Friend of GATA 2 (FOG2) is a transcriptional cofactor involved mostly in cardiac function. The aim of this study was to investigate the role of hepatic FOG2 in insulin sensitivity and lipid accumulation. FOG2 overexpression by adenovirus-expressing FOG2 (Ad-FOG2) significantly attenuates insulin signaling in hepatocytes in vitro. Opposite effects were observed when FOG2 was knocked down through adenovirus-expressing small hairpin RNA for FOG2 (Ad-shFOG2). Furthermore, FOG2 knockdown by Ad-shFOG2 ameliorated insulin resistance in leptin receptor–mutated (db/db) mice, and FOG2 overexpression by Ad-FOG2 attenuated insulin sensitivity in C57BL/6J wild-type (WT) mice. In addition, Ad-FOG2 reduced, whereas Ad-shFOG2 promoted, hepatic triglyceride (TG) accumulation in WT mice under fed or fasted conditions, which was associated with increased or decreased hepatic peroxisome proliferator–activated receptor α (PPARα) expression, respectively. Moreover, the improved insulin sensitivity and increased hepatic TG accumulation by Ad-shFOG2 were largely reversed by adenovirus-expressing PPARα (Ad-PPARα) in WT mice. Finally, we generated FOG2 liver-specific knockout mice and found that they exhibit enhanced insulin sensitivity and elevated hepatic TG accumulation, which were also reversed by Ad-PPARα. Taken together, the results demonstrate a novel function of hepatic FOG2 in insulin sensitivity and lipid metabolism through PPARα.
Insulin resistance is a risk factor for type 2 diabetes (T2D), and dysregulated hepatic lipid metabolism is the major cause of nonalcoholic fatty liver disease. Many studies have demonstrated a relationship between T2D and nonalcoholic fatty liver disease. For example, insulin resistance has been shown to accompany liver steatosis (1–3). On the other hand, improved insulin sensitivity sometimes is not enough to ameliorate disrupted lipid metabolism (4–6). These results suggest a complex relationship between the regulation of insulin sensitivity and hepatic lipid metabolism. Because the liver is critical in the regulation of glucose and lipid metabolism, the function of many genes expressed in the liver needs to be explored.
Friend of GATA 2 (FOG2, also known as ZFPM2) was first cloned from mouse embryonic brain (7) and heart (8). FOG2 is expressed in many tissues (8,9) and functions as a coregulator of the transcriptional factor GATA family (7,8). Studies have shown that FOG2 is involved in the regulation of many physiological processes, including coronary vascular development and heart morphology (10–12). However, some studies have implied a possible role of FOG2 in the regulation of insulin signaling and lipid metabolism such that FOG2 can bind to the regulatory unit p85 of phosphatidylinositol 3-kinase (PI3K) and attenuate phosphorylation of protein kinase B (AKT), the major component of insulin signaling in various cell types (13). Furthermore, FOG2 expression is increased in liver of insulin-resistant mice induced by interleukin-6 injection or with mutation in leptin receptor (db/db) (14). Moreover, overexpression of FOG2 blocks adipogenesis (15), a process closely related to lipid metabolism (16). Although FOG2 expression is not that abundant in normal liver (7,9), hepatic FOG2 may play an important role in the regulation of insulin sensitivity and lipid metabolism. The aim of the current study was to investigate this possibility.
Research Design and Methods
Animals and Treatment
Male C57BL/6J wild-type (WT) and leptin receptor–mutated (db/db) mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). FOG2flox/flox mice (12) backcrossed to the C57BL/6J background for at least four generations were crossed with albumin-Cre mice to generate FOG2 liver-specific knockout (FLKO) mice. Eight- to 10-week-old mice were maintained on a 12-h light/dark cycle at 25°C and provided free access to commercial rodent chow and tap water before initiation of the experiments. Food intake and body weight were measured daily. Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences.
Plasmid Construction and Cell Treatments
The FOG2 cDNA was amplified from FOG2 overexpression vector provided by N. Kim (Seoul National University, Seoul, Korea). The DNA fragment encoding nuclear receptor subfamily 2, group F, member 2 (NR2F2) was amplified from mouse hepatic cDNA. Double-stranded small interfering RNA (siRNA) targeting mouse NR2F2 was purchased from Shanghai GenePharma (Shanghai, China). The siRNA sequence specific for mouse NR2F2 was 5′-ACUGGCCAUAUAUGGCAAUUCAAUAUAUUGAAUUGCCAUAUAUGGCAGU-3′. Mouse primary hepatocytes were prepared by collagenase perfusion (17), and Hep1-6 and 293T cells (Cell Centre, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences) were maintained in DMEM as described previously (17). Primary hepatocytes were transfected with NR2F2-expressing plasmid by Effectene Transfection Reagent (Qiagen, Hilden, Germany) or NR2F2 siRNA by X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany).
Generation and Administration of Recombinant Adenoviruses
Recombinant adenovirus-expressing (Ad) human FOG2 was generated by using the AdEasy Adenoviral Vector System (Qbiogene, Irvine, CA), and adenovirus-expressing scrambled or small hairpin RNA (shRNA) for mouse FOG2 was generated by using the BLOCK-iT Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The scrambled sequence is 5′-TTCTCCGAACGTGTCACGT-3′. The shRNA sequence for mouse FOG2 is 5′-GCAAGATCAGCTTCAATAAGG-3′. The Ad-peroxisome proliferator–activated receptor α (Ad-PPARα) was provided by Y. Liu (Institute for Nutritional Sciences, Shanghai, China). Purified high-titer stocks of amplified recombinant adenoviruses (17) were diluted in PBS and administered at a dose of 1 × 107 plaque-forming units/well in 12-well plates or injected at a dose of 1 × 109 plaque-forming units/mice through the tail vein for a single injection.
Blood Glucose, Serum Insulin, Glucose Tolerance Tests, Insulin Tolerance Tests, and HOMA for Insulin Resistance Index
Levels of blood glucose and serum insulin were measured with a Glucometer Elite monitor or Mercodia Ultrasensitive Rat Insulin ELISA kit (ALPCO Diagnostics, Salem, NH), respectively. Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed by intraperitoneal injection of 2 g/kg glucose after overnight fasting and 0.75 units/kg insulin after 4 h of fasting, respectively. The HOMA for insulin resistance (HOMA-IR) index was calculated according to Eq. 1:
Glucose Output and Glycogen Content Assays
Glucose output and glycogen content were determined as described previously (17).
Measurements of Triglyceride, Cholesterol, and Free Fatty Acids
Hepatic and cellular lipids were extracted with chloroform methanol (18), and triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) levels were determined by the appropriate kits (TG and TC [SSUF-C, Shanghai, China], FFA [Wako Pure Chemical Industries, Osaka, Japan]), according to the manufacturers’ instructions.
Histological Analysis of Tissues
Frozen sections of liver were stained with Oil Red O, and paraformaldehyde-fixed, paraffin-embedded sections of liver were stained with hematoxylin-eosin (H-E).
In Vivo Insulin Signaling Assay
Mice were fasted for 4 h before insulin injection as previously described (18).
Western Blot Analysis
Western blot analysis was performed as previously described (17) with primary antibodies obtained as follows: anti-p-insulin receptor (IR) (tyr1150/1151), anti-IR, anti-p-IRS1 (tyr612/608 [human/mouse]), anti–insulin receptor substrate 1 (IRS1), anti-p-AKT (ser473), anti-p-AKT (thr308), anti-AKT, anti-p–glycogen synthase kinase (GSK)-3β (ser9), anti-GSK-3β and anti-NR2F2 (Cell Signaling Technology, Beverly, MA); anti-PPARα (Abcam, Cambridge, U.K.); anti-CPT1A (Proteintech, Chicago, IL); anti-FOG2 (Santa Cruz Biotechnology, Dallas, TX); and anti-HA (Sigma-Aldrich, St. Louis, MO).
RNA Isolation and Relative Quantitative RT-PCR
All data are expressed as mean ± SEM. Significant differences were assessed either by two-tailed Student t test or one-way ANOVA followed by the Student-Newman-Keuls test as indicated. For GTTs and ITTs, t test or one-way ANOVA was used to compare the difference between or among groups of mice at each time point examined. P < 0.05 was considered statistically significant.
FOG2 Regulates Insulin Sensitivity In Vitro
To investigate the role of FOG2 in insulin sensitivity, we infected nonimmunogenic murine hepatocellular carcinoma Hep1-6 cells and mouse primary hepatocytes with Ad-FOG2 or control green fluorescent protein (Ad-GFP) and examined their effects on insulin-stimulated phosphorylation of IR (tyr1150/1151), IRS1 (tyr612/608 [human/mouse]), AKT on ser473 and thr308, and GSK-3β (ser9) (19). FOG2 overexpression impaired insulin-stimulated phosphorylation of AKT and GSK-3β in both cell lines (Fig. 1A). Opposite effects were observed when FOG2 was knocked down by Ad-shRNA specific for FOG2 (Ad-shFOG2) compared with control cells infected with scrambled adenovirus (Ad-scrambled) (Fig. 1B). Insulin-stimulated phosphorylation of IR and IRS1, however, were not affected under either condition (Fig. 1A and B). In addition, glycogen content was significantly decreased by Ad-FOG2 or increased by Ad-shFOG2 in the presence or absence of insulin in primary mouse hepatocytes, whereas glucose output was decreased by Ad-FOG2 but not affected by Ad-shFOG2 (Fig. 1C–F). Furthermore, genes related to glycogenolysis (glycogen phosphorylase [Pygl]) (20) were increased by Ad-FOG2 or decreased by Ad-shFOG2, whereas genes related to glycogen synthesis (glycogen synthase [Gs]) or gluconeogenesis (glucose-6-phosphatase [G6pase], phosphoenolpyruvate carboxylase [Pepck], pyruvate carboxylase [Pc], fructose1,6-bisphosphatase [Fbp1], and glucokinase [Gk]) (20) were either not affected or showed discordant changes in primary hepatocytes infected with Ad-FOG2 or Ad-shFOG2 (Supplementary Fig. 1).
Ad-shFOG2 Ameliorates Insulin Resistance in db/db Mice
As observed previously (14), we also found that FOG2 expression is higher in the livers of db/db mice compared with control mice (Fig. 2A). However, FOG2 expression was lower in the adipose tissue, with no difference in the muscle of db/db mice (Supplementary Fig. 2A). We then injected db/db mice with Ad-shFOG2 or Ad-scrambled and examined whether decreased FOG2 expression could reverse insulin resistance in these mice. As expected, Ad-shFOG2 significantly decreased FOG2 expression in the livers of db/db mice (Fig. 2B) but had no effect on body weight or food intake (Supplementary Table 2). Ad-shFOG2 also significantly reduced levels of fed and fasting blood glucose and fasting serum insulin as well as HOMA-IR index (except for levels of fed serum insulin) in db/db mice (Fig. 2C–E). Blood glucose levels decreased much more quickly after challenge with glucose or insulin in db/db mice injected with Ad-shFOG2 than in control mice as measured by GTT and ITT comparing the difference between groups of mice at each time point (Fig. 2F). The improved effect of Ad-shFOG2 on glucose tolerance is most likely caused by inhibition of glycogenolysis rather than by altered gluconeogenesis based on analysis for genes related to these processes or the change of insulin secretion during the GTT (Supplementary Fig. 3). No significant difference on pyruvate tolerance tests was observed between db/db mice injected with Ad-shFOG2 or Ad-scrambled at any time point (Supplementary Fig. 4). Additionally, insulin-stimulated phosphorylation of AKT and GSK-3β, but not IR and IRS1, were increased in the livers of db/db mice injected with Ad-shFOG2 (Fig. 2G). Moreover, no changes in FOG2 expression and insulin signaling were observed in white adipose tissue (WAT) and muscle of db/db mice with FOG2 knockdown (Supplementary Fig. 2B–E). Similarly, Ad-shFOG2 improved insulin sensitivity in C57BL/6J WT mice (Supplementary Fig. 5) without changing their body weight and food intake (Supplementary Table 3).
Ad-FOG2 Attenuates Insulin Sensitivity in WT Mice
To further confirm the role of FOG2 in insulin sensitivity, we injected male C57BL/6J WT mice with Ad-FOG2 or Ad-GFP and found increased hepatic FOG2 expression in Ad-FOG2 mice compared with control mice (Fig. 3A). Although body weight and food intake remained unchanged (Supplementary Table 4), fed and fasting blood glucose levels were significantly increased in Ad-FOG2 mice (Fig. 3B). Fasting serum insulin levels were not changed; however, fed serum insulin levels were significantly elevated by Ad-FOG2 (Fig. 3C). Furthermore, the HOMA-IR index was also increased in Ad-FOG2 mice (Fig. 3D). Consistent with these changes, Ad-FOG2 mice displayed attenuated glucose tolerance and clearance as measured by GTT and ITT comparing the difference between groups of mice at each time point (Fig. 3E).
FOG2 Regulates Hepatic Lipid Metabolism in WT Mice
Because insulin resistance often is associated with dysregulated lipid metabolism (1), we explored the possible involvement of FOG2 in lipid metabolism in WT mice injected with Ad-FOG2 or Ad-GFP under fed state. Liver weight but not fat mass was decreased by Ad-FOG2 compared with control mice (Supplementary Table 4). Moreover, lipid accumulation was significantly decreased in the livers of Ad-FOG2 mice as demonstrated by H-E and Oil Red O staining (Fig. 4A). Consistent with these changes, levels of hepatic TG and FFAs and serum FFAs were decreased in Ad-FOG2 mice (Fig. 4B and C). However, levels of hepatic TC and serum TG and TC were not changed by Ad-FOG2 (Fig. 4B and C).
Consistently, liver weight was increased in Ad-shFOG2 mice compared with mice injected with Ad-scrambled, with no change in fat mass under fed state (Supplementary Table 3). Meanwhile, Ad-shFOG2 mice developed more significant TG accumulation than control mice as demonstrated by H-E and Oil Red O staining (Fig. 4D). Accordingly, levels of hepatic TG, TC, and FFAs were significantly increased, whereas serum levels of TC and FFAs were decreased, by Ad-shFOG2 (Fig. 4E and F). Serum TG levels, however, were not affected in Ad-shFOG2 mice (Fig. 4F).
Hepatic PPARα Expression Is Regulated by FOG2 in an NR2F2-Dependent Manner
Hepatic lipid metabolism is regulated by four processes, including fatty acid synthesis, fatty acid oxidation, fatty acid uptake, and TG secretion (21). We therefore examined the effects of FOG2 on genes related to these four processes in livers of Ad-FOG2 or Ad-shFOG2 mice and their corresponding control mice. Except for genes related to fatty acid oxidation (Pparα and carnitine palmitoyltransferase 1a [Cpt1a]) (22), other genes related to lipogenesis (fatty acid synthase [Fas], stearoyl-CoA desaturase 1 [Scd1], acetyl CoA carboxylase [Acc], Srebp1c, malic enzyme [Me], carbohydrate-responsive element–binding protein [Chrebp], Pparγ, Srebp2, and 3-hydroxy-3-methylglutaryl-CoA synthase 2 [Hmgcs2]) and fatty acid uptake (Cd36 and fatty acid–binding protein [Fabp]) (22) either were not affected or showed discordant changes by Ad-FOG2 or Ad-shFOG2 (Fig. 5A and B). Consistently, serum levels of β-hydroxybutyrate, which reflects a state of fatty acid oxidation (23), were markedly reduced in db/db mice injected with Ad-shFOG2 and increased in WT mice injected with Ad-FOG2 compared with control mice under the fasted state (Supplementary Fig. 6). Moreover, genes related to TG secretion (apolipoprotein B [ApoB] and apolipoprotein E [ApoE]) (22) were also examined, and only ApoB displayed corresponding changes under both conditions (Fig. 5A and B).
On the basis of these results, we speculated that PPARα might be involved in FOG2-regulated lipid metabolism. As predicted, protein levels of hepatic PPARα and its downstream CPT1A were significantly increased by Ad-FOG2 or decreased by Ad-shFOG2 (Fig. 5C and D). Because PPARα-null mice accumulated more TGs in the liver under fasting conditions (24), we speculated that Ad-shFOG2 mice have similar phenotypes. Indeed, Ad-shFOG2 mice had more significantly elevated hepatic lipid accumulation compared with control mice after fasting for 24 h as shown by H-E and Oil Red O staining (Supplementary Fig. 7A). Meanwhile, increased levels of hepatic TG, TC, and FFAs and reduced levels of serum TG and TC without a change in serum FFA levels were observed in Ad-shFOG2 mice compared with control mice (Supplementary Fig. 7B and C). Consistently, overexpression of FOG2 significantly decreased hepatic lipid accumulation compared with control mice fasted for 24 h (Supplementary Fig. 7D–F).
The expression of PPARα is regulated by NR2F2 (25), which binds directly to FOG2 (26), suggesting that FOG2 might regulate the expression of PPARα through NR2F2. As expected, FOG2 had no effect on expression of NR2F2 but significantly changed NR2F2-stimulated expression of insulin-like 3 (INSL3) and NR2F2-inhibited expression of angiotensin (AGT) (Fig. 5E and F) (27), suggesting regulatory effects of FOG2 on NR2F2 activity. NR2F2 overexpression blocked Ad-FOG2–stimulated PPARα expression, and an opposite effect was observed for Ad-shFOG2–inhibited PPARα expression when NR2F2 was knocked down in primary cultured mouse hepatocytes (Fig. 5G and H). The role of NR2F2 was then investigated in WT mice injected with Ad-shFOG2 and/or Ad-shNR2F2. Knockdown of NR2F2 (as demonstrated by Western blot) significantly reversed the effects of Ad-shFOG2 on improved insulin sensitivity and disrupted hepatic lipid accumulation (Supplementary Fig. 8).
The Effect of Ad-shFOG2 on Improved Insulin Sensitivity and Promoted TG Accumulation Is Reversed by Ad-PPARα in WT Mice
PPARα is the key regulator for fatty acid oxidation (28), and PPARα deficiency protects high-fat diet–induced insulin resistance (29), implying that PPARα may mediate the effects of FOG2. To test this possibility, we examined insulin-stimulated phosphorylation of AKT and GSK-3β in Hep1-6 cells infected with Ad-shFOG2 or Ad-scrambled and Ad-PPARα or Ad-GFP. Overexpression of PPARα significantly blocked the beneficial effects of Ad-shFOG2 on improving insulin-stimulated phosphorylation of AKT and GSK-3β (Supplementary Fig. 9). Insulin-stimulated phosphorylation of IR and IRS1, however, were not affected by any treatment (Supplementary Fig. 9). We injected C57BL/6J WT mice with Ad-PPARα and examined its effect on Ad-shFOG2–improved insulin sensitivity. Expression of hepatic PPARα and its downstream target CPT1A (22) was significantly increased by Ad-PPARα (Fig. 6A). All mice exhibited normal body weight, food intake, and adipose tissue weight (Supplementary Table 5). As expected, Ad-PPARα significantly elevated fed blood glucose, fasting glucose, and fasting insulin levels in Ad-shFOG2 mice (Fig. 6B and C). The HOMA-IR index was also reversed by Ad-PPARα in Ad-shFOG2 mice (Fig. 6D). Moreover, the improved glucose clearance and insulin sensitivity by FOG2 knockdown was markedly reversed by Ad-PPARα compared with the difference in blood glucose levels among various groups of mice at each time point (Fig. 6E).
Because TRB3 is a direct target of PPARα that inhibits AKT activation and impairs insulin sensitivity (30,31), we speculated that it may mediate FOG2/PPARα regulation of insulin sensitivity. This possibility was confirmed by the observation that TRB3 expression was regulated by FOG2 in a PPARα-dependent pathway and that TRB3 is required for FOG2 regulation of insulin signaling and insulin sensitivity in vitro and in vivo (Supplementary Figs. 10 and 11).
In addition, Ad-PPARα prevented Ad-shFOG2–induced lipid accumulation under the fasting condition, as demonstrated by H-E and Oil Red O staining (Fig. 6F). Moreover, Ad-shFOG2–increased levels of hepatic TG and FFAs, but not TC, were significantly reversed by Ad-PPARα (Fig. 6G). Additionally, levels of serum FFAs remained the same, whereas levels of serum TG and TC were significantly decreased by Ad-PPARα in Ad-shFOG2 mice (Fig. 6H). Similar reversal effects by Ad-PPARα were observed in Ad-shFOG2 mice under the fed state (Supplementary Fig. 12).
FLKO Mice Exhibit Improved Insulin Sensitivity and Increased Hepatic TG Accumulation Through PPARα
To gain more insight into the impact of FOG2 on insulin sensitivity and fatty acid metabolism, we generated FLKO mice. The efficiency of FLKO mice was demonstrated by the almost completely abolished expression of FOG2 in primary hepatocytes isolated from FLKO mice compared with those from control mice (Fig. 7A). Again, hepatic NR2F2 expression was not altered in FLKO mice (Supplementary Fig. 13). There was no difference in food intake and body weight between FLKO and control mice (Supplementary Fig. 14). Fed and fasting blood glucose decreased dramatically in FLKO mice (Fig. 7B), with no difference in serum insulin levels (Fig. 7C). The HOMA-IR index was also decreased in these mice (Fig. 7D). Moreover, FLKO mice showed improved glucose tolerance and clearance as measured by GTT and ITT comparing the difference between groups of mice at each time point and insulin signaling in primary hepatocytes (Fig. 7E and F). On the other hand, FLKO mice showed more significant TG accumulation during fasting as demonstrated by H-E and Oil Red O staining and lipid parameter measurements (Fig. 7G–I). Again, the improved insulin sensitivity and promoted hepatic TG accumulation in FLKO mice were largely reversed by Ad-PPARα (Fig. 8 and Supplementary Table 6).
FOG2 exists mainly in heart, testis, and brain, and most known FOG2 functions are related to these three tissues (9,10). FOG2 is also expressed in liver (7), an important tissue integral to glucose and lipid metabolism (1), but the function of hepatic FOG2 is poorly understood. We show that FOG2 overexpression and knockdown impairs and improves insulin signaling in vitro, respectively. Furthermore, FOG2 knockdown ameliorates insulin resistance in db/db mice, and FOG2 overexpression attenuates insulin sensitivity in WT mice. In addition, FOG2 overexpression reduces hepatic TG accumulation under fed and fasted conditions, whereas FOG2 knockdown has the opposite effect. The effects of adenovirus-mediated FOG2 expression are specific for FOG2 because control adenovirus had no effect on insulin sensitivity, hepatic lipid metabolism, or inflammation (Supplementary Figs. 15–17). Finally, FLKO mice exhibit improved insulin sensitivity and increased hepatic TG accumulation. These results highlight a critical role of hepatic FOG2 in the regulation of insulin sensitivity and hepatic TG accumulation under both pathological and physiological conditions. The results also suggest that FOG2 attenuates insulin sensitivity by promoting glycogenolysis but not by stimulating gluconeogenesis because glucose output was decreased as a compensatory response to the possible increase in glucose levels or regulated in an insulin-independent pathway. Insulin integrates glucose and lipid metabolism together, and insulin resistance is normally closely linked with liver steatosis (1,3). However, the current results suggest that this is not always the case because FOG2 promotes insulin resistance but ameliorates liver steatosis. Inconsistent insulin resistance and liver steatosis were also observed in various mouse models (4,6). The current finding of FOG2 in an uncoupling relationship of insulin resistance and liver steatosis suggests that attention should be paid to the opposite function of genes as drug targets when treating patients with these two metabolic symptoms.
The current results suggest that processes related to lipogenesis and fatty acid uptake are unlikely to contribute to the protective effect of FOG2 on hepatic TG accumulation. Instead, expression of the key regulator for fatty acid oxidation PPARα was elevated by Ad-FOG2 or decreased by Ad-shFOG2. Because PPARα is the key modulator of fatty acid oxidation that plays a key role in liver steatosis (28), we speculated that FOG2 regulates hepatic TG accumulation through influencing fatty acid oxidation. Consistently, PPARα overexpression ameliorated liver steatosis induced by adenovirus-mediated knockdown or genetic deletion of FOG2. The expression of fatty acid secretion gene ApoB was also increased by FOG2 overexpression or decreased by FOG2 knockdown, indicating that altered TG secretion might also be involved in FOG2 regulation of hepatic TG accumulation.
The insulin signaling pathway comprises three major components: the IR and related IRS, PI3K, and AKT (19). We found that FOG2 affects phosphorylation of AKT but not IR, IRS1, or IRS2 (Supplementary Fig. 18). Adenovirus-mediated or agonist-induced activation of PPARα reportedly protects from insulin resistance in various animal models or humans (32–36). In contrast, other studies have shown that hepatic PPARα activation causes insulin resistance (31,37) and that PPARα deficiency reduces insulin resistance (38,39). On the basis of these results, we hypothesized that one possibility is that FOG2 regulates insulin signaling by PPARα. Consistently, PPARα downstream target fibroblast growth factor 21 (Fgf21) expression was regulated by FOG2 in a similar manner as PPARα (Supplementary Fig. 19). PPARα overexpression attenuated insulin sensitivity in WT mice with FOG2 knockdown or in FLKO mice. TRB3 is a direct target of PPARα that inhibits AKT activation and impairs insulin sensitivity (30,31). In the current study, we found that TRB3 expression was regulated by FOG2 in a PPARα-dependent pathway and that TRB3 is required for FOG2 regulation of insulin signaling and insulin sensitivity in vitro and in vivo. In addition, FOG2 may inhibit PI3K activity by directly binding to its regulatory subunit P85 (13), suggesting the possible involvement of PI3K in mediating FOG2 regulation of insulin sensitivity, which requires further investigation.
FOG2 normally binds to other transcription factors exerting its function (9). A previous study showed that the PPARα promoter has a binding site for hepatocyte nuclear factor 4 α regulatory element (HNF4α-RE) (40), which can be recognized by a set of nuclear receptor superfamily members, including NR2F2 (25). NR2F2 is a nuclear orphan receptor that belongs to the steroid/thyroid hormone receptor superfamily and plays an important role in metabolism (41) and development (42). NR2F2 can be a repressor of transcription for other nuclear hormone receptors, including PPARα (25). FOG2 can bind to NR2F2 directly and repress its transcription activity (26). In the current study, we show that FOG2 regulation of PPARα expression, as well as insulin sensitivity and hepatic lipid metabolism, is mediated in an NR2F2-dependent manner. Because GATA4 is also known to be a cofactor for FOG2, the possible involvements of HNF4α and GATA4 in mediating FOG2 regulation of PPARα require future study.
Of note, we found that FOG2 expression was decreased in WAT of db/db mice, which might be mediated by transforming growth factor β-regulated (43) or interleukin-6–regulated (14) microRNAs, including miR200 or miR8 (13). In 3T3-L1 cells, FOG2 is shown to control adipogenesis (15), a key process that determines the mass of adipose tissue. These results are consistent with other studies that showed FOG2 to be an important regulator for development in various tissues (11,12). The function of decreased FOG2 expression in WAT of db/db mice, however, requires further study.
In summary, as described in our working model (Fig. 8I), we observed an important role of hepatic FOG2 in insulin sensitivity and liver steatosis through PPARα. These results should provide novel insights into the understanding of the molecular mechanisms and potential treatment targets for T2D and liver steatosis.
Funding. This work was supported by grants from the National Natural Science Foundation of China (81130076, 81325005, 31271269, 81390350, 81300659, 81471076, 81400792, 81500622, and 81570777), Basic Research Project of Shanghai Science and Technology Commission (13JC1409000), and International S&T Cooperation Program of China (Singapore 2014DFG32470). This research was also supported by the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs international partnership program for creative research teams. F.G. was supported by the One Hundred Talents Program of the Chinese Academy of Sciences.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. Y.G. and J.Y. researched data and contributed to the writing, review, and editing of the manuscript. J.D., K.L., F.X., and F.Y. researched data. B.L., Y.X., Y.L., and S.C. provided research material. F.G. directed the project and contributed to the discussion and writing, review, and editing of the manuscript. F.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.