In Aotearoa New Zealand, approximately 1 in 3 adults and 1 in 8 children are classified as obese, with Māori and Pacific communities disproportionately affected (1) . While maternal nutrition has been extensively studied, paternal impacts and the combined effect of both parents’ obesogenic environments on offspring health remain underexplored (2) . The primary objective of this study is to characterise the metabolic phenotype of parent rats fed a High Fat High Sugar (HFHS) diet and investigate the birth characteristics of their offspring, from a factorial mating design. Eighty female and 40 male Sprague-Dawley rats were randomised to a standard chow diet (SD) (24% protein, 18% fat, 58% carbohydrates) or HFHS diet (Specialty Feeds SF23-120: 16% protein, 41% fat, 43% carbohydrates) for five weeks prior to mating. Females were then continued on their respective diets throughout pregnancy and lactation. Four mating combinations were established: SDmum-SDdad, SDmum-HFHSdad, HFHSmum-SDdad, and HFHSmum-HFHSdad. A subset of parents (n=38) underwent body composition assessments using dual-energy X-ray absorptiometry (DEXA). Additionally, a subgroup (n=23) was evaluated for metabolic profiles using Prometheon metabolic cages. Offspring birth weights and body lengths were recorded. The HFHS diet’s efficacy was confirmed in both male and female rats, with HFHS groups showing higher body weight (females: 327.1 g ± 19.7 vs. 288.2 g ± 20.1; males: 575.8 g ± 39.8 vs. 532.6 g ± 50.3; p < 0.05), greater fat percentage (females: 46.8% ± 5.6 vs. 29.2% ± 5.6; males: 40.5% ± 7.2 vs. 28.7% ± 6.8; p < 0.001), and a lower respiratory exchange ratio (RER) (females: 0.8108 ± 0.0275 vs. 0.8679 ± 0.0288; males: 0.8257 ± 0.0304 vs. 0.8759 ± 0.0266; p < 0.05) compared to the SD group. In male offspring, birth weights in HFHSmum-SDdad (6.3 g ± 0.9) and HFHSmum-HFHSdad (6.0 g ± 0.9) groups were significantly lower (p < 0.0001) than in SDmum-SDdad (6.980 g ± 0.7753) and SDmum-HFHSdad (7.0 g ± 0.7) groups. Birth weights were further reduced in HFHSmum-HFHSdad versus HFHSmum-SDdad (Mean Diff. = 0.3g; p < 0.05).Body lengths in HFHSmum-HFHSdad males were shorter (43.1 mm ± 3.2; p < 0.0001) compared to other groups (≥ 45.3 mm). Female offspring birth weights were lower in the HFHSmum-SDdad (5.8g ± 0.8) and HFHSmum-HFHSdad groups (5.8 g ± 0.9; p<0.0001) compared to the other groups (means ≥ 6.4g) but paternal HFHS diet had no additional effect on birth weight. As with males, body lengths in the HFHSmum-HFHSdad female offspring were significantly shorter (4 mm ± 3; p<0.0001) compared to all other groups (≥44mm). Parental HFHS diets synergistically reduce offspring birth length and weight, with stronger effects in males. These findings underscore the importance of inclusive dietary guidelines for both parents to reduce intergenerational obesity risk and support long-term health.

Interleukin-6 (IL-6) is produced and secreted by skeletal muscle cells during exercise and plays an important role in mediating metabolic responses to exercise. The promoter region of the IL-6 gene contains a common genetic variant (-174 G/C, rs1800795) which may alter responses to exercise training. To isolate the impact of this gene variant on exercise-induced IL-6 expression and skeletal muscle transcription responses following exercise we generated knock-in mice with a GG or variant CC genotype for the murine homolog of rs1800795. The overall gross metabolic phenotype of resting mice was similar between genotypes; however, following acute treadmill running the variant CC genotype was associated with a greater increase in skeletal muscle IL-6 mRNA and circulating IL-6. Furthermore, we observed that mice with the variant CC genotype exhibited sex-specific differences in skeletal muscle master metabolism regulatory genes, and had greater increases in genes controlling mitochondrial biogenesis in skeletal muscle post-exercise. However, there was no effect of genotype on exercise-induced skeletal muscle glycogen depletion, circulating free fatty acids, blood glucose and lactate production, or exercise-responsive gene expression in subcutaneous fat. These findings suggest that the IL-6 promoter variant -174 G/C may result in enhanced skeletal muscle adaptations in response to exercise training, and could mean that individuals with the ‘C’ allele may more readily gain improvements in metabolic health in response to exercise training.

There is some evidence for temperature-dependent stimulation of mitochondrial biogenesis; however, the role of elevated muscle temperature during exercise in mitochondrial adaptation to training has not been studied in humans in vivo. The purpose of this study was to determine the role of elevating muscle temperature during exercise in temperate conditions through the application of mild, local heat stress on mitochondrial adaptations to endurance training. Eight endurance-trained males undertook 3 weeks of supervised cycling training, during which mild (~ 40 °C) heat stress was applied locally to the upper-leg musculature of one leg during all training sessions (HEAT), with the contralateral leg serving as the non-heated, exercising control (CON). Vastus lateralis microbiopsies were obtained from both legs before and after the training period. Training-induced increases in complex I (fold-change, 1.24 ± 0.33 vs. 1.01 ± 0.49, P = 0.029) and II (fold-change, 1.24 ± 0.33 vs. 1.01 ± 0.49, P = 0.029) activities were significantly larger in HEAT than CON. No significant effects of training, or interactions between local heat stress application and training, were observed for complex I–V or HSP70 protein expressions. Our data provides partial evidence to support the hypothesis that elevating local muscle temperature during exercise augments training-induced adaptations to mitochondrial enzyme activity.

Clinically, there is a positive correlation between BMI and the risk of tendinopathy. However, the underlying mechanisms are not understood. Dyslipidaemia and increased circulating free fatty acids (FFA) are associated with increased BMI. We hypothesised that increased FFA concentrations negatively affect rat tendon-derived cells (rTDCs) through mitochondrial-mediated mechanisms. rTDCs were isolated and treated with oleic acid (OA), stearic acid (SA), and palmitic acid (PA). Cell viability was assessed using AlamarBlue™ assay, and gene expression using real-time PCR. Cell respiration and reactive oxygen species (ROS) production were measured using high-resolution respirometry and MitoSox staining. PA transport into the mitochondria was blocked by pre-treatment with 50µM etomoxir. Treatment with SA and PA at 10 µg/ml decreased rTDC viability by 40% and 60%, respectively. PA decreased the gene expression of the tendon markers Scx and Tnmd , and increased the expression of Mmp3, Mmp13 , and Ptgs2 (encoding Cox-2). FFA treatment increased the expression of Cpt1 and Pdk4 , indicating an increase in mitochondrial FFA oxidation. PA, at 10 µg/ml, increased cellular respiration and ROS production. Pre-treatment with etomoxir partially inhibited the effects of PA on cell viability, Mmp3 gene expression, ROS production, and cell respiration, but did not affect PA-induced inhibition of Scx or Tnmd expression. We found that increased saturated FFA concentrations in the microenvironment reduce cell viability and alter ROS production, respiration, and gene expression. Blocking PA transport into mitochondria partially reversed the negative effects of PA. Overall, an increase in saturated FFA concentrations may contribute to poor tendon health.

While oxygen is essential for oxidative phosphorylation, O2 can form reactive species (ROS) when interacting with electrons of mitochondrial electron transport system. ROS is dependent on O2 pressure (PO2) and has traditionally been assessed in O2 saturated media, PO2 at which mitochondria do not typically function in vivo. Mitochondrial ROS can be significantly elevated by the respiratory complex II substrate succinate, which can accumulate within hypoxic tissues, and this is exacerbated further with reoxygenation. Intertidal species are repetitively exposed to extreme O2 fluctuations, and have likely evolved strategies to avoid excess ROS production. We evaluated mitochondrial electron leakage and ROS production in permeabilized brain of intertidal and subtidal triplefin fish species from hyperoxia to anoxia, and assessed the effect of anoxia reoxygenation and the influence of increasing succinate concentrations. At typical intracellular PO2, net ROS production was similar among all species; however at elevated PO2, brain tissues of the intertidal triplefin fish released less ROS than subtidal species. In addition, following in vitro anoxia reoxygenation, electron transfer mediated by succinate titration was better directed to respiration, and not to ROS production for intertidal species. Overall, these data indicate that intertidal triplefin fish species better manage electrons within the ETS, from hypoxic-hyperoxic transitions.

Cold water immersion (CWI) following intense exercise is a common athletic recovery practice. However, CWI impacts muscle adaptations to exercise training, with attenuated muscle hypertrophy and increased angiogenesis. Tissue temperature modulates the abundance of specific miRNA species and thus CWI may affect muscle adaptations via modulating miRNA expression following a bout of exercise. The current study focused on the regulatory mechanisms involved in cleavage and nuclear export of mature miRNA, including DROSHA, EXPORTIN-5, and DICER. Muscle biopsies were obtained from the vastus lateralis of young males (n = 9) at rest and at 2, 4, and 48 h of recovery from an acute bout of resistance exercise, followed by either 10 min of active recovery (ACT) at ambient temperature or CWI at 10°C. The abundance of key miRNA species in the regulation of intracellular anabolic signaling (miR-1 and miR-133a) and angiogenesis (miR-15a and miR-126) were measured, along with several gene targets implicated in satellite cell dynamics (NCAM and PAX7) and angiogenesis (VEGF and SPRED-1). When compared to ACT, CWI suppressed mRNA expression of DROSHA (24 h p = 0.025 and 48 h p = 0.017), EXPORTIN-5 (24 h p = 0.008), and DICER (24 h p = 0.0034). Of the analyzed miRNA species, miR-133a (24 h p < 0.001 and 48 h p = 0.007) and miR-126 (24 h p < 0.001 and 48 h p < 0.001) remained elevated at 24 h post-exercise in the CWI trial only. Potential gene targets of these miRNA, however, did not differ between trials. CWI may therefore impact miRNA abundance in skeletal muscle, although the precise physiological relevance needs further investigation.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. beta-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle beta-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated beta-catenin(S552) phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific beta-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype x diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower beta-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated beta-catenin phosphorylation(S552) and actin remodeling (interaction effect of insulin x palmitate p < 0.05). Finally, beta-catenin(S552) phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total beta-catenin expression was unchanged. These findings suggest that beta-catenin dysfunction is associated with the development of insulin resistance.

Diminished insulin and insulin-like growth factor-1 signaling extends the lifespan of invertebrates1-4; however, whether it is a feasible longevity target in mammals is less clear5-12. Clinically utilized therapeutics that target this pathway, such as small-molecule inhibitors of phosphoinositide 3-kinase p110α (PI3Ki), provide a translatable approach to studying the impact of these pathways on aging. Here, we provide evidence that dietary supplementation with the PI3Ki alpelisib from middle age extends the median and maximal lifespan of mice, an effect that was more pronounced in females. While long-term PI3Ki treatment was well tolerated and led to greater strength and balance, negative impacts on common human aging markers, including reductions in bone mass and mild hyperglycemia, were also evident. These results suggest that while pharmacological suppression of insulin receptor (IR)/insulin-like growth factor receptor (IGFR) targets could represent a promising approach to delaying some aspects of aging, caution should be taken in translation to humans.

The anaesthetic isoeugenol has been used as metabolic suppressant for commercial transport of live lobsters in order to decrease energy expenditure and improve survival. Given the central role of mitochondria in metabolism and structural similarities of isoeugenol to the mitochondrial electron carrier coenzyme Q, we explored the influence on mitochondrial function of isoeugenol. Mitochondrial function was measured using high-resolution respirometry and saponin-permeabilised heart fibres from the Australasian red spiny lobster, Jasus edwardsii. Relative to vehicle (polysorbate), isoeugenol inhibited respiration supported by complex I (CI) and cytochrome c oxidase (CCO). While complex II (CII), which also reduces coenzyme Q, was largely unaffected by isoeugenol, respiration supported by CII when uncoupled was depressed. Titration of isoeugenol indicates that respiration through CI has a half-maximal inhibitory concentration (IC50) of 2.4±0.1 µmol l-1, and a full-maximal inhibitory concentration (IC100-) of approximately 6.3 µmol l-1. These concentrations are consistent with those used for transport and euthanasia of J. edwardsii and indicate that CI is a possible target of isoeugenol, like many other anaesthetics with quinone-like structures.

Mitochondrial dynamics are regulated by the differing molecular pathways variously governing biogenesis, fission, fusion, and mitophagy. Adaptations in mitochondrial morphology are central in driving the improvements in mitochondrial bioenergetics following exercise training. However, there is a limited understanding of mitochondrial dynamics in response to inactivity. Skeletal muscle biopsies were obtained from middle-aged males (n = 24, 49.4 ± 3.2 years) who underwent sequential 14-day interventions of unilateral leg immobilisation, ambulatory recovery, and resistance training. We quantified vastus lateralis gene and protein expression of key proteins involved in mitochondrial biogenesis, fusion, fission, and turnover in at baseline and following each intervention. PGC1α mRNA decreased 40

Exercise induces adaptions which improve cellular function and exercise performance and help to protect from a host of non-communicable diseases. Accumulating evidence suggests that reactive oxygen species (ROS) produced during exercise co-ordinate adaptive transcriptional responses. In the rested state, ATP is supplied to most cells through mitochondrial oxidative phosphorylation, and during this process, electrons can interact with oxygen at certain sites in electron transport complexes to form superoxide. To meet increased energy demands during exercise, skeletal muscle mitochondrial oxygen consumption increases dramatically. However, the efficiency of the mitochondria to produce ATP increases under exercise conditions resulting in lower rate of overall mitochondrial ROS being produced per unit of oxygen. That exercise-induced ROS seems to be primarily derived from non-mitochondrial sources questions whether mitochondrial ROS regulate exercise-induced adaptations. This chapter describes the potential specific mitochondrial sites that produce ROS during exercise and provides an overview of localised mechanisms through which discrete mitochondrial redox signalling may contribute to coordinating molecular responses to acute exercise. We discuss technological advances that allow organelle-specific monitoring of ROS and development of mitochondrial-targeted antioxidants and genetic models to manipulate endogenous redox pathways, which will allow systematic assessment of the role of mitochondrial ROS in exercise responsiveness.

Neutrophils accumulate in peripheral tissues during obesity and are critical coordinators of tissue inflammatory responses. Here, we provide evidence that inhibition of the primary neutrophil protease, neutrophil elastase, with α1-antitrypsin A (A1AT) can improve insulin sensitivity and glucose homeostasis of mice fed a high-fat diet. This was attributed to improved insulin-induced IRS-1 phosphorylation in white adipose tissue and provides further support for a role of neutrophils in mediating diet-induced peripheral tissue insulin resistance.

BYL719 (alpelisib) is a small molecule inhibitor of PI3K p110α developed for cancer therapy. Targeted suppression of PI3K has led to lifespan extension in rodents and model organisms. If PI3K inhibitors are to be considered as an aging therapeutic, it is important to understand the potential consequences of long-term exposure, and the most practical way to achieve this is through diet administration. Here, we investigated the pharmacokinetics of BYL719 delivered in diet and the efficacy of BYL719 to suppress insulin signaling when administered in the diet of 8-month-old male and female mice. Compared to oral gavage, diet incorporation resulted in a lower peak plasma BYL719 (3.6 vs. 9.2 μM) concentration but similar half-life (~1.5 h). Consuming BYL719 resulted in decreased insulin signaling in liver and muscle within 72 h, and mice still showed impaired glucose tolerance and insulin sensitivity following 6 weeks of access to a diet containing 0.3 g/kg BYL719. However, consuming BYL719 did not affect food intake, body mass, muscle function (rotarod and hang time performance) or cognitive behaviors. This provides evidence that BYL719 has long-term efficacy without major toxicity or side effects, and suggests that administering BYL719 in diet is suitable for studying the effect of pharmacological suppression of PI3K p110α on aging and metabolic function.

To investigate within the one study potential molecular and cellular changes associated with mitochondrial biogenesis following 15 days of exposure to moderate hypoxia. Eight males underwent a muscle biopsy before and after 15 days of hypoxia exposure (FiO2 = 0.140–0.154; ~ 2500–3200 m) in a hypoxic hotel. Mitochondrial respiration, citrate synthase (CS) activity, and the content of genes and proteins associated with mitochondrial biogenesis were investigated. Our main findings were the absence of significant changes in the mean values of CS activity, mitochondrial respiration in permeabilised fibers, or the content of genes and proteins associated with mitochondrial biogenesis, after 15 days of moderate normobaric hypoxia. Our data provide evidence that 15 days of moderate normobaric hypoxia have negligible influence on skeletal muscle mitochondrial content and function, or genes and proteins content associated with mitochondrial biogenesis, in young recreationally active males. However, the increase in mitochondrial protease LON content after hypoxia exposure suggests the possibility of adaptations to optimise respiratory chain function under conditions of reduced O2 availability.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neutrophil elastase (NE) is a serine protease expressed by neutrophils which is inhibited endogenously by α1-Antitrypsin A (AAT). Obesity is associated with increased plasma NE:AAT ratios and adipose tissue NE accumulation, which may drive obesity-associated metabolic dysfunction. Exogenous AAT is now an FDA approved treatment for AAT deficiency, therefore we investigated whether AAT treatment may also have potential therapeutic benefits for diet induced metabolic dysfunction. Standard Chow and high-fat diet (HFD) fed male C57Bl6 mice were randomized to receive 3x weekly IP injections of either AAT (2mg) or vehicle (PBS) for 10 weeks. Chow fed mice treated with recombinant AAT showed no differences in plasma NE, body weight or metabolic phenotype compared to vehicle treated control mice. However, during HFD feeding AAT treatment attenuated increases in plasma NE and white adipose tissue (WAT) NE and neutrophil accumulation without affecting circulatory neutrophil levels or body weight. Consistent with NE knockout mice being partially protected from HFD glucose intolerance, treatment of HFD fed mice with AAT enhanced whole body insulin sensitivity which was attributed to higher insulin-dependent p-AktSer473 and reduced inflammation markers in WAT but no other peripheral tissues. Treatment of 3T3L1 adipocytes with recombinant NE impaired insulin-stimulated glucose uptake andp-AktSer473, suggesting that inhibition of NE in WAT is the primary mechanism through which AAT treatment enhances insulin sensitivity. Collectively, our data suggests AAT may play a potential role in mitigating diet-induced neutrophil infiltration in WAT insulin resistance. Disclosure R.F. D’Souza: None. J.S.T. Woodhead: None. S.W. Masson: None. S.L. James: None. C. Hedges: None. T.L. Merry: None. Funding Health Research Council of New Zealand

Genetic inhibition of the p110α isoform of phosphatidylinositol-3-kinase (PI3K) can increase murine lifespan, enhance mitochondrial function and alter tissue-specific oxidative balance. Here, we investigated whether pharmacological inhibition of the p110α isoform of PI3K induces similar enhancement of mitochondrial function in middle-aged mice. Eight-month-old male and female mice were fed a diet containing 0.3 g/kg of the p110α-selective inhibitor BYL-719 (BYL) or a vehicle diet (VEH) for 6 weeks. Mice consuming BYL-719 had higher blood glucose and insulin, and tended towards decreased body weight. After 72 h, gene expression of the mitochondrial biogenesis mediators Pgc1α, Tfam and Nrf1 was greater in liver of BYL-719 males only, but unchanged in skeletal muscle of either sex. Six weeks of BYL-719 treatment did not affect mitochondrial content or function in the liver or skeletal muscle of either sex. In livers of males only, the expression of the antioxidant genes Nfe2l2, Cat, Sod1 and Sod2 increased within 72 h of BYL-719 treatment, and remained higher after 6 weeks. This was associated with an increase in hepatic GSH content and catalase protein expression, and lower H2O2 levels. Our results suggest that pharmacological inhibition of p110α in adult mice does not affect liver or skeletal muscle mitochondrial function, but does show sex- and tissue-specific effects on up-regulation of antioxidant response.

Mitochondria putatively regulate the aging process, in part, through the small regulatory peptide, mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) that is encoded by the mitochondrial genome. Here we investigated the regulation of MOTS-c in the plasma and skeletal muscle of healthy aging men. Circulating MOTS-c reduced with age, but older (70-81 y) and middle-aged (45-55 y) men had ~1.5-fold higher skeletal muscle MOTS-c expression than young (18-30 y). Plasma MOTS-c levels only correlated with plasma in young men, was associated with markers of slow-type muscle, and associated with improved muscle quality in the older group (maximal leg-press load relative to thigh cross-sectional area). Using small mRNA assays we provide evidence that MOTS-c transcription may be regulated independently of the full length 12S rRNA gene in which it is encoded, and expression is not associated with antioxidant response element (ARE)-related genes as previously seen in culture. Our results suggest that plasma and muscle MOTS-c are differentially regulated with aging, and the increase in muscle MOTS-c expression with age is consistent with fast-to-slow type muscle fiber transition. Further research is required to determine the molecular targets of endogenous MOTS-c in human muscle but they may relate to factors that maintain muscle quality.

Small regulatory peptides encoded within the mitochondrial genome (mitochondrial-derived peptides) have been shown to have cellular cyto- and metabolo-protective roles that parallel those of exercise. Here we provide evidence that humanin and SHLP6 are exercise-sensitive mitochondrial derived peptides. Studies to determine whether mitochondrial-derived peptides play a role in regulating exercise-induced adaptations are warranted.

Advances in medicine have contributed to an ageing population, and various age-associated pathologies contribute to loss of independence and quality of life. Calorie restriction and genetic inhibition of insulin signalling pathway intermediates such as phosphatidylinositol-3-kinase (PI3K), have shown promise in improving lifespan but are difficult to implement clinically. Therefore, we sought to test whether pharmacological inhibition of PI3K using an orally ingested p110α-selective inhibitor, BYL-719, promotes changes in glucose homeostasis and physical function in adult (8-month old) male and female mice. Three days of exposure to a standard chow diet containing 0.30 g/kg BYL-719 elevated blood glucose in response to a meal challenge, however, following four weeks of BYL-719 diet consumption blood glucose response to a meal was normalised in females, but not males. Despite this, both male and female mice fed the BYL-719 diet were glucose intolerance and had impaired insulin sensitivity compared to mice fed a control diet. There was no difference between groups in measures of physical performance (treadmill, rotarod or hang time performance) or stress/anxiety behaviour assessed through open field test. However, female mice fed the BYL-719 diet showed greater exploratory behaviour in an object recognition test. Collectively, these data indicate that short-term pharmacological inhibition of PI3K p110α alters glucose homeostasis, but has only mild effect with respect to the physiological stimulus of a meal, and without deterioration of physical or cognitive function in adult mice. This indicates BYL-719 is safely tolerated in adult mice, and longer-term study is warranted to test the efficacy of BYL-719 in prolonging lifelong health in an aged population. Disclosure C. Hedges: None. J. Boix: None. T.L. Merry: None. Funding Health Research Council of New Zealand