Paternal High Fat Diet/Aerobic Exercise Reprograms The Hepatic Steatosis In The Offspring Of C57BL/6 Mice

Objective: There is emerging evidence that parental lifestyle and environmental exposure, such as exercise, high-fat diet(HFD), and chemical exposure during embryogenesis, lead to variations in offspring and, in some cases, grand-offspring development. Thus far, most research in this area has focused on maternal contributions. Human and animal studies have shown that maternal obesity and weight gain during pregnancy are related to high body mass index(BMI) in childhood and subsequent obesity, insulin resistance, and hyperinsulinemia in adulthood. Moreover, exercise during pregnancy can have positive effects on offspring. Recent observations have shown that developmental programming can be influenced by the paternal line. For example, paternal smoking, HFD and occupational chemical exposure are associated with an increased risk of cancer, obesity and mental health disorders in children. Non-alcoholic fatty liver disease(NAFLD) is an increasingly common chronic liver disease worldwide. Maternal lifestyle and associated risk of type 2 diabetes mellitus(T2DM), obesity and NAFLD in offspring has been well characterized. However, little information exists about the effects of paternal lifestyle on HFD-induced hepatic steatosis in offspring. It is therefore of great interest to define the transgenerational effects of paternal environmental conditions on mammals and the underlying mechanisms that may mediate these effects. Here, in well-established animal models of paternal HFD/treadmill exercise to test the hypothesis in mice that HFD-induced hepatic steatosis in the offspring would be influenced by paternal lifestyle.Methods:(1) Animal model: Three-week-old sibling C57BL/6 male mice were divided into three groups: the control(C), the exercise group(E) and the high fat diet group(HF). After intervention, each male mouse mated with a female sibling fed standard-chow before and during pregnancy. All offspring(F1) were weaned at 3 weeks of age and given normal chow for 1week following HFD for 4 weeks.(2) Training protocol: EM group were given exercise training on motorized treadmill at 12m/min(75% VO2 max) on a 0% grade, training took place 5 days per week, 60 min per day, maintained for 6 weeks.(3) High fat diet protocol: HF group with the high-fat diet using Research Diet Inc formula(calorie ratio: 20% protein, 20% carbohydrate, 60% fat) for 12 weeks.(4) Oral glucose tolerance test(OGTT): OGTT was performed at 4 and 8 weeks of age in male offspring. After a 6-h fast, glucose(10?l/g) was given orally at time 0; tail blood was collected at fasting and then at 15, 30, 60, 90 and 120 min after glucose determination.(5) Histologic Analysis: Oil red O(ORO) staining was performed. Images of positive staining were captured by an Olympus BX50 microscope and the staining area was measured in integrated optical density(IOD) by Image-Pro Plus 6.0.(6) Real-time PCR was used to detect SREBP-1, Fasn, Ch REBP, Pgc-1?, Tfam, Nrf1, Cox4 m RNA. ?-actin quantification was used as an internal control. Relative expression level of target gene was determined as 2 –??CT.(7) Western blot was used to detect AMPK, p AMPK-T172, ACC, p ACC-S79, LC3A/B, P62, CPT-1, PGC-1?, SREBP-1 protein level in liver.Results:(1) Male offspring body length and body weight: There were no significant effect of paternal HFD/exercise on male offspring body length was observed. The male pups from obese fathers were significantly heavier(HFM vs. CM, P<0.05), whereas the males from exercise fathers were lighter(EM vs. CM, P<0.001).(2) OGTT and Lipid profile: There was no difference in OGTT analyses and AUC at 4 weeks of age. After 4 weeks, HFD-induced, HFM group displayed glucose intolerance with greater AUC compared with CM group(P<0.05), whereas paternal exercise had moderate beneficial effects on male EM mice, which had lower fasting blood glucose and at 60 min later than CM(P<0.01 or P<0.001). However, EM did not exhibit a lowered AUC relative to control offspring at 8 weeks of age. Lipid profile, plasma TG, T-CHO, LDL-C, and FFA were significantly higher in male pups from obese fathers(P<0.05 or P<0.001), whereas they were all significantly lower in pups from exercised fathers(P<0.05, P<0.01 or P<0.001).(3) Hepatic oil red O staining: HFM group had significantly increased hepatic TAG accumulation compared to CM group(P<0.01). Remarkably, male offspring from exercise father were completely protected against the hepatic steatosis(EM vs. CM, P<0.001).(4) Markers of de novo lipogenesis in liver: Paternal HFD increased hepatic SREBP-1 and Fasn expression in male offspring at 8 weeks of age(HFM vs. CM, P<0.05). Paternal exercise decreased hepatic SREBP-1 and Fasn expression expression(EM vs. CM, P<0.05). No differences were observed in CHREBP m RNA and ACC protein expression among the three groups.(5) Markers of fatty acid oxidation and mitochondrial function in liver: Paternal HFD resulted in a significantly(P<0.05) decreased in hepatic AMPK?, p AMPK?-T172 and p ACC-S79 protein levels compared with C male offspring(P<0.05). Paternal exercise increased hepatic AMPK?, p AMPK?-T172 and p ACC-S79 protein levels(P<0.05). However, expression of CPT-1, a rate-limiting step in mitochondrial fatty acid entry, was no different among the three groups. Paternal HFD decreased hepatic expression of PGC-1? in 8-week male offsprings compared to counterparts from C funders(P<0.05). Similarly, downstream target gene Nrf1 and Tfam m RNA were decreased by paternal obesity(P<0.05), whereas they were all significantly higher in pups from exercised fathers. However, m RNA level of Cox4 was no different among the three groups.(5) Markers of autophagy in liver:Hepatic autophagy was inhibited by paternal obesity, whereas that was increased by the paternal exercise.Conclusions:(1) After 4 weeks of HFD exposure, male offspring(F1) of HF had significantly obesity, impaired glucose tolerance, hyperlipidemia, and increased hepatic TAG accumulation compared with control, whereas paternal exercise ameliorated the HFD-induced metabolic disorders in male F1;(2) Paternal HFD aggravated male offspring HFD-induced hepatic steatosis, which may be associated with increased lipogenesis, decreased hepatic mitochondrial biogenesis and autophagy;(3) Paternal exercise protected male offspring against HFD-induced hepatic steatosis, which may be associated with decreased levels of de novo lipogenesis, increased markers of mitochondrial biogenesis and autophagy.