E. Matthew Morris

Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model

BACKGROUND & AIMS In this study, we sought to determine the temporal relationship between hepatic mitochondrial dysfunction, hepatic steatosis and insulin resistance, and to examine their potential role in the natural progression of non-alcoholic fatty liver disease (NAFLD) utilising a sedentary, hyperphagic, obese, Otsuka Long-Evans Tokushima Fatty (OLETF) rat model. METHODS OLETF rats and their non-hyperphagic control Long-Evans Tokushima Otsuka (LETO) rats were sacrificed at 5, 8, 13, 20, and 40 weeks of age (n=6-8 per group). RESULTS At 5 weeks of age, serum insulin and glucose and hepatic triglyceride (TG) concentrations did not differ between animal groups; however, OLETF animals displayed significant (p<0.01) hepatic mitochondrial dysfunction as measured by reduced hepatic carnitine palmitoyl-CoA transferase-1 activity, fatty acid oxidation, and cytochrome c protein content compared with LETO rats. Hepatic TG levels were significantly elevated by 8 weeks of age, and insulin resistance developed by 13 weeks in the OLETF rats. NAFLD progressively worsened to include hepatocyte ballooning, perivenular fibrosis, 2.5-fold increase in serum ALT, hepatic mitochondrial ultrastructural abnormalities, and increased hepatic oxidative stress in the OLETF animals at later ages. Measures of hepatic mitochondrial content and function including beta-hydroxyacyl-CoA dehydrogenase activity, citrate synthase activity, and immunofluorescence staining for mitochondrial carbamoyl phosphate synthetase-1, progressively worsened and were significantly reduced at 40 weeks in OLETF rats compared to LETO animals. CONCLUSIONS Our study documents that hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance in the OLETF rats. This evidence suggests that progressive mitochondrial dysfunction contributes to the natural history of obesity-associated NAFLD.

Angiotensin II-induced NADPH Oxidase Activation Impairs Insulin Signaling in Skeletal Muscle Cells

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.

Rats selectively bred for low aerobic capacity have reduced hepatic mitochondrial oxidative capacity and susceptibility to hepatic steatosis and injury

Fatty liver has been linked to low aerobic fitness, but the mechanisms are unknown. We previously reported a novel model in which rats were artificially selected to be high capacity runners (HCR) and low capacity runners (LCR) that in a sedentary condition have robustly different intrinsic aerobic capacities. We utilized sedentary HCR/LCR rats (generation 17; max running distance equalled 1514 ± 91 vs. 200 ± 12 m for HCR and LCR, respectively) to investigate if low aerobic capacity is associated with reduced hepatic mitochondrial oxidative capacity and increased susceptibility to hepatic steatosis. At 25 weeks of age, LCR livers displayed reduced mitochondrial content (reduced citrate synthase activity and cytochrome c protein) and reduced oxidative capacity (complete palmitate oxidation in hepatic mitochondria (1.15 ± 0.13 vs. 2.48 ± 1.1 nm g−1 h, P < 0.0001) and increased peroxisomal activity (acyl CoA oxidase and catalase activity) compared to the HCR. The LCR livers also displayed a lipogenic phenotype with higher protein content of both sterol regulatory element binding protein‐1c and acetyl CoA carboxylase. These differences were associated with hepatic steatosis in the LCR including higher liver triglycerides (6.00 ± 0.71 vs. 4.20 ± 0.39 nmol g−1, P= 0.020 value), >2‐fold higher percentage of hepatocytes associated with lipid droplets (54.0 ± 9.2 vs. 22.0 ± 3.5%, P= 0.006), and increased hepatic lipid peroxidation compared to the HCR. Additionally, in rats aged to natural death, LCR livers had significantly greater hepatic injury (fibrosis and apoptosis). We provide novel evidence that selection for low intrinsic aerobic capacity causes reduced hepatic mitochondrial oxidative capacity that increases susceptibility to both hepatic steatosis and liver injury.

Daily exercise vs. caloric restriction for prevention of nonalcoholic fatty liver disease in the OLETF rat model

The maintenance of normal body weight either through dietary modification or being habitually more physically active is associated with reduced incidence of nonalcoholic fatty liver disease (NAFLD). However, the means by which weight gain is prevented and potential mechanisms activated remain largely unstudied. Here, we sought to determine the effects of obesity prevention by daily exercise vs. caloric restriction on NAFLD in the hyperphagic, Otsuka Long-Evans Tokushima Fatty (OLETF) rat. At 4 wk of age, male OLETF rats (n = 7-8/group) were randomized to groups of ad libitum fed, sedentary (OLETF-SED), voluntary wheel running exercise (OLETF-EX), or caloric restriction (OLETF-CR; 70% of SED) until 40 wk of age. Nonhyperphagic, control strain Long-Evans Tokushima Otsuka (LETO) rats were kept in sedentary cage conditions for the duration of the study (LETO-SED). Both daily exercise and caloric restriction prevented obesity and the development of type 2 diabetes observed in the OLETF-SED rats, with glucose tolerance during a glucose tolerance test improved to a greater extent in the OLETF-EX animals (30-50% lower glucose and insulin areas under the curve, P < 0.05). Both daily exercise and caloric restriction also prevented excess hepatic triglyceride and diacylglycerol accumulation (P < 0.001), hepatocyte ballooning and nuclear displacement, and the increased perivenular fibrosis and collagen deposition that occurred in the obese OLETF-SED animals. However, despite similar hepatic phenotypes, OLETF-EX rats also exhibited increased hepatic mitochondrial fatty acid oxidation, enhanced oxidative enzyme function and protein content, and further suppression of hepatic de novo lipogenesis proteins compared with OLETF-CR. Prevention of obesity by either daily exercise or caloric restriction attenuates NAFLD development in OLETF rats. However, daily exercise may offer additional health benefits on glucose homeostasis and hepatic mitochondrial function compared with restricted diet alone.

Angiotensin II-induced non-alcoholic fatty liver disease is mediated by oxidative stress in transgenic TG(mRen2)27(Ren2) rats.

BACKGROUND/AIMS Non-alcoholic fatty liver disease (NAFLD) is a common health problem and includes a spectrum of hepatic steatosis, steatohepatitis and fibrosis. The renin-angiotensin system (RAS) plays a vital role in blood pressure regulation and appears to promote hepatic fibrogenesis. We hypothesized that increased RAS activity causes NAFLD due to increased hepatic oxidative stress. METHODS We employed the transgenic TG(mRen2)27(Ren2) hypertensive rat, harboring the mouse renin gene with elevated tissue Angiotensin II (Ang II). RESULTS Compared with normotensive Sprague-Dawley (SD) control rats, Ren2 developed significant hepatic steatosis by 9 weeks of age that progressed to marked steatohepatitis and fibrosis by 12 weeks. These changes were associated with increased levels of hepatic reactive oxygen species (ROS) and lipid peroxidation. Accordingly, 9-week-old Ren2 rats were treated for 3 weeks with valsartan, an angiotensin type 1 receptor blocker, or tempol, a superoxide dismutase/catalase mimetic. Hepatic indices for oxidative stress, steatosis, inflammation and fibrosis were markedly attenuated by both valsartan and tempol treatment. CONCLUSIONS This study suggests that Ang II causes development and progression of NAFLD in the transgenic Ren2 rat model by increasing hepatic ROS. Our findings also support a potential role of RAS in prevention and treatment of NAFLD.

Albumin Activation of NAD(P)H Oxidase Activity Is Mediated via Rac1 in Proximal Tubule Cells

Background: Rac1 is a Rho-family small GTP-ase, when activated is pivotal in NAD(P)H oxidase (NOX) activation and generation of reactive oxygen species (ROS). Evidence links Rac1 activation to receptor-mediated albumin endocytosis in the proximal tubule cell (PTC). Thus in states of albumin overload, Rac1 activation could lead to NOX activation and ROS formation in the PTC. Furthermore, accumulating evidence supports that HMG-CoA reductase inhibition may reduce oxidative stress and albuminuria. Methods: To investigate the role of HMG-CoA reductase inhibition of Rac1 and oxidative stress we used the opossum kidney PTC. ROS generation in the PTC was confirmed using oxidative fluorescent dihydroethidium staining. Results: We observed time-dependent increases in NOX activity with bovine serum albumin (albumin) stimulation (500 µg/dl, maximum at 20 min, p < 0.05) that was inhibited in a concentration-dependent manner with the HMG-CoA reductase inhibitor rosuvastatin (1 µM, p < 0.05). Additionally, the Rac1 inhibitor NSC23766 (100 ng/ml) attenuated albumin activation of NOX. Western blot analysis confirmed Rac1 translocation to plasma membrane in the PTC following albumin stimulation and subsequent inhibition by rosuvastatin and NSC23766. Conclusions: These data demonstrate that albumin-mediated increases in NOX activity and ROS in PTC are reversed by inhibition of Rac1 signaling with the use of rosuvastatin.

PGC-1α overexpression results in increased hepatic fatty acid oxidation with reduced triacylglycerol accumulation and secretion

Studies have shown that decreased mitochondrial content and function are associated with hepatic steatosis. We examined whether peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression and a subsequent increase in mitochondrial content and function in rat primary hepatocytes (in vitro) and Sprague-Dawley rats (in vivo) would comprehensively alter mitochondrial lipid metabolism, including complete (CO(2)) and incomplete (acid-soluble metabolites) fatty acid oxidation (FAO), tricarboxylic acid cycle flux, and triacylglycerol (TAG) storage and export. PGC-1α overexpression in primary hepatocytes produced an increase in markers of mitochondrial content and function (citrate synthase, mitochondrial DNA, and electron transport system complex proteins) and an increase in FAO, which was accompanied by reduced TAG storage and TAG secretion compared with control. Also, the PGC-1α-overexpressing hepatocytes were protected from excess TAG accumulation following overnight lipid treatment. PGC-1α overexpression in hepatocytes lowered expression of genes critical to VLDL assembly and secretion (apolipoprotein B and microsomal triglyceride transfer protein). Adenoviral transduction of rats with PGC-1α resulted in a liver-specific increase in PGC-1α expression and produced an in vivo liver phenotype of increased FAO via increased mitochondrial function that also resulted in reduced hepatic TAG storage and decreased plasma TAG levels. In conclusion, overexpression of hepatic PGC-1α and subsequent increases in FAO through elevated mitochondrial content and/or function result in reduced TAG storage and secretion in the in vitro and in vivo milieu.

Skeletal muscle mitochondrial and metabolic responses to a high-fat diet in female rats bred for high and low aerobic capacity.

Rats selected artificially to be low-capacity runners (LCR) possess a metabolic syndrome phenotype that is worsened by a high-fat diet (HFD), whereas rats selected to be high-capacity runners (HCR) are protected against HFD-induced obesity and insulin resistance. This study examined whether protection against, or susceptibility to, HFD-induced insulin resistance in the HCR-LCR strains is associated with contrasting metabolic adaptations in skeletal muscle. HCR and LCR rats (generation 20; n = 5-6; maximum running distance approximately 1800 m vs. approximately 350 m, respectively (p < 0.0001)) were divided into HFD (71.6% energy from fat) or normal chow (NC) (16.7% energy from fat) groups for 7 weeks (from 24 to 31 weeks of age). Skeletal muscle (red gastrocnemius) mitochondrial-fatty acid oxidation (FAO), mitochondrial-enzyme activity, mitochondrial-morphology, peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha), and peroxisome proliferator-activated receptor delta (PPARdelta) expression and insulin sensitivity (intraperitoneal glucose tolerance tests) were measured. The HFD caused increased adiposity and reduced insulin sensitivity only in the LCR and not the HCR strain. Isolated mitochondria from the HCR skeletal muscle displayed a 2-fold-higher rate of FAO on NC, but both groups increased FAO following HFD. PGC-1alpha mRNA expression and superoxide dismutase activity were significantly reduced with the HFD in the LCR rats, but not in the HCR rats. PPARdelta expression did not differ between strains or dietary conditions. These results do not provide a clear connection between protection of insulin sensitivity and HFD-induced adaptive changes in mitochondrial function or transcriptional responses but do not dismiss the possibility that elevated mitochondrial FAO in the HCR may play a protective role.

Changes in skeletal muscle mitochondria in response to the development of type 2 diabetes or prevention by daily wheel running in hyperphagic OLETF rats

The temporal changes in skeletal muscle mitochondrial content and lipid metabolism that precede type 2 diabetes are largely unknown. Here we examined skeletal muscle mitochondrial fatty acid oxidation (MitoFAOX) and markers of mitochondrial gene expression and protein content in sedentary 20- and 40-wk-old hyperphagic, obese Otsuka Long-Evans Tokushima fatty (OLETF-SED) rats. Changes in OLETF-SED rats were compared with two groups of rats who maintained insulin sensitivity: age-matched OLETF rats given access to voluntary running wheels (OLETF-EX) and sedentary, nonobese Long-Evans Tokushima Otsuka (LETO-SED) rats. As expected, glucose tolerance tests revealed insulin resistance at 20 wk that progressed to type 2 diabetes at 40 wk in the OLETF-SED, whereas both the OLETF-EX and LETO-SED maintained whole body insulin sensitivity. At 40 wk, complete MitoFAOX (to CO(2)), beta-hydroxyacyl-CoA dehydrogenase activity, and citrate synthase activity did not differ between OLETF-SED and LETO-SED but were significantly (P < 0.05) higher in OLETF-EX compared with OLETF-SED rats. Genes controlling skeletal muscle MitoFAOX (PGC-1alpha, PPARdelta, mtTFA, cytochrome c) were not different between OLETF-SED and LETO-SED at any age. Compared with the OLETF-SED, the OLETF-EX rats had significantly (P < 0.05) higher skeletal muscle PGC-1alpha, cytochrome c, and mtTFA mRNA levels at 20 and 40 wk and PPARdelta at 40 wk; however, protein content for each of these markers did not differ between groups at 40 wk. Limited changes in skeletal muscle mitochondria were observed during the transition from insulin resistance to type 2 diabetes in the hyperphagic OLETF rat. However, diabetes prevention through increased physical activity appears to be mediated in part through maintenance of skeletal muscle mitochondrial function.

Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats

Here, we sought to compare the efficacy of combining exercise and metformin for the treatment of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) in hyperphagic, obese, type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (age: 20 wk, hyperglycemic and hyperinsulinemic; n = 10/group) were randomly assigned to sedentary (O-SED), SED plus metformin (O-SED + M; 300 mg·kg(-1)·day(-1)), moderate-intensity exercise training (O-EndEx; 20 m/min, 60 min/day, 5 days/wk treadmill running), or O-EndEx + M groups for 12 wk. Long-Evans Tokushima Otsuka (L-SED) rats served as nonhyperphagic controls. O-SED + M, O-EndEx, and O-EndEx + M were effective in the management of type 2 diabetes, and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, O-EndEx lowered liver triglyceride content and fasting hyperglycemia more than O-SED + M. In addition, exercise elicited greater improvements compared with metformin alone on postchallenge glycemic control, liver diacylglycerol content, hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activities and in the attenuation of markers of hepatic fatty acid uptake and de novo fatty acid synthesis. Surprisingly, combining metformin and aerobic exercise training offered little added benefit to these outcomes, and in fact, metformin actually blunted exercise-induced increases in complete mitochondrial palmitate oxidation and β-HAD activity. In conclusion, aerobic exercise training was more effective than metformin administration in the management of type 2 diabetes and NAFLD outcomes in obese hyperphagic OLETF rats. Combining therapies offered little additional benefit beyond exercise alone, and findings suggest that metformin potentially impairs exercise-induced hepatic mitochondrial adaptations.