Laura H. Tetri

Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent

The aims of this study were to determine whether combining features of a western lifestyle in mice with trans fats in a high-fat diet, high-fructose corn syrup in the water, and interventions designed to promote sedentary behavior would cause the hepatic histopathological and metabolic abnormalities that characterize nonalcoholic steatohepatitis (NASH). Male C57BL/6 mice fed ad libitum high-fat chow containing trans fats (partially hydrogenated vegetable oil) and relevant amounts of a high-fructose corn syrup (HFCS) equivalent for 1-16 wk were compared with mice fed standard chow or mice with trans fats or HFCS omitted. Cage racks were removed from western diet mice to promote sedentary behavior. By 16 wk, trans fat-fed mice became obese and developed severe hepatic steatosis with associated necroinflammatory changes. Plasma alanine aminotransferase levels increased, as did liver TNF-alpha and procollagen mRNA, indicating an inflammatory and profibrogenic response to injury. Glucose intolerance and impaired fasting glucose developed within 2 and 4 wk, respectively. Plasma insulin, resistin, and leptin levels increased in a profile similar to that seen in patients with NASH. The individual components of this diet contributed to the phenotype independently; isocaloric replacement of trans fats with lard established that trans fats played a major role in promoting hepatic steatosis and injury, whereas inclusion of HFCS promoted food consumption, obesity, and impaired insulin sensitivity. Combining risk factors for the metabolic syndrome by feeding mice trans fats and HFCS induced histological features of NASH in the context of a metabolic profile similar to patients with this disease. Because dietary trans fats promoted liver steatosis and injury, their role in the epidemic of NASH needs further evaluation.

Protective role of angiotensin II type 2 receptor signaling in a mouse model of pancreatic fibrosis.

The renin-angiotensin system contributes to pathological processes in a variety of organs. In the pancreas, blocking the angiotensin II (AII) type 1 receptor (AT1) attenuates pancreatic fibrogenesis in animal models of pancreatitis. Because the role of the AII type 2 receptor (AT2) in modulating pancreatic injury is unknown we investigated the role of AT2 in pancreatic injury and fibrosis. Pancreatic fibrosis was induced by repetitive cerulein administration in C57BL/6 wild-type (WT) or AT2-deficient (AT2-/-) mice and assessed by morphology and gene expression at 10 days. There was no difference between WT and AT2-/- mice in the degree of acute pancreatic injury as assessed by amylase release at 9 and 12 h and by histological examination of the pancreas at 12 h. In contrast, parenchymal atrophy and fibrosis were more pronounced in AT2-/- mice compared with WT mice at 10 days. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for alpha-smooth muscle actin and by immunocytochemistry; PSC activation was further increased in AT2-/- mice compared with WT mice. The level of pancreatic transforming growth factor-beta1 mRNA and protein after repetitive cerulein treatment was higher in AT2-/- mice than in WT mice. Our results demonstrate that, in contrast to AT1 receptor signaling, AT2 receptor signaling modulates protective antifibrogenic effects in a mouse model of cerulein-induced pancreatic fibrogenesis. We propose that the effects of AII on injury-induced pancreatic fibrosis may be determined by the balance between AT1 and AT2 receptor signaling.

Dietary trans-Fatty Acid Induced NASH is Normalized Following Loss of trans-Fatty Acids from Hepatic Lipid Pools

Previous experiments in mice showed that dietary trans-fats could play a role in non-alcoholic steatohepatitis (NASH) yet little is known about the accumulation trans-fats in hepatic lipid pools in relationship to liver injury. NASH is also associated with obesity yet improves with only modest weight loss. To distinguish the role of obesity versus sustained consumption of a trans-fat containing diet in causing NASH, mice with obesity and NASH induced by consuming a high trans-fat diet for 16xa0weeks were subsequently fed standard chow or maintained on trans-fat chow for another 8xa0weeks. The accumulation, partitioning and loss of trans-fats in the major hepatic lipid pools during and after trans-fat consumption were determined. Obese mice switched to standard chow remained obese but steatohepatitis improved. trans-fats were differentially incorporated into the major hepatic lipid pools and the loss of trans-fats after crossover to control chow was greatest in the cholesteryl ester pool. In summary, dietary changes can improve the biochemical and histopathological changes of NASH despite persistent obesity in mice. Analysis of hepatic lipids confirmed that dietary trans-fats accumulate in the major lipid pools and are released differentially with diet normalization. The substantial loss of trans-fats from the cholesteryl ester pool in parallel with improvement in NASH suggests that this pool of trans-fats could play a role in the pathogenesis of NASH.

A mechanism by which dietary trans fats cause atherosclerosis

Dietary trans fats (TFs) have been causally linked to atherosclerosis, but the mechanism by which they cause the disease remains elusive. Suppressed transforming growth factor (TGF)-β responsiveness in aortic endothelium has been shown to play an important role in the pathogenesis of atherosclerosis in animals with hypercholesterolemia. We investigated the effects of a high TF diet on TGF-β responsiveness in aortic endothelium and integration of cholesterol in tissues. Here, we show that normal mice fed a high TF diet for 24 weeks exhibit atherosclerotic lesions and suppressed TGF-β responsiveness in aortic endothelium. The suppressed TGF-β responsiveness is evidenced by markedly reduced expression of TGF-β type I and II receptors and profoundly decreased levels of phosphorylated Smad2, an important TGF-β response indicator, in aortic endothelium. These mice exhibit greatly increased integration of cholesterol into tissue plasma membranes. These results suggest that dietary TFs cause atherosclerosis, at least in part, by suppressing TGF-β responsiveness. This effect is presumably mediated by the increased deposition of cholesterol into cellular plasma membranes in vascular tissue, as in hypercholesterolemia.

Biallelic Mutations in MITF Cause Coloboma, Osteopetrosis, Microphthalmia, Macrocephaly, Albinism, and Deafness

Human MITF is, by convention, called the microphthalmia-associated transcription factor because of previously published seminal mouse genetic studies; however, mutations in MITF have never been associated with microphthalmia in humans. Here, we describe a syndrome that we term COMMAD, characterized by coloboma, osteopetrosis, microphthalmia, macrocephaly, albinism, and deafness.xa0COMMAD is associated with biallelic MITF mutant alleles and hence suggests a role for MITF in regulating processes such as optic-fissure closure and bone development or homeostasis, which go beyond what is usually seen in individuals carrying monoallelic MITF mutations.

Postnatal Hyperoxia Exposure Durably Impairs Right Ventricular Function and Mitochondrial Biogenesis

&NA; Prematurity complicates 12% of births, and young adults with a history of prematurity are at risk to develop right ventricular (RV) hypertrophy and impairment. The long‐term risk for pulmonary vascular disease, as well as mechanisms of RV dysfunction and ventricular‐vascular uncoupling after prematurity, remain poorly defined. Using an established model of prematurity‐related lung disease, pups from timed‐pregnant Sprague Dawley rats were randomized to normoxia or hyperoxia (fraction of inspired oxygen, 0.85) exposure for the first 14 days of life. After aging to 1 year in standard conditions, rats underwent hemodynamic assessment followed by tissue harvest for biochemical and histological evaluation. Aged hyperoxia‐exposed rats developed significantly greater RV hypertrophy, associated with a 40% increase in RV systolic pressures. Although cardiac index was similar, hyperoxia‐exposed rats demonstrated a reduced RV ejection fraction and significant RV‐pulmonary vascular uncoupling. Hyperoxia‐exposed RV cardiomyocytes demonstrated evidence of mitochondrial dysregulation and mitochondrial DNA damage, suggesting potential mitochondrial dysfunction as a cause of RV dysfunction. Aged rats exposed to postnatal hyperoxia recapitulate many features of young adults born prematurely, including increased RV hypertrophy and decreased RV ejection fraction. Our data suggest that postnatal hyperoxia exposure results in mitochondrial dysregulation that persists into adulthood with eventual RV dysfunction. Further evaluation of long‐term mitochondrial function is warranted in both animal models of premature lung disease and in human adults who were born preterm.

Impaired autonomic function in adolescents born preterm

Preterm birth temporarily disrupts autonomic nervous system (ANS) development, and the long‐term impacts of disrupted fetal development are unclear in children. Abnormal cardiac ANS function is associated with worse health outcomes, and has been identified as a risk factor for cardiovascular disease. We used heart rate variability (HRV) in the time domain (standard deviation of RR intervals, SDRR; and root means squared of successive differences, RMSSD) and frequency domain (high frequency, HF; and low frequency, LF) at rest, as well as heart rate recovery (HRR) following maximal exercise, to assess autonomic function in adolescent children born preterm. Adolescents born preterm (less than 36 weeks gestation at birth) in 2003 and 2004 and healthy age‐matched full‐term controls participated. Wilcoxon Rank Sum tests were used to compare variables between control and preterm groups. Twenty‐one adolescents born preterm and 20 term‐born controls enrolled in the study. Preterm‐born subjects had lower time‐domain HRV, including SDRR (69.1 ± 33.8 vs. 110.1 ± 33.0 msec, respectively, P = 0.008) and RMSSD (58.8 ± 38.2 vs. 101.5 ± 36.2 msec, respectively, P = 0.012), with higher LF variability in preterm subjects. HRR after maximal exercise was slower in preterm‐born subjects at 1 min (30 ± 12 vs. 39 ± 9 bpm, respectively, P = 0.013) and 2 min (52 ± 10 vs. 60 ± 10 bpm, respectively, P = 0.016). This study is the first report of autonomic dysfunction in adolescents born premature. Given prior association of impaired HRV with adult cardiovascular disease, additional investigations into the mechanisms of autonomic dysfunction in this population are warranted.

Early Pulmonary Vascular Disease in Young Adults Born Preterm

Rationale: Premature birth affects 10% of live births in the United States and is associated with alveolar simplification and altered pulmonary microvascular development. However, little is known about the long‐term impact prematurity has on the pulmonary vasculature. Objectives: Determine the long‐term effects of prematurity on right ventricular and pulmonary vascular hemodynamics. Methods: Preterm subjects (n = 11) were recruited from the Newborn Lung Project, a prospectively followed cohort at the University of Wisconsin‐Madison, born preterm with very low birth weight (≤1,500 g; average gestational age, 28 wk) between 1988 and 1991. Control subjects (n = 10) from the same birth years were recruited from the general population. All subjects had no known adult cardiopulmonary disease. Right heart catheterization was performed to assess right ventricular and pulmonary vascular hemodynamics at rest and during hypoxic and exercise stress. Measurements and Main Results: Preterm subjects had higher mean pulmonary arterial pressures (mPAPs), with 27% (3 of 11) meeting criteria for borderline pulmonary hypertension (mPAP, 19‐24 mm Hg) and 18% (2 of 11) meeting criteria for overt pulmonary hypertension (mPAP ≥ 25 mm Hg). Pulmonary vascular resistance and elastance were higher at rest and during exercise, suggesting a stiffer vascular bed. Preterm subjects were significantly less able to augment cardiac index or right ventricular stroke work during exercise. Among neonatal characteristics, total ventilatory support days was the strongest predictor of adult pulmonary pressure. Conclusions: Young adults born preterm demonstrate early pulmonary vascular disease, characterized by elevated pulmonary pressures, a stiffer pulmonary vascular bed, and right ventricular dysfunction, consistent with an increased risk of developing pulmonary hypertension.

Sex-Specific Skeletal Muscle Fatigability and Decreased Mitochondrial Oxidative Capacity in Adult Rats Exposed to Postnatal Hyperoxia

Premature birth affects more than 10% of live births, and is characterized by relative hyperoxia exposure in an immature host. Long-term consequences of preterm birth include decreased aerobic capacity, decreased muscular strength and endurance, and increased prevalence of metabolic diseases such as type 2 diabetes mellitus. Postnatal hyperoxia exposure in rodents is a well-established model of chronic lung disease of prematurity, and also recapitulates the pulmonary vascular, cardiovascular, and renal phenotype of premature birth. The objective of this study was to evaluate whether postnatal hyperoxia exposure in rats could recapitulate the skeletal and metabolic phenotype of premature birth, and to characterize the subcellular metabolic changes associated with postnatal hyperoxia exposure, with a secondary aim to evaluate sex differences in this model. Compared to control rats, male rats exposed to 14 days of postnatal hyperoxia then aged to 1 year demonstrated higher skeletal muscle fatigability, lower muscle mitochondrial oxidative capacity, more mitochondrial damage, and higher glycolytic enzyme expression. These differences were not present in female rats with the same postnatal hyperoxia exposure. This study demonstrates detrimental mitochondrial and muscular outcomes in the adult male rat exposed to postnatal hyperoxia. Given that young adults born premature also demonstrate skeletal muscle dysfunction, future studies are merited to determine whether this dysfunction as well as reduced aerobic capacity is due to reduced mitochondrial oxidative capacity and metabolic dysfunction.

24 PARADOXICAL CHANGES IN INSULIN TOLERANCE IN MICE WITH NONALCOHOLIC FATTY LIVER DISEASE INDUCED BY SEDENTARY ACTIVITY AND AN AMERICAN FAST FOOD-TYPE DIET.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States. Insulin resistance plays a central role in both the development and progression of NAFLD. Contributing factors to this may include the increasingly sedentary lifestyle of the population and increased consumption of a high-fat diet and high-fructose corn syrup (HFCS). The aim of this study was to characterize the glucose and insulin tolerance of sedentary mice fed a diet similar in composition to commonly consumed fast food (FFD). Methods Male C57/BL6 mice (n = 10 in each treatment group) were fed ad libitum a FFD diet containing trans-fats (Harlan-Teklad, 43% of calories from fat) and water containing HFCS equivalent (6 g/kg/d) or standard chow and water. To promote sedentariness in the FFD group, the cages wire racks were removed. The insulin tolerance tests were performed after 6 hours of food deprivation at 4, 8, and 12 weeks of feeding; regular human insulin (1 U/kg) was injected intraperitoneally, and blood glucose was measured at 0, 20, 40, and 60 minutes. Glucose tolerance test was performed at 8 weeks by the administration of glucose 1 g/kg intraperitoneally after 12 hours of food deprivation. Blood glucose was measured at 0, 20, 40, 60, and 150 minutes. Results Hepatic steatosis increased progressively over 8 weeks in a distinctly zone 1 to zone 3 distribution pattern, similar to pediatric NAFLD. At 8 weeks, the triglyceride content of the FFD livers was 24 μg/mg (SD 8.0) and the control triglyceride content was 8.2 μg/mg (SD 1.6) (p < .01). Baseline fasting glucose levels were higher in the FFD mice than controls throughout the study period (at weeks 4, 8, and 12; p < .05). As shown in the Table below, the blood glucose levels after insulin injection were paradoxically lower in FFD mice than controls in mice fed for 4 and 8 weeks; in contrast, the blood glucose levels after insulin injection in mice fed the FFD for 12 weeks were higher than controls, suggesting impaired glucose tolerance developed by this later time point in sedentary mice fed the FFD. Glucose tolerance testing showed substantially higher glucose levels in the FFD mice after 8 weeks of feeding, indicating earlier onset of impaired glucose tolerance than insulin resistance. The differences were significant at 20, 40, 60, and 150 minutes (p < .01). Conclusions The increased insulin responsiveness following 4 or 8 weeks but not 12 weeks of sedentary activity and feeding FFD might indicate initially increased insulin sensitivity or, alternatively, impaired insulin clearance or impaired counterregulatory mechanisms against low glucose levels. After 12 weeks, impaired insulin responsiveness was found. These findings might be explained by the following: (1) the unique metabolism of fructose in the liver because fructose is a precursor for triglyceride synthesis and only a small amount of fructose enters into the systemic circulation, (2) impaired insulin clearance, (3) increased oxidant stress in the liver, (4) impaired islet cell function in the pancreas, or (5) insulin resistance develops in the liver and adipose tissue earlier than in muscle. Because insulin tolerance testing is a measure of muscle glucose uptake, it is also possible that increasing fat content in muscle over time might cause peripheral insulin resistance by week 12 in this model. Insulin Tolerance Test Glucose, % of Time 0 (± SD)