Mary P. Moore, Rory P. Cunningham, Taylor J. Kelty, Nhu Y. Nguyen

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Ketogenic diet in combination with voluntary exercise impacts markers of hepatic metabolism and
oxidative stress in male and female Wistar rats
Mary P. Moore1.2, Rory P. Cunningham1,2, Taylor J. Kelty4, Luigi R. Boccardi1,2,3, Nhu Y. Nguyen1,2,
Frank W. Booth4, and R. Scott Rector1,2,3
1Research Service, Harry S Truman Memorial Veterans Medical Center, Columbia, MO, 65201;
Downloaded from www.nrcresearchpress.com by BOSTON UNIVERSITY on 05/26/19. For personal use only.
2Department of Nutrition and Exercise Physiology, University of Missouri, Columbia, MO; 3Medicine-
Division of Gastroenterology and Hepatology, and 4Biomedical Sciences, University of Missouri,
Columbia, MO 65211
Address of Correspondence:
R. Scott Rector, PhD
Research Health Scientist and Associate Professor
Harry S Truman Memorial VA Hospital
Departments of Nutrition and Exercise Physiology and Medicine -
Division of Gastroenterology and Hepatology
University of Missouri-Columbia
Columbia, MO 65212
Appl. Physiol. Nutr. Metab.
Tel: 573-884-0979
Fax: 573-884-4595
Email: rectors@health.missouri.edu
Contact information for other authors:

Mary P. Moore: mpmxn5@mail.missouri.edu
Rory P. Cunningham: rcw4b@mail.missouri.edu
Taylor J. Kelty: taylor.kelty@mail.missouri.edu
Luigi Boccardi: lrb5z5@mail.missouri.edu
Nhu Nguyen: nynb5b@mail.missouri.edu
Frank W. Booth: boothf@missouri.edu
R. Scott Rector: rectors@health.missouri.edu
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Abstract
Ketogenic diets (KD) are shown to benefit hepatic metabolism; however, their effect on the liver when
combined with exercise are unknown. We investigated the effects of a KD vs a ‘Western’ diet (WD) on
markers of hepatic lipid metabolism and oxidative stress in exercising rats. Male and female Wistar rats
with access to voluntary running wheels were randomized to three groups (n= 8-14 per group): standard
chow (SC; 17% Fat), WD (42% Fat) or KD (90.5% Fat) for 7 weeks. Body fat percentage (BF%) was
increased in WD and KD vs SC, although KD females displayed lower BF% vs WD (p≤0.05). Liver
triglycerides were higher in KD and WD vs SC, but were attenuated in KD females vs WD (p≤0.05). KD
suppressed hepatic markers of de novo lipogenesis (FAS, ACC) and increased markers of mitochondrial
biogenesis/content (PGC-1α, TFAM and citrate synthase activity). KD also increased the hepatic GPx1
and lowered oxidized glutathione. Females rats exhibited elevated hepatic markers of mitochondrial
biogenesis (TFAM), mitophagy (LC3II/I ratio, ATG 12:5) and cellular energy homeostasis
(pAMPK/AMPK) vs males. These data highlight that KD and exercise beneficially impacts hepatic
metabolism and oxidative stress and merits further investigation.
 Ketogenic diet feeding combined with exercise improved hepatic oxidative stress,
suppressed markers of de novo lipogenesis, increased markers of mitochondrial content vs
western diet feeding.
 Males and females responded similarly to combined ketogenic diet feeding and exercise.
 Female rats exhibited elevated hepatic markers of autophagy/mitophagy and energy
homeostasis compared with male rats.
Key words: ketogenic diet; mitochondria; oxidative stress; exercise; liver; sex; lipid metabolism
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Introduction
In recent decades, rates of obesity and its associated conditions, including insulin resistance, type
2 diabetes and nonalcoholic liver disease (NAFLD) have markedly increased and contribute to
cardiovascular disease (CVD) (Flegal et al. 2016; Fox et al. 2007). NAFLD is a common chronic disease,
comprised of a spectrum of liver injury ranging from simple steatosis to steatohepatitis (NASH), fibrosis
and end stage cirrhosis. The prevalence of NAFLD in the general population ranges from 10% to 30%
and as high as 80% – 100% in obese populations (Chalasani et al. 2012; Vernon et al. 2011). Currently,
there are no pharmacological treatments available for NAFLD. Recently, attention has shifted to
alternative dietary regimes to combat metabolic diseases such as NAFLD. Ketogenic diets (KD), low in
carbohydrate and very high in fat, have traditionally been used to treat neurodegenerative disorders. More
recently, KD have been used therapeutically to treat a multitude of health disorders ranging from obesity
to type 2 diabetes and NAFLD (Partsalaki et al. 2012; Samaha et al. 2003; Schugar and Crawford 2012;
York et al. 2009). With the increasing therapeutic application of low-carbohydrate diets, further
understanding of the range of metabolic responses observed in the liver are timely.
Although KD are accepted as an efficacious approach to weight loss (Hession et al. 2009;
Nordmann et al. 2006; Santos et al. 2012; Shai et al. 2008), there still remains significant controversy
about their metabolic effects. KD have been shown to improve glucose tolerance, insulin sensitivity and
decrease markers of hepatic de novo lipogenesis in male mice compared to mice maintained on a high fat,
high sucrose ‘western’ diet (WD) (Badman et al. 2009; Kennedy et al. 2007). Interestingly, improved
mitochondrial quantity and quality and increased antioxidant defense systems are potential mechanisms
through which KD feeding may also result in physiological benefits (Hutfles et al. 2017; Hyatt et al.
2016; Kennedy et al. 2007; Milder and Patel 2012; Milder et al. 2010). Conversely, others have shown
that KD feeding can increase fat mass, reduce lean mass and increase insulin resistance (Garbow et al.
2011). Elevations in hepatic inflammation and steatosis in male rat and mouse models have also been
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reported (Asrih et al. 2015; Garbow et al. 2011; Jornayvaz et al. 2010). Clearly, further research is
needed in order to address the current disparities in the literature.
Regular exercise aids in the management and prevention of obesity, insulin resistance, type 2
diabetes, and NAFLD. Studies investigating the synergistic effects of combining exercise and KD
feeding on liver health are limited. To our knowledge only one group has assessed the impact of a KD
(67% kcal fat) combined with exercise (Holland et al. 2016), reporting that 6 weeks of resistance training
+ KD in male rats reduced hepatic steatosis and improved serum insulin, glucose and lipid profiles
compared to WD and standard chow (SC) fed groups. However, there is a paucity of research examining
the physiological impact of a traditional KD (i.e. > 90% kcal fat) in conjunction with aerobic exercise on
markers of hepatic metabolism and any sex differences that may exist. Given the current knowledge gaps,
we tested the hypothesis that very high fat KD (> 90% kcal fat) feeding in conjunction with voluntary
wheel running exercise would significantly impact hepatic markers of de novo lipogenesis and
mitochondrial health compared to high fat, high sucrose WD and SC feeding in male and female Wistar
rats.
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Materials and Method
Animal protocol - Experimental protocols were approved by the University of Missouri Animal
Care and Use Committee. Rats were maintained on a 12:12-h light/dark cycle at 21–22 °C. Male and
female Wistar rats were pair housed (with same sex) until 7 weeks of age. At 7 weeks of age, male and
female rats were randomly assigned to one of three groups (n = 8-14/group), and provided access to
standard chow (SC; 26.5%, 56.5%, 17%-PRO, CHO, FAT; #5008; Formulab Diet Purina; St. Louis, MO,
USA), a ‘Western-style’ diet (WD; 15.2%, 42.7%, 42% & 0.2% cholesterol by weight; #88137; Envigo
Teklad Diets; Madison, WI, USA ) or a ketogenic diet (KD; 9.2%, 0.3%, 90.5%; #96355; Envigo Teklad
Diets) and water ad libitum for 7 weeks. See Table 1 for macronutrient breakdown of diets. Females were
matched for body mass at the beginning of the study. However, due to availability of male rats during
randomization, the WD male rats had a higher starting body mass compared with SC and KD male rats.
Males and females were considered as separate groups for a total of 6 groups. All animals were
individually housed for the duration of the study. Rats were fasted 3 to 4 hours prior to euthanasia via
CO2 asphyxiation.
Wheel Running - To assess voluntary physical activity, all groups were given free access to
voluntary running wheels 24 h/d for the duration of the study (7 weeks). Running distance was monitored
daily using BC 800 bicycle computers (Sigma Sport, St. Charles, IL, USA). Running wheels were locked
48 hours prior to euthanasia.
Body composition - Body composition was measured within 5 minutes after euthanasia, using a
Hologic QDR-1000/W dual-energy x-ray absorptiometry machine.
Ketone Body measurement – Whole blood β-hydroxybutyrate levels were measured using a
Precision-Xtra ketone body meter (#B075RQS6B2; Abbott Laboratories, Santa Clara, CA) and blood
ketone test strips (#B010MPEOOK; Abbott Laboratories) via tail vein stick. Ketone bodies were
measured at week 3 and 7 with the average reported.
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Hepatic triacylglycerol content and enzyme activity - Livers were quickly excised from
euthanized rats, flash frozen in liquid nitrogen and stored at -80C. Liver triacylglycerol (TAG) content,
citrate synthase activity were measured as described previously (Rector et al. 2008).
Reduced glutathione (GSH) and oxidized glutathione (GSSG) - GSH and GSSG concentrations
were determined by fluorometric methods as previously described (Rivera et al. 2008), utilizing methods
of Hissin and Hilf (Hissin and Hilf 1976).
Serum analyses – Blood was collected via cardiac puncture immediately after euthanasia and
allowed to clot before centrifugation at 1,300rpm for 10 minutes. Serum was extracted and stored at -
80C. Serum glucose, insulin, triglycerides (TAGs), free fatty acids (FFA) and cholesterol concentrations
were measured as previously described (Laye et al. 2009a; Rector et al. 2008). Serum alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) assays were performed by Comparative
Clinical Pathology Services (Columbia, MO) using commercially available assays. Serum TBARS (Item
No. 10009055, Cayman Chemical, Ann Arbor, Michigan) and Total Antioxidant Capacity (TAC; Item
No.709001, Cayman Chemical) were measured according to the manufacturer’s instructions.
Western Blotting - Western blot analysis was completed in liver homogenate. Triton X-100 cell
lysates were prepared and Laemmli gel loading buffer (#161-0737, Bio-Rad Laboratories, Hercules, CA)
was added to the lysate to produce Western blot-ready Laemmli samples. The microtube containing this
mixture was then boiled at 100 °C for 10 minutes. For each sample, 10-20 g of protein was added to
each well when running a Western blot gel. Proteins were separated by tris-glycine sodium dodecyl
sulfate polyacrylamide gel electrophoresis and transferred to a PVDF transfer membrane (#88518,
Thermo Scientific, Rockford, IL). The membrane was then incubated overnight in blocking solution (5%
dry milk in Tris-buffered saline (TBS-) Tween 20 buffer) followed by overnight incubation in the primary
antibody. After incubation, the membranes were washed with TBS-Tween 20 buffer and placed in a
horseradish peroxidase-conjugated secondary antibody (#7074, #7076, Cell Signaling Technology,
Danvers, MA). Blots (n = 8/group) and individual protein bands were quantified using a densitometer
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(Image Lab 3.0). For Western blot analysis, each sample was adjusted to the average intensity of all
samples on the gel. The total protein staining for each lane, quantified by laser densitometry was used to
correct for any differences in protein loading or transfer of all band densities. 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 (FASN; #3189, Cell
Signaling), autophagy-related gene 12 (ATG12, #4180, Cell Signaling), 5’ adenosine monophosphate
activated protein kinase (AMPK; #2603, Cell Signaling), T172 phosphorylated AMPK (p-AMPK; #2531,
Cell Signaling), nucleoporin 62 (p62; #5114, Cell Signaling), nuclear factor E2–related factor 2
(NFE2L2/NRF2; #137550, Abcam), Kelch-like ECH-associated protein 1 (Keap1; #12721, Cell
Signaling), Light chain 3 (LC3) A/B (#12741, Cell Signaling), cluster of differentiation 36 (CD36;
#133625, Abcam, Cambridge, MA), mitochondrial transcription factor A (TFAM; #8076, Cell Signaling),
oxidative phosphorylation (OXPHOS; #110413, Abcam, Cambridge, MA), peroxisome proliferator
activated receptor- (PGC-1, #3242, Millipore, St. Louis, MO), Sirtuin 1 (SIRT1; #1540, Santa Cruz, ),
Sirtuin 3 (SIRT3; #2627, Cell Signaling), superoxide dismutase 1 (SOD1, #13498, Abcam), superoxide
dismutase 2 (SOD2, #13194, Cell Signaling), Cytochrome c (COX1; #4280, Cell Signaling), Glutathione
peroxidase 1 (GPx1; #22604, Abcam), Sterol regulatory element-binding protein 1 (SREBP1; #367, Santa
Cruz), Tumor necrosis factor- (TNF-; ##11948; Cell Signaling), CD11b ( #NB110-89474; Novus
Biologicals; Centennial, CO, USA), CD68 (#17832; Santa Cruz).
Statistical Analysis - Main effects of diet (standard chow vs western diet vs ketogenic diet) and
sex (male vs female) were examined via a two‐way ANOVA in SPSS (IBM SPSS Statistics for Windows,
Version 24.0. Armonk, NY), and significant main effects (p ≤ 0.05) were followed up with Fisher's
LSD post hoc comparisons. When a significant interaction explained a main effect(s), only the
interaction was interpreted. All values are reported as means ± SE, and a p value of ≤0.05 denotes a
statistically significant difference.
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Results
Animal and Serum Characteristics – Male rats weighed significantly more than females (main
effect of sex, p = 0.009; Fig. 1A). WD fed males were heavier than SC and KD fed males at the
beginning of the study (Fig. 1A). WD and KD males gained less weight across the 7 week intervention
compared with SC males (Fig. 1B). Final body mass of SC and WD males were greater than KD males
(main effect of diet, p = 0.0009; Fig. 1A). Body fat percentage at the end of the study was significantly
higher in WD and KD fed rats vs SC in both sexes (p<0.001). Interestingly, KD fed females had a
significantly lower body fat percentage vs WD females (p0.05; Fig. 1C) and this was not observed in the
males. Food and energy intake are presented as grams/day (Fig. 1D) and kcal/day (Fig. 1E), respectively.
Food and energy intake were greater in male vs female rats (main effect of sex, Fig. 1D and E; p ≤ 0.041).
Energy intake (kcal/day) was significantly higher in the WD males vs SC and KD fed males, while WD
and KD fed females consumed more energy vs SC females (sex*diet interaction, p = 0.0106; Fig. 1E).
Food and energy intake were not significantly different between KD fed males and females (Fig. 1D and
E; p > 0.05). In addition, female rats ran significantly more than their male counterparts (main effect of
sex, p < 0.0001; Fig. 1G) and WD male ran significantly more than SC males.
As expected, blood ketone bodies were significantly elevated in KD fed rats (main effect of diet,
p < 0.0001), with greater elevations observed in males vs females (sex*diet interaction, p < 0.0001; Table
2). Serum TAGs, cholesterol, glucose, and insulin concentrations were higher in WD fed animals vs SC
and KD fed groups (main effect of diet, p < 0.0001; Table 2). No differences in serum NEFA
concentrations were observed among groups (Table 2). We also assessed serum markers of liver injury,
inflammation, and oxidative stress. There was no effect of diet on serum AST or ALT concentrations (p
> 0.05; Table 2), although male rats had higher serum AST concentrations vs females (main effects of
sex, p = 0.0064; Table 2). Surprisingly, serum TBARS concentrations were elevated in females vs males
(main effect of sex, p=0.0005), which was driven by dramatic elevations in KD fed females (sex*diet
interaction, p=0.0008; Table 2). No differences in TAC levels were observed among groups (p > 0.05,
Table 2).
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Liver Phenotype - To further explore the impact of a KD on the liver in exercising male and
female rats, we assessed hepatic markers of lipid content, oxidative stress and inflammation. Both WD
and KD feeding displayed significantly elevated liver triglycerides (TAGs) (~4.6 and 4.5-fold vs SC,
respectively; main effect of diet, p < 0.0001; Fig. 2A) even though rats were voluntary wheel running;
whereas, hepatic TAGs were 30% lower in KD females vs WD females (p<0.05) and significantly lower
vs KD fed males (sex*diet interaction, p = 0.0235; Fig. 2A). Although hepatic TAGs were elevated, KD
feeding decreased oxidized glutathione (GSSG) levels vs SC (main effect of diet, p = 0.0378; Fig. 2C)
and increased glutathione peroxidase 1 (GPx1) protein content vs SC and WD in both sexes (main effect
of diet, p < 0.0001; Fig. 2D). Hepatic nuclear factor E2–related factor 2 (NRF2) content, another protein
involved in the oxidative stress response was significantly lower in KD fed males vs SC fed males
(sex*diet interaction, p ≤ 0.05), while no differences in NRF2 protein content was noted within female
groups. Furthermore, male rats exhibited elevated hepatic NRF2 protein content compared with females
(main effect for sex, p = 0.0008; Fig. 2E). The repressor of NRF2, Kelch-like ECH-associated protein 1
(KEAP1), did not differ based on diet, but was significantly elevated in the liver of female vs male rats (p
< 0.0001, Fig. 2E). There were no differences in hepatic superoxide dismutase (SOD)1 or SOD2 protein
content (p > 0.05; Supplementary Figure S11).
The proinflammatory mediators, tumor necrosis factor- (TNF-) and cluster of differentiation
(CD)11b protein content were significantly decreased in KD vs WD, with decreases in CD11b for KD vs
SC fed animals also observed (main effect of diet, p  0.05; Fig. 2F). Interestingly, females displayed
greater hepatic CD11b (driven by SC) and CD68 (driven by SC and KD) protein content, while males
displayed greater TNF- content, largely due to the dramatic elevations in WD fed males (main effect of
sex, p  0.05; Fig. 2F).
5' AMP-activated protein kinase (AMPK) is a marker of cellular energy homeostasis and
regulator of both hepatic fatty acid oxidation and de novo lipogenesis. Total hepatic AMPK content was
1 Supplementary data are available with the article through the journal Web site.
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significantly elevated with WD feeding and suppressed with KD feeding (main effect of diet, p<0.0001;
Fig. 3A). pAMPK/AMPK ratio, an indicator of AMPK activation, was significantly lower for WD males
vs SC and KD fed males (sex*diet interaction, p = 0.0458; Fig. 3A). Interestingly, Thr 172
phosphorylated pAMPK and pAMPK/AMPK ratio was significantly elevated in females vs males (main
effect of sex, p = 0.0118 and p < 0.0001, respectively; Fig. 3A). Total SREBP-1c (120kDa), an upstream
transcriptional regulator of de novo lipogenesis, was elevated in WD females vs SC females and KD
females; these differences were not observed in males (sex*diet interaction, p = 0.004; Fig. 3B).
Furthermore, females displayed elevated levels of the mature transcriptionally active fragment (68kDa
isoform) of SREBP-1c vs males (main effect for sex, p  0.05; Fig. 3B). KD feeding dramatically
suppressed downstream markers of de novo lipogenesis, including ACC and FASN, as well as increasing
pACC/ACC ratio (marker of inactivation) (main effect of diet, p = 0.001; Fig. 3B). Additionally, the lipid
transporter CD36, was significantly lowered in females vs males (main effect of sex, p = 0.0007).
Finally, KD females displayed elevated hepatic CD36 content vs. SC females (sex*diet interaction,
p0.05; Fig. 3C), which was not observed in males.
Hepatic Mitochondrial Markers- Hepatic citrate synthase activity, a surrogate measure of
mitochondrial mass was significantly higher with KD and WD feeding compared with SC in male and
female rats (main effect of diet, p = 0.015; Fig. 4A). Cytochrome c protein content, a marker of
mitochondrial content, was also higher with a KD, driven by KD fed males (main effect of diet, p =
0.015; Fig. 4B). To further examine mitochondrial content, we assessed the protein content of individual
complexes in the electron transport chain (OXPHOS). In contrast to the above, complexes I, II and III
were lowered with a KD vs SC, while complex IV was elevated (main effect of diet p  0.05; Fig. 4C).
Additionally, KD fed animals displayed decreased complex I and II content vs WD (main effect of diet
p0.05; Fig. 4C). No differences in complex V were observed among groups.
PGC-1α and mitochondrial transcription factor A (TFAM), markers of mitochondrial biogenesis,
were significantly increased with a KD and WD vs SC, with greater increases in KD vs WD for PGC-1α
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(main effect of diet, p  0.05; Fig. 5A). Males displayed greater PGC-1α vs females (main effect of sex, p
= 0.026; Fig. 5A), while females displayed greater TFAM protein content vs males (main effect of sex, p
 0.05; Fig 5A). Sirtuin (SIRT)1 content, a protein responsible for deacetylating and increasing PGC-1α
activation was significantly lower in KD vs SC and WD fed rats (main effect of diet, p = 0.0018; Fig.
5A). SIRT3, another sirturin involved in mitochondrial biogenesis regulation, displayed a significant
main effect for diet (p = 0.0001; Fig 5A), likely driven by WD males (not observed in females) (sex*diet
interaction, p = 0.0497; Fig 5A).
Finally, we investigated whether markers of hepatic autophagy and mitophagy were altered by
KD feeding. Light chain 3 (LC3) II/LC3 I ratio, an indicator of autophagosome formation, was
significantly decreased in WD vs SC groups (main effect of diet, p = 0.0308; Fig. 5B), but was not
different for KD vs SC groups. Both WD and KD increased Sequestosome-1 (p62), a scaffold binding
protein for LC3, vs SC (main effect of diet, p < 0.0001; Fig. 5B). In addition, LC3II/LC3I ratio and
ATG12:5 conjugate were elevated in female compared with male rats (main effect for sex, p < 0.0001 and
p = 0.0446, respectively; Fig 5B).
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Discussion
High fat, very low carbohydrate, ketogenic diets (KD) have received recent attention in the
management of obesity, type 2 diabetes, and NAFLD (Partsalaki et al. 2012; Samaha et al. 2003; Schugar
and Crawford 2012; York et al. 2009). Hepatic ketogenesis plays an important role in maintaining whole
body TCA cycle homeostasis and supplies extrahepatic organs with energy substrates in glucose-limiting
states. However, limited studies have examined the impact of KD on hepatic health, particularly when
combined with exercise training, and none to date have examined potential sex differences in the
responses. Here, we report a number of unique and novel observations pertaining to KD feeding when
combined with wheel running exercise in male and female Wistar rats. Namely, KD feeding did not
abnormally elevate serum lipids, insulin or glucose concentrations compared with a lower fat (though still
high fat), western diet. KD feeding also reduced markers of hepatic oxidative stress and inflammation
despite elevating hepatic TAGs. KD feeding also dramatically suppressed markers of hepatic de novo
lipogenesis, as well as increased select markers of hepatic mitochondrial content and biogenesis. On the
other hand, KD feeding increased serum lipid peroxidation in females, increased hepatic TAG levels, and
downregulated several mitochondrial electron transport chain proteins in the liver.
Here we report that a KD resulted in a more favorable body composition vs WD feeding in
exercising female rats, without observing similar improvements in male rats. Others have also reported
improvements in body composition vs WD feeding in exercising rats (Holland et al. 2016). Though not
assessed in the current study, it has been speculated that mechanisms behind these observations are
caused by factors such as a reduced appetite, hormonal changes caused by a KD (Gibson et al. 2015; Paoli
et al. 2015) and increased energy expenditure (Kennedy et al. 2007). In addition, we report that KD
feeding attenuated elevations in serum TAGs, cholesterol, glucose, and insulin concentrations compared
with WD feeding in exercising male and female rats. These improvements in glucose and insulin are in
agreement with some (Holland et al. 2016; Kennedy et al. 2007) but not all (Garbow et al. 2011; Grandl et
al. 2018) previous studies. The exact explanation for the discrepancies in the literature are unknown, but
could be related to the % fat in the KD and/or the duration of the feeding studies. Further research is
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needed to determine what a healthy adaptation to KD feeding entails (de Oliveira Caminhotto and Lima
2013).
The previously reported effects of KD on measures of oxidative stress and antioxidant benefits
seem to be largely equivocal (Greco et al. 2016; Hyatt et al. 2016; Jarrett et al. 2008; Kephart et al. 2017;
Sullivan et al. 2004). Here, KD feeding reduced hepatic GSSG (the oxidized form of glutathione) and
increased the antioxidant enzyme GPx1 with a corresponding decrease in NRF2 in KD males, suggesting
a reduced need for a NRF2 antioxidant response. Similarly, long-term KD feeding (75% kcal fat) has
been shown to up-regulate GSH biosynthesis and enhance mitochondrial antioxidant status in the
hippocampus of male rats vs SC (Jarrett et al. 2008). Conversely, a recent study demonstrated no
improvements in liver, skeletal muscle, or brain oxidative stress markers in KD fed rats (67% kcal fat) vs
SC (Parry et al. 2018). In addition to improvements in oxidative stress measures, we found that KD
feeding reduced hepatic inflammatory markers TNF and CD11b compared with WD feeding, findings
which were in agreement with other reports of reduced hepatic inflammation (phosphorylated/pan-p65)
and serum ALTs in exercised male rats fed a KD (67% kcal fat) vs WD and SC (Holland et al. 2016).
Collectively, these data suggest that a KD improves antioxidant defense and reduces oxidative stress and
inflammation. Further research is warranted to elucidate the mechanisms involved.
KD (93.3% kcal fat) feeding is known to reduce hepatic de novo lipogenesis and increase hepatic
fatty acid oxidation, while high-fat (~40%kcal), high sucrose, WD feeding increases hepatic de novo
lipogenesis and impairs fatty acid oxidation (Garbow et al. 2011; Kennedy et al. 2007). Similarly, we
demonstrate that KD feeding downregulates markers of hepatic de novo lipogenesis (ACC, pACC,
FASN) vs WD feeding. On the other hand, six weeks of KD (67% kcal fat) feeding in resistance trained
male rats failed to alter hepatic markers of lipogenesis when compared with SC and WD (Holland et al.
2016). These differences are likely due to differing macronutrient composition (23% PRO, 10% CHO,
67% FAT) compared with the current study.
In the current study, KD feeding increased measures of mitochondrial mass (citrate synthase
activity and cytochrome c protein content), despite downregulation in hepatic protein content of the
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electron transport chain complexes I, II and III. The observed increases in mitochondrial mass are
consistent with past literature in which KD feeding (67% kcal fat) (Hyatt et al. 2016) and exercise (Rector
et al. 2011) alone are shown to increase hepatic mitochondrial volume and function. The impact of KD
diet on hepatic electron transport chain complex content is mixed (Hutfles et al. 2017; Hyatt et al. 2016;
Kephart et al. 2017), with further research needed in this area.
The mitochondrial deacetylase enzyme, SIRT 3, has been shown to be induced by increases in
PGC-1 and normally functions to catalyze NAD+ dependent deacetylation of numerous substrates in the
mitochondrial matrix in order to maintain mitochondrial function (Ansari et al. 2017; Kim et al. 2010).
Chronic high fat, high sucrose, WD feeding has been associated with increases in mitochondrial protein
hyperacetylation, reductions in deacetylase activity, in particular SIRT3, and subsequent decreased
mitochondrial function in sedentary mice with a fatty liver (Kendrick et al. 2011; Morris et al. 2013)
Here, we observe that a high fat, high sucrose, WD resulted in a significant increases in hepatic SIRT3
protein content vs SC in exercising male rats. These differences in findings may possibly be due to the
confounding effect of exercise in our study. Conversely, a KD did not result in significant changes in
hepatic SIRT3 protein content vs SC fed exercising male rats, despite increases in PGC-1 protein
content in response to a KD. Although acetylation status and sirtuin activity were not assessed in the
current study, the reductions in ETC complex protein content, in particular complex I and II, in KD fed
animals vs SC and WD fed animals may be associated with decreased SIRT3 mediated deacetylation at
the mitochondrial matrix in response to a KD. Future studies should explore the impact of a KD on
acetylation status and sirtuin activity on the liver and their subsequent downstream effects on
mitochondrial function.
PGC-1α and TFAM coregulate multiple transcription factors involved in mitochondrial
biogenesis and content (Wu et al. 1999), and we have previously reported that PGC-1α and TFAM
expression are increased in the liver with exercise training in SC fed rats (Laye et al. 2009b). Here,
hepatic mitochondrial biogenesis markers PGC-1α and TFAM were upregulated with WD and KD
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compared with SC feeding in exercising rats. While some studies have reported increased PGC-1α
expression in response to a KD (Jornayvaz et al. 2010), others have reported no effects in sedentary male
rodents (Kennedy et al. 2007; Parry et al. 2018). These differing observations compared to the current
study may be due to the fact that all of our animals were exercising. In fact, while exercise was shown to
increase skeletal muscle PGC-1α in KD fed mice, KD feeding alone did not increase PGC-1 vs SC
(Hyatt et al. 2016). The observed increases in hepatic PGC-1α content occurred despite lower hepatic
SIRT1 content in KD compared with SC and WD groups. SIRT1 is a major cytosolic deacetylase and a
primary function is to deacetylate PGC-1α, thus increasing its transcriptional activity (Amat et al. 2009).
Others also report increases in PGC-1α transcription independent of SIRT1 and also in the presence of
exercise (Cantó and Auwerx 2009; Terada et al. 2002). Future studies should explore the acetylation
status of hepatic PGC-1α and its transcriptional activity in response to KD and exercise, as well as in
sedentary conditions, to fully elucidate the impacts of a KD and exercise on hepatic mitochondrial
biogenesis.
Mitochondrial processes of autophagy, termed mitophagy, is the clearance of damaged or poorly
functioning mitochondria. Mitophagy and biogenesis are intricately linked in an effort to maintain
mitochondrial homeostasis (Kim et al. 2007). With increases in markers of hepatic mitochondrial
mass/content and mitochondrial biogenesis, we suspected that mitophagy may be blunted in exercising
WD and KD fed animals. Indeed, a marker of mitophagy, LC3II/LC3I ratio, was decreased with WD and
KD feeding, along with elevated p62, indicative of impaired mitophagic activity (Yoshii and Mizushima
2017). As these are surrogate measures of mitophagy, future research should investigate the impact of a
KD on mitophagic flux in hepatic tissue.
Female rats displayed a higher hepatic LC3II/LC3I ratio, as well as increased TFAM and
ATG12:5, key players in mitochondrial biogenesis and mitophagy. Female rats also exhibited greater
increases in the content of markers related to hepatic lipid import (CD36) and decreases in de novo
lipogenesis (inactivation of ACC through increased pACC) vs males, findings likely linked to greater
increases in pAMPK/AMPK ratio. These data suggest that females may have a greater propensity for
15
Page 16 of 33
increased hepatic fatty acid oxidation, potentially serving as a protective mechanism against NAFLD,
which has been shown by others previously (Pramfalk et al. 2015). These data are in agreement with
previous studies from our lab and others, in which females exhibited greater capacity for hepatic
mitophagy (Cunningham et al. 2018; Sheldon et al. 2016; Von Schulze et al. 2018). Although
speculative, this may be due in part to elevated levels of circulating sex hormones, which have been
shown to be protective against increased hepatic steatosis (Gutierrez-Grobe et al. 2010; Kamada et al.
2011; Lonardo et al. 2006).
In summary, very high fat, low carbohydrate KD feeding in conjunction with wheel running
exercise exerts a number of beneficial effects on hepatic health in male and female rats. Specifically, KD
feeding favorably changed serum markers of lipids, glucose and insulin and reduced markers of hepatic
oxidative stress, inflammation and lipogenesis. Furthermore, KD feeding increased select markers of
hepatic mitochondrial content and mitochondrial biogenesis. However, compared with low fat SC, KD
feeding also increased hepatic TAGs and downregulated hepatic proteins involved in electron transport
chain despite rats undergoing voluntary physical activity. KD are prescribed with increasing frequency to
treat metabolic disease including obesity, T2D and NAFLD. This study provides novel insight into the
impact of changes in dietary macronutrient content in conjunction with exercise on liver health in both
male and female rats. The metabolic effects of KD are not yet completely understood and warrant further
investigation.
16
Page 17 of 33
Acknowledgements: Support: VA-Merit Grant I01BX003271 (R.S.R.). This work was supported with
resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO.
Involved in the study concept and design (M. P. M., R. S. R., T. J. K., and F. W. B.); acquisition of data
(M. P. M., R. P. C., L. R. B., N. N., V. J., T. J. K., F. W. B., and R. S. R.); analysis and interpretation of
data (M. P. M., R. P. C., L. R. B., N. N., and R. S. R.); drafting of the manuscript (M. P. M. and R. S. R.);
critical revision of the manuscript for important intellectual content (M. P. M., R. P. C., T. J. K., F. W. B.,
and R. S. R.); statistical analysis (M. P. M., R. P. C., and R. S. R.); obtained funding (R. S. R.).
The authors have no conflicts of interest to report.
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Table 1. Macronutrient content of each respective diet

Diet SC WD KD
Calorie (per gram) 3.2 4.5 6.7

Carbohydrate (% of kcal) 56.5 42.7 0.3
Protein (% of kcal) 26.5 15.2 9.2
Fat (% of kcal) 17.0 42.0 90.5
Fatty acid profile (% of fat)
Saturated fat 26.3 61.8 25.0
Monounsaturated fat 29.4 27.3 20.8

Polyunsaturated fat 22.1 4.7 50.0
Unknown 22.2 6.2 4.2
Cholesterol (% by weight) trace 0.2 -
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Table 2. Serum Biomarkers

Biomarker Male Female Significance
SC WD KD SC WD KD
Glucose, mg/dL 286.9±33.7 446.8±83.9* 234.2±28.0# 333.1±40.0 307.5±20.5* 217.0±11.8# Diet, p=0.004
Insulin, ng/dL 4.5±0.4 5.2±0.7 3.5±0.5*# 4.0±0.3 7.6±0.5*$ 2.8±0.3# Sex*Diet, p=0.004
Diet, p<0.0001
Ketone Bodies, mmol/L 0.98±0.05 0.94±0.06 2.77±0.24*# 0.81±0.04 0.80±0.09 1.66±0.13*#$ Sex*Diet, p<0.0001
Sex, p<0.0001
Diet, p<0.0001
Triglycerides, mg/dL 112.6±17.1 182.3±35.1* 117.1±23.5# 66.9±8.0 216.4±35.0* 58.3±6.5# Diet, p<0.0001
Total Cholesterol, 0.75±0.02 0.96±0.10* 0.79±0.05# 0.67±0.03 1.07±0.04* 0.63±0.04# Diet, p<0.0001
mmol/L
Non-esterified Fatty 0.55±0.13 0.34±0.13 0.49±0.20 0.44±0.09 0.61±5 0.58±0.16
Acids, mg/dL
ALT, U/L 50.0±14.8 35.0±2.5 58.0±3.9 41.6±5.4 35.6±3.3 41.7±4.0 Sex, p=0.006
AST, U/L 155.6±40.7 159.1±28.0 212.1±43.1 109.1±53.4 90.4±8.5 128.4±13

Total Antioxidant 1.35±0.2 1.64±0.2 1.45±0.2 1.93±0.3 1.42±0.2 1.21±0.2
Capacity, mM
TBARS, µm 7.4±0.6 7.7±0.8 5.2±0.7 9.0±0.8 7.4±0.3 14.0±2.3*#$ Sex*Diet, p=0.0008
Diet, p=0.0005
(* significant difference (p≤ 0.05) from SC. # significant difference (p≤ 0.05) between WD and KD. $
significant difference (p ≤ 0.05) for male vs. female within diet. Values are presented as mean ± SE. n =
7-12/group).
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Figures
Figure 1 - Effects of diet and sex on animal characteristics: Body mass changes over time (A), mean
Change in Body Mass (B), End Body Fat Percentage (C), Food Intake (grams/day) (D), Energy Intake
(kcals/day) (E), Distance ran/night (weekly) (F), Average distance ran/night (overall) (G). Main effects,
interactions and p values reported in text above graphs. * significant difference (p≤0.05) from SC. #
significant difference (p ≤0.05) between WD and KD. $ significant difference (p ≤0.05) for male vs.
female within diet. Values are presented as mean ± SE (n = 7-14/group).
Figure 2 - Effects of diet and sex on liver triglycerides hepatic oxidative stress and inflammation:
Liver triglycerides (A), Hepatic GSH (B), Hepatic GSSG (C), Hepatic GPX1 (D), Hepatic oxidative stress
(E), Hepatic Inflammation (F), Representative western blots (G). Main effects, interactions and p values
reported in text above graphs. * significant difference (p ≤ 0.05) from SC. # significant difference (p ≤
0.05) between WD and KD. $ significant difference (p ≤ 0.05) for male vs. female within diet. Values are
presented as mean ± SE (n = 7-14/group).
Figure 3 - Effects of diet and sex on hepatic lipid regulation: Hepatic AMPK content (A), Hepatic de
novo lipogenesis (B), Hepatic CD36 (C), Representative western blots (D). Main effects, interactions and
p values reported in text above graphs. * significant difference (p ≤ 0.05) from SC. # significant
difference (p ≤ 0.05) between WD and KD. $ significant difference (p ≤ 0.05) for male vs. female within
diet. Values are presented as mean ± SE (n = 7-14/group).
Figure 4 - Effects of diet and sex on hepatic mitochondrial content: Hepatic citrate synthase activity
(A), Hepatic cytochrome c protein content (B), Hepatic mitochondrial content (C), Representative
western blots (D). Main effects, interactions and p values reported in text above graphs. * significant
difference (p ≤ 0.05) from SC. # significant difference (p ≤ 0.05) between WD and KD. $ significant
difference (p ≤ 0.05) for male vs. female within diet. Values are presented as mean ± SE (n = 7-14/group).
Figure 5 - Effects of diet and sex on hepatic mitochondrial biogenesis and hepatic
autophagy/mitophagy: Hepatic mitochondrial biogenesis proteins (A), Hepatic mitochondrial
autophagy/mitophagy protein content (B), Representative western blots (C). Main effects, interactions and
p values reported in text above graphs. * significant difference (p ≤ 0.05) from SC. # significant
difference (p ≤ 0.05) between WD and KD. $ significant difference (p ≤ 0.05) for male vs. female within
diet. Values are presented as mean ± SE (n = 7-14/group).
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Figure 1 - Effects of diet and sex on animal characteristics: Body mass changes over time (A), mean Change
in Body Mass (B), End Body Fat Percentage (C), Food Intake (grams/day) (D), Energy Intake (kcals/day)
(E), Distance ran/night (weekly) (F), Average distance ran/night (overall) (G). Main effects, interactions and
p values reported in text above graphs. * significant difference (p≤0.05) from SC. # significant difference (p
≤0.05) between WD and KD. $ significant difference (p ≤0.05) for male vs. female within diet. Values are
476x289mm (300 x 300 DPI)

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Figure 2 - Effects of diet and sex on liver triglycerides hepatic oxidative stress and inflammation: Liver
triglycerides (A), Hepatic GSH (B), Hepatic GSSG (C), Hepatic GPX1 (D), Hepatic oxidative stress (E),
Hepatic Inflammation (F), Representative western blots (G). Main effects, interactions and p values reported
in text above graphs. * significant difference (p ≤ 0.05) from SC. # significant difference (p ≤ 0.05)
between WD and KD. $ significant difference (p ≤ 0.05) for male vs. female within diet. Values are
262x131mm (300 x 300 DPI)

Page 31 of 33
Figure 3 - Effects of diet and sex on hepatic lipid regulation: Hepatic AMPK content (A), Hepatic de novo
lipogenesis (B), Hepatic CD36 (C), Representative western blots (D). Main effects, interactions and p values
reported in text above graphs. * significant difference (p ≤ 0.05) from SC. # significant difference (p ≤
0.05) between WD and KD. $ significant difference (p ≤ 0.05) for male vs. female within diet. Values are
272x140mm (300 x 300 DPI)

Downloaded from www.nrcresearchpress.com by BOSTON UNIVERSITY on 05/26/19. For personal use only. Page 32 of 33
Figure 4 - Effects of diet and sex on hepatic mitochondrial content: Hepatic citrate synthase activity (A),
Hepatic cytochrome c protein content (B), Hepatic mitochondrial content (C), Representative western blots
(D). Main effects, interactions and p values reported in text above graphs. * significant difference (p ≤ 0.05)
from SC. # significant difference (p ≤ 0.05) between WD and KD. $ significant difference (p ≤ 0.05) for
male vs. female within diet. Values are presented as mean ± SE (n = 7-14/group).
217x126mm (300 x 300 DPI)

Page 33 of 33
Figure 5 - Effects of diet and sex on hepatic mitochondrial biogenesis and hepatic autophagy/mitophagy:
Hepatic mitochondrial biogenesis proteins (A), Hepatic mitochondrial autophagy/mitophagy protein content
(B), Representative western blots (C). Main effects, interactions and p values reported in text above graphs.
* significant difference (p ≤ 0.05) from SC. # significant difference (p ≤ 0.05) between WD and KD. $
significant difference (p ≤ 0.05) for male vs. female within diet. Values are presented as mean ± SE (n = 7-
14/group).
385x270mm (300 x 300 DPI)

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metabolism and oxidative stress and merits further investigation.

suppressed markers of de novo lipogenesis, increased markers of mitochondrial content vs

western diet feeding.

 Female rats exhibited elevated hepatic markers of autophagy/mitophagy and energy

homeostasis compared with male rats.

needed in order to address the current disparities in the literature.

Materials and Method

48 hours prior to euthanasia.

Hologic QDR-1000/W dual-energy x-ray absorptiometry machine.

measured at week 3 and 7 with the average reported.

of Hissin and Hilf (Hissin and Hilf 1976).

No.709001, Cayman Chemical) were measured according to the manufacturer’s instructions.

horseradish peroxidase-conjugated secondary antibody (#7074, #7076, Cell Signaling Technology,

Signaling), autophagy-related gene 12 (ATG12, #4180, Cell Signaling), 5’ adenosine monophosphate

(NFE2L2/NRF2; #137550, Abcam), Kelch-like ECH-associated protein 1 (Keap1; #12721, Cell

oxidative phosphorylation (OXPHOS; #110413, Abcam, Cambridge, MA), peroxisome proliferator

Biologicals; Centennial, CO, USA), CD68 (#17832; Santa Cruz).

statistically significant difference.

content (p > 0.05; Supplementary Figure S11).

sex, p  0.05; Fig. 2F).

transcriptional regulator of de novo lipogenesis, was elevated in WD females vs SC females and KD

p0.05; Fig. 3C), which was not observed in males.

Hepatic Mitochondrial Markers- Hepatic citrate synthase activity, a surrogate measure of

p0.05; Fig. 4C). No differences in complex V were observed among groups.

PGC-1α and mitochondrial transcription factor A (TFAM), markers of mitochondrial biogenesis,

interaction, p = 0.0497; Fig 5A).

p = 0.0446, respectively; Fig 5B).

downregulated several mitochondrial electron transport chain proteins in the liver.

Here we report that a KD resulted in a more favorable body composition vs WD feeding in

inflammation. Further research is warranted to elucidate the mechanisms involved.

67% FAT) compared with the current study.

Kephart et al. 2017), with further research needed in this area.

hyperacetylation, reductions in deacetylase activity, in particular SIRT3, and subsequent decreased

PGC-1α and TFAM coregulate multiple transcription factors involved in mitochondrial

Mitochondrial processes of autophagy, termed mitophagy, is the clearance of damaged or poorly

KD on mitophagic flux in hepatic tissue.

2011; Lonardo et al. 2006).

The authors have no conflicts of interest to report.

SIRT1 controls the transcription of the peroxisome proliferator-activated receptor-gamma Co-

activator-1alpha (PGC-1alpha) gene in skeletal muscle through the PGC-1alpha autoregulatory

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