast, miR-181c overexpression did not alter IDH2 expression at either the mRNA (Fig 9C) or protein level (Fig 9D) after 10 weeks of HF. Given our findings of the potential role for miR-181c in IDH1-regulated lipogenesis, we measured the expression of genes involved in lipogenesis in the liver. In mice that received 10 weeks of HF diet, treatment with miR-181c resulted in lower liver mRNA expression of genes involved in long chain fatty acid synthesis (FASN, p = 0.03; SREBP1, p = 0.003; Fig 9E).

Fig 9

Effect of liver-specific miR-181c overexpression on lipogenesis.

Western blot (A) and quantitative polymerase chain reaction (qRT-PCR, B) of IDH1 in the liver from AAV-8 vector-injected mice. Western blot (C) and qRT-PCR (D) of IDH2 in the liver of AAV-8 vector-injected mice. (E) qRT-PCR was used to assess liver mRNA expression of sterol regulatory element binding transcription factor 1 (SREBP1), fatty acid synthase (FASN), and ATP citrate lyase (ACLY) genes, which are involved in lipogenesis. OE, overexpression; Scr, scrambled oligonucleotide. Data are expressed as mean ± SEM; n = 8 per group. * p<0.05 by t-test analysis. *p<0.05, **p<0.01 by two-sample t-test.

The miR-181c-overexpressing mice and their AAV-8 scramble-injected controls exhibited no difference in liver weight after 10 weeks of a HF diet (Fig 10A). Histology showed elevated lipid accumulation in the liver of AAV-8 scramble-injected mice by Oil-Red-O staining, Masson-Trichrome, and H&E (Fig 10B upper panel) after 10 weeks of HF diet; however, AAV-8-miR-181c-injected mice had significantly lower lipid droplet accumulation (Fig 10B lower panel). No other changes were observed in either group by Masson-Trichrome or H&E staining (Fig 10B). Post hoc analysis revealed that within the HF groups, liver triglyceride levels were significantly lower in the miR-181c-overexpressing mice than in the controls (Fig 10C). Finally, among the HF group, liver-specific overexpression of miR-181c lowered the plasma triglyceride (Fig 10D) and plasma leptin (Fig 10E) levels compared to those of AAV-8 scramble injected mice.

Fig 10

Effect of liver-specific miR-181c overexpression on metabolic consequences of a high-fat (HF) diet.

AAV-8-miR-181c-injected and AAV-8-scramble-injected mice were compared after 10 weeks of a HF diet. (A) Total liver weight. (B) Liver histology, Oil-Red-O staining, Masson-Trichrome, and H&E staining. (C) Liver triglyceride content. (D) Plasma triglyceride levels. (E) Plasma leptin levels. OE, overexpression; Scr, scrambled oligonucleotide. Data are expressed as mean ± SEM; n = 8 per group. *p<0.05 by Bonferroni post hoc analysis after intergroup differences were found by 1-way ANOVA. *p<0.05, **p<0.01, ***p<0.001 by two-sample t-test.

Discussion

The ability of miR-181c overexpression to confer robust protection in the obesity paradigm has two important implications. First, these results support miR-181c as a potential therapeutic target to combat obesity and lipogenesis. As it has already been established that miR-181c can be delivered in vivo using a nanovector [3], these findings identify a paradigm useful for testing the efficacy of miR-181c overexpression and determining its role in fat inhibition and cholesterol biosynthesis. Second, these findings demonstrate that compensation for the loss of miR-181c can confer protection against obesity when mice are exposed to a HF diet. c/d KO mice have a severe obese phenotype when fed a HF diet. Plasma leptin level is considered one of the key biomarkers for obesity and metabolic diseases [31,32]. We found significantly higher levels of plasma leptin in c/d KO mice than in WT mice after 26 weeks of HF diet (Fig 4B). However, liver-specific delivery of miR-181c significantly lowered the plasma leptin level compared to that of the scramble-injected group after 26 weeks of HF diet (Fig 10E). The data from the c/d KO mice and mice with liver-specific overexpression of miR-181c suggest that miR-181c can offer protection in the liver from HF exposure by inhibiting lipogenesis.

The nuclear-encoded miR-181 family members play an important role in cardiac function by regulating target genes both in the cytoplasm and within mitochondria. miR-181a/b regulates PTEN expression in the cytoplasm, whereas miR-181c regulates the mt-COX1 gene in mitochondria [5]. We have previously shown, both in vitro [4] and in vivo [3], a pivotal role for mitochondrial miR-181c in cardiac dysfunction. We [3-5] and others [6] have identified a significant role for miR-181c in the heart during end-stage heart failure. Unlike in heart, however, here we identified a nuclear-encoded cytoplasmic target, IDH1, for miR-181c in the liver (another mitochondria-enriched organ). Mature miR-181c can be expressed mainly in the mitochondrial fraction of cardiomyocytes; however, in hepatocytes, miR-181c directly binds to the 3′-UTR of IDH1 in the cytoplasm. By downregulating IDH1 during DIO, miR-181c can regulate lipid metabolism. These findings raise an important question: Why does miR-181c translocate into the mitochondria in the heart? Is it because cardiomyocytes lack IDH1 expression? Our current study can provide some important hints in the field of mitochondrial miRNA.

Acetyl-CoA and NADPH are essential common precursors and cofactors for lipid biosynthesis. IDH1, a key NADPH producer, contributes to the activation of triglyceride and cholesterol synthesis by the liver [33]. Similar to the metabolic phenotype of IDH1 transgenic mice [33], c/d KO mice have an increase in body fat composition and insulin insensitivity after 26 weeks of a HF diet. These two mouse models highlight the potential for IDH1 to promote induction of fatty liver, hyperlipidemia, and obesity by altering lipid biosynthesis in the liver, especially under HF-induced stress. Furthermore, IDH1 has been shown to activate lipogenesis under hypoxic conditions by reductive glutamine metabolism [34]. IDH1 can also regulate the lipogenic pathways by activating the transcriptional factors SREBP1 and SREBP2 [35]. Alternatively, Chu et al. [2] showed that knockdown of IDH1 downregulates lipid synthesis-related gene expression and upregulates β-oxidation and cholesterol transport-related gene expression, thereby inhibiting lipid synthesis in the liver. Therefore, liver-specific overexpression of miR-181c may be a potential therapeutic target for abnormal fat synthesis during DIO. miR-181c can attenuate lipid biosynthesis by directly binding to the 3′-UTR of IDH1 mRNA and perhaps overexpressing miR-181c in liver may play that therapeutic approach against obesity (Fig 11).

Fig 11

Role of miR-181c in regulating high-fat diet-induced lipogenesis.

This schematic diagram illustrates key steps in the signaling pathway linking a high fat diet with increased lipogenesis. According to this model, high fat triggers lipogenesis by activating IDH1 though SREBF, ACLY, and FASN pathways. Liver-specific miR-181c can directly bind to the 3′-UTR of IDH 1 mRNA and mitigate lipogenesis.

In this study, we observed that c/d KO mice exhibit a severe obese phenotype with significant hyperinsulinemia and hyperglycemia when they are fed a diet composed of 60% fat. These data also suggest that the miR-181c-IDH1 pathway plays an important role in dietary stress. Thus, liver-specific overexpression of miR-181c can protect against dietary stress by inhibiting lipogenesis. miRNA therapeutics may be a potential treatment regimen; however, the tissue-targeted delivery is challenging with the potential for off-target side effects [36]. In the obesity field, miR-143~145 cluster delivery in the liver has been shown to have a protective effect against obesity-associated diabetes [37]. Previously, we showed that miR-181c can be overexpressed in vivo using an electrostatic complex: nanovector with positively charged liposomal nanoparticles and negatively charged plasmid DNA. Previous work also showed no immune response during this nanovector treatment regimen [3]. However, nanovector-based miR-181c delivery can distribute miR-181c in all the tissues, including heart. Overexpression of miR-181c can cause severe cardiac dysfunction via mitochondrial mechanisms [3-5]. In this study, we packaged miR-181c construct in AAV-8 and injected 1011 viral particles/mouse through retro-orbital injection, which did not cause miR-181c overexpressing in heart, lung, spleen, or kidney (Fig 7). Thus, future technologies should focus on liver-specific miR-181c delivery to prevent DIO complications, such as nonalcoholic fatty liver disease. Such interventions might be useful for patients with nonalcoholic fatty liver disease.

Conclusions

In summary, in this study we demonstrated that miR-181c plays an essential role in inhibiting triglyceride and cholesterol biosynthesis by targeting IDH1 mRNA in the cytoplasm. c/d KO mice have a severe obese phenotype during HF. Overexpression of miR-181c during DIO can protect against the metabolic consequences of HF exposure by altering lipid metabolism.

(PPTX)

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(PPTX)

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8 Sep 2021

PONE-D-21-26341Role of miR-181c in Diet-Induced Obesity Through Regulation of Lipid Synthesis in LiverPLOS ONE

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Reviewer #1: In this manuscript, Akiyoshi et al explored the role of miR-181c in diet induced obesity in vivo through both loss and gain of function assays. In miR-181c/d knockout mouse model, the authors have shown that inactivation of miR-181c/d led to glucose intolerance post 26 weeks high fat diet challenge associated with more lipid accumulation in liver and up-regulation of lipid synthesis genes potentially through up-regulation of IDH1 at protein level. Similarly, the authors have also tested the response of HFD in miR-181c overexpressing mouse model through AAV mediated gene delivery. Although no change in body weight, the authors claimed expression of miR-181c can attenuate lipid accumulation in liver post high fat diet challenge.

Previously the authors have shown inactivation of miR-181c/d protected mice against ischemia-reperfusion injury, therefore, the results presented in this manuscript are un-expected. Here are several concerns:

1. The regulatory role of miR-181c/d on IDH1

No sufficient data supporting that miR-181c directly regulates IDH1 in liver. For example, the authors have not shown mRNA expression level of IDH1 and IDH2 in miR-181c/d knockout animals or AAV8-miR-181c injected animals. There is no luciferase assay suggesting miR-181c indeed regulate IDH1 3’UTR activity. Without this evidence, the changes of IDH1 protein expression could be secondary effect.

2. Relevance of miR-181c in liver disease

The authors hypothesized that c/d KO mice would be protected from metabolic stress, so the results shown here are unexpected. Is there any change of miR-181c or miR-181d in NASH or other liver disease?

3. Characterization of mouse phenotype post high fat diet challenge

The authors should provide better phenotype characterization of the mouse model. For example, there is only one-time GTT and ITT. The authors should show baseline GTT and ITT in the mouse models (before HFD). There is no liver weight; H&E and liver fibrosis results.

Minor issue

1. The oil-red staining images have poor quality. No scale bar, Figure 4A is not white balanced.

Reviewer #2: The authors have explored the role of miR-181c in a DIO mouse model using a miR-181c -/- mouse and rescue with a AAV-8 delivering miR-181c.

Endpoints are:

Changes in target gene expression using qPCR

Indirect calorimetric measurements

Glucose tolerance tests/insulin levels

Body fat

TGs

Concluding that IDH1 is important in coordinating the changes in lipid biology related to 181c and propose that overexpression of 181c is protective

I do think the observations are of merit and warrant publication but I do have I number of reservations/comments:

- Be consistent in 181c nomenclature ie use 181c throughout

- The main text describing Figure 8, I personally found confusing in regards to glucose tolerance test and would just state no blood glucose or insulin levels were measured

- The authors measure leptin but no rationale as to why measured

- Why did the authors chose a retro-orbital route for AAV delivery, especially if aiming for liver expression, admittedly this was achieved, but tail vein injection is often used to optimize delivery to the liver.

- Could the authors explain why the numbers of mice in each group or observations, varied so much.

- Figure 4+10 (when any histology) need to include magnification

- Figure 5B +5C can you explain why in whole heart tissue you have low levels of 181c but then can find it in the mito fraction. Whole tissue still has the mito fraction within it, so would it not express 181c too?

- For first use of gene name in the manuscript define, as definition of gene names occurs later in the manuscript or in figure legends

- The evidence for IDH1 is mainly through association based on experimental observations and the literature. It would have been good if the authors had demonstrated that 181c is directly acting on the IDH1 3’UTR in a liver cell to provide more supporting evidence for the authors conclusion.

- Discussion, I would present some ideas about why 181c has a cytosolic v. mitochondrial location in the liver in contrast to the heart. Are any gender differences anticipated/known, as authors have used a male mouse model. I would be more reserved on the use of 181c as a protective agent for obesity as in the context of the liver it can act as an Oncomir too.

**********

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1 Nov 2021

To,

Dr. Aijun Qiao, Ph.D.

Academic Editor

PLOS ONE

Dear Dr. Qiao:

We thank the reviewers for their careful consideration, favorable comments, and many helpful suggestions for improving our manuscript. In addition, we would also like to thank you for giving us the opportunity to re-submit our manuscript to PLOS ONE.

We have addressed all of the reviewers’ comments and their requests for additional data. Particularly, we have included qPCR results for IDH1 and IDH2 expression after miR-181c overexpression using the liver tissues; along with proper histopathological evaluation. Additionally, we have also included the liver weight after miR-181c overexpression using AAV-8 vector to respond to the concern raised by Reviewer #1. We have also included the gender difference in terms of miR-181c expression in the liver to respond to one of the points raised by Reviewer #2. We agree that gender differences are very important, and we are planning to further study the implications of higher miR-181c levels in female livers in our future studies. We hope that the reviewers agree that we have addressed all of their concerns, and trust this manuscript is now acceptable for publication in PLOS ONE. We look forward to hearing favorably from you.

We have included detailed responses to each of the reviewer’s comments below. Their comments are in black and our responses are in red.

Reviewer #1:

1. The regulatory role of miR-181c/d on IDH1

No sufficient data supporting that miR-181c directly regulates IDH1 in liver. For example, the authors have not shown mRNA expression level of IDH1 and IDH2 in miR-181c/d knockout animals or AAV8-miR-181c injected animals. There is no luciferase assay suggesting miR-181c indeed regulate IDH1 3’UTR activity. Without this evidence, the changes of IDH1 protein expression could be secondary effect.

Thank you for the suggestion. In our knock-out mouse model, miR-181c is deleted from birth. Therefore, it is highly possible that a compensatory mechanism exists which directly or indirectly influences IDH1 expression in the liver, as evidenced by our previous studies 1,2. To validate whether miR-181c regulates IDH1 expression in the liver, we performed qPCR for IDH1 and IDH2 in the liver of AAV8-miR-181c injected mice. As shown in Fig. 9A-B, miR-181c overexpression significantly downregulates IDH1 protein expression, and there are no changes in IDH1 mRNA expression, suggesting that miR-181c translationally inhibits IDH1 expression by directly binding to the IDH1 3’-UTR region. We did not observe any changes in IDH2 expression (Fig. 9C-D).

The reviewer is correct that the biochemical assay using dual luciferase would determine the direct association of a miRNA and its target mRNA. This assay was already performed in hepatocytes to determine whether the 3’-UTR of IDH1 is a direct target for miR-181a 3. The putative site (UGAAUGU) for miR-181a and miR-181c is identical, and that is the “seed” sequence for the miR-181 family 1. Chu et al., 3 has demonstrated that the miR-181 “seed” sequence can directly bind to the 3’-UTR of IDH1 mRNA. Even though the conclusion of the Chu et al. study3 is different than our current study, the dual-luciferase assay is identical to what the reviewer has suggested.

2. Relevance of miR-181c in liver disease

The authors hypothesized that c/d KO mice would be protected from metabolic stress, so the results shown here are unexpected. Is there any change of miR-181c or miR-181d in NASH or other liver disease?

Thank you for the great suggestion. This would indeed suggest that miR-181c is a therapeutic candidate for liver diseases. In our study, we have provided strong evidence for the therapeutic role of miR-181c in obesity-induced lipogenesis. Furthermore, Mukherjee et al.4, has demonstrated the projective role of miR-181c in chronic liver disease. Similar to our study, Mukherjee et al., observed (in chronic liver disease due to Hepatitis C viral infection) significant downregulation of pre-miR-181c and miR-181c in the liver. In their study, the authors have proposed that miR-181c can be used as a therapeutic target for end-stage liver disease, including hepatocellular carcinoma (HCC), based on the protective role of miR-181c delivery to the liver 4. Non-Alcoholic SteatoHepatitis (NASH) is also considered an end-stage liver disease. It would be interesting to examine miR-181c regulation in NASH samples. However, NASH is beyond the scope of this current study.

3. Characterization of mouse phenotype post high fat diet challenge

The authors should provide better phenotype characterization of the mouse model. For example, there is only one-time GTT and ITT. The authors should show baseline GTT and ITT in the mouse models (before HFD). There is no liver weight; H&E and liver fibrosis results.

Thank you for raising this important point. Like humans, until we observe the early signs of diabetes in post prandial glucose tests or urine tests using dipsticks, we do not perform the oral glucose tolerance test (for human or rats) or GTT and ITT for mouse. Under baseline conditions on a normal chow diet, miR-181c/d KO mice do not show any sign of hyperglycemia by regular urine test using dipstick or by random blood glucose test 1. We performed blood glucose measurement during the mid-light cycle in miR-181c KO mice (6-8 weeks of age) before switching their diet to the HFD. We did not observe any signs of hyperglycemia in miR-181c/d KO mice at this stage. We have now included the data in the results (page 10, second paragraph). Additionally, As shown in Figs. 4B and 4Cs, miR-181c KO mice show no obese phenotype and no changes in plasma triglyceraldehyde with WT mice under aged-matched normal chow diet. Therefore, we anticipate there will be no changes in GTT and ITT at the normal chow condition.

In line 239: “Under baseline conditions when all mice were 6-8 weeks old and on a normal chow diet, light cycle postprandial blood glucose levels did not differ between c/d KO (149.0±4.4 mg/dl) and WT mice (147.4±7.4 mg/dl).”.

To focus on the role of miR-181c in the liver, we have measured liver weights and performed histology 6 weeks post AAV8-Scr and AAV8-miR-181c injection. As shown in Fig. 10A, there were no differences in the dry liver weights between the two groups. Consistent with Oil-Red-O staining, both the H&E and Mason Trichrome staining show significant lipid droplet accumulation in hepatocytes of animals fed 26 weeks of the HFD (Fig. 10B); but AAV8-miR-181c treatment can normalize lipid accumulation in hepatocytes. Apart from these changes, there were no significant differences in the H&E staining between the AAV8-Scr and AAV8-miR-181c groups. Mason Trichrome staining also shows no significant liver fibrosis (Fig. 10B).

Minor issue

1. The oil-red staining images have poor quality. No scale bar, Figure 4A is not white balanced.

We apologize for the oversight. We now have included the magnification scale in all pictures. Additionally, we have re-taken all pictures after white balancing. Please see Figs. 4A and 10B.

Reviewer #2:

The authors have explored the role of miR-181c in a DIO mouse model using a miR-181c -/- mouse and rescue with a AAV-8 delivering miR-181c.

I do think the observations are of merit and warrant publication but I do have I number of reservations/comments:

Thank you.

- Be consistent in 181c nomenclature i.e. use 181c throughout

We apologize for the oversight. We now have corrected this I n the manuscript.

- The main text describing Figure 8, I personally found confusing in regards to glucose tolerance test and would just state no blood glucose or insulin levels were measured

We apologize for the confusion. We did measure blood glucose and insulin levels after 10 weeks of the HFD. The first time-point (0 min) of Figs. 8B and 8C represent the fasting blood glucose level and insulin level, respectively. We now have clarified in the text, please see page 14, 2nd paragraph.

The rational for using the glucose tolerance test (IPGTT) is to check pancreatic-beta-cell function in the AAV8-miR-181c injected group. Because of high adiposity, isolating the pancreas from 10-week HFD-fed mice is difficult. This is why we did not check miR-181c expression in the pancreas of AAV8-miR-181c injected mice (Fig. 7). However, despite using the AAV8 vector, it is possible to overexpress miR-181c in the pancreatic beta-cells. To confirm miR-181c delivery in a liver-specific manner, we have performed IPGTT.

- The authors measure leptin but no rationale as to why measured

The goal of the current study is to establish the role of miR-181c in diet-induced obesity. Plasma leptin level is considered one of the key biomarkers for obesity and metabolic diseases 5,6 which is why we have also measured leptin levels after 26 weeks of the HFD. In order to conclude the protective effects of miR-181c delivery, we have shown that delivery of miR-181c can lower the plasma leptin level compared to the AAV8-Scr injected group (Fig. 10E). We now have included this rationale in the results section (page 11, first paragraph) and again in the discussion section (first paragraph).

- Why did the authors choose a retro-orbital route for AAV delivery, especially if aiming for liver expression, admittedly this was achieved, but tail vein injection is often used to optimize delivery to the liver.

We use both retro-orbital and tail-vein injections on a regular basis. In our hand, the efficiency of drug delivery into a mouse is better with the retro-orbital route than tail-vein. Therefore, we chose retro-orbital. It has been shown by multiple groups that retro-orbital and tail-vein injection can both be equally effective for delivery into the liver 7,8.

- Could the authors explain why the numbers of mice in each group or observations, varied so much.

Thank you for this important question. This is a very long protocol – 26 weeks of the HF diet, and we used retro-orbital injection during this time period. During this long-term study, we encountered numerous animal health issues from self-inflected wounds, aggressive behavior with each other or spontaneous dermatitis. In line with the regulations issued by the animal research ethics committee, animals with injuries were euthanized in order to limit suffering. Therefore, even though we start our experiments with equal number of mice (8-10 mice/group), by the end of 26 weeks, we have lost 1 or 2 animals.

- Figure 4+10 (when any histology) need to include magnification

Sorry for the oversight. We have now included the magnification scale for Figure 4A and 10B.

- Figure 5B +5C can you explain why in whole heart tissue you have low levels of 181c but then can find it in the mito fraction. Whole tissue still has the mito fraction within it, so would it not express 181c too?

Thank you for raising this important point here. The reviewer is absolutely correct that mitochondrial miR-181c expression should be observed in the total heart tissue. However, in Fig. 5B we have compared miR-181c expression in heart and liver tissues. Our data suggest that liver tissue expresses a very high level of miR-181c compared to the heart, and thus, the levels are much higher in Fig. 5B compared to Fig. 5C. Additionally, miR-181c exclusively localizes to the mitochondrial fraction in the heart 9. In Fig. 5C, when we used 1 ng of total RNA from the mitochondrial pellet, miR-181c copy number is significantly higher than 1 ng of total RNA from heart tissue. The total heart tissue derived RNA will have nuclear, cytoplasmic and other cellular organelle fractions apart from mitochondrial RNA.

- For first use of gene name in the manuscript define, as definition of gene names occurs later in the manuscript or in figure legends

We apologize for the oversight. We have now included the full gene name when they are initially used in the manuscript.

- The evidence for IDH1 is mainly through association based on experimental observations and the literature. It would have been good if the authors had demonstrated that 181c is directly acting on the IDH1 3’UTR in a liver cell to provide more supporting evidence for the authors conclusion.

We agree with the reviewer. However, the dual-luciferase assay to demonstrate the direct association of the miR-181 “seed” sequence and the 3’-UTR of IDH1 mRNA in hepatocytes has already been performed 3. The putative site (UGAAUGU) for miR-181a and miR-181s is identical - that is the “seed” sequence for the miR-181 family 1. Chu et al., 3 has demonstrated that the miR-181 “seed” sequence can directly bind to the 3’-UTR of IDH1 mRNA. Even though the conclusion of the Chu et al., study3 is different than our current study, the dual-luciferase assay is identical to what the reviewer has suggested we do.

- Discussion, I would present some ideas about why 181c has a cytosolic v. mitochondrial location in the liver in contrast to the heart. Are any gender differences anticipated/known, as authors have used a male mouse model. I would be more reserved on the use of 181c as a protective agent for obesity as in the context of the liver it can act as an Oncomir too.

Thank you for the suggestion. We have now discussed this important issue on pages 16-17 within the discussion section.

Additionally, thank you for raising the important question. We now have performed qPCR for miR-181c expression in both age-matched male and female mouse livers. Our results showed that miR-181c expression in female mouse livers is significantly higher than the age-matched male group.

While it is possible that miR-181c can act as an OncomiR, several studies have shown that miR-181c overexpression can attenuate tumor growth and metastasis for hepatocellular carcinoma (HCC) 4,10.

References

1. Das S, Kohr M, Dunkerly-Eyring B, et al. Divergent Effects of miR-181 Family Members on Myocardial Function Through Protective Cytosolic and Detrimental Mitochondrial microRNA Targets. J Am Heart Assoc. 2017;6(3).

2. Banavath HN, Roman B, Mackowski N, et al. miR-181c Activates Mitochondrial Calcium Uptake by Regulating MICU1 in the Heart. J Am Heart Assoc. 2019;8(24):e012919.

3. Chu B, Wu T, Miao L, Mei Y, Wu M. MiR-181a regulates lipid metabolism via IDH1. Sci Rep. 2015;5:8801.

4. Mukherjee A, Shrivastava S, Bhanja Chowdhury J, Ray R, Ray RB. Transcriptional suppression of miR-181c by hepatitis C virus enhances homeobox A1 expression. J Virol. 2014;88(14):7929-7940.

5. Ghadge AA, Khaire AA. Leptin as a predictive marker for metabolic syndrome. Cytokine. 2019;121:154735.

6. Boutari C, Mantzoros CS. Adiponectin and leptin in the diagnosis and therapy of NAFLD. Metabolism. 2020;103:154028.

7. Wang F, Nojima M, Inoue Y, Ohtomo K, Kiryu S. Assessment of MRI Contrast Agent Kinetics via Retro-Orbital Injection in Mice: Comparison with Tail Vein Injection. PLoS One. 2015;10(6):e0129326.

8. Price JE, Barth RF, Johnson CW, Staubus AE. Injection of cells and monoclonal antibodies into mice: comparison of tail vein and retroorbital routes. Proc Soc Exp Biol Med. 1984;177(2):347-353.

9. Das S, Ferlito M, Kent OA, et al. Nuclear miRNA regulates the mitochondrial genome in the heart. Circ Res. 2012;110(12):1596-1603.

10. Ai J, Gong C, Wu J, et al. MicroRNA181c suppresses growth and metastasis of hepatocellular carcinoma by modulating NCAPG. Cancer Manag Res. 2019;11:3455-3467.

Submitted filename: Final Response to reviewers comments 10 12 2021.docx

Click here for additional data file.

11 Nov 2021

Role of miR-181c in Diet-Induced Obesity Through Regulation of Lipid Synthesis in Liver

PONE-D-21-26341R1

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Reviewer #2: The authors have addressed all my major concerns. Though the author's discussion on the organ specific differences of 181c ie heart and liver, would of benefit by being expanded on eg the metabolic function of the liver v the heart primary function of producing sufficient ATP for cardiomyocyte contractility.

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24 Nov 2021

PONE-D-21-26341R1

Role of miR-181c in Diet-induced obesity through regulation of lipid synthesis in liver

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Table 1

Primers used in qPCR analysis for gene candidates of interest.

Gene	Primer sequence (5′ → 3′)
Mouse	Forward	Reverse
16S rRNA	ACC GCA AGG GAA AGA TGA AA	GCC ACA TAG ACG AGT TGA TTC
ACLY	ACC AGA AGG GAG TGA CCA TC	GAT GTT GTC CAG CAT TCC AC
β-Actin	GGC TGT ATT CCC CTC CAT CG	CCA GTT GGT AAC AAT GCC ATG T
FASN	TGA GAT CCC AGC ACT TCT TG	TGA CAT GAA CAT TGG AGC CT
GAPDH	AGG TCG GTG TGA ACG GAT TTG	TGT AGA CCA TGT AGT TGA GGT CA
SREBP1	AGC AGG AGA ACC TGA CCC TA	TTT CAT GCC CTC CAT AGA CA
5S rRNA	TCT CGT CTG ATC TCG GAA GC	AGC CTA CAG CAC CCG GTA TT
IDH1	ATG CAA GGA GAT GAA ATG ACA CG	GCA TCA CGA TTC TCT ATG CCT AA
IDH2	GAC AAG CAC TAT AAG ACT GAC	TCT GGT GTT CTC GGT AAT G