Paula M. Miotto

Maternal High Fat Feeding Does Not Have Long-Lasting Effects on Body Composition and Bone Health in Female and Male Wistar Rat Offspring at Young Adulthood

High fat diets adversely affect body composition, bone mineral and strength, and alter bone fatty acid composition. It is unclear if maternal high fat (HF) feeding permanently alters offspring body composition and bone health. Female rats were fed control (CON) or HF diet for 10 weeks, bred, and continued their diets throughout pregnancy and lactation. Male and female offspring were studied at weaning and 3 months, following consumption of CON diet. At weaning, but not 3 months of age, male and female offspring from dams fed HF diet had lower lean mass and higher fat and bone mass, and higher femur bone mineral density (females only) than offspring of dams fed CON diet. Male and female offspring femurs from dams fed HF diet had higher monounsaturates and lower n6 polyunsaturates at weaning than offspring from dams fed CON diet, where females from dams fed HF diet had higher saturates and lower n6 polyunsaturates at 3 months of age. There were no differences in strength of femurs or lumbar vertebrae at 3 months of age in either male or female offspring. In conclusion, maternal HF feeding did not permanently affect body composition and bone health at young adulthood in offspring.

In the absence of phosphate shuttling, exercise reveals the in vivo importance of creatine-independent mitochondrial ADP transport.

The transport of cytosolic adenosine diphosphate (ADP) into the mitochondria is a major control point in metabolic homeostasis, as ADP concentrations directly affect glycolytic flux and oxidative phosphorylation rates within mitochondria. A large contributor to the efficiency of this process is thought to involve phosphocreatine (PCr)/Creatine (Cr) shuttling through mitochondrial creatine kinase (Mi-CK), whereas the biological importance of alterations in Cr-independent ADP transport during exercise remains unknown. Therefore, we utilized an Mi-CK knockout (KO) model to determine whether in vivo Cr-independent mechanisms are biologically important for sustaining energy homeostasis during exercise. Ablating Mi-CK did not alter exercise tolerance, as the time to volitional fatigue was similar between wild-type (WT) and KO mice at various exercise intensities. In addition, skeletal muscle metabolic profiles after exercise, including glycogen, PCr/Cr ratios, free ADP/adenosine monophosphate (AMP), and lactate, were similar between genotypes. While these data suggest that the absence of PCr/Cr shuttling is not detrimental to maintaining energy homeostasis during exercise, KO mice displayed a dramatic increase in Cr-independent mitochondrial ADP sensitivity after exercise. Specifically, whereas mitochondrial ADP sensitivity decreased with exercise in WT mice, in stark contrast, exercise increased mitochondrial Cr-independent ADP sensitivity in KO mice. As a result, the apparent ADP Km was 50% lower in KO mice after exercise, suggesting that in vivo activation of voltage-dependent anion channel (VDAC)/adenine nucleotide translocase (ANT) can support mitochondrial ADP transport. Altogether, we provide insight that Cr-independent ADP transport mechanisms are biologically important for regulating ADP sensitivity during exercise, while highlighting complex regulation and the plasticity of the VDAC/ANT axis to support adenosine triphosphate demand.

Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

Combined high-fat-resveratrol diet and RIP140 knockout mice reveal a novel relationship between elevated bone mitochondrial content and compromised bone microarchitecture, bone mineral mass, and bone strength in the tibia.

SCOPE While resveratrol (RSV) is associated with the prevention of high-fat (HF) diet-induced insulin resistance, the effects on bone health combined with an HF-diet is unknown. Therefore, we determined the effect of RSV on bone microarchitecture in the presence of an HF-diet, while also elucidating molecular adaptations within bone that could contribute to bone health status. METHODS AND RESULTS Male C57BL6 mice were provided control (10% fat) or HF-diet (60% fat) in the presence or absence of RSV for 12 weeks. While RSV prevented HF diet-induced glucose intolerance, HF-RSV compromised tibial microarchitecture, mineral mass, and strength. The compromised outcomes following HF-RSV corresponded with higher markers of osteoclast-activation and bone-resorption (decreased OPG/RANKL ratio; increased cathepsin K), as well as higher markers of tibial mitochondrial content. A molecular model of elevated mitochondrial content (RIP140 knock out (KO) mice) was utilized to determine proof-of-principle that increasing mitochondrial content coincides with decrements in bone health. RIP140 KO mice displayed higher markers of mitochondrial content, and similar to HF-RSV, had compromised bone microarchitecture, lower BMD/strength, and higher markers of osteoclast-activation/bone-resorption. CONCLUSION These data show that in the presence of an HF-diet, RSV negatively alters bone health, a process associated with increased mitochondrial content and markers of bone resorption.

α-Linolenic acid supplementation and exercise training reveal independent and additive responses on hepatic lipid accumulation in obese rats

α-Linolenic acid (ALA) supplementation or exercise training can independently prevent hepatic lipid accumulation and reduced insulin signaling; however, this may occur through different mechanisms of action. In the current study, obese Zucker rats displayed decreased phospholipid (PL) content in association with hepatic lipid abundance, and therefore, we examined whether ALA and exercise training would prevent these abnormalities differently to reveal additive effects on the liver. To achieve this aim, obese Zucker rats were fed control diet alone or supplemented with ALA and were sedentary or exercise trained for 4 wk (C-Sed, ALA-Sed, C-Ex, and ALA-Ex). ALA-Sed rats had increased microsomal-triglyceride transfer protein (MTTP), a protein required for lipoprotein assembly/secretion, as well as modestly increased PL content in the absence of improvements in mitochondrial content, lipid accumulation, or insulin sensitivity. In contrast, C-Ex rats had increased mitochondrial content and insulin sensitivity; however, this corresponded with minimal improvements in PL content and hepatic lipid accumulation. Importantly, ALA-Ex rats demonstrated additive improvements in PL content and hepatic steatosis, which corresponded with increased mitochondrial content, MTTP and apolipoprotein B100 content, greater serum triacylglyceride, and insulin sensitivity. Overall, these data demonstrate additive effects of ALA and exercise training on hepatic lipid accumulation, as exercise training preferentially increased mitochondrial content, while ALA promoted an environment conducive for lipid secretion. These data highlight the potential for combination therapy to mitigate liver disease progression.

Exercise-induced reductions in mitochondrial ADP sensitivity contribute to the induction of gene expression and mitochondrial biogenesis through enhanced mitochondrial H2O2 emission

Acute exercise rapidly induces mitochondrial gene expression, however, the intracellular events regulating this process remain incompletely understood. The purpose of this study was to determine whether reductions in mitochondrial ADP sensitivity during exercise have a biological role in regulating mitochondrial-derived reactive oxygen species (ROS) production and the induction of mitochondrial biogenesis. Mitochondrial creatine kinase wildtype (WT) and knockout (KO) mice have divergent responses in ADP sensitivity during exercise, and we therefore used these mice to determine the relationship between mitochondrial ADP sensitivity, ROS production, and mitochondrial adaptations to exercise. In WT mice, acute exercise reduced mitochondrial ADP respiratory sensitivity and the ability of ADP to suppress ROS production, while increasing mitochondrial gene transcription (PGC-1α, PGC-1β and PDK4). In stark contrast, in KO mice, exercise increased ADP sensitivity, reduced mitochondrial ROS emission, and did not induce gene transcription. Despite the divergence in mRNA responses, exercise similarly induced calcium/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK) phosphorylation in WT and KO mice, however only WT mice were associated with redox stress (4HNE). These data implicate acute changes in ADP sensitivity in mitochondrial adaptations to exercise. To further examine this we chronically exercise trained mice. While training increased mitochondrial content and reduced ADP sensitivity in WT mice, KO mice did not exhibit adaptations to exercise. Combined, these data suggest that exercise-induced attenuations in mitochondrial ADP sensitivity mediate redox signals that contribute to the induction of acute and chronic mitochondrial adaptations.

High-Fat Diet Causes Mitochondrial Dysfunction as a Result of Impaired ADP Sensitivity

Although molecular approaches altering mitochondrial content have implied a direct relationship between mitochondrial bioenergetics and insulin sensitivity, paradoxically, consumption of a high-fat (HF) diet increases mitochondrial content while inducing insulin resistance. We hypothesized that despite the induction of mitochondrial biogenesis, consumption of an HF diet would impair mitochondrial ADP sensitivity in skeletal muscle of mice and therefore manifest in mitochondrial dysfunction in the presence of ADP concentrations indicative of skeletal muscle biology. We found that HF consumption increased mitochondrial protein expression; however, absolute mitochondrial respiration and ADP sensitivity were impaired across a range of biologically relevant ADP concentrations. In addition, HF consumption attenuated the ability of ADP to suppress mitochondrial H2O2 emission, further suggesting impairments in ADP sensitivity. The abundance of ADP transport proteins were not altered, but the sensitivity to carboxyatractyloside-mediated inhibition was attenuated after HF consumption, implicating alterations in adenine nucleotide translocase (ANT) ADP sensitivity in these observations. Moreover, palmitoyl-CoA is known to inhibit ANT, and modeling intramuscular palmitoyl-CoA concentrations that occur after HF consumption exacerbated the deficiency in ADP sensitivity. Altogether, these data suggest that an HF diet induces mitochondrial dysfunction secondary to an intrinsic impairment in mitochondrial ADP sensitivity that is magnified by palmitoyl-CoA.

Sex differences in mitochondrial respiratory function in human skeletal muscle

Mitochondrial bioenergetic contributions to sex differences in human skeletal muscle metabolism remain poorly defined. The primary aim of this study was to determine whether mitochondrial respiratory kinetics differed between healthy young men and women in permeabilized skeletal muscle fibers. While men and women displayed similar ( P > 0.05) maximal respiration rates and abundance of mitochondrial/adenosine diphosphate (ADP) transport proteins, women had lower ( P < 0.05) mitochondrial ADP sensitivity (+30% apparent Km) and absolute respiration rates at a physiologically relevant ADP concentration (100 μM). Moreover, although men and women exhibited similar carnitine palmitoyl transferase-I protein content- and palmitoyl-CoA-supported respiration, women displayed greater sensitivity to malonyl-CoA-mediated respiratory inhibition. These data establish baseline sex differences in mitochondrial bioenergetics and provide the foundation for studying mitochondrial function within the context of metabolic perturbations and diseases that affect men and women differently.

Prior exercise training improves cold tolerance independent of indices associated with non-shivering thermogenesis

Mammals defend against cold‐induced reductions in body temperature through both shivering and non‐shivering thermogenesis. The activation of non‐shivering thermogenesis is primarily driven by uncoupling protein‐1 in brown adipose tissue and to a lesser degree by the browning of white adipose tissue. Endurance exercise has also been shown to increase markers of white adipose tissue browning. This study aimed to determine whether prior exercise training would alter the response to a cold challenge and if this would be associated with differences in indices of non‐shivering thermogenesis. It is shown that exercise training protects against cold‐induced weight loss by increasing food intake. Exercise‐trained mice were better able to maintain their core temperature, independent of differences in markers of non‐shivering thermogenesis.

The importance of exercise intensity, volume and metabolic signalling events in the induction of mitochondrial biogenesis

A classical consequence of exercise training is the induction of mitochondrial biogenesis, a highly coordinated process acting to enhance oxidative capacity and aerobic energy production. Mediated by acute transcriptional events following each individual bout of exercise, a key factor initiating mitochondrial biogenesis is the transient upregulation of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) expression, activating several downstream transcription factors implicated in the control of mitochondrial content and structure. However, our understanding of the signalling events inducing the exercise-mediated increase in skeletal muscle PGC-1α mRNA expression remains incomplete. Traditionally, literature has largely focused on the role of intracellular metabolites (AMP, ADP, Ca2+) during exercise as potent activators of AMP-activated protein kinase (AMPK), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation events mediating cellular adaptations. Furthermore, other lines of evidence suggest glycogen depletion and lactate accumulation, representing a metabolic profile indicative of energetic stress, are linked to the upregulation of PGC-1α mRNA content (Egan et al. 2010). It is therefore conceivable that the mitochondrial biogenic responses to exercise are dependent on the degree of metabolic stress; however, this has remained a contentious subject in human studies as a graded relationship between exercise-mediated signalling events and PGC-1α expression has not been consistently established (Brandt et al. 2016). This is of particular interest with respect to low-volume sprint interval training, in which the extent of energetic demands could be important in overcoming the reduction in volume to elicit mitochondrial adaptations classically observed following continuous prolonged exercise. To provide novel insight into the role of energetic stress in PGC-1α expression during intense exercise, a recent paper published by Fiorenza et al. in The Journal of Physiology (Fiorenza et al. 2018) examined the metabolic perturbations and mitochondrial biogenic responses to three distinct exercise protocols. Specifically, 12 trained men completed an acute bout of repeated-sprint exercise (RS, 18 × 5 s all out with 30 s recovery), work-matched speed endurance exercise (SE, 6 × 20 s all out with 120 s recovery), and traditional continuous moderate intensity exercise (CM, 50 min at 70% V̇O2max). Fiorenza et al. reported that (1) between high intensity exercise bouts, metabolic stress was a predictor of skeletal muscle mRNA responses indicative of mitochondrial biogenesis, while in contrast, (2) PGC-1α mRNA expression following moderate intensity exercise was not dependent on energetic perturbations when these responses were directly compared to high intensity exercise bouts. Specifically, while all types of exercise utilized by Fiorenza et al. increased skeletal muscle PGC-1α mRNA expression at a 3 h post-exercise time point, this response was greater following SE and CM compared to RS. In a similar trend, the associated transcription factors Tfam and Nrf2, mediating mitochondrial DNA transcription, and Mfn2 and Drp1, implicated in mitochondrial structural remodelling, were only upregulated with SE and CM, but not RS. Despite the work-matched nature of SE and RS exercise bouts, greater CaMKII and p38MAPK phosphorylation, and a greater rise in muscle lactate concentrations, as well as higher plasma adrenaline levels, were identified following SE, likely contributing to the enhanced transcriptional responses to SE exercise. In addition, the novel finding that heat shock protein 72 (HSP72) mRNA levels were elevated to a greater extent following SE exercise compared to both CM and RS further indicates a heightened metabolic stress response to this low-volume intense exercise bout. This evidence would therefore implicate metabolic stress as a key factor required for high intensity exercise to overcome the reduction in training volume and elicit cellular transcription events mediating downstream mitochondrial adaptations. However, in contrast to these findings, AMPK phosphorylation was elevated to a similar extent in skeletal muscle obtained immediately following all three exercise bouts, regardless of the different PGC-1α transcriptional responses. In addition, CaMKII phosphorylation, p38 MAPK phosphorylation, and the accumulation of cellular metabolites were generally lower following CM despite the substantial elevation in PGC-1α mRNA content. This finding is consistent with previous work by the same authors (Brandt et al. 2016) in which a relationship between energetic stress and PGC-1α mRNA induction was not identified following various 60 min endurance-based exercise protocols interspersed with very brief periods at a higher intensity. Combined, these data provide interesting evidence that while PGC-1α mRNA expression is dependent on the degree of metabolic stress associated with different intensities and volumes of exercise, it also appears that other intracellular mechanisms are important for the induction of PGC-1α mRNA expression. In this respect, the increased superoxide dismutase 2 (SOD2) mRNA content observed by Fiorenza et al. (2018) following SE and CM exercise, and the enhanced HSP72 mRNA response to SE exercise, could occur as a result of greater reactive oxygen species (ROS) production, supporting a rationale to further examine the role of ROS as a cellular signalling event. Specifically, an increase in mitochondrial-derived ROS has been reported in response to acute exercise (Place et al. 2015), further influencing several redox-sensitive pathways implicated in mitochondrial adaptations. While the exact mechanistic links remain unknown, recent work has shown that mitochondrial transcriptional events and PGC-1α expression following treadmill running are blunted in mice with attenuated exercise-mediated ROS production in the presence of ADP, without changes