A Polynesian-specific SLC22A3 variant associates with low plasma lipoprotein(a) concentrations independent of apo(a) isoform size in men.

Lipoprotein(a) (Lp(a)) is a low-density lipoprotein (LDL)-like particle in which the apolipoprotein B component is covalently linked to apolipoprotein(a). Lp(a) is a well-established independent risk factor for cardiovascular diseases. Plasma lipoprotein(a) concentrations vary enormously between individuals and ethnic groups. Several nucleotide polymorphisms in the SLC22A3 gene associate with Lp(a) concentration in people of different ethnicities. We investigated the association of a Polynesian-specific (Maori and Pacific peoples) SLC22A3 gene coding variant  p.Thr44Met) with the plasma concentration of Lp(a) in a cohort of 302 healthy Polynesian males. An apolipoprotein(a)-size independent assay assessed plasma lipoprotein(a) concentrations, all other lipid and apolipoprotein concentrations were measured using standard laboratory techniques. Quantitative real-time polymerase chain reaction was used to determine apolipoprotein(a) isoforms. The range of metabolic (HbA1c, blood pressure and blood lipids) and blood lipid variables were similar between the non-carriers and carriers in age, ethnicity and BMI adjusted models. However, rs8187715 SLC22A3 variant was significantly associated with lower lipoprotein(a) concentrations. Median lipoprotein(a) concentration was 10.60 nmol/L (IQR 5.40 to 41.00) in non-carrier group, and was 7.60 nmol/L (IQR 5.50 to 12.10) in variant carrier group (p <0.05). Lp(a) concentration  inversely correlated with apolipoprotein(a) isoform size. After correction for apolipoprotein(a) isoform size, metabolic parameters and ethnicity, the association between the SLC22A3 variant and plasma Lp(a) concentration remained. This study is the first to identify the association of this gene variant and low plasma Lp(a) concentrations. This provides evidence for better guidance on ethnic specific cut-offs when defining "elevated" and "normal" plasma Lp(a) concentrations in clinical applications.

Locally applied heat stress during exercise training may promote adaptations to mitochondrial enzyme activities in skeletal muscle.

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.

A high quality Aotearoa New Zealand dietary pattern adapting a Mediterranean diet for metabolic health: a feasibility study.

AIM: To assess the feasibility of a family-based dietary intervention study using a meal kit home delivery service, in people at risk of cardio-metabolic disease. METHODS: A 12-week dietary intervention feasibility study of adults (termed the index participants) at increased risk of metabolic and cardiovascular disease, enriched for Maori who are indigenous New Zealanders. The study sample also included the household/whanau members living with the index participant. All participants received a 12 week intervention using weekly home delivery of meal kits and groceries consistent with a Mediterranean dietary pattern. Outcomes were the metabolic syndrome severity score (MetSSS); feasibility and acceptability of the intervention; dietary intake; and other clinical and anthropometric measures. RESULTS: There were 29 index participants recruited and in addition, 50 household/whanau members took part in the feasibility study. The mean (SD) household/whanau size was 3.45 (1.4) people, and the mean (SD) number of people in each household/whanau who participated in the study was 2.84 (1.2). The feasibility of intervention to households/whanau was proven in this context. The mean (SD) change in MetSSS was 0.03 (0.33), N = 27, P = 0.69 and there was a statistically significant decrease in body weight of 1.37 kg (95% CI 0.11 to 2.62), p = 0.034. The food deliveries were well received, the dinner kits more so than the grocery items. CONCLUSION: It is feasible to recruit individuals and households/whanau to a family-based dietary intervention. Use of a meal kit home delivery service to provide food which is consistent with the intervention dietary pattern was well received. This feasibility study identified improvements to be made such as nutrition behaviour change support, more variety in food provided, more recipes, and better matching of food quantity to family size. TRIAL REGISTRATION: ANZCTR-ACTRN12621000856819p registered 2.JUN.2021 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=382021&isReview=true.

He Rourou Whai Painga, an Aotearoa New Zealand dietary pattern for metabolic health and whanau wellbeing: protocol for a randomized controlled trial.

Background: Cardiometabolic diseases are highly prevalent in Aotearoa New Zealand. Dietary intake is a modifiable risk factor for such diseases and certain dietary patterns, specifically the Mediterranean diet (MedDiet), are associated with improved metabolic health. This study aims to test whether an intervention including a Mediterranean dietary pattern incorporating high quality New Zealand foods (NZMedDiet pattern) and behavior change science can improve the metabolic health of participants and their household/whanau. Methods and analysis: This is a multi-center, three-stage trial with two parallel group superiority randomized controlled trials (RCTs), and a longitudinal cohort study embedded within the trial design. The first RCT (RCT 1) is a comparison of the NZMedDiet pattern compared to usual diet for 12 weeks. The Behavior Change Wheel was used to select and implement strategies to support participant adherence to the NZMedDiet, such as web-based nutrition education on healthy shopping and cooking. The second (RCT 2) compares online social support to no online social support for 12 weeks, administered to participants immediately following RCT 1. The third stage is a longitudinal cohort study where all participants are followed from the beginning of their start of the active intervention for 12 months in total. The primary outcome measure for each stage is the metabolic syndrome severity score (MetSSS). The duration of enrolment is 12-15 months. The total recruitment target is 200 index participants and their household/whanau members who participate with them, and the primary analyses will be intention to treat on index participants. Discussion: The trial will test whether the NZMedDiet pattern and behavior change support improves the cardiometabolic health of people in Aotearoa New Zealand. Clinical trial registration: https://www.anzctr.org.au/Default.aspx, identifier ACTRN12622000906752 and https://www.isrctn.com/, identifier ISRCTN89011056 (Spirit 2).

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Habitual Dietary Patterns, Nutrient Intakes, and Adherence to the Mediterranean Diet among New Zealand Adults: The NZ MED Cross-Sectional Study.

There is increasing evidence that adherence to a Mediterranean dietary pattern reduces the incidence of diet-related diseases. To date, the habitual dietary intake of New Zealand (NZ) adults has not been examined in relation to its alignment with a Mediterranean-style dietary pattern. This study aimed to define the habitual dietary patterns, nutrient intakes, and adherence to the Mediterranean Diet in a sample of 1012 NZ adults (86% female, mean age 48 ± 16 years) who had their diabetes risk defined by the Australian Type 2 Diabetes Risk Assessment Tool (AUSDRISK). Dietary intakes were collected using a validated semi-quantitative NZ food frequency questionnaire, and dietary patterns were identified using principal component analysis. Reported intakes from the FFQ were used in conjunction with the Mediterranean-Style Dietary Pattern Score (MSDPS) to determine adherence to a Mediterranean dietary pattern. Mixed linear models were used to analyze the association between dietary patterns and MSDPS with demographics, health factors, and nutrient intakes. Two distinct dietary patterns were identified: Discretionary (positive loadings on processed meat, meat/poultry, fast food, sweet drinks, and sugar, sweets, and baked good) and Guideline (positive loadings on vegetables, eggs/beans, and fruits). Adherence to dietary patterns and diet quality was associated with age and ethnicity. Dietary patterns were also associated with sex. Adherence to a Mediterranean dietary pattern defined by the MSDPS was low, indicating that a significant shift in food choices will be required if the Mediterranean Diet is to be adopted in the NZ population.

A role for ß-catenin in diet-induced skeletal muscle insulin resistance.

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. ß-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 ß-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated ß-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific ß-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower ß-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated ß-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, ß-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total ß-catenin expression was unchanged. These findings suggest that ß-catenin dysfunction is associated with the development of insulin resistance.

The effects of sugar in drinking water on Streptococcus pyogenes colonisation in a murine nasopharyngeal infection model.

The number of sugar-sweetened beverages consumed per day has been associated with an increased risk of acute rheumatic fever, an autoimmune disease triggered by superficial Streptococcus pyogenes infection. To explore if there could be a biological basis for this association, we used a mouse model of S. pyogenes nasopharyngeal colonisation combined with a dietary intervention. We observed an increased bacterial load in the nasopharynx of mice receiving sucrose drinking water post-infection, suggesting that high sucrose intake promotes S. pyogenes growth and/or survival. This provides new insight into the potential biological basis behind the association seen in humans.

Metabolic syndrome severity score (MetSSS) associates with metabolic health status in multi-ethnic Aotearoa New Zealand cohorts.

AIM: To investigate the relationship of metabolic syndrome severity score (MetSSS) with glucose regulatory and cardiovascular disease (CVD) status in Aotearoa New Zealand. METHODS: MetSSS and MetSSS component coefficients were calculated for participants from the cross-sectional Workforce Diabetes Study (WDS) (n = 5,806) and Diabetes, Heart and Health Survey (DHAH) (n = 4,010) and compared by ethnicity (European, Maori, Pacific and Asian), glucose regulatory status [impaired fasting glucose, impaired glucose tolerance and type 2 diabetes) and history of cardiovascular disease. RESULTS: MetSSS positively associated with impaired glucose regulatory status and history of cardiovascular disease for all ethnic groups. Ethnicity significantly affected different coefficients of the MetSSS components, however all ethnicities had an approximately normal MetSSS distribution, with Maori and Pacific curves being right-shifted compared to European. While the MetSSS thresholds that capture 80% of participant with type 2 diabetes (T2D) were higher for Maori and Pacific, the difference in MetSSS between those participants with and without type 2 diabetes within an ethnicity group was similar across ethnicities. CONCLUSION: MetSSS may have utility as a tool to quantify an individual's cardiometabolic disease risk within the multi-ethnic population of Aotearoa New Zealand, however ethnic-specific categories for disease risk are likely to be required.

The minor allele of the CREBRF rs373863828 p.R457Q coding variant is associated with reduced levels of myostatin in males: Implications for body composition.

OBJECTIVE: The minor allele (A) of the rs373863828 variant (p.Arg457Gln) in CREBRF is restricted to indigenous peoples of the Pacific islands (including New Zealand Maori and peoples of Polynesia), with a frequency of up to 25% in these populations. This allele associates with a large increase in body mass index (BMI) but with significantly lower risk of type-2 diabetes (T2D). It remains unclear whether the increased BMI is driven by increased adiposity or by increased lean mass. METHODS: We undertook body composition analysis using DXA in 189 young men of Maori and Pacific descent living in Aotearoa New Zealand. Further investigation was carried out in two orthologous Arg458Gln knockin mouse models on FVB/NJ and C57BL/6j backgrounds. RESULTS: The rs373863828 A allele was associated with lower fat mass when adjusted for BMI (p < 0.05) and was associated with significantly lower circulating levels of the muscle inhibitory hormone myostatin (p < 0.05). Supporting the human data, significant reductions in adipose tissue mass were observed in the knockin mice. This was more significant in older mice in both backgrounds and appeared to be the result of reduced age-associated increases in fat mass. The older male knockin mice on C57BL/6j background also had increased grip strength (p < 0.01) and lower levels of myostatin (p < 0.05). CONCLUSION: Overall, these results prove that the rs373863828 A-allele is associated with a reduction of myostatin levels which likely contribute to an age-dependent lowering of fat mass, at least in males.

The Impact of Exogenous Insulin Input on Calculating Hepatic Clearance Parameters.

OBJECTIVE: Model-based metabolic tests require accurate identification of subject-specific parameters from measured assays. Insulin assays are used to identify insulin kinetics parameters, such as general and first-pass hepatic clearances. This study assesses the impact of intravenous insulin boluses on parameter identification precision. METHOD: Insulin and C-peptide data from two intravenous glucose tolerance test (IVGTT) trials of healthy adults (N = 10 × 2; denoted A and B), with (A) and without (B) insulin modification, were used to identify insulin kinetics parameters using a grid search. Monte Carlo analysis (N = 1000) quantifies variation in simulation error for insulin assay errors of 5%. A region of parameter values around the optimum was identified whose errors are within variation due to assay error. A smaller optimal region indicates more precise practical identifiability. Trial results were compared to assess identifiability and precision. RESULTS: Trial B, without insulin modification, has optimal parameter regions 4.7 times larger on average than Trial A, with 1-U insulin bolus modification. Ranges of optimal parameter values between trials A and B increase from 0.04 to 0.12 min-1 for hepatic clearance and from 0.07 to 0.14 for first-pass clearance on average. Trial B's optimal values frequently lie outside physiological ranges, further indicating lack of distinct identifiability. CONCLUSIONS: A small 1-U insulin bolus improves identification of hepatic clearance parameters by providing a smaller region of optimal parameter values. Adding an insulin bolus in metabolic tests can significantly improve identifiability and outcome test precision. Assay errors necessitate insulin modification in clinical tests to ensure identifiability and precision.

Skeletal muscle NOX4 is required for adaptive responses that prevent insulin resistance.

Reactive oxygen species (ROS) generated during exercise are considered integral for the health-promoting effects of exercise. However, the precise mechanisms by which exercise and ROS promote metabolic health remain unclear. Here, we demonstrate that skeletal muscle NADPH oxidase 4 (NOX4), which is induced after exercise, facilitates ROS-mediated adaptive responses that promote muscle function, maintain redox balance, and prevent the development of insulin resistance. Conversely, reductions in skeletal muscle NOX4 in aging and obesity contribute to the development of insulin resistance. NOX4 deletion in skeletal muscle compromised exercise capacity and antioxidant defense and promoted oxidative stress and insulin resistance in aging and obesity. The abrogated adaptive mechanisms, oxidative stress, and insulin resistance could be corrected by deleting the H2O2-detoxifying enzyme GPX-1 or by treating mice with an agonist of NFE2L2, the master regulator of antioxidant defense. These findings causally link NOX4-derived ROS in skeletal muscle with adaptive responses that promote muscle function and insulin sensitivity.

Effect of immune modulation on the skeletal muscle mitochondrial exercise response: An exploratory study in mice with cancer.

Cancer causes mitochondrial alterations in skeletal muscle, which may progress to muscle wasting and, ultimately, to cancer cachexia. Understanding how exercise adaptations are altered by cancer and cancer treatment is important for the effective design of exercise interventions aimed at improving cancer outcomes. We conducted an exploratory study to investigate how tumor burden and cancer immunotherapy treatment (anti-PD-1) modify the skeletal muscle mitochondrial response to exercise training in mice with transplantable tumors (B16-F10 melanoma and EO771 breast cancer). Mice remained sedentary or were provided with running wheels for ~19 days immediately following tumor implant while receiving no treatment (Untreated), isotype control antibody (IgG2a) or anti-PD-1. Exercise and anti-PD-1 did not alter the growth rate of either tumor type, either alone or in combination therapy. Untreated mice with B16-F10 tumors showed increases in most measured markers of skeletal muscle mitochondrial content following exercise training, as did anti-PD-1-treated mice, suggesting increased mitochondrial content following exercise training in these groups. However, mice with B16-F10 tumors receiving the isotype control antibody did not exhibit increased skeletal muscle mitochondrial content following exercise. In untreated mice with EO771 tumors, only citrate synthase activity and complex IV activity were increased following exercise. In contrast, IgG2a and anti-PD-1-treated groups both showed robust increases in most measured markers following exercise. These results indicate that in mice with B16-F10 tumors, IgG2a administration prevents exercise adaptation of skeletal muscle mitochondria, but adaptation remains intact in mice receiving anti-PD-1. In mice with EO771 tumors, both IgG2a and anti-PD-1-treated mice show robust skeletal muscle mitochondrial exercise responses, while untreated mice do not. Taken together, we postulate that immune modulation may enhance skeletal muscle mitochondrial response to exercise in tumor-bearing mice, and suggest this as an exciting new avenue for future research in exercise oncology.

α1-Antitrypsin A treatment attenuates neutrophil elastase accumulation and enhances insulin sensitivity in adipose tissue of mice fed a high-fat diet.

Neutrophils accumulate in insulin-sensitive tissues during obesity and may play a role in impairing insulin sensitivity. The major serine protease expressed by neutrophils is neutrophil elastase (NE), which is inhibited endogenously by α1-antitrypsin A (A1AT). We investigated the effect of exogenous (A1AT) treatment on diet-induced metabolic dysfunction. Male C57Bl/6j mice fed a chow or a high-fat diet (HFD) were randomized to receive intraperitoneal injections three times weekly of either Prolastin (human A1AT; 2 mg) or vehicle (PBS) for 10 wk. Prolastin treatment did not affect plasma NE concentration, body weight, glucose tolerance, or insulin sensitivity in chow-fed mice. In contrast, Prolastin treatment attenuated HFD-induced increases in plasma and white adipose tissue (WAT) NE without affecting circulatory neutrophil levels or increases in body weight. Prolastin-treated mice fed a HFD had improved insulin sensitivity, as assessed by insulin tolerance test, and this was associated with higher insulin-dependent IRS-1 (insulin receptor substrate) and AktSer473 phosphorylation, and reduced inflammation markers in WAT but not liver or muscle. In 3T3-L1 adipocytes, Prolastin reversed recombinant NE-induced impairment of insulin-stimulated glucose uptake and IRS-1 phosphorylation. Furthermore, PDGF mediated p-AktSer473 activation and glucose uptake (which is independent of IRS-1) was not affected by recombinant NE treatment. Collectively, our findings suggest that NE infiltration of WAT during metabolic overload contributes to insulin resistance by impairing insulin-induced IRS-1 signaling.NEW & NOTEWORTHY 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.

Mitochondrial-derived peptides and exercise.

Acute exercise, and in particular aerobic exercise, increases skeletal muscle energy demand causing mitochondrial stress, and mitochondrial-related adaptations which are a hallmark of exercise training. Given that mitochondria are central players in the exercise response, it is imperative that they have networks that can communicate their status both intra- and inter-cellularly. Peptides encoded by short open-reading frames within mitochondrial DNA, mitochondrial-derived peptides (MDPs), have been suggested to form a newly recognised branch of this retrograde signalling cascade that contribute to coordinating the adaptive response to regular exercise. Here we summarise the recent evidence that acute high intensity exercise in humans can increase concentrations of the MDPs humanin and MOTS-c in skeletal muscle and plasma, and speculate on the mechanisms controlling MDP responses to exercise stress. Evidence that exercise training results in chronic changes in MDP expression within tissues and the circulation is conflicting and may depend on the mode, duration, intensity of training plan and participant characteristics. Further research is required to define the effect of these variables on MDPs and to determine whether MDPs other than MOTS-c have exercise mimetic properties. MOTS-c treatment of young and aged mice improves exercise capacity/performance and leads to adaptions that are similar to that of being physically active (weight loss, increased antioxidant capacity and improved insulin sensitivity), however, studies utilising a MOTS-c inactivating genetic variant or combination of exercise + MOTS-c treatment in mice suggest that there are distinct and overlapping pathways through which exercise and MOTS-c evoke metabolic benefits. Overall, MOTS-c, and potentially other MDPs, may be exercise-sensitive myokines and further work is required to define inter- and intra-tissue targets in an exercise context.

Plasma mitochondrial derived peptides MOTS-c and SHLP2 positively associate with android and liver fat in people without diabetes.

Mitochondrial-derived peptides (MDPs) are encoded by the mitochondrial genome and hypothesised to form part of a retrograde signalling network that modulates adaptive responses to metabolic stress. To understand how metabolic stress regulates MDPs in humans we assessed the association between circulating MOTS-c and SHLP2 and components of metabolic syndrome (MS), as well as depot-specific fat mass in participants without overt type 2 diabetes or cardiovascular disease. One-hundred and twenty-five Chinese participants (91 male, 34 female) had anthropometry, whole body dual-energy X-ray absorptiometry scans and fasted blood samples analysed. Chinese female participants and an additional 34 European Caucasian female participants also underwent magnetic resonance imaging and spectroscopy (MRI/S) for visceral, pancreatic and liver fat quantification. In Chinese participants (age = 41 ± 1 years, BMI = 27.8 ± 3.9 kg/m2), plasma MOTS-c (315 ± 27 pg/ml) and SHLP2 (1393 ± 82 pg/ml) were elevated in those with MS (n = 26). While multiple components of the MS sequelae positively associated with both MOTS-c and SHLP2, including blood pressure, fasting plasma glucose and triglycerides, the most significant of these was waist circumference (p < 0.0001). Android fat had a greater effect on increasing plasma MOTS-c (p < 0.004) and SHLP2 (p < 0.009) relative to whole body fat. Associations with MRI/S parameters corrected for total body fat mass revealed that liver fat positively associated with plasma MOTS-c and SHLP2 and visceral fat with SHLP2. Consistent with hepatic stress being a driver of circulating MDP concentrations, plasma MOTS-c and SHLP2 were higher in participants with elevated liver damage markers and in male C57Bl/6j mice fed a diet that induces hepatic lipid accumulation and damage. Our findings provide evidence that in the absence of overt type 2 diabetes, components of the MS positively associated with levels of MOTS-c and SHLP2 and that android fat, in particular liver fat, is a primary driver of these associations. MOTS-c and SHLP2 have previously been shown to have cyto- and metabolo-protective properties, therefore we suggest that liver stress may be a mitochondrial peptide signal, and that mitochondrial peptides are part of a hepatic centric-hormetic response intended to restore metabolic balance.

The CREBRF diabetes-protective rs373863828-A allele is associated with enhanced early insulin release in men of Maori and Pacific ancestry.

AIMS/HYPOTHESIS: The minor A allele of rs373863828 (CREBRF p.Arg457Gln) is associated with increased BMI, but reduced risk of type 2 and gestational diabetes in Polynesian (Pacific peoples and Aotearoa New Zealand Maori) populations. This study investigates the effect of the A allele on insulin release and sensitivity in overweight/obese men without diabetes. METHODS: A mixed meal tolerance test was completed by 172 men (56 with the A allele) of Maori or Pacific ancestry, and 44 (24 with the A allele) had a frequently sampled IVGTT and hyperinsulinaemic-euglycaemic clamp. Mixed linear models with covariates age, ancestry and BMI were used to analyse the association between the A allele of rs373863828 and markers of insulin release and blood glucose regulation. RESULTS: The A allele of rs373863828 is associated with a greater increase in plasma insulin 30 min following a meal challenge without affecting the elevation in plasma glucose or incretins glucagon-like polypeptide-1 or gastric inhibitory polypeptide. Consistent with this point, following an i.v. infusion of a glucose bolus, participants with an A allele had higher early (p < 0.05 at 2 and 4 min) plasma insulin and C-peptide concentrations for a similar elevation in blood glucose as those homozygous for the major (G) allele. Despite increased plasma insulin, rs373863828 genotype was not associated with a significant difference (p > 0.05) in insulin sensitivity index or glucose disposal during hyperinsulinaemic-euglycaemic clamp. CONCLUSIONS/INTERPRETATION: rs373863828-A allele associates with increased glucose-stimulated insulin release without affecting insulin sensitivity, suggesting that CREBRF p.Arg457Gln may increase insulin release to reduce the risk of type 2 diabetes.

ß-Catenin is required for optimal exercise- and contraction-stimulated skeletal muscle glucose uptake.

KEY POINTS: Loss of ß-catenin impairs in vivo and isolated muscle exercise/contraction-stimulated glucose uptake. ß-Catenin is required for exercise-induced skeletal muscle actin cytoskeleton remodelling. ß-Catenin675 phosphorylation during exercise may be intensity dependent. ABSTRACT: The conserved structural protein ß-catenin is an emerging regulator of vesicle trafficking in multiple tissues and supports insulin-stimulated glucose transporter 4 (GLUT4) translocation in skeletal muscle by facilitating cortical actin remodelling. Actin remodelling may be a convergence point between insulin and exercise/contraction-stimulated glucose uptake. Here we investigated whether ß-catenin is involved in regulating exercise/contraction-stimulated glucose uptake. We report that the muscle-specific deletion of ß-catenin induced in adult mice (BCAT-mKO) impairs both exercise- and contraction (isolated muscle)-induced glucose uptake without affecting running performance or canonical exercise signalling pathways. Furthermore, high intensity exercise in mice and contraction of myotubes and isolated muscles led to the phosphorylation of ß-cateninS675 , and this was impaired by Rac1 inhibition. Moderate intensity exercise in control and Rac1 muscle-specific knockout mice did not induce muscle ß-cateninS675 phosphorylation, suggesting exercise intensity-dependent regulation of ß-cateninS675 . Introduction of a non-phosphorylatable S675A mutant of ß-catenin into myoblasts impaired GLUT4 translocation and actin remodelling stimulated by carbachol, a Rac1 and RhoA activator. Exercise-induced increases in cross-sectional phalloidin staining (F-actin marker) of gastrocnemius muscle was impaired in muscle from BCAT-mKO mice. Collectively our findings suggest that ß-catenin is required for optimal glucose transport in muscle during exercise/contraction, potentially via facilitating actin cytoskeleton remodelling.

Sirtuin 1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle.

While it has long been known that contraction robustly stimulates skeletal muscle glucose uptake, the molecular steps regulating this increase remain incompletely defined. The mammalian ortholog of Sir2, sirtuin 1 (SIRT1), is an NAD+-dependent protein deacetylase that is thought to link perturbations in energy flux associated with exercise to subsequent cellular adaptations. Nevertheless, its role in contraction-stimulated glucose uptake has not been described. The objective of this study was to determine the importance of SIRT1 to contraction-stimulated glucose uptake in mouse skeletal muscle. Using a radioactive 2-deoxyglucose uptake (2DOGU) approach, we measured ex vivo glucose uptake in unstimulated (rested) and electrically stimulated (100 Hz contraction every 15 s for 10 min; contracted) extensor digitorum longus (EDL) and soleus from ∼15-wk-old male and female mice with muscle-specific knockout of SIRT1 deacetylase activity and their wild-type littermates. Skeletal muscle force decreased over the contraction protocol, although there were no differences in the rate of fatigue between genotypes. In EDL and soleus, loss of SIRT1 deacetylase activity did not affect contraction-induced increase in glucose uptake in either sex. Interestingly, the absolute rate of contraction-stimulated 2DOGU was ∼1.4-fold higher in female compared with male mice, regardless of muscle type. Taken together, our findings demonstrate that SIRT1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle. Moreover, to our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in mouse skeletal muscle.NEW & NOTEWORTHY Here, we demonstrate that glucose uptake in response to ex vivo contractions is not affected by the loss of sirtuin 1 (SIRT1) deacetylase function in muscle, regardless of sex or muscle type. Interestingly, however, similar to studies on insulin-stimulated glucose uptake, we demonstrate that contraction-stimulated glucose uptake is robustly higher in female compared with the male skeletal muscle. To our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in skeletal muscle.

Efficacy of Providing the PI3K p110α Inhibitor BYL719 (Alpelisib) to Middle-Aged Mice in Their Diet.

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.