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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2024 Jan 23;326(3):G264–G273. doi: 10.1152/ajpgi.00153.2023

NADPH oxidase 1 promotes hepatic steatosis in obese mice and is abrogated by augmented skeletal muscle mass

Sebastian Larion 1,2, Caleb A Padgett 1, James D Mintz 1, Jennifer A Thompson 3, Joshua T Butcher 1, Eric J Belin de Chantemèle 1,2, Stephen Haigh 1, Sandeep Khurana 4, David J Fulton 1,5, David W Stepp 1,6,✉
PMCID: PMC11211036  PMID: 38258487

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Keywords: NAFLD, NOX1, myostatin, PTP1B, SREBP1, SREBP

Abstract

Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.

NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western hemisphere, a condition largely driven by the epidemic of obesity (1). Hepatic steatosis is the first stage of NAFLD, representing the gateway to more advanced disease states such as nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis (2). Although initially perceived as benign, simple steatosis is increasingly appreciated as an important marker of elevated hepatic and cardiovascular-specific risk. Simple steatosis progresses to liver fibrosis at about half the rate of NASH, with progression more likely in patients with greater cardiometabolic burden (3–6). Hepatic steatosis is an independent risk factor for adverse cardiovascular outcomes even after adjusting for traditional covariates such as smoking, diabetes, and obesity (7–11). Indeed, cardiovascular disease is the leading cause of death in patients with NAFLD (12). As NAFLD affects more than 90 million patients in the United States (1), a greater understanding of disease-causing steatotic mechanisms is needed to address the upcoming challenges of the NAFLD pandemic.

Exercise as a means for sustained weight loss is first-line therapy for NAFLD (12). However, significant improvements in NAFLD have been reported even in individuals who undergo exercise but do not lose weight (13, 14). For instance, Johnson et al. (13) reported that 4 wk of aerobic exercise results in >20% reduction in hepatic triglyceride (TAG) content in obese patients with NAFLD but did not lead to significant weight loss. Resistance exercise training, which primarily increases muscle mass, reduces intrahepatic lipid content in patients with NAFLD, independent of changes in body weight (15, 16). These emerging data suggest that the beneficial effects of exercise on NAFLD may not derive strictly from reductions in weight per se but rather improvement in an as-yet unidentified component of metabolic health, including potentially a result of increased muscle. Although uncoupling the metabolic effects of increased muscle from weight loss is difficult to assess experimentally, a greater understanding of the physiological adaptions of resistance exercise may lead to more effective prescriptive regimens in NAFLD.

A characteristic feature of hepatic steatosis is elevated oxidant stress (17–19), where exuberant reactive oxygen species (ROS) production overwhelms antioxidant defenses, inhibits physiological redox signaling, induces direct cellular injury, and impairs liver function. Interestingly, physical exercise decreases serum markers of hepatic oxidant stress (20–22), proposing the intriguing hypothesis that exercise improves NAFLD via mechanisms that abate hepatic oxidant stress. However, the enzymatic source of elevated hepatic oxidants in NAFLD is intensely debated. One possible source is NADPH oxidase 1 (NOX1), an enzyme expressed in hepatocytes, whose only known function is superoxide (O2•−) production (23, 24). The bulk of the literature overwhelmingly investigates NOX1 in late-stage NAFLD, where pharmacological or genetic NOX1 inhibition prevents inflammatory signaling and fibrosis development in chronic liver injury models (23, 25, 26). Consistent with experimental data, hepatic Nox1 is increased in humans with NASH or cirrhotic liver disease (25, 27). Moreover, as one of the dreaded complications of NAFLD, Liang et al. (28) recently described that NOX1 promotes tumorigenesis and hepatocellular carcinoma in diethylnitrosamine (DEN)-induced liver injury. However, there is a paucity of data regarding the specific role of Nox1 in obese mice with extensive metabolic liver disease, with scant literature available providing seemingly contradictory information concerning the relative importance of NOX1 in early steatosis. For instance, although hepatic NOX1 is reportedly upregulated in hyperphagic db/db mice, Nox1 knockout mice are not protected from high-fat, high-cholesterol diet-induced intrahepatic triglyceride accumulation (27, 29). As simple steatosis in the setting of obesity is the most common human NAFLD phenotype, a greater understanding of early disease-causing mechanisms may provide novel insights on NAFLD pathogenesis.

In this study, we mimic components of exercise by producing a phenotype of hypermuscularity without weight loss in the db/db obese mouse model of NAFLD through deletion of myostatin, a potent negative regulator of muscle growth. We determine the effect of increased muscle mass on hepatic steatosis, lipogenic pathways, and oxidant stress and further probe specific links between these variables by deletion of NOX1 in db/db mice and cultured hepatocytes with adenoviral transfection of NOX1. Finally, we use to genetic and diet-induced animal models to probe the relationship between serum insulin (Ins), Nox1, and sterol regulatory element binding protein 1 (Srebp1), identifying NOX1 as a novel determinant of hepatic steatosis in db/db mice.

MATERIALS AND METHODS

Animal Studies

All studies described were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited animal program and all studies were approved by the Institutional Animal Care and Use Committee (IACUC). Lean and obese (hyperphagic db/db) mice with and without Nox1, Myostatin, or protein tyrosine phosphatase 1B (Ptp1b) gene disruption were created by crossing C57BL6 mice heterozygous for leptin receptor mutation (db+/−, Jackson Laboratory, strain 000697) with constitutive knockout mice to create four possible animal groups per genotype: lean controls, lean knockout mice, obese controls, and obese knockout mice as previously described (30–32). Detailed methods are provided in the online Supplemental Material, which is available at https://doi.org/10.6084/m9.figshare.24584787.

RESULTS

Hypermuscularity Resolves Steatosis and Hepatic Nox1 Expression in db/db Mice

A cardinal aspect of exercise physiology is increase in muscle mass with sustained effort. To mimic this effect, lean and obese (db/db) mice were crossed with myostatin knockout mice as previously described (31). Myostatin is a negative regulator of muscle growth and myostatin deletion results in 70% increase in gastrocnemius (Gastrocn.) mass in db/db mice (Supplemental Fig. S1). Hypermuscularity was associated with significantly improved fasting blood glucose (FBG) and hemoglobin A1c (HgbA1c) in db/db mice, but pancreatic hormones, such as insulin, that were elevated in db/db mice showed no improvement (Supplemental Table S1). Additional metabolic parameters of obesity, such as body weight and visceral adiposity, were unaffected by myostatin deletion, demonstrating that weight loss does not explain the benefits of increased muscle mass in this model.

The effect of increased muscle mass on the liver is listed in Fig. 1 and Supplemental Fig. S2, showing hypermuscularity with myostatin deletion dramatically improved liver morphology, liver size, and hepatic steatosis in db/db mice. Lipid analysis revealed that hypermuscularity significantly reduced intrahepatic fat (%), triglyceride species, and cholesterol content in db/db mice. Quantitative PCR revealed that hypermuscularity significantly decreased obesity-induced hepatic Srebp1, the master regulator of hepatic lipogenesis, and downstream targets stearoyl-CoA desaturase (Scd1), fatty acid synthase (Fasn), and acetyl-CoA carboxylase α (Acaca) in db/db mice (Fig. 2). As resistance exercise attenuates hepatic oxidant stress and NADPH oxidases are implicated in hepatic superoxide production, we tested the hypothesis that hypermuscularity in obesity prevents hepatic expression of the most common NOX isoforms (23, 33). Interestingly, obesity-induced hepatic NOX1 was completely restored to control levels by hypermuscularity in db/db mice with no impact on NOX2 (Supplemental Fig. S3) or NADPH oxidase 4 (NOX4) (Fig. 2C), linking the beneficial effects of hypermuscularity on steatosis with hepatic NOX1.

Figure 1.

Figure 1.

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Hypermuscularity with myostatin (Myo) deletion improves hepatic steatosis in db/db mice (KO). A: myostatin deletion reduces hepatic steatosis in db/db mice as assessed by representative oil-red-O staining. Myostatin deletion reduces total (B) and normalized (C) indices of liver size (n = 8–10), intrahepatic fat by NMR (n = 6) (D), and triglyceride content (E) by GC/MS (n = 6). Scale bar: 200 μm. Data are shown as means ± SE. *P < 0.05 vs. lean controls; #P < 0.05 vs. obese controls. KO, knockout; NMR, nuclear magnetic resonance; TAG, triacylglycerol.

Figure 2.

Figure 2.

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Hypermuscularity with myostatin (Myo) deletion reduces hepatic Srebp1 and NOX1 expression in db/db mice (KO). Hypermuscularity with myostatin deletion reverses obesity-induced hepatic Srebp1 expression (A) and downstream targets (B) involved in lipogenesis. C: myostatin deletion specifically reverses hepatic Nox1 expression but not NOX4 in db/db mice. n = 4–7. Data are shown as means ± SE. *P < 0.05 vs. lean controls; #P < 0.05 vs. obese controls. KO, knockout; NOX1, NADPH oxidase 1.

NOX1 Deletion Abolishes Hepatic Oxidant Stress in db/db Mice

To directly investigate the role of NOX1 in steatosis, we examined oxidant stress and hepatic lipid accumulation in db/db mice in which the NOX1 gene had been deleted (32). Confocal microscopy using 8-hydroxy-2-deoxyguanosine (8-OhDG) and nitrotyrosine immunofluorescence staining revealed that NOX1 deletion completely abolished obesity-induced oxidized DNA and peroxynitrite toxin formation in the liver of db/db mice, respectively (Fig. 3, A and B and Supplemental Fig. S4). NOX1 deletion also significantly decreased plasma thiobarbituric acid reactive substance (TBARS) concentration, a marker of whole body lipid oxidation in db/db mice (Fig. 3C), implicating NOX1 as a major source of endogenous oxidants not only in the liver but obesity in general. Hepatic NOX4 was unaffected by NOX1 deletion to suggest compensatory changes in other major NOX isoforms (Fig. 3D), identifying NOX1 as the dominant source of hepatic oxidants in db/db mice.

Figure 3.

Figure 3.

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NOX1 deletion abolishes hepatic oxidant stress in db/db mice. NOX1 deletion abolishes obesity-induced hepatic oxidant stress as evidenced by representative 8-hydroxydeoxyguanosine (8-OhDG) (A) and nitrotyrosine immunofluorescence staining (B). C: NOX1 deletion reduces plasma thiobarbituric acid reactive substance (TBARS) concentration in db/db mice (KO) (n = 5 or 6). D: NOX1 deletion did not result in compensatory changes in hepatic NOX4 expression (n = 7 or 8). Scale bar: 100 μM. Data are shown as means ± SE. *P < 0.05 vs. lean controls; #P < 0.05 vs. obese controls; ns, no groups significantly different. 8-OhDG, 8-hydroxy-2-deoxyguanosine; KO, knockout; NOX1, NADPH oxidase 1.

Nox1 Deletion Resolves Hepatic Steatosis and Srebp1 Expression in db/db Mice

The effect of NOX1 deletion in fatty liver is listed in Fig. 4 and Supplemental Fig. S5, demonstrating that NOX1 deletion improved liver morphology, size indices, and hepatic steatosis in db/db mice. Lipid analysis confirmed that NOX1 deletion profoundly attenuated obesity-induced intrahepatic fat accumulation (%) in db/db mice including a remarkable 60% reduction in triglycerides species (Fig. 4, B and C). Furthermore, NOX1 deletion significantly decreased obesity-induced hepatic Srebp1, Scd1, and Fasn expression in db/db mice (Fig. 4, D and E), paralleling gene expression changes noted with hypermuscularity (Fig. 2, A and B). Downstream of Srebp1, NOX1 deletion also significantly reduced hepatic oleic acid (18:1n-9) concentration, the primary end product of de novo lipogenesis (DNL), in all three major lipid species in db/db mice (Fig. 4F). To evaluate the potential extrahepatic effects of NOX1, we performed comprehensive metabolic phenotyping of db/db mice with NOX1 deletion. NOX1 deletion did not improve visceral adiposity, glucose tolerance, or hypertriglyceridemia in db/db mice (Supplemental Table S2). Metabolic chambers indicated that NOX1 deletion had no effect on carbon dioxide production (V̇co2)/CO2 respiratory exchange ratio (RER) in db/db mice, suggesting similar rates of whole body fatty acid oxidation. Moreover, NOX1 deletion had no effect on gastrocnemius muscle triglyceride or free fatty acid concentration in db/db mice, arguing against significant ectopic lipid accumulation. Taken together, these data support a direct role of NOX1 as a novel determinant of hepatic steatosis in db/db mice, which are reversed by hypermuscularity.

Figure 4.

Figure 4.

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NOX1 deletion reduces hepatic steatosis and Srebp1 expression in db/db mice (KO). NOX1 deletion reduces hepatic steatosis in db/db mice as assessed by representative oil-red-O staining (A), total hepatic fat (n = 10) (B), and intrahepatic triglyceride content (n = 5 or 6) (C). NOX1 deletion also prevents obesity-induced hepatic Srebp1 (D) and downstream target genes involved in lipogenesis (E) (n = 4–7). Nox1 deletion attenuates obesity-induced hepatic oleic acid concentration in free fatty acid (FFA) (F), triglyceride (TAG), and cholesterol ester (CE) species (n = 6). Scale bar: 200 μm. Data are shown as means ± SE. *P < 0.05 vs. lean controls; #P < 0.05 vs. obese controls. NOX1, NADPH oxidase 1; KO, knockout.

NOX1 Overexpression Increases SREBP1 in Insulin-Treated HepG2 Cells

Although SREBP1 is associated with elevated oxidant stress, a potential causal role has not been described. To directly investigate the role of NOX1 on insulin-mediated SREBP1 expression, HepG2 cells were treated with NOX1-, NADPH oxidase organizer 1 (NOXO1)-, and NADPH oxidase activator 1 (NOXA1)-overexpressing adenoviral vectors, NOX1-complex subunits required for NOX1 activation, or β-galactosidase control virus. Predictably, SREBP1 expression was significantly increased in insulin-treated HepG2 cells versus control conditions in β-galactosidase-overexpressing cells (Fig. 5A). Furthermore, downstream products of SREBP1 were also increased in insulin-treated HepG2 cells, strengthening the association between NOX1 and SREBP1 expression (Supplemental Fig. S6). However, NOX1, NOXO1, and NOXA1 coexpression significantly enhanced insulin-mediated SREBP1 expression as compared with control virus. Further imaging studies indicated that NOX1 treatment further stimulated SREBP1 colocalization to the nucleus in insulin-treated HepG2 cells (Supplemental Fig. S7). As the only known function of NOX1 is superoxide production, this suggests that superoxide is a signaling intermediary responsible for the functions of NOX1 on SREBP1. To directly evaluate the stimulatory role of superoxide on SREBP1 expression, the experiment was repeated in NOX5-overexpressing HepG2 cells, a related NOX homologue that also generates superoxide. Remarkably, NOX5-overexpression similarly increased SREBP1 expression by insulin versus control virus (Fig. 5B), confirming superoxide as the key mechanistic agent for the actions of NOX1 on SREBP1. Thus, we conclude that NOX1 is a novel regulator of SREBP1 expression.

Figure 5.

Figure 5.

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NOX1 overexpression increases insulin-induced SREBP1 expression in HepG2 cells, and NOX1 expression remains elevated in obese mice with elevated insulin signaling. Insulin-mediated SREBP1 expression was significantly increased in NOX1, NOXO1, and NOXA1 (A) or NOX5-overexpressing HepG2 cells vs. β-galactose control virus (n = 3) (B). Increasing insulin signaling with Ptp1b deletion increases hepatic steatosis (C) as assessed by representative oil-red-O sections, intrahepatic triglyceride content (n = 4) (D), lipogenic gene expression (n = 5–8) (E), and preserves elevated hepatic NOX1 expression (F) in db/db mice (n = 4). Scale bar: 200 μm. Data are shown as means ± SE. *P < 0.05 vs. lean controls; #P < 0.05 vs. obese controls. NOX1, NADPH oxidase 1; NOXO1, NADPH oxidase organizer 1; Ptp1b, protein tyrosine phosphatase 1B; SREBP1, sterol regulatory element binding protein 1.

Insulin as a Metabolic Driver of Hepatic Nox1

Although hepatic NOX1 has been reliably shown to be elevated in db/db mice (29), the metabolic controllers of NOX1 in obesity are unclear. To characterize the upstream drivers, we evaluated NOX1 expression in animal models that modulated endogenous insulin and glucose signaling in vivo. We first investigated NOX1 expression in lean and obese (db/db) mice crossed with control or Ptp1b knockout mice. Ptp1b is a negative regulator of insulin receptor tyrosine kinase, and Ptp1b deletion results in enhanced insulin signaling (30, 34, 35). We and others have demonstrated that Ptp1b deletion improves glucose homeostasis as indicated by significantly reduced hemoglobin A1c in db/db mice (Supplemental Table S3) (30). Other indices of obesity, such as body weight and adiposity, however, remain unaffected by Ptp1b deletion. As a readout of hepatic insulin signaling, Ptp1b deletion profoundly upregulated hepatic steatosis, triglyceride content, and lipogenic programming as indicated by significantly increased Scd1, Fasn, and Acaca expression (Fig. 5, C–E). Ptp1b deletion also significantly enhanced SCD1-16 and SCD1-18 product:precursor enzyme activity indices in obese mice (Supplemental Fig. S8E), suggesting elevated hepatic lipogenesis. However, NOX1 expression remained elevated in obese mice despite improved glucose control with Ptp1b knockout (Fig. 5F), indicating that increased hepatic insulin signaling maintains NOX1 expression even as it improves glycemia. Interestingly, Ptp1b deficiency did not further upregulate Srebp1 expression in db/db mice (Supplemental Fig. S8F), consistent with the hypothesis that hyperglycemia in the presence of elevated insulin is required for Srebp1 induction.

To further interrogate metabolic drivers, we next investigated NOX1 expression in control or streptozotocin (STZ)-treated C57B6 mice. As a model of type 1 diabetes, STZ abolishes pancreatic β-cell insulin release, resulting in severely elevated fasting glucose (366 ± 19 vs. 152 ± 10 mg/dL; P < 0.001). As shown in Fig. 6, STZ treatment significantly increased hepatic Scd1, Fasn, and Acaca expression in fed mice but not in fasted mice, indicating that only-insulin-dependent, and not nutrient-dependent, Srebp1 expression is attenuated by STZ (Fig. 6, A and B). However, despite severe hyperglycemia, hepatic NOX1 was not induced by STZ under either fasting or fed conditions (Fig. 6C), indicating that sustained hyperglycemia does not induce NOX1 expression in the absence of elevated insulin.

Figure 6.

Figure 6.

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Hepatic NOX1 expression is not induced in the absence of elevated serum insulin. Abolishing serum insulin with streptozotocin (STZ) preserves feeding (A) but not fasted increase (B) in lipogenic genes as evaluated by PCR (n = 5–7). C: hepatic NOX1 expression is unchanged (n = 4–6). D: representative hepatic steatosis in db/db, HFD, and HFHC mice. HFHC did not increase serum insulin (E) or NOX1 (F) as compared with controls (n = 5–7). Data are shown as means ± SE. *P < 0.05 vs. other groups; ns, no groups significantly different. NOX1, NADPH oxidase 1; Srebp1, sterol regulatory element binding protein 1.

Two additional diet-induced obesity models of high-fat diet (60%; HFD) or high-fat, high-carbohydrate (HFHC) diets were compared with db/db mice (36). Although both diets resulted in obesity, the HFD model produced hepatic steatosis and insulin resistance as indicated by elevated serum insulin levels, whereas the HFHC model, for reasons unclear, did not produce hepatic steatosis and insulin resistance (Fig. 6, D and E). Interestingly, in both db/db and HFD-fed mice with hepatic steatosis, NOX1 expression was increased in the liver, whereas in HFHC mice without hepatic steatosis or elevated serum insulin, NOX1 expression remained at control levels (Fig. 6F). Taken together, each of these models point to insulin as the driving metabolic factor of injurious hepatic NOX1 overexpression in obesity.

Hepatic Steatosis Specifically Increases NOX1 Expression in Obese Humans

Finally, we sought to increase the translatability of this study by determining whether hepatic NOX1 is increased in obese humans with steatosis. Using a previously published GEO dataset containing microarray profiles from humans with biopsy-proven NAFLD (37), we found that hepatic NOX1, NOXO1, and NOXA1 expression to be significantly increased in obese humans with steatosis versus obese controls without steatosis (Fig. 7, A and B). Other NOX isoforms did not differ between groups (Fig. 7C; Supplemental Fig. S9), strongly suggesting elevated NOX1 is a ubiquitous feature of hepatic steatosis.

Figure 7.

Figure 7.

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NOX1 predicts hepatic steatosis in obese humans. Hepatic expression of NOX1 (A) and coactivators NOXO1 and NOXA1 (B) in obese humans with (n = 10) and without (n = 15) biopsy-proven hepatic steatosis. C: expression of other NOX-family genes is not different between groups. *P < 0.05 vs. control group; ns, not significantly different. Data obtained from GEO [GDS4881]. NOX1, NADPH oxidase 1; NOXO1, NADPH oxidase organizer 1.

DISCUSSION

The major finding of this study is that hypermuscularity with myostatin deletion resolves hepatic steatosis in db/db mice via mechanisms involving NOX1. There are numerous metabolic benefits with increased muscle including improvements in insulin resistance, glycemic control, and whole body adiposity, and disentangling the specific components beneficial in NAFLD is experimentally challenging. To uncouple the metabolic benefits of increased muscle from weight loss, we deleted myostatin in db/db mice. Myostatin is a secreted myokine in the TGF-β superfamily that potently inhibits muscle growth. Genetic or pharmacological myostatin inhibition significantly increases muscle mass (38, 39), and myostatin blockade has been extensively used as a resistance exercise mimetic to investigate the physiological adaptions of increased muscle. Importantly, myostatin inhibition does not affect body weight or visceral adiposity in db/db mice (31, 40–42), indicating that the hepatic benefits of hypermuscularity in our model are independent of weight loss. Although myostatin inhibition is known to protect against hepatic triglyceride accumulation (38, 43), underlying mechanisms are unclear. For instance, myostatin deletion has been shown to attenuate fatty acid uptake (44), increase fatty acid oxidation (45), and prevent hepatic insulin resistance (46) in mice with diet-induced obesity. We show here that hypermuscularity with myostatin deletion attenuates hepatic Srebp1 and downstream targets in db/db mice. Consistent with our myostatin model, Akt1-mediated muscle hypertrophy similarly downregulated hepatic steatosis and Scd1 expression in obese mice (47). Although a more exact mechanism remains to be defined, these data nevertheless demonstrate metabolic cross talk between muscle and liver tissue whereby an intervention in muscle with myostatin deletion indirectly alleviates triglyceride accumulation in the liver. This circuit suggests that resistance exercise programs that focus on increasing muscle mass may be effective prescriptive regimens in NAFLD. Moreover, pharmacological myostatin inhibition may be a particularly attractive therapeutic strategy for patients with NAFLD who are unable or unwilling to tolerate strenuous exercise.

Although the benefits of exercise are incontrovertible, underlying mechanisms conferring the metabolic adaptations of increased muscle in NAFLD are unclear. Moreover, clinical data reporting the effect of physical exercise on hepatic oxidant stress are surprisingly scarce. We report that hypermuscularity abolishes NOX1 upregulation in db/db mice, suggesting that increased muscle mass modulates NOX1 to represent a novel potential mechanism regulating intrahepatic lipid accumulation. Our data indicate that NOX1 transcription is a reliable indicator of activity and oxidant production; however, the regulation of component assembly in obesity and its pathological significance remain unexplored. Thus, in addition to reducing metabolic risk factors such as peripheral insulin resistance NOX1 may yet be another physiological adaption of increased muscle, explaining the benefits of resistance exercise in NAFLD.

To directly evaluate the role of NOX1 in steatosis, NOX1 was deleted in db/db mice, revealing that deletion abolished hepatic oxidant stress, triglyceride content, and Srebp1 programming in obese mice. Conversely, NOX1 transfection potentiated SREBP1 expression in insulin-stimulated HepG2 cells, providing complementary in vitro and in vivo evidence linking Srebp1 expression with NOX1. As the only known function of NOX1 is superoxide production, this suggests that NOX1-derived superoxide impacts Srebp1 activation. To directly investigate the role of superoxide, SREBP1 expression was assessed in NOX5-overexpressing HepG2 cells, a superoxide-producing NOX homologue (48). Remarkably, NOX5 overexpression similarly potentiated SREBP1 expression as with NOX1, confirming superoxide as the key mechanistic agent for the action of NOX1 on SREBP1. As the canonical locus of hepatic lipogenesis, this is an important conceptual advance as it implies a novel level of Srebp1 regulation mediated by superoxide-sensing mechanisms. However, Srebp1 activation is extremely complex, involving multiple transcriptional and posttranscriptional regulatory systems, many of which are incompletely understood. Hepatic Srebp1 induction by NOX1-derived superoxide, or oxidant stress-related mechanisms in general, is even less understood. A potential role for NOX1-induced lipid accumulation was recently suggested by a report published during completion of this study, stating pCMV-mediated NOX1 overexpression upregulates insulin-stimulated glucose uptake in AML12 hepatocytes (29), and our study does not rule out other potential effects of NOX1 knockout on lipid metabolism. NOX1 expression has also been reported in all major cell types of the liver including hepatocytes, stellate cells, and sinusoidal endothelial cells (23, 26, 49), and a limitation of our study is that the global knockout model precludes identification of the predominant cell type responsible for NOX1 function. Cell-specific strategies and quantitative hepatic oxidant stress measurement will be required to further our understanding of the impact of NOX1 in obesity. Although the precise mechanisms for NOX1 induction of Srebp1 remain to be defined, these data hint at an exciting new form of Srebp1 regulation governed by the signal-transduction properties of oxidant stress and potentially redox biology. Targeting these mechanisms may broaden therapeutic approaches to NAFLD.

We conclude by hypothesizing that hepatic NOX1 is controlled by metabolic drivers (50). Obesity is characterized by a number of intimately related metabolic derangements including hyperglycemia and hyperinsulinemia-related mechanisms, and identifying specific components exacerbating NOX1 is challenging. In this study, we modulate endogenous glucose and insulin signaling to dissect the obesity phenotype to discern upstream NOX1 drivers. We determined that increased muscle mass with myostatin deletion reduced steatosis in parallel with hepatic NOX1 expression in db/db mice. In contrast, enhancing insulin signaling with Ptp1b deletion preserved hepatic steatosis and NOX1 expression even as it improved hyperglycemia in obese mice. Sustained hyperglycemia with STZ failed to induce hepatic NOX1 expression in the absence of serum insulin, and similarly, NOX1 expression and hepatic steatosis were prevented in mice with diet-induced obesity in the absence of elevated serum insulin. Finally, hepatic NOX1 predicts steatosis in obese humans, identifying hyperinsulinemia as the central metabolic derangement explaining NOX1-mediated steatosis in obesity.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Material, Supplemental Tables S1–S3, and Supplemental Figs. S1–S9: https://doi.org/10.6084/m9.figshare.24584787.

GRANTS

This work was supported by National Institutes of Health Grants HL124733, 1R01HL147159 (to D.W.S. and D.J.F.), HD087527, and 15POST25730006 (to J.A.T.), 1F31HL154646 (to C.A.P.), and 17POST33410322 to (J.T.B.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.L. and D.W.S. conceived and designed research; S.L., C.A.P., J.D.M., J.A.T., J.T.B., E.J.B.d.C., S.H., and D.W.S. performed experiments; S.L., J.D.M., J.A.T., J.T.B., E.J.B.d.C., S.H., and D.W.S. analyzed data; S.L., J.A.T., J.T.B., E.J.B.d.C., S.K., and D.W.S. interpreted results of experiments; S.L., C.A.P., J.A.T., and D.W.S. prepared figures; S.L., C.A.P., and D.W.S. drafted manuscript; S.L., C.A.P., and D.W.S. edited and revised manuscript; S.L., C.A.P., J.D.M., J.A.T., J.T.B., E.J.B.d.C., S.H., S.K., D.J.F., and D.W.S. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors are grateful to the Augusta University Histology Core and Vanderbilt Mouse Metabolic Phenotyping Center (DK059637) for sample processing. S. Larion thanks the Augusta University Internal Medicine–Research Pathway, the AASLD Emerging Liver Scholars program, and the AASLD Foundation for presentation assistance.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material, Supplemental Tables S1–S3, and Supplemental Figs. S1–S9: https://doi.org/10.6084/m9.figshare.24584787.

Data Availability Statement

Data will be made available upon reasonable request.

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