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Maternal Physical Activity and Sex Impact Markers of Hepatic Mitochondrial Health

CUNNINGHAM, RORY P.1,2; MOORE, MARY P.1,2; MEERS, GRACE M.1,2,3; RUEGSEGGER, GREGORY N.4; BOOTH, FRANK W.2,4; RECTOR, R. SCOTT1,2,3

Author Information
Medicine & Science in Sports & Exercise: October 2018 - Volume 50 - Issue 10 - p 2040-2048
doi: 10.1249/MSS.0000000000001675

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a common chronic disease, with its prevalence increasing along with growing obesity rates. NAFLD is composed of a spectrum of liver injury ranging from simple fat accumulation in the liver (steatosis), to steatohepatitis (NASH), to its end stage of cirrhosis. It has been reported that the prevalence of NAFLD in the general population ranges from 10% to 30% and as high as 80%–100% in obese populations (1,2). Strikingly, similar rates are seen in children (3,4). NAFLD is a serious public health concern as it is considered an independent risk factor for cardiovascular, liver-related, and all-cause mortality (5,6). Currently, there are no well-established pharmacological treatments.

A potential contributor to the rise in NAFLD is maternal obesity, as the gestational environment during crucial periods of fetal growth and development can have a significant effect on susceptibility to chronic diseases later in life. It is possible that circulating lipids from a poor maternal diet can enter the fetal circulation and alter DNA via epigenetic modifications (7). Indeed, poor maternal diet and maternal obesity have contributed to trans-generational increases in risk for type 2 diabetes, obesity, and NAFLD (8–12). In the United States, more than one in five pregnant women are obese (13), and as many as 60% of women are overweight at the time of conception (14). This highlights the importance of understanding the consequences of maternal obesity, and targeting interventions to such crucial periods of fetal development may be an avenue of therapy to curb the rise of NAFLD in both adults and children.

One such therapeutic avenue to attenuate rising NAFLD rates may be maternal exercise. In rodent models, physical activity while pregnant has been shown to have beneficial effects on metabolic outcomes in the offspring, such as reduced adiposity, improved glucose tolerance, and decreased hepatic lipid accumulation (15–18). Moreover, exercising before and during the gestational period may in fact offset some of the metabolic perturbations caused by a maternal high-fat diet (HFD), including the attenuation in hepatic lipid accumulation in older adult offspring (15). In addition, our group has previously demonstrated that HFD-fed male offspring from exercised (voluntary wheel running) dams had reduced susceptibility to hepatic steatosis development compared with HFD-fed offspring from sedentary dams when studied at 8 months old (16). These offspring from exercising dams displayed increased markers of hepatic mitochondrial biogenesis and autophagy, potentially contributing to their reduced hepatic steatosis. However, as NAFLD progression is associated with hepatic mitochondrial dysfunction (19) and little is known about the effect of maternal exercise/physical activity on hepatic mitochondrial health in the offspring, this continues to be an important area of investigation.

Given our obesigenic environment and rising rates of obesity during pregnancy, it is important to examine the effects of maternal physical activity under conditions of maternal high-fat western-style dietary feeding conditions on offspring health. In addition, although maternal physical activity has been shown to confer beneficial effects on hepatic health in older adult offspring, it is largely unknown whether these effects are seen in younger offspring. Given the current knowledge gaps in the literature, we tested the hypothesis that maternal physical activity could protect against maternal western diet (WD)–induced hepatic steatosis in young, sedentary adult rats fed a normal low fat diet.

METHODS

Animal protocol

Virgin female Wistar rats (7–8 wk; n = 4 per group) were subjected to normal chow diet (ND; Formulab Diet 5008; Nestle Purina, Indianapolis, IN) or WD (42% kcal from fat, 27% kcal from sucrose; Teklad TD.88137; Envigo, Indianapolis, IN) for 5 wk before and throughout pregnancy ad libitum. A study time line is shown in Figure 1. For mating, a male rat (14–16 wk old) eating an ND was placed in the cage for up to 4 d. Besides this mating window, all male breeder rats were maintained in sedentary cage conditions. Pregnancy was confirmed by formation of a copulatory plug. Female rats continued the same experimental diet intervention until term, at which time all rats received ND. After birth, litter sizes were culled to 10 pups (5 males and 5 females) when necessary to keep pup body masses more homogeneous. Body weight was recorded weekly from 3 to 18 wk of age. From exercised (RUN) or sedentary (SED) dams on either an ND or a WD, male and female offspring were randomly selected from each litter, resulting in eight total offspring groups: ND/SED, WD/SED, ND/RUN, and WD/RUN for both male and female offspring. It is important to note that offspring groups were dictated by maternal conditions, as all offspring were maintained in ND/SED conditions. At 18 wk of age (n = 7–12), they were euthanized by CO2 asphyxiation before body fat assessment and tissue collection. Rats were maintained in a 12-h light–12-h dark cycle at 21°C–22°C, with food and water provided ad libitum. The Institutional Animal Care and Use Committee of the University of Missouri approved all procedures. A subset of SED animal characteristic has been reported previously (20).

FIGURE 1
FIGURE 1:
Study design.

Dam wheel running

To assess physical activity, dams placed in the RUN condition were given free access to voluntary running wheels for 24 h·d−1 5 wk before breeding and through gestation. Wheels were removed when the pups were born. Running distance was monitored using BC 800 Bicycle Computers (Sigma Sports, St. Charles, IL). The voluntary running wheel approach represents a less stressful model of increasing physical activity versus forced treadmill exercise training.

Body composition

Body composition was measured in dams immediately before breeding, and in 18-wk-old offspring using a Hologic QDR-1000/W dual-energy x-ray absorptiometry machine.

Hepatic triacylglycerol, histology, and enzyme activities

Livers were quickly excised from anesthetized rats, with sections flash frozen in liquid nitrogen or placed in 10% formalin. Liver triacylglycerol (TAG) content, citrate synthase activity, and 3-hydroxyacyl-CoA dehydrogenase (β-HAD) activity were measured as described previously (21). Formalin-fixed livers were embedded in paraffin, serially sliced, and stained with hematoxylin and eosin as previously referenced (21).

Western blots

Western blot analysis was completed in liver homogenate. The primary antibodies used are as follows: acetyl coenzyme A carboxylase (ACC; #3662, Cell Signaling, Danvers, MA), S79 phosphorylation-specific ACC (p-ACC; #3661, Cell Signaling); fatty acid synthase (FAS; #3189, Cell Signaling), microsomal triglyceride transfer protein (#135994; Santa Cruz Biotechnology, Dallas, TX), peroxisome proliferator activator receptor γ (PPARγ; #7273, Santa Cruz Biotechnology), autophagy-related gene 12 (ATG12, #4180, Cell Signaling), autophagy-related gene 5 (ATG5; #12994, Cell Signaling), adenosine monophosphate activated protein kinase α (AMPK; #2603, Cell Signaling), T172 phosphorylated AMPK (p-AMPK; #2531, Cell Signaling), PTEN-induced putative kinase 1 (#6946, Cell Signaling), nucleoporin 62 (P62; #5114, Cell Signaling), BCL2-interacting protein 3 (BNIP3; #3769, Cell Signaling), Kelch-like ECH-associated protein 1 (Keap1; #12721, Cell Signaling), LC3 A/B (#12741, Cell Signaling), CD36 (#133625, Abcam, Cambridge, MA), mitochondrial transcription factor A (TFAM; #8076, Cell Signaling), Parkin (Parkin; #2132, Cell Signaling), oxidative phosphorylation (OXPHOS; #110413, Abcam), nuclear factor E2–related factor 2 (NFE2L2/NRF2; #137550, Abcam), and peroxisome proliferator-activated receptor-gamma (PGC1α, #3242, Millipore, St. Louis, MO). Blots (n = 7–12 per group) were analyzed via densometric analysis (Image Lab 3.0). Amido-black staining was used to control for differences in protein loading and transfer as previously described (21).

Statistical analysis

Statistical analyses were completed in SPSS (IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY) with P < 0.05 used to determine statistical significance of all comparisons. Comparisons across all variables were assessed via three-way ANOVA (maternal condition–maternal diet–offspring sex). Significant interactions were followed post hoc with a Fisher’s LSD test with pooled standard deviation to determine individual group differences. Data are presented as mean ± SE.

RESULTS

Dam characteristics

WD feeding significantly increased (P < 0.05) food intake (Fig. 2A) and body fat percentage (Fig. 2C) compared with ND-fed dams, but there was no difference in body weight among the groups (Fig. 2B). Wheel running significantly increased (P < 0.05) both food intake (Fig. 2A) and body fat percentage (Fig. 2C) compared with SED dams, the latter of which is likely due to the increase in food consumed in the RUN group. There is no effect of diet or exercise on dam fat-free mass (Fig. 2D). ND-fed dams ran an average of 9.7 km·d−1, whereas WD-fed dams ran an average of 8.43 km·d−1 over the total 8 wk, including the gestational period (Fig. 2E), which was not statistically significantly different. There were no changes in litter size among any of the groups (data not shown).

FIGURE 2
FIGURE 2:
Effect of diet and exercise on dam food intake (A), body weight (B), body fat percentage (C), fat-free mass (D), and running distance (E). Effects of diet, maternal exercise, and sex on offspring food intake (F), body weight (G), body fat percentage (H), and fat-free mass (I). Main effects and P values reported in text above graphs. ∞, sex–exercise interaction. *Significant difference (P < 0.05) between ND and WD. #Significant difference (P < 0.05) between RUN and SED within WD. Values are presented as mean ± SE. A–E, n = 4; F, n = 6, WD/RUN female n = 2; G–I, n = 7–12.

Offspring characteristics

Male offspring’s food intake was greater than females (main effect of sex, P < 0.05, Fig. 2F), whereas offspring from RUN dams had reduced food intake compared with their SED counterparts (main effect of physical activity, P < 0.05, Fig. 2F). Male offspring had markedly higher body weight than females (main effect of sex, P < 0.05, Fig. 2G), whereas maternal RUN decreased (P < 0.05) the body weight of male WD-fed offspring compared with their WD/SED counterparts (Fig. 2G). Similarly, male offspring had increased fat-free mass compared with female offspring (main effect of sex, P < 0.05, Fig. 2I). WD feeding significantly increased (P < 0.05) offspring body fat percentage (Fig. 2H), an effect due to the increase in the males. Interestingly, there was a sex–RUN interaction for body fat percentage, with maternal RUN decreasing body fat percentage in males (driven by the attenuated increase in body fat percentage in WD/RUN compared with WD/SED), but with an increased body fat percentage in females (Fig. 2H).

Liver phenotype

Histological representation demonstrates that WD feeding elevated liver TAG accumulation in the offspring (Fig. 3B). This was confirmed by biochemical liver TAG assessment, with offspring from WD-fed dams having significantly increased liver TAG accumulation (main effect of diet, P < 0.05, Fig. 3A), although this was primarily driven by the increase in male offspring. Female offspring had markedly lower liver TAG accumulation than their male counterparts (main effect of sex, P < 0.05, Fig. 3A), and they did not develop WD-induced steatosis as witnessed in the male offspring fed the WD. Within ND-fed males, maternal wheel running tended (P = 0.07) to lower liver TAG accumulation compared with their ND-SED counterparts. Given this trend, we performed power calculations and determined an n = 10 per group would allow us to achieve statistical significance with an effect size of 0.80 and significance set at P < 0.05 for maternal RUN to lower liver TAG in maternal ND feeding conditions in 4-month-old male offspring. However, under maternal WD feeding conditions, an N > 100 is estimated to be necessary to see a maternal physical activity effect on hepatic TAG in male offspring at 4 months of age.

FIGURE 3
FIGURE 3:
Effects of diet, maternal exercise, and sex on offspring (A) liver triglycerides (TAG); (B) histological hepatic steatosis in offspring. Main effects and P values reported in text above graphs. Values are presented as mean ± SE (n = 6–12).

We have previously demonstrated that maternal exercise did not affect hepatic mitochondrial content in the offspring (16). Here, hepatic citrate synthase, a surrogate marker of mitochondrial content, also did not differ among offspring groups (data not shown). Moreover, maternal wheel running had no effect on protein content of the complexes of the electron transport chain (ETC), although complex II and complex III were elevated and decreased, respectively, in female offspring only compared with males (main effect of sex, P < 0.05, Fig. 4B and C). In addition, complex III was decreased with maternal WD in both sexes (main effect of diet, P < 0.05, Fig. 4C) and significantly increased in WD RUN versus WD SED in males only (interaction, P < 0.05, Fig. 4C). Hepatic β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity also was reduced in female rats (main effect of sex, P < 0.05, Fig. 4A), perhaps reflective of reduced liver TAG in female offspring. Interestingly, WD-fed offspring from the RUN dams exhibited reduced β-HAD activity compared with SED (P < 0.05, Fig. 4A).

FIGURE 4
FIGURE 4:
Effects of diet, maternal exercise, and sex of offspring on Beta-HAD activity (A), electron transport chain complexes complex II (B), complex III (C), and representative western blots (D). Main effects and P values reported in text above graphs. #Significant difference (P < 0.05) between RUN vs SED within WD. Values are presented as mean ± SE (n = 7–12).

Further exploration of the hepatic mitochondrial pathways found that maternal exercise increased hepatic mitochondrial biogenesis markers PPARγ and TFAM (main effect of activity, Fig. 5B and C), although PGC1α was not affected by maternal physical activity (Fig. 5A). The maternal physical activity–induced increases in TFAM protein content in females were attenuated with maternal WD feeding (P < 0.05, Fig. 5C). Interesting effects of sex on mitochondrial biogenesis were also observed, with female offspring having decreased PPARγ protein content, with elevated TFAM content (main effect of sex, P < 0.05, Fig. 5B and C). Contrastingly, maternal WD feeding decreased PGC1α while increasing PPARγ in the offspring (main effect of diet, P < 0.05, Fig. 5A and B).

FIGURE 5
FIGURE 5:
Effects of diet, maternal exercise, and sex of offspring on protein markers of mitochondrial biogenesis in offspring, PGC1α (A), PPARγ (B), TFAM (C). D, Representative western blots. Main effects and P values reported in text above graphs. *Significant difference (P < 0.05) between ND and WD within RUN. Values are presented as mean ± SE (n = 7–12).

With elevated markers of mitochondrial biogenesis in offspring of RUN dams, yet no differences in content, this led us to investigate whether markers of mitochondrial turnover were elevated via autophagy and mitophagy. Maternal RUN upregulated hepatic NRF2 (main effect of physical activity, P < 0.05, Fig. 6H), while also increasing BNIP3 within the WD group, and ATG12:ATG5 conjugation within WD-fed male offspring (P < 0.05, Fig. 6D and E). In addition, maternal RUN also reduced Keap1 proteins levels within the ND-fed female rats (P < 0.05, Fig. 6G). Female offspring displayed marked increases in several of these markers such as P62, BNIP3, ATG12:5, LC3 II/I, and Keap 1 (main effect of sex, P < 0.05, Fig. 6C–F). Interestingly, Parkin (a precursor to P62) was decreased in females (main effect of sex, P < 0.05, Fig. 6B). Overall, maternal WD feeding had little effect on markers of mitochondrial turnover assessed, with WD increasing BNIP3 in RUN male offspring (P < 0.05, 6D), LC3 II/I ratio in SED male offspring (P < 0.001, Fig. 6F), and decreasing LC3 II/I ratio in SED female offspring (P < 0.05, Fig. 6F) and hepatic NRF2 content within the RUN female offspring (P < 0.05, Fig. 6H).

FIGURE 6
FIGURE 6:
Effects of diet, maternal exercise, and sex on protein markers of autophagy and mitophagy in offspring. Pink (A), Parkin (B), P62 (C), BNIP3 (D), ATG12:5 (E), LC3 II/I (F), Keap1 (G), NRF2 (H), representative western blots (I). Main effects and P values reported in text above graphs. #Significant difference (P < 0.05) between RUN vs SED. *Significant difference (P < 0.05) between RUN vs SED. Values are presented as mean ± SE (n = 7–12).

Although maternal RUN had no significant effect on liver TAG accumulation, we examined protein markers of hepatic lipid regulators for confirmation. Maternal RUN had no effect on pAMPK/AMPK, pACC/ACC, FAS, CD36, or microsomal triglyceride transfer protein (see Supplemental Figure 1A–E, Supplemental Digital Content 1, Hepatic lipid regulators, http://links.lww.com/MSS/B287). However, female offspring displayed increased potential for de novo lipogenesis, with reduced protein markers of pAMPK/AMPK and pACC/ACC and elevated FAS (main effect of sex, P < 0.05, see Supplemental Figure 1A–C, Supplemental Digital Content 1, Hepatic lipid regulators, http://links.lww.com/MSS/B287). The reduction in pAMPK/AMPK was driven by the elevation of pAMPK in male offspring (data not shown). Hepatic CD36 was decreased in female offspring (main effect of sex, P < 0.05, see Supplemental Figure 1D, Supplemental Digital Content 1, Hepatic lipid regulators, http://links.lww.com/MSS/B287).

DISCUSSION

There is mounting evidence suggesting that a poor maternal diet can cause metabolic disturbances in offspring and contribute to diseases such as obesity, type 2 diabetes, and NAFLD (8–12). Maternal HFD can increase the susceptibility of offspring for developing hepatic steatosis in rats and nonhuman primates (9,22), even in offspring fed low fat diets postweaning (8,15). The rapidly rising rates of children and adults presenting with NAFLD is an area of concern, and preclinical animal studies suggest that maternal obesity may be contributing to this trend (23). Regular exercise before and during pregnancy may be a useful tool to defend against the effects of maternal obesity on the hepatic health of offspring, with reduced liver TAG accumulation observed in older, adult rodent offspring (15,16). Our current findings suggest that increasing maternal physical activity improves markers of mitochondrial biogenesis and autophagy/mitophagy in young adult rats, which may lay the foundation for the improvements in hepatic steatosis observed in older rodents. In addition, this article highlights marked sex differences in female offspring’s capacity for hepatic mitochondrial turnover, providing a potential explanation as to why female rodents are often more protected from hepatic steatosis development. These data shed light on the time line of hepatic steatosis progression in rodents under the influence of maternal physical activity, and provide insight into the sex differences on NAFLD development.

Interestingly, male offspring were more affected by maternal conditions than their female counterparts. Maternal diet had a significant effect on steatosis (driven by the males), as male offspring from WD-fed dams displayed marked increases in liver TAG and steatosis compared with ND offspring, in conjunction with previous reports (8,9,24). Moreover, male offspring of WD-fed dams displayed attenuated increases in both body weight and adiposity due to maternal RUN conditions. This has been attributed to excess fuels in the gestational environment caused by a high-fat or WD being used with maternal exercise, and decreasing the transfer of lipids to the fetus (15). In addition, maternal RUN decreased food intake in the offspring, also contributing to the attenuated WD-induced increases in body weight and adiposity in the males.

Maternal exercise/physical activity has also been shown to reduce steatosis in the liver of 8- and 12-month-old offspring (15,16), but not at 4 months (16). However, the offspring at 4 and 8 months were HFD fed, and changes in liver phenotype may not be fully attributable to maternal conditions. The current data echo these previous findings, as liver TAG were not affected by increasing maternal physical activity for both male and female 4-month-old offspring, suggesting the benefits of maternal physical activity on liver lipid accumulation may not become more apparent until later in life. Male offspring from ND-RUN dams displayed a trend for reduced liver TAG, compared with their ND-SED counterparts. It is possible that the lack of effect of maternal RUN on hepatic TAG in offspring was due to the activity modality (maternal voluntary wheel running) not being a strong enough stimuli to elicit a favorable adaptation as compared with standard forced treadmill aerobic exercise training. However, this seems unlikely given the daily running distances of the dams (avg 6–8 km per night). Moreover, voluntary wheel running is less stressful on the dam and allows for undisturbed gestation, removing added potential confounders with treadmill training. Furthermore, the activity-induced attenuation may have been more pronounced at 8 or 12 months, as Stanford et al. (15) reported liver TAG were 10-fold higher in their SED-ND offspring at 12 months compared with the conditions here at 4 months.

NAFLD progression is related to the decline in function of hepatic mitochondria (19,25), including reduced activity in electron transport chain (ETC) complexes, impaired fatty acid β-oxidation, and a decrease in mitochondrial DNA and content. HFD feeding of dams has led to offspring displaying decreased hepatic mitochondrial DNA content (26), as well as impaired expression of genes involved in hepatic mitochondrial biogenesis (12,15). Accordingly, we sought to determine whether maternal WD would alter mitochondrial content and function in the offspring, and if maternal physical activity might prevent any deleterious effects. Mitochondrial content, measured by citrate synthase activity, was not altered by RUN, diet, or sex in 4-month-old offspring (data not shown), similar to what we have previously reported (16). ETC complexes are mitochondrial enzymes that regulate ATP production by oxidative phosphorylation, and both their activity and content are downregulated by a HFD and NAFLD phenotype (27–29). Here, cytochrome reductase (complex III) was downregulated with maternal WD, potentially indicative of a reduced hepatic oxidative capacity. Interestingly, β-HAD activity, the rate-limiting step in mitochondrial β-oxidation, was lower in the offspring exposed to maternal WD and RUN compared with offspring from SED dams. These findings are in discordance with previous studies that found no differences of maternal exercise on β-oxidation (16), although this may be due to the offspring being HFD fed, which is known to increase β-oxidation (30).

Maternal exercise has been shown to influence markers of hepatic mitochondrial biogenesis in an effort to manufacture new, high-quality mitochondria (16,17,31), and the current findings support this notion. Protein abundance of PPARγ and TFAM, key transcription factors for mitochondrial biogenesis, were significantly elevated in offspring of maternally physically active dams. A vital transcriptional coactivator of these proteins and fundamental component of mitochondrial biogenesis, PGC1α, was surprisingly not affected by maternal RUN. Although seeming counterintuitive, these findings are validated by previous work by our group and others where either PGC1α expression or methylation status was not altered in 4-month-old offspring exposed to maternal exercise, yet were significantly altered at 8 and 12 months of age (16,17). This delay may be due to the methylation status of the PGC1α promotor (17), with maternal exercise decreasing methylation and allowing gene transcription in a time dependent manner. Future studies assessing the epigenetic alterations during embryonic development would shed light on how maternal exercise/activity affects the gestational environment.

With mitochondrial biogenesis elevated without concurrent increases in markers of content, we suspected that mitochondrial autophagy/mitophagy may be elevated in RUN offspring to improve mitochondrial turnover. This turnover hinges on the intimately linked processes of biogenesis and autophagy, as dysfunctional, aberrant mitochondria are removed via selected autophagy (mitophagy) and replaced in an effort to maintain mitochondrial homeostasis (32). We have previously demonstrated that hepatic protein levels of TFAM and ATG12:5 conjugation, key players in the turnover of mitochondria, are elevated with maternal exercise in older offspring (16). In the current study, we show similar findings in younger adult offspring. NRF2, an integral protein in mitochondrial turnover and antioxidant defense, was significantly increased with maternal RUN in all groups, whereas its negative regulator, Keap1, was concurrently decreased in female ND rats only. In addition, the mitophagy protein BNIP3 in the WD group and ATG12:ATG5 conjugation in WD male group was elevated with maternal RUN. Knockdown of Keap1 promotes mitochondrial hyperfusion via elevated NRF2, improving mitochondrial efficiency and decreasing reactive oxygen species (33). Similarly, NRF2-KO and BNIP3-KO rodent models have shown suppression of the autophagosome formation along with swollen mitochondria and reduced cristae, respectively (34,35), whereas ATG12:5 conjugate is also required for autophagosome formation (36). Together, these data highlight the potential effects of maternal physical activity on the offspring’s ability to regulate mitochondrial turnover to prevent reactive oxygen species accumulation, and ultimately contribute to improved hepatic and mitochondrial health. Furthermore, although these markers of autophagy/mitophagy were not assessed in skeletal muscle in the current study but given that increased basal autophagy is required for endurance exercise training-induced skeletal muscle adaptation and improvement of physical performance (37), it is possible that this improved phenotype may allow for more favorable adaptations to future exercise training in the offspring. This speculation warrants future investigation.

Increased de novo lipogenesis, impaired β-oxidation, increased lipid import, and decreased export all contribute to hepatic steatosis development (38). In the current study, there were no effects of maternal RUN or WD on our assessed markers of hepatic lipid regulation (see Supplemental Figure 1, Supplemental Digital Content 1, Hepatic lipid regulators, http://links.lww.com/MSS/B287). This is in contrast to previous reports, where maternal exercise increased markers of hepatic TAG export and upregulated genes involved in β-oxidation (16,17). However, these discrepancies may be due to the age and diet of the offspring, as in the previous reports offspring were HFD fed. Interestingly, female offspring displayed decreased pACC/ACC, likely driven by the decrease in pAMPK/AMPK, and elevated FAS protein. This suggests that de novo lipogenesis may be elevated in female offspring, despite lower hepatic TAG content.

As mentioned previously, female offspring were protected against maternal WD-induced hepatic steatosis at 4 months of age. Moreover, females displayed profound elevations in markers for mitochondrial biogenesis and autophagy/mitophagy compared with male littermates. These sex differences observed are likely due to differences in hormonal milieu; indeed, elevated levels of circulating estrogen species have been shown to be protective against the development of NAFLD, demonstrated by elevated risk for the development of NAFLD in both postmenopausal women and ovariectomized rodents (39–41). In addition, estrogen-related receptor α is a known transcription factor for mitochondrial biogenesis (42). Here, we provide novel evidence that perhaps the protection from maternal WD-induced hepatic steatosis in female rats is due in part to increases in hepatic mitochondrial biogenesis and autophagy/mitophagy. More detailed interrogation of these pathways are beyond the scope of this report but are strongly warranted.

In summary, here we provide evidence that while increasing maternal physical activity through wheel running does not prevent maternal WD-induced hepatic steatosis in 4-month-old rodents, it increased markers of hepatic mitochondrial biogenesis and autophagy/mitophagy. We suspect this improvement in mitochondrial health may lay the foundation for the reduced hepatic steatosis observed in older rats. These findings shed further light on the time line of hepatic steatosis development under maternal physical activity and dietary influences and may encourage earlier therapeutic interventions among children and young adults in either the prevention and/or treatment of NAFLD.

This work was supported by the American Heart Association Grant AHA 16PRE271500 (GNR), and partially supported by the VA-Merit Grant I01BX003271-01 (RSR). This work was supported with resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO. The authors have no conflicts of interest to disclose.

Involved in the study concept and design (R. P. C., R. S. R., G. N. R., and F. W. B.); acquisition of data (R. P. C., M. P. M., G. M. M., G. N. R., F. W. B., and R. S. R.); analysis and interpretation of data (R. P. C., M. P. M., G. M. M., and R. S. R.); drafting of the manuscript (R. P. C. and R. S. R.); critical revision of the manuscript for important intellectual content (R. P. C., M. P. M., G. M. M., G. N. R., F. W. B., and R. S. R.); statistical analysis (R. P. C., M. P. M., and R. S. R.); obtained funding (G. N. R., F. W. B., and R. S. R.).

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Keywords:

MATERNAL OBESITY; MATERNAL EXERCISE; PHYSICAL ACTIVITY; NAFLD; MITOCHONDRIA; BIOGENESIS; AUTOPHAGY

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© 2018 American College of Sports Medicine