Hepatic Ago2 regulates expression of specific miRNAs involved in energy metabolism

Ago1 and Ago2 are the predominant Ago family members expressed in the liver25. To investigate if RNA silencing is associated with energy and metabolic homeostasis, we generated liver-specific Ago1-deficient (L-Ago1 KO: Ago1fl/fl Alb-CreTg/0) and Ago2-deficient (L-Ago2 KO: Ago2fl/fl Alb-CreTg/0) mice (Supplementary Fig. 1a and b). On control diet (CD), both L-Ago1 KO and L-Ago2 KO mice gained weight similarly to their controls, L-Ago1 WT (Ago1fl/fl Alb-Cre0/0) or L-Ago2 WT (Ago2fl/fl Alb-Cre0/0), and showed no obvious abnormalities during development or in adulthood. In addition, levels of serum alanine aminotransferase (ALT) were comparable between the groups (Supplementary Fig. 1c and d). These results suggest that general functions of the liver are likely unaffected by the absence of Ago1 or Ago2 during development in regular feeding conditions.

Among the Ago proteins, Ago2 uniquely possesses a slicer activity known to contribute to the expression of specific miRNA21,22,23 and mRNA cleavage18,26. In the Ago2-deficient livers, expression levels of Ago1 are increased (Supplementary Fig. 1b), and therefore Ago1 may compensate for Ago2’s non-slicer activity-dependent function. To gain insight into the specific roles of Ago2’s activity in the regulation of liver function, we first assessed the effect of hepatic Ago2-deficiency on miRNA expression profile. Expression profile analyses revealed that the expression levels of 25 miRNAs were significantly reduced in L-Ago2 KO liver, while 8 miRNAs were significantly increased (Fig. 1a, b). Among these significant miRNAs, miR-148a is one of the most abundant miRNAs expressing in the WT liver (Fig. 1b and Supplementary Fig. 2a). In addition, 17 out of 33 total significant miRNAs abundantly expressed were in the top 10 percent of miRNAs expressing in the WT liver (Fig. 1c and Supplementary Table 1). Intriguingly, this group contained miRNAs known to be associated with metabolic diseases (MD-miRNAs) and detrimental to glucose and lipid metabolism, including miR-802, miR-103/107, miR-130a, and miR-148a12,13,27,28,29, which were downregulated in the L-Ago2 KO liver (Fig. 1c). We additionally utilized the list of significant miRNAs altered in Ago2 KO liver through the miRNA enrichment pathway analysis by the biological processes and molecular function categories (Fig. 1d). Hepatic Ago2 deficiency affected the functional clades associated with energy metabolism including fatty acid biosynthesis, AMPK signaling, protein processing, and insulin signaling. Taken together, these results imply the unique role of Ago2 in the expressional regulation of a specific repertoire of MD-miRNAs for energy metabolism. Consistent with these observations, the reduction of MD-miRNAs in L-Ago2 KO liver is accompanied by increased expression of their known target mRNAs, such as hepatocyte nuclear factor 1 homeobox B (Hnf1β), caveolin-1 (Cav1), peroxisome proliferator-activated receptor gamma coactivator 1α (Pgc1α), and low-density lipoprotein receptor (Ldlr), which regulates energy metabolism (Fig. 1e)12,13,29.

Fig. 1 Effects of hepatic Ago2-deficiency on MD-miRNA expression in the liver. a–c A heatmap diagram illustrating the differential expression of hepatic mature miRNAs (a), raw counts of significant miRNAs normalized by DESeq2 and transformed by Log2 (b), and fold changes of significant miRNAs whose expression levels are in the top 10 percent in the WT liver (c) in the liver of L-Ago2 WT (n = 3) and L-Ago2 KO (n = 3) mice fed NCD at 9 weeks of age. Significant miRNAs differentially expressed between L-Ago2 WT and L-Ago2 KO groups were identified using DESeq2 (|fold change| > 2x and adjusted p < 0.05) and plotted as a heatmap using z-score. d Metabolic pathway enrichment analysis of miRNAs significantly downregulated (blue) and upregulated (red) miRNAs in the liver of L-Ago2 KO mice fed NCD at 9 weeks of age. These miRNAs were queried to calculate the most enriched KEGG pathways using DIANA-mirPath web-server (p < 0.05 and MicroT threshold < 0.8). Pathways unrelated to hepatic functions were excluded in this pyramid plot. e–g Expression levels of MD-miRNAs’ target mRNAs (e), selective MD-miRNAs (f), and their pri-miRNAs (g) in the liver of L-Ago2 WT (n = 8) and L-Ago2 KO (n = 8) mice fed NCD at 25 weeks of age. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

To examine the mechanism by which hepatic Ago2 regulates expression of specific miRNAs, we measured the expression levels of each mature and primary miRNA (pri-miRNA) employing TaqMan probe-based gene expression analysis. Mature miRNA levels of miR-802, miR-107/miR-103, miR-130a, and miR-148a/148b/152, were reduced in L-Ago2 KO liver (Fig. 1f), despite intact expression levels of their pri-miRNAs (Fig. 1g). These results suggest that the miRNA maturation process is impaired in the Ago2-deficient liver. To further confirm this regulation, we utilized Ago2-deficient mouse embryonic fibroblasts (MEFs) reconstituted with wild type Ago2 (Ago2 WT) or slicer-defective mutant Ago2 (Ago2 D669A, or “DA,” containing an aspartate to alanine substitution at residue 669)22,23,24. Expression levels of these MD-miRNAs were increased by reconstitution of Ago2 WT (Supplementary Fig. 2b). Importantly, Ago2 D669A mutant only partially induced expression of key MD-miRNAs including miR-107, miR-103, and miR-130a, while expression of miR-148b required Ago2 but not its slicer activity (Supplementary Fig. 2b). While the loss of Dicer also caused a reduction of MD-miRNAs (Supplementary Fig. 2c), these results support a crucial role of Ago2 in the expression of a subset of MD-miRNAs.

While Dicer recognizes the 5′ phosphate end and 2-nucleotide 3′ overhang structure of precursor miRNA for precise and effective biogenesis of miRNAs30,31, recent studies have provided different mechanistic insights into the Ago2-mediated processing of miRNA. One of the proposed characteristics of miRNAs processed by Ago2 is that their precursors have a relatively shorter loop size that likely prevents recognition by Dicer. Moreover, these precursors have no mis-matching at position 10 or 11 between guide and passenger strands32. The miRNAs with reduced expression in L-Ago2 KO liver tended to have shorter loop sizes than those induced by L-Ago2 KO liver, and had no mis-matching at positions 10 or 11 (Supplementary Fig. 2d–f). This information suggests that there may be structural similarities among MD-miRNAs that require Ago2 for maturation.

We next asked if any of the 25 significant miRNAs, Ago1, or Ago2 might have disease associations proximal to their orthologous human miRNAs and genes. To this end, we first identified mouse-human orthologous miRNAs using data from miRBase33 and miROrtho34, identifying a total of 27 human miRNAs (some mouse miRNAs do not have clear orthologs or map to two human miRNAs) (Supplementary Table 2). We then examined a large collection of genetic variants associated with 213 diseases and phenotypes collected from the National Human Genome Research Institute (NHGRI) Catalog of Published Genome-Wide Association Studies (GWAS)35 that have been expanded to include additional variants in strong linkage disequilibrium (r2 > 0.8) with the tagged variants36. This analysis revealed that several of the orthologous human miRNAs have proximal GWAS signal, often for relevant phenotypes (Supplementary Table 3). For example, a genetic variant (rs6953596) located 245 bases away from the gene encoding miR-148a is strongly associated with body mass index (BMI) in African Americans and thus might act by altering gene regulatory mechanisms controlling the expression of miR-148a. In addition, while there is no detectable GWAS signal near Ago1, there is suggestive GWAS signal (p = 10−6) located within an intron of Ago2 for “Thiazide-induced adverse metabolic effects in hypertensive patients” in African Americans37. These results suggest a possible association of Ago2 and miRNAs whose expression is regulated by Ago2 with human metabolic diseases.

Inactivation of hepatic Ago2 improves systemic glucose metabolism

Considering the miRNA enrichment pathway analysis indicated that hepatic Ago2 is implicated in glucose metabolism, we then investigated Ago2’s role in regulating this regard. We observed that L-Ago2 KO mice fed normal chow diet (NCD) exhibited enhanced capacities for glucose metabolism, as assessed by glucose, insulin, and pyruvate tolerance tests (GTT, ITT, and PTT) after 20 weeks of age (Fig. 2a–c). These results suggest that hepatic Ago2 deficiency improves insulin sensitivity and inhibits gluconeogenesis, leading to glucose tolerance. To investigate how Ago2 regulates glucose metabolism, we first examined capacities of hepatic gluconeogenesis in Ago2-deficient primary hepatocytes. Glucose production was similarly induced between genotypes (Fig. 2d). We then asked if Ago2 regulates the fundamental catabolic capacities of hepatocytes. To examine the glycolytic rate, the extracellular acidification rate (ECAR) was determined in primary hepatocytes upon addition of glucose. In the presence of oligomycin, Ago2-deficient hepatocytes showed a higher increase in ECAR compared to controls (Fig. 2e). To determine whether Ago2 regulates oxidation of pyruvate, we measured mitochondrial oxygen consumption rate (OCR) in WT and Ago2-deficient hepatocytes in the presence of pyruvate. Upon the addition of the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), Ago2-deficient hepatocytes greatly upregulated oxygen consumption compared to WT controls (Fig. 2f). In a complementary approach, we also measured ATP content/ADP content in these hepatocytes in the absence or presence of pyruvate. The basal ATP/ADP ratio in Ago2-deficient hepatocytes was higher than that in WT controls, and this difference was even greater upon addition of pyruvate (Fig. 2g). Taken together, these data indicate that hepatic Ago2 functions to enhance gluconeogenesis and suppress glucose oxidation while its inactivation results in increased glucose-driven energy production.

Fig. 2 Hepatic Ago2-deficiency improves glucose metabolism. a Glucose tolerance test performed in L-Ago2 WT (n = 10) and KO (n = 8) mice fed NCD at 20 weeks of age. b Insulin tolerance test performed in L-Ago2 WT (n = 9) and KO (n = 6) mice fed NCD at 21 weeks of age. c Pyruvate tolerance test performed in L-Ago2 WT (n = 9) and KO (n = 10) mice fed NCD at 24 weeks of age. d Glucose production in primary hepatocytes isolated from L-Ago2 WT and L-Ago2 KO incubated in the absence (n = 4 for L-Ago2 WT and L-Ago2 KO, respectively) or presence of 100 or 200 μM Bt-cAMP or pCPT-cAMP (n = 2 for L-Ago2 WT and L-Ago2 KO, respectively). e Extracellular acidification (ECAR) in the absence or presence of 10 mM glucose in primary hepatocytes isolated from L-Ago2 WT (n = 3 for control, n = 6 for glucose) and L-Ago2 KO mice (n = 3 for control, n = 6 for glucose). f Mitochondrial oxygen consumption rate (OCR) in the absence or presence of 2 mM pyruvate in primary hepatocytes isolated from L-Ago2 WT (n = 3 for control and pyruvate, respectively) and L-Ago2 KO mice (n = 3 for control and pyruvate, respectively). g The ATP/ADP ratio in primary hepatocytes isolated from L-Ago2 WT and L-Ago2 KO mice in the absence (n = 6 for L-Ago2 WT and L-Ago2 KO, respectively) or presence of 5 mM pyruvate (n = 6 for L-Ago2 WT and L-Ago2 KO, respectively). Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

Hepatic Ago2 regulates glucose metabolism in insulin insufficiency

If hepatic Ago2-deficiency improves systemic glucose metabolism by suppressing gluconeogenesis and accelerating glucose oxidation in the liver, other diabetic conditions may also be improved by hepatic Ago2-deficiency. To examine this possibility, we employed a pharmacological model by administering the insulin antagonist peptide, S961 (43 amino acids in length). S961 binds to the insulin receptor and blocks insulin signaling in vivo to acutely induce hyperglycemia38. This model allowed us to further assess the role of hepatic Ago2 in glycemic control without the potential confounding effects of body weight and adiposity. After infusing S961 into L-Ago2 WT and L-Ago2 KO mice fed NCD at 12 weeks of age, we examined the effect of hepatic Ago2-deficiency on S961-induced deterioration of glucose metabolism. At this age, while glucose metabolism assessed by GTT was comparable between the genotypes in a PBS-treated control group, S961 treatment caused hyperglycemia in WT mice 1 week after treatment (Fig. 3a), and hyperinsulinemia and glycogenolysis in WT mice after 2 weeks of treatment (Fig. 3b, c). Remarkably, L-Ago2 KO mice are resistant to S961-induced glucose intolerance after 1 week of treatment (Fig. 3a) and S961 treatment of L-Ago2 KO mice resulted in a lower induction of plasma insulin levels and higher hepatic glycogen contents compared with control mice (Fig. 3b, c). These data indicate that inactivation of hepatic Ago2 improves systemic glucose metabolism in the condition of insulin insufficiency.

Fig. 3 Hepatic Ago2-deficiency prevents S961-induced acute glucose intolerance. a Glucose tolerance tests at one-week post treatment of S961 or phosphate-buffered saline (PBS). L-Ago2 WT (n = 10 for PBS and n = 13 for S961) and KO (n = 11 for PBS and n = 14 for S961) mice fed NCD at 9 weeks of age were continuously treated with S961 (10 nM/week) via osmotic pumps. The graph on the right shows an integrated area under the glucose disposal curves (AUC) for each condition. b Serum insulin levels after daytime food withdrawal for 6 h in L-Ago2 WT and KO mice at 2 weeks post S961 (n = 7, each genotype) or PBS (n = 6, each genotype) treatment. c Hepatic glycogen contents in L-Ago2 WT and KO mice at 2 weeks post S961 (n = 7, each genotype) or PBS (n = 5, each genotype) treatment. d A heatmap diagram illustrating the differential expression of mature miRNAs in the liver of L-Ago2 WT and KO mice treated with PBS or S961 for 2 weeks. Significant miRNAs differentially expressed between genotypes were identified using DESeq2 (|fold change| > 1.25x and adjusted p < 0.0005). Clusters I and IV are miRNAs differentially expressed between L-Ago2 WT (n = 6) and L-Ago2 KO (n = 6) groups. Clusters II and III are miRNAs differentially expressed by S961 treatment in WT and KO groups, respectively. The log2 expression values were scaled by z-score. e, f Effect of S961-treatment on expression of MD-miRNAs (e) and genes regulating energy metabolism (f) in the liver of L-Ago2 WT and KO mice treated with PBS or S961 (n = 6, each group) for 2 weeks. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

Expression profiles that assessed the effect of insulin insufficiency on miRNAs in the liver categorized four different classes of miRNAs; (I) dominantly expressed in L-Ago2 WT, (II) induced by S961 in both L-Ago2 WT and KO, (III) suppressed by S961 in both L-Ago2 WT and KO, and (IV) dominantly expressed in L-Ago2 KO (Fig. 3d). Importantly, S961 treatment did not modulate expression levels of miRNAs categorized in class (IV) in both L-Ago2 WT and KO liver. Conversely, several miRNAs in class (I) that contains MD-miRNAs including miR-802, miR-103/107, and miR-130a were strikingly induced by S961 treatment in the WT liver in an Ago2-dependent manner (Fig. 3d, e). These analyses implicate hepatic Ago2-dependent miRNAs play a role in the disruption of glucose metabolism under the condition of insulin insufficiency. Despite the decrease of miR-802 and miR-103/107 expression in the Ago2-deficient condition, expression levels of their known targets, Hnf1β and Cav1, were comparable between the genotypes even in PBS-treated groups at this age (Fig. 3f). While expression of glucose-6-phosphatase (G6Pase), a gluconeogenic gene, was similarly induced between the genotypes (Fig. 3f), expressions of genes critical for mitochondrial function, such as Pgc1α, peroxisome proliferator-activated receptor alpha (Pparα), mitochondrial transcription factor (Tfam), and citrate synthase (Cs) were higher in the liver of L-Ago2 KO mice. These results, along with the Ago2-deficient hepatocyte analyses, suggest that Ago2 regulates the program of a subset of miRNAs involved in energy metabolism, which may be associated with enhancement of gluconeogenesis and suppression of glycolysis and hepatic mitochondrial oxidation induced by insulin insufficiency.

Critical roles of hepatic Ago2 in energy metabolism on high-fat-diet challenge

We next asked if nutrient challenge might accentuate Ago2’s role in metabolic regulation. We thus employed a high-fat diet (HFD)-induced obesity model that induces insulin resistance, glucose intolerance, and hepatic steatosis. We placed L-Ago2 KO, and L-Ago1 KO, and control WT mice on HFD or a control diet (CD), commencing at 4 weeks of age (Fig. 4a and Supplementary Fig. 3a). Of note, the body weights of the HFD-fed L-Ago2 KO mice became lower than that of controls and the difference reached statistical significance at 22 weeks of age (Fig. 4a). Conversely, the body weight of L-Ago1 KO mice was comparable to controls in the HFD condition (Supplementary Fig. 3a). The improvements in glucose metabolism observed in the HFD-fed L-Ago2 KO were even more pronounced when compared to the CD-feeding condition, as assessed by GTT, ITT, and PTT (Fig. 4b–d, Supplementary Fig. 4a and b). These improvements were not observed in L-Ago1 KO fed HFD (Supplementary Fig. 3b–f). Consistent with improved systemic glucose tolerance and insulin sensitivity in L-Ago2 KO mice, HFD-induced pancreatic β-cell proliferation and islet hypertrophy were attenuated compared to L-Ago2 WT mice (Supplementary Fig. 4c–g), supporting that hepatic Ago2-deficiency improves insulin sensitivity in the pathogenesis of obesity.

Fig. 4 Ago2-deficiency in the liver improves glucose metabolism in obesity. a Body weights of L-Ago2 WT (n = 16) and KO (n = 17) mice fed HFD, and L-Ago2 WT (n = 15) and KO (n = 11) mice fed CD, starting in 4 weeks of age. b GTT performed in L-Ago2 WT (n = 16) and KO (n = 17) mice fed HFD at 20 weeks of age. c ITT performed in L-Ago2 WT (n = 16) and KO (n = 17) mice fed HFD at 14 weeks of age. d PTT performed in L-Ago2 WT (n = 8) and KO (n = 8) mice fed HFD at 17 weeks of age. e–h Hyperinsulinemic-euglycemic clamp studies performed in L-Ago2 WT (n = 6) and L-Ago2 KO (n = 10) mice fed HFD for 20 weeks. e Glucose infusion rates (GIR) throughout the clamp procedures. The graph on the right shows an integrated area under curves (AUC) of GIR. f Insulin clearance levels during the clamp. g Tissue glucose uptakes in gastrocnemius muscle, visceral fat, and subcutaneous fat tissues. h Suppression of hepatic glucose production (sHGP) during the clamp. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

To further demonstrate the role of hepatic Ago2, we performed the hyperinsulinemic-euglycemic clamp study to examine the whole-body glucose metabolism and insulin sensitivity. Glucose infusion rates (GIR) during the clamp studies indicated that L-Ago2 KO mice required significantly higher levels of glucose infusion to maintain blood glucose consistent with increased insulin sensitivity (Fig. 4e and Supplementary Fig. 4h). Insulin clearance and glucose uptakes in gastrocnemius muscle, visceral and subcutaneous fat, brown adipose tissue, and heart were comparable (Fig. 4f, g and Supplementary Fig. 4j). Conversely, hepatic glucose production was significantly suppressed in L-Ago2 KO mice (Fig. 4h and Supplementary Fig. 4k). These studies indicate that the liver is the main locus responsible for improving systemic insulin sensitivity and glucose metabolism in L-Ago2 KO mice.

Importantly, the liver of L-Ago2 KO mice was characterized by lowered liver weights and triglyceride content, accompanied by lower serum ALT levels on HFD (Fig. 5a–c). In addition, plasma triglyceride levels were lower in L-Ago2 KO mice fed HFD, while those of cholesterol, phospholipids, and free fatty acids were comparable between the genotypes (Supplementary Fig. 5a–d). There was also a reduction in hepatic fatty infiltration as visualized by haematoxylin and eosin (H&E) staining (Fig. 5d). While levels of genes involved in lipid biosynthesis such as stearoyl-CoA desaturase-1 (Scd1) and fatty acid synthease (Fasn) were comparable between the genotypes, those of carnitine palmitoyltransferase 1A (Cpt1a) and acetyl-coenzyme A synthetase 2-like (Acss1) that mediate catabolic processes of fatty acids were increased in the livers of L-Ago2 KO mice (Fig. 5e). Consistently, mitochondrial OCR in response to palmitate and acetate whose circulating levels are positively correlated with obesity and its related sequelae39 was significantly higher in Ago2-deficient hepatocytes compared to controls (Fig. 5f). These cellular phenotypes could promote reduction of hepatic triglyceride accumulation and lowered hepatic steatosis levels we have observed in the liver of L-Ago2 KO mice fed HFD. Quantitative Magnetic Resonance technology (EchoMRI) analyses revealed that total body fat mass in L-Ago2 KO mice is lower than in WT controls (44.3% fat reduction in L-Ago2 KO: p < 0.05, t-test) (Supplementary Fig. 5e), while lean mass composition of L-Ago2 KO mice was slightly higher than that of control mice on HFD at 20 weeks of age (Supplementary Fig. 5f). In addition, the ability to utilize fat mass, which was calculated by measurements of fat and lean compositions before and after an overnight fast, was higher in L-Ago2 KO mice on HFD compared to WT controls (1.675-fold higher in L-Ago2 KO: p < 0.01, t-test) (Fig. 5g). In support of these observations, there was an increased copy number of mitochondrial-DNA (mtDNA) in the Ago2-deficient liver in obesity (Fig. 5h). Consistently, rates of energy expenditure of L-Ago2 KO mice fed HFD for 12 weeks were significantly higher than those of controls (Fig. 5i and Supplementary Fig. 5g and h), despite no significant changes in body weight, total physical activity, food intake, or amounts of fecal lipids between genotypes (Supplementary Fig. 5i–l). We performed similar experiments with L-Ago1 WT and L-Ago1 KO mice fed HFD and found that there were no differences in the regulation of energy homeostasis between the genotypes (Supplementary Fig. 3g–m). Taken together, these data indicate that inactivation of Ago2, but not Ago1, in the liver increases mitochondrial capacity and energy expenditure, which appears to link to improvement of obesity-associated pathophysiology.

Fig. 5 Ago2-deficiency in the liver prevents hepatic steatosis with enhanced energy expenditure. a Liver weight in L-Ago2 WT mice fed CD (n = 5), L-Ago2 KO mice fed CD (n = 4), L-Ago2 WT mice fed HFD (n = 5), and L-Ago2 KO mice fed HFD (n = 5) at 30 weeks of age. b, c Liver triglyceride (TG) contents (b) and serum ALT levels (c) in L-Ago2 WT (n = 7) and L-Ago2 KO (n = 5) mice fed HFD at 23 weeks of age. d H&E-stained sections of the liver in each genotype at 30 weeks of age. Scale bar, 100 μm. e Expression levels of key mRNAs involved in energy metabolism in the liver of L-Ago2 WT (n = 8) and L-Ago2 KO (n = 5) mice fed HFD at 23 weeks of age. f Levels of β-oxidation in the presence of 0.12 mM palmitate and mitochondrial OCR in the presence of 5 mM acetate in primary hepatocytes isolated from L-Ago2 WT (n = 3 for control, n = 3 palmitate and acetate, respectively) and L-Ago2 KO mice (n = 3 for control, n = 3 palmitate and acetate, respectively). g Effects of a 14-h fast on fat mass, and lean body mass in L-Ago2 WT (n = 9) and KO (n = 10) mice fed HFD at 20 weeks of age. h Copy numbers of mtDNA in L-Ago2 WT (n = 7) and KO (n = 5) mice fed HFD at 23 weeks of age. i Energy expenditure in L-Ago2 WT (n = 8) and KO (n = 8) mice fed HFD at 16 weeks of age. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

Ago2-mediated RNA silencing regulates expression of genes involved in energy metabolism

To investigate molecular mechanisms by which Ago2 orchestrates hepatic energy metabolism, we additionally profiled hepatic miRNA expression under the condition of HFD (Fig. 6a). Utilizing the list of significant miRNAs on HFD, the miRNA target pathway enrichment analysis revealed that hepatic Ago2 deficiency affected several functional clades such as glucose and lipid metabolism and protein translation regulation (Fig. 6b). While Ago2 protein levels were slightly decreased in the liver of mice fed HFD (Supplemental Fig. 6a), several MD-miRNAs were highly induced in the liver of L-Ago2 WT mice fed HFD and a leptin-deficient (ob/ob) obese mice (Fig. 6c and Supplemental Fig. 6b). Importantly, these miRNAs are constantly decreased in that of L-Ago2 KO mice (Fig. 6a, c). Consistent with these observations, expression levels of their known target mRNAs, such as Hnf1β, Cav1, and Pgc1α, are increased in L-Ago2 KO liver (Fig. 6d). Of note, expression levels of these genes were comparable between L-Ago1 WT and KO liver (Supplementary Fig. 3n).

Fig. 6 Hepatic Ago2 regulates expression of MD-miRNAs and Ampka1 in obesity. a A heatmap diagram illustrating the differential expression of mature miRNAs in the liver of L-Ago2 WT (n = 3) and L-Ago2 KO (n = 3) mice fed HFD for 16 weeks. Significant miRNAs differentially expressed between L-Ago2 WT and L-Ago2 KO groups were identified using DESeq2 (|fold change| > 2x and adjusted p < 0.05) and plotted as a heatmap using z-score. b Metabolic pathway enrichment analysis of miRNAs significantly down-regulated (blue) and up-regulated (red) miRNAs in the liver of L-Ago2 KO mice fed HFD for 16 weeks. These miRNAs were queried to calculate the most enriched KEGG pathways using DIANA-mirPath web-server (p < 0.05 and MicroT threshold < 0.8). Pathways unrelated to liver functions were excluded in this pyramid plot. c Expression levels of specific MD-miRNAs in the liver of L-Ago2 WT mice (n = 8) fed NCD at 25 weeks of age, and L-Ago2 WT (n = 7) and L-Ago2 KO (n = 5) mice fed HFD at 23 weeks of age. d Expression levels of MD-miRNAs’ target mRNAs and pri-miRNAs in the liver of L-Ago2 WT (n = 7) and L-Ago2 KO (n = 5) mice fed HFD at 25 weeks of age. e Compared expression levels of Ampka1, miR-148/152, and their pri-miRNAs in primary hepatocytes isolated from L-Ago2 WT and L-Ago2 KO mice. f Relative luciferase activity by which Ampka1 3'﻿ UTR with or without harboring a mutation at miR148/152 putative target site was assessed in primary hepatocytes isolated from L-Ago2 WT (n = 4 for control, n = 6 for Ampka1 3′ UTR and Ampka1 3′ UTR-M, respectively) and L-Ago2 KO mice (n = 4 for control, n = 6 for Ampka1 3′ UTR and Ampka1 3′ UTR-M, respectively). g Quantification of Ampka1, Cs, and β-actin mRNA levels in fractions collected from polysome profiles of primary hepatocytes isolated from L-Ago2 WT and L-Ago2 KO mice. The graphs show the quantification of the results. h A proposed role of hepatic Ago2 in the regulation of glucose and lipid metabolism in the liver. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

To explore targets of Ago2-dependent MD-miRNAs for metabolic regulation, we then took a bioinformatics approach with miRNA sequencing data obtained under lean, HFD, and S961-treated conditions (Supplementary Fig. 6c and d). With the Ago2-dependent miRNAs, we extracted lists of predicted conserved target genes involved in energy metabolism from the widely used TargetScan 7.1 website. This analysis identified a subset of genes, including Ampka1 (also known as Prkaa1, a catalytic subunit of AMPK that plays a critical role in AMPK activation) and Cs, that have 3′ untranslated region (UTR) containing multiple target sites for Ago2-dependent miRNAs including miR-148a/152 known to evoke hyperlipidemia, hypercholesteremia, and atherosclerosis14,15 (Supplementary Fig. 6e and f). As AMPK is known as a critical regulator of energy metabolism, we further assessed the role of Ago2 in Ampka1 expression. Analyzing a public database of photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) with Ago240 revealed that Ago2 binds to the region of the Ampka1 3′ UTR that contains binding sites of Ago2-dependent miRNAs including miR-148/152 and miR-130a (Supplementary Fig. 6g). Consistent with these findings, we confirmed the induction of Ampka1 expression, accompanied by the reduction of miR-148a, miR-148b, and miR-152, in the liver of L-Ago2 KO mice fed HFD and in Ago2-deficient primary hepatocytes (Fig. 6d, f). We next conducted luciferase assays in which Ampka1’s 3′ UTR, with or without harboring a mutation at the miR-148/152 target site, was sub-cloned into luciferase expression vector. The luciferase activity of each was measured in primary hepatocytes isolated from L-Ago2 WT and KO mice. Luciferase activity was higher in Ago2 KO hepatocyte transfected with Ampkα1 3′ UTR without a mutation compared with Ago2 WT hepatocyte, but the induction in Ago2 KO hepatocyte disappeared in the setting with mutated Ampka1 3' UTR (Fig. 6f). These analyses demonstrated that miR-148/152 are involved in suppression of Ampka1 expression in a manner dependent on Ago2 and a miR-148/152 target site (Fig. 6f). As miRNA inhibits the translation of target mRNAs through RNA silencing, we additionally asked if Ago2-deficiency affects translation of genes having target sites of MD-miRNAs by investigating polysome-bound mRNA expression patterns. Expression levels of polysome-bound Ampka1 and Cs were enriched in Ago2-deficient primary hepatocytes, while those of β-Actin were comparable (Fig. 6g). Taken together, these findings further confirm that hepatic Ago2-mediated MD-miRNA expression and RNA silencing are linked to expressional regulation of genes involved in energy metabolism (Fig. 6h).

Hepatic Ago2 regulates energy consumption associated with AMPK activation

We next examined AMPKα1 protein levels and noticed that Ago2 deficiency increased not only the protein levels but also activity of AMPK, assessed by phosphorylation levels of AMPKα, AMPKβ, and an AMPK substrate, Acetyl-CoA carboxylase (ACC), in the liver of L-Ago2 KO mice fed HFD and treated with S961 (Fig. 7a and Supplementary Fig. 7a and b). In agreement with the activation of AMPK, other AMPK substrates, UNC-51-like kinase 1 (ULK1), and Mitochondrial fission factor (MFF) are increased in the liver of L-Ago2 KO mice (Supplementary Fig. 7a), suggesting enhanced autophagy/mitophagy and improved mitochondrial quality in the Ago2-deficient liver in obesity. Consistently, expression levels of the Tfam-mitochondrial gene pathway are increased in the liver of L-Ago2 KO mice fed HFD (Fig. 7b).

Fig. 7 Hepatic Ago2-deficiency enhances energy expenditure associated with protein synthesis and AMPK activation. a, b Western blot analyses of AMPK expression and activation (a) and mRNA expression of the Tfam-mitochondrial genes (b) in the liver of L-Ago2 WT (n = 5) and L-Ago2 KO (n = 5) fed HFD at 25 weeks of age. c ATP, ADP, and ATP/ADP ratio levels in L-Ago2 WT (n = 5) and L-Ago2 KO (n = 5) mice fed HFD at 25 weeks of age. ATP/ADP ratio levels were independently measured with a distinct procedure from the ATP and ADP assays. d Western blot analysis of total and specific protein levels normalized by 12S-genomic DNA in the liver of L-Ago2 WT (n = 5) and KO (n = 5) mice fed HFD at 30 weeks of age. e Serum albumin levels in L-Ago2 WT (n = 8) and L-Ago2 KO (n = 8) mice fed HFD at 25 weeks of age. f Energy consumption rate measured in primary hepatocytes isolated from L-Ago2 WT (n = 4, n = 4, n = 3 for 0, 2 h, 5 h, respectively) and L-Ago2 KO (n = 4, n = 4, n = 4 for 0, 2 h, 5 h, respectively) mice in the presence of 1 mM metformin. g Effect of Ago2-deficiency on expression of AMPK activation in primary hepatocytes isolated from L-Ago2 WT (n = 8, n = 8, n = 8 for 0, 2 h, 5 h, respectively) and L-Ago2 KO (n = 8, n = 8, n = 8 for 0, 2, 5 h, respectively) mice in the presence of 1 mM metformin. The graphs show the quantification of the results. h Effect of Ago2 on nascent protein synthesis. Primary hepatocytes isolated from L-Ago2 WT and L-Ago2 KO mice were treated with or without 200 μM Phenformin (Phen) or 10 μM Rotenone (Rote) for 5 h (n = 15 for control, n = 9 for Phen, n = 6 for Rote). i A proposed role of hepatic Ago2 in suppression of protein translation, leading to AMPK activation. The graphs show the quantification of the results. Data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 Full size image

AMPK is activated when cellular energy level becomes low. Indeed, we found a profound induction of ADP levels in the Ago2-deficient liver, while ATP levels are comparable between the genotypes, leading to a reduction of ATP/ADP ratio in the liver of L-Ago2 KO mice fed HFD (Fig. 7c). While Ago2-deficiency enhances capacity for mitochondrial oxidation and ATP production in hepatocytes (Figs. 2g, 5f), systemic energy expenditure is also increased (Fig. 5i). Therefore, we hypothesized that both energy production and consumption are enhanced in the liver of L-Ago2 KO mice compared to their controls. Since a main function of Ago2 is to suppress protein translation, which is one of the most energy consuming cellular processes, through RNA silencing, we investigated the effect of Ago2-deficiency on protein synthesis in the liver. Levels of total and specific proteins normalized by DNA contents were higher in the liver of L-Ago2 KO mice (Fig. 7d and Supplementary Fig. 7c). Similarly, examination of the levels of hepatic and serum albumin, one of the most abundant circulating proteins produced by the liver, revealed that the albumin levels were increased in L-Ago2 KO mice compared to their controls (Fig. 7d, e). These observations suggest that enhanced protein synthesis in the liver may result in a lowered ATP/ADP ratio and AMPK activation in L-Ago2 KO mice.

To further examine if Ago2 deficiency accelerates cellular energy consumption associated with protein synthesis, we treated primary hepatocytes with metformin, which is an anti-diabetic drug and inhibits mitochondrial respiratory-chain complex I activity restricting ATP generation41, and measured ATP/ADP levels. Consistent with enhanced capacity for energy production in Ago2-deficiency (Fig. 2g), relative ATP/ADP levels were higher in Ago2-deficient hepatocytes compared to controls, however, the levels were rapidly decreased post-metformin treatment, suggesting a higher energy consumption rate in Ago2-deficiency (Fig. 7f). Consistently, levels of metformin-induced AMPK activation in Ago2-deficient hepatocytes were significantly higher than that in controls (Fig. 7g). To directly investigate the effect of Ago2-deficiency on protein synthesis in hepatocytes, we measured the levels of nascent protein synthesis. Compared to WT controls, the levels were significantly increased in Ago2-deficient hepatocytes (Fig. 7h). By restricting energy supply using phenformin and rotenone, both of which inhibit mitochondrial respiratory-chain complex I activity, the levels of nascent protein synthesis in Ago2-deficient hepatocytes were equivalent or still higher compared to those in controls (Fig. 7g).

Since Ago2's slicer activity uniquely regulates RNA silencing, we then asked if the slicer activity is involved in the regulation of energy consumption and AMPK activation. Ago2-deficient MEFs were characterized by enhanced expression of Ampka1, and reconstitution of the MEFs with WT Ago2 suppressed expression of both Ampka1 mRNA and AMPKα protein, whereas the Ago2 D669A mutant did not (Supplementary Fig. 8a). We also observed that AMPK activity, assessed by phosphorylation levels of AMPKα and ACC, was higher in Ago2-deficient MEFs under serum starvation condition (Supplementary Fig. 8a). While activated AMPKα is known to suppress mRNA translation42, levels of nascent protein synthesis in Ago2-deficient MEFs are increased compared to the cells reconstituted with WT Ago2 (Supplementary Fig. 8b). To investigate if enhanced protein synthesis reasons AMPK activation in Ago2 deficiency, we treated MEFs with cycloheximide (CHX), a protein synthesis inhibitor, and monitored AMPK activation. Inhibition of protein synthesis resulted in reduction of AMPK activation in WT MEFs, and the effect became more robust in Ago2-deficient MEFs, indicating that Ago2 suppresses protein synthesis-mediated energy consumption (Supplementary Fig. 8c). Taken together, these results indicate that, in addition to an increase of Ampka1 expression, there is enhanced energy consumption associated with protein synthesis, leading to the lowered ATP/ADP ratio, which appears to enhance AMPK activation in Ago2-deficient conditions (Fig. 7i).

Hepatic Ago2 deficiency improves glucose metabolism in a hepatic Ampka1-deficient condition

While hepatic Ago2-deficiency reduces expression of a specific repertoire of MD-miRNAs of which some of them target Ampka1, it is obvious that changes in expression of these miRNAs also affects translation of other target mRNAs. Similarly, enhanced protein synthesis in the liver of L-Ago2 KO mice must influence not only AMPK activation but also other cellular events linked metabolic regulation. To clarify the role of Ampka1 in the metabolic alterations in L-Ago2 KO mice, we generated liver-specific Ampka1- and Ago2-deficient mice (L-DKO mice) and placed them and their control groups, L-Ampka1 WT and L-Ampka1 KO mice, on HFD for analyses of glucose metabolism. While no significant difference was observed in body weight and fasting blood glucose levels among these three groups, L-DKO mice exhibited enhanced glucose tolerance in the condition of HFD feeding for 5 weeks (Fig. 8a–c). Plasma insulin levels of L-Ampka1 KO mice were higher than those in L-Ampka1 WT mice and the levels were drastically decreased in L-DKO mice on HFD (Fig. 8d). These results indicate that inactivation of hepatic Ago2 can improve glucose metabolism even in an Ampka1-deficient condition where insulin resistance occurs (Fig. 8d–f). Consistently, expression levels of a specific repertoire of MD-miRNAs were constantly decreased in the liver of L-DKO mice, while levels of their targets, Hnf1β, Cav1, and Pgc1α, and genes critical for enhancing mitochondrial function were higher in the liver of L-DKO mice compared to L-Ampka1 WT or L-Ampka1 KO mice (Fig. 8g). Taken together, these results suggest that the effect of hepatic Ago2-deficiency on glucose metabolism overrides that of AMPKα1 functions and that a cellular condition which leads to activation of AMPK, but not a direct activation per se, is an important factor that improves glucose metabolism observed in L-Ago2 KO mice.