Paternal Aerobic Exercise/high Fat Diet Reprograms The Metabolic Phenotype In The Offspring Of C57BL/6 Mice

Objective:Obesity is the result of energy imbalance, when the body’s energy intake exceeds energy consumption, excess fat storage in the body, or the higher proportion of adipose tissue to body weight. Conventional wisdom holds that heredity,changes in dietary factors, lack of exercise is the main cause of obesity. However, this doctrine of obesity has been difficult to interpret the global obesity; especially childhood obesity is rapidly increasing. Animal experiments and human observation show that animals fed with identical diet(E.g. high-fat high-calorie diet), partial obesity prone subjects under certain dietary environment, while another part of the object can maintain a normal body weight, indicating that other factors may also affect obesity susceptibility. During the past decades, a large number of studies basis of environmental factors of obesity. Researches have focused on genetic mechanisms and environmental factors induce the pathological mechanisms of obesity, and the obese related metabolic disorders. The latest study found that paternal(F0) epigenetic modifications may pass transgenerational inheritance cause offspring(F1) susceptible to obesity and T2 DM. Recently study showed exercise or high fat diet during pregnancy impacted fetal size and metabolism are well established. However, knowledge about the extent of contributions of paternal aerobic exercise or diet before mating and the long-term consequences on offspring is minimal; particularly the role of non-genetic factors in the causal pathway. Paternal high-fat-diet(HFD) exposure programs β-cell ’dysfunction’ in female offspring. This is the first report in mammals of non-genetic, intergenerational transmission of metabolic sequelae of a HFD from father to offspring. In the present study, our goals were to determine the effects of paternal 6 weeks aerobic exercise or high fat diet on offspring metabolic phenotype and to investigate the mechanisms associated with such effects. Thus, we assessed visceral fat mass, the Lee’s index, the growth and development, the OGTT and the activity of PPARγ in the male offspring.Methods:(1) Animal model: Sibling males were divided into three groups: the control(C), the exercise group(E) and the high fat diet group(HF). Sibling females were kept sedentary. The E group mice in the running group were exercised on a motor-driven rodent treadmill for 5 days a week for a total of 6 weeks; the HF group mice feeding 6 weeks high fat diet. After 6 weeks, one male and one female were mated. To minimize male-female interactions, which may impact on maternal investment or care and since affect offspring development, pregnancy was assured by observing a copulation plug(identified each morning within 1 h after lights on) and signaled to remove the female to her own cage. F1 male mice of exercised group,high fat diet group and sedentary group were named for EO,HFO and CO;(2) Exercise training protocol: E group were given exercise training on motorized treadmill at 12m/min(75% VO2max) for 60 min/day, once per day for 6 weeks(5 days per week);(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 6 weeks;(4) Morphological characteristics: The offspring of each group were weighed and measured before sacrificed under anesthesia. Visceral fat from the epididymal and perirenal fat depots of the male mice were quickly harvested, weights of the organs were also recorded;(5) Oral glucose tolerance test(OGTT): Mice were fasted for 16 hours before experiment. The body weight of each mouse was measured and documented. The serum fasting glucose concentration was measured by using One Touch meter at the time point of T0. Immediately 20% glucose solution was rapidly perfused via mouth based on body weight at 10μl/g, Blood glucose concentration at T15, T30, T60 and T120 min were measured respectively;(6) Enzyme immunoassay: Plasma lipid profile and insulin of the offspring were measured by using mice enzyme immunoassay(ELISA) kit following the manufacturer’s protocol;(7) H&E staining: The H&E staining is a classic nucleic acid staining method traditionally used on tissue observe in morphology. All pups were weighed and killed the morning following the last treatment day and were fasted overnight(14 h) with free access to water. The pancreatic islet was removed from euthanized mice and ?xed in 10% of neutral buffered formalin, embedded in paraffin. The paraffin was blocked 6 mm sections and stained with hematoxylin and eosin and then photographed at 20× and 40× magni?cations;(8) Real-time PCR was used to detect PPARγ, TNFα and Adiponectin m RNA levels in epididymal fat of CO, EO and HFO mice;(9) Bisulfite conversion pyrosequencing was used to examine the level of methylatd CpG sites of PPARγ at position-437 bp.Results:(1) Gender Differences: Here we show that paternal aerobic exercise/high fat diet reprograms the metabolic phenotype in mice F1 male offspring;(2) Growth and Development characteristics: There was no difference in the body length and body weight of male offspring from CO, HFO and EO groups under normal diet environment. After 4 weeks high fat diet intervention, the body weight in EO group were significantly lower compared with CO group(P<0.05);while mice in HFO group were significantly higher(P<0.05).but the body length of CO, EO and HFO groups has no difference;(3) Changes of Visceral Fat weight: Compared with CO group, the weight of epididymal fat, perirenal fat and total fat were 39.86%, 55.97%, 43.25% lower in EM group(P<0.01), and 42.47%, 50.28%, 44.31% higher in HFO group(P<0.05);(4) Changes of Fat weight/Body weight: Compared with CO group, EF/W, PF/W and TF/W were 34.64%, 52.32%, 38.37% lower in EO group(P<0.01), and 36.26%, 44.94%, 38.31% higher in HFO group(P<0.05);(5) OGTT: There was no difference of OGTT curve in CO, HFO and EO groups under normal diet environment; After 4 weeks high fat diet intervention, the peak value of OGTT curve is higher in HFO group than CO group, and blood glucose level decreased slowly after 30 minutes. Furthermore the glucose level at T120 min was still higher than that of fasting level, showed that HFO group was sensitive to metabolic abnormalities caused by high-fat diet. However, we found that the time point of OGTT peak shifted back in EO group, and blood glucose level decreased quickly after 30 minutes, showed that EO group can resist on metabolic abnormalities caused by high-fat diet;(6) Plasma lipid profile: Compared with CO group, plasma TG, T-CHD, LDL-C and FFA level were 67.49%, 17.76%, 64.50%,10.16% lower in EO group(P<0.05), meanwhile the HDL-C level was 19.69% higher in EO group(P<0.05); but in HFO group, plasma TG, T-CHD, LDL-C and FFA level were 32.12%, 15.03%, 20.85%, 13.43% higher compared with CO group, meanwhile the HDL-C level was 17.61% lower in HFO group(P<0.05);(7) Fasting Plasma Glucose and Serum Insulin: Fasting Plasma Glucose level was 3.15% higher in HFO group comparing with CO group and 26.49% lower in EO group; Serum Insulin level was 8.60% higher in HFO group comparing with CO group and 28.62% higher in EO group;(8) The pancreatic islet: There was no difference in the pancreatic islet in CO, EO and HFO groups;(9) PPAR-γ, TNF-α and Adiponectin m RNA levels in epididymal fat of male offsprings: PPARγ m RNA level was 29.37% lower in HFO group comparing with CO group and 23.23%(P<0.05) higher in EO group; TNFα m RNA level was 57.42%(P<0.05) higher in HFO group comparing with CO group and 19.16% lower in EO group; Adiponectin m RNA level was 9.14% lower in HFO group comparing with CO group and 45.88%(P<0.05) higher in EO group;(10) Bisulfite conversion pyrosequencing: Compared with CO group, the fraction of methylated Cp G sites of PPARγ at-437 bp was 11.54% higher in HFO group(P<0.05); methylation of the Cp G at position-437 in the PPARγ promoter region was 5.41% higher in EO group compare to the level observed in CO group, however, the difference has no statistical significance.Conclusions:(1) Here we show that paternal aerobic exercise/high fat diet reprograms the metabolic phenotype in mice F1 male offspring;(2) Compared with CO group, there was no difference of metabolic phenotype in EO group under normal diet environment; but after 4 weeks high fat diet intervention, EO group of metabolic phenotype appears significant difference.OGTT results also suggest that EO group of mice can resist on high fat diet induced abnormal glucose tolerance;(3) Compared with CO group, there was no difference of metabolic phenotype in HFO group under normal diet environment; but after 4 weeks high fat diet intervention, HFO group of metabolic phenotype appears significant difference.OGTT results also suggest that HFO group of mice was sensitive to abnormal glucose tolerance caused by high fat diet;(4) The decrease of EO group’s visceral fat may associated with elevated PPARγ and Adiponectin gene expression in offspring epididymal fat, and reduced TNFα gene expression;(5) The increase of HFO group’s visceral fat may associated with reduced PPARγ and Adiponectin gene expression in offspring epididymal fat, and elevated TNFα gene expression;(6) The decrease of PPARγ gene expression possibly via increased methylation of the PPARγ promoter at position-437. The underlying mechanisms seem to include epigenetic modifications, but further studies are needed to explore.