Metabolic plasticity and obesity-associated changes in diurnal postexercise metabolism in mice.

BACKGROUND: Circadian disruption is widespread and increases the risk of obesity. Timing of therapeutic interventions may promote coherent and efficient gating of metabolic processes and restore energy homeostasis. AIM: To characterize the diurnal postexercise metabolic state in mice and to identify the influence of diet-induced obesity on identified outcomes. METHODS: C57BL6/NTac male mice (6 wks of age) were fed a standard chow or high-fat diet for 5 weeks. At week 5, mice were subjected to a 60-min (16 m/min, 5 % incline) running bout (or sham) during the early rest (day) or early active (night) phase. Tissue and serum samples were collected immediately post-exercise (n = 6/group). In vivo glucose oxidation was measured after oral administration of 13C-glucose via 13CO2 exhalation analysis in metabolic cages. Basal and isoproterenol-stimulated adipose tissue lipolysis was assessed ex vivo for 1 h following exercise. RESULTS: Lean mice displayed exercise-timing-specific plasticity in metabolic outcomes, including phase-specificity in systemic glucose metabolism and adipose-tissue-autonomous lipolytic activity depending on time of day. Conversely, obesity impaired temporal postexercise differences in whole-body glucose oxidation, as well as the phase- and exercise-mediated induction of lipolysis in isolated adipose tissue. This obesity-induced alteration in diurnal metabolism, as well as the indistinct response to exercise, was observed concomitant with disruption of core clock gene expression in peripheral tissues. CONCLUSIONS: Overall, high-fat fed obese mice exhibit metabolic inflexibility, which is also evident in the diurnal exercise response. Our study provides physiological insight into exercise timing-dependent aspects in the dynamic regulation of metabolism and the influence of obesity on this biology.

Exercise induces tissue-specific adaptations to enhance cardiometabolic health.

The risk associated with multiple cancers, cardiovascular disease, diabetes, and all-cause mortality is decreased in individuals who meet the current recommendations for physical activity. Therefore, regular exercise remains a cornerstone in the prevention and treatment of non-communicable diseases. An acute bout of exercise results in the coordinated interaction between multiple tissues to meet the increased energy demand of exercise. Over time, the associated metabolic stress of each individual exercise bout provides the basis for long-term adaptations across tissues, including the cardiovascular system, skeletal muscle, adipose tissue, liver, pancreas, gut, and brain. Therefore, regular exercise is associated with a plethora of benefits throughout the whole body, including improved cardiorespiratory fitness, physical function, and glycemic control. Overall, we summarize the exercise-induced adaptations that occur within multiple tissues and how they converge to ultimately improve cardiometabolic health.

Time of day determines postexercise metabolism in mouse adipose tissue.

The circadian clock is a cell-autonomous transcription-translation feedback mechanism that anticipates and adapts physiology and behavior to different phases of the day. A variety of factors including hormones, temperature, food-intake, and exercise can act on tissue-specific peripheral clocks to alter the expression of genes that influence metabolism, all in a time-of-day dependent manner. The aim of this study was to elucidate the effects of exercise timing on adipose tissue metabolism. We performed RNA sequencing on inguinal adipose tissue of mice immediately following maximal exercise or sham treatment at the early rest or early active phase. Only during the early active phase did exercise elicit an immediate increase in serum nonesterified fatty acids. Furthermore, early active phase exercise increased expression of markers of thermogenesis and mitochondrial proliferation in inguinal adipose tissue. In vitro, synchronized 3T3-L1 adipocytes showed a timing-dependent difference in Adrb2 expression, as well as a greater lipolytic activity. Thus, the response of adipose tissue to exercise is time-of-day sensitive and may be partly driven by the circadian clock. To determine the influence of feeding state on the time-of-day response to exercise, we replicated the experiment in 10-h-fasted early rest phase mice to mimic the early active phase metabolic status. A 10-h fast led to a similar lipolytic response as observed after active phase exercise but did not replicate the transcriptomic response, suggesting that the observed changes in gene expression are not driven by feeding status. In conclusion, acute exercise elicits timing-specific effects on adipose tissue to maintain metabolic homeostasis.

Protocol to assess arteriovenous differences across the liver and hindlimb muscles in mice following treadmill exercise.

The tissue-specific release and uptake of metabolites in response to exercise is incompletely understood. Here, we detail a protocol to assess arteriovenous differences across the liver and hindlimb muscles in response to treadmill exercise in mice. We describe steps for the treadmill running of mice and the region-specific sampling of blood from the liver and hindlimb. This procedure is particularly relevant for the study of tissue-specific metabolism in response to exercise. For complete details on the use and execution of this protocol, please refer to Sato et al. (2022).1.

Pterostilbene Fails to Rescue Insulin Secretion and Sensitivity in Multiple Murine Models of Diabetes.

Diabetes incidence is rising globally at an accelerating rate causing issues at both the individual and societal levels. However, partly inspired by Ayurvedic medicine, a naturally occurring compound called pterostilbene has been demonstrated to protect against diabetes symptoms, though mainly in rats. The purpose of this study was to investigate the putative protective effect of pterostilbene on the two main aspects of diabetes, namely insulin resistance and decreased insulin secretion, in mice. To accomplish this, we employed diet-induced obese as well as streptozotocin-induced diabetic C57BL/6NTac mice for fasting glucose homeostasis assessment, tolerance tests and pancreas perfusions. In addition, we used the polygenic model of diabetes TALLYHO/JngJ to assess for prevention of ß-cell burnout. We found that the diet-induced obese C57BL/6NTac mice were insulin resistant, but that pterostilbene had no impact on this or on overall glucose regulation. We further found that the reported protective effect of pterostilbene against streptozotocin-induced diabetes was absent in C57BL/6NTac mice, despite a promising pilot experiment. Lastly, we observed that pterostilbene does not prevent or delay onset of ß-cell burnout in TALLYHO/JngJ mice. In conjunction with the literature, our findings suggest variations in the response to pterostilbene between species or between strains of species.

Comparative analysis of oral and intraperitoneal glucose tolerance tests in mice.

OBJECTIVE: The glucose tolerance test (GTT) is widely used in preclinical research to investigate glucose metabolism, but there is no standardised way to administer glucose. The aim of this study was to directly compare the effect of the route of glucose administration on glucose and insulin kinetics during a GTT in mice. METHODS: A GTT was performed in lean male and female mice and obese male mice and glucose was administered via the oral or intraperitoneal (I.P.) route. Samples were collected frequently during the GTT to provide a full time-course of the insulin and glucose excursions. In another cohort of lean male mice, plasma concentrations of insulin, c-peptide, and incretin hormones were measured at early time points after glucose administration. A stable-isotope labelled GTT (SiGTT) was then performed to delineate the contribution of exogenous and endogenous glucose to glycemia during the GTT, comparing both methods of glucose administration. Finally, we present a method to easily measure insulin from small volumes of blood during a GTT by directly assaying whole-blood insulin using ELISA and show a good concordance between whole-blood and plasma insulin measurements. RESULTS: We report that I.P. glucose administration results in an elevated blood glucose excursion and a largely absent elevation in blood insulin and plasma incretin hormones when compared to oral administration. Utilising stable-isotope labelled glucose, we demonstrate that the difference in glucose excursion between the two routes of administration is mainly due to the lack of suppression of glucose production in I.P. injected mice. Additionally, rates of exogenous glucose appearance into circulation were different between lean and obese mice after I.P., but not after oral glucose administration. CONCLUSION: Reflecting on these data, we suggest that careful consideration be given to the route of glucose administration when planning a GTT procedure in mice and that in most circumstances the oral route of glucose administration should be preferred over the I.P. route to avoid possible artifacts originating from a non-physiological route.

Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis.

Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.

Diet-induced vitamin D deficiency reduces skeletal muscle mitochondrial respiration.

Vitamin D deficiency is associated with symptoms of skeletal muscle myopathy including muscle weakness and fatigue. Recently, vitamin D-related metabolites have been linked to the maintenance of mitochondrial function within skeletal muscle. However, current evidence is limited to in vitro models and the effects of diet-induced vitamin D deficiency upon skeletal muscle mitochondrial function in vivo have received little attention. In order to examine the role of vitamin D in the maintenance of mitochondrial function in vivo, we utilised an established model of diet-induced vitamin D deficiency in C57BL/6J mice. Mice were either fed a control diet (2200 IU/kg i.e. vitamin D replete) or a vitamin D-deplete (0 IU/kg) diet for periods of 1, 2 and 3 months. Gastrocnemius muscle mitochondrial function and ADP sensitivity were assessed via high-resolution respirometry and mitochondrial protein content via immunoblotting. As a result of 3 months of diet-induced vitamin D deficiency, respiration supported via complex I + II (CI + IIP) and the electron transport chain (ETC) were 35 and 37% lower when compared to vitamin D-replete mice (P < 0.05). Despite functional alterations, citrate synthase activity, AMPK phosphorylation, mitofilin, OPA1 and ETC subunit protein content remained unchanged in response to dietary intervention (P > 0.05). In conclusion, we report that 3 months of diet-induced vitamin D deficiency reduced skeletal muscle mitochondrial respiration in C57BL/6J mice. Our data, when combined with previous in vitro observations, suggest that vitamin D-mediated regulation of mitochondrial function may underlie the exacerbated muscle fatigue and performance deficits observed during vitamin D deficiency.

Nicotinamide riboside supplementation does not alter whole-body or skeletal muscle metabolic responses to a single bout of endurance exercise.

KEY POINTS: Acute nicotinamide riboside (NR) supplementation does not alter substrate metabolism at rest, during or in recovery from endurance exercise. NR does not alter NAD+ -sensitive signalling pathways in human skeletal muscle. NR supplementation and acute exercise influence the NAD+ metabolome. ABSTRACT: Oral supplementation of the NAD+ precursor nicotinamide riboside (NR) has been reported to alter metabolism alongside increasing sirtuin (SIRT) signalling and mitochondrial biogenesis in rodent skeletal muscle. However, whether NR supplementation can elicit a similar response in human skeletal muscle is unclear. This study assessed the effect of 7-day NR supplementation on whole-body metabolism and exercise-induced mitochondrial biogenic signalling in skeletal muscle. Eight male participants (age: 23 ± 4 years, V̇O2peak 46.5 ± 4.4 ml kg-1  min-1 ) received 1 week of NR or cellulose placebo (PLA) supplementation (1000 mg day-1 ). Muscle biopsies were collected from the medial vastus lateralis prior to supplementation and pre-, immediately post- and 3 h post-exercise (1 h of 60% Wmax cycling) performed following the supplementation period. There was no effect of NR supplementation on substrate utilisation at rest or during exercise or on skeletal muscle mitochondrial respiration. Global acetylation, auto-PARylation of poly ADP-ribose polymerase 1 (PARP1), acetylation of Tumour protein 53 (p53)Lys382 and Manganese superoxide dismutase (MnSOD)Lys122 were also unaffected by NR supplementation or exercise. NR supplementation did not increase skeletal muscle NAD+ concentration, but it did increase the concentration of deaminated NAD+ precursors nicotinic acid riboside (NAR) and nicotinic acid mononucleotide (NAM) and methylated nicotinamide breakdown products (Me2PY and Me4PY), demonstrating the skeletal muscle bioavailability of NR supplementation. In summary, 1 week of NR supplementation does not alter whole-body metabolism or skeletal muscle signal transduction pathways implicated in the mitochondrial adaptation to endurance exercise.

The mechanisms of skeletal muscle atrophy in response to transient knockdown of the vitamin D receptor in vivo.

KEY POINTS: Reduced vitamin D receptor (VDR) expression prompts skeletal muscle atrophy. Atrophy occurs through catabolic processes, namely the induction of autophagy, while anabolism remains unchanged. In response to VDR-knockdown mitochondrial function and related gene-set expression is impaired. In vitro VDR knockdown induces myogenic dysregulation occurring through impaired differentiation. These results highlight the autonomous role the VDR has within skeletal muscle mass regulation. ABSTRACT: Vitamin D deficiency is estimated to affect ∼40% of the world's population and has been associated with impaired muscle maintenance. Vitamin D exerts its actions through the vitamin D receptor (VDR), the expression of which was recently confirmed in skeletal muscle, and its down-regulation is linked to reduced muscle mass and functional decline. To identify potential mechanisms underlying muscle atrophy, we studied the impact of VDR knockdown (KD) on mature skeletal muscle in vivo, and myogenic regulation in vitro in C2C12 cells. Male Wistar rats underwent in vivo electrotransfer (IVE) to knock down the VDR in hind-limb tibialis anterior (TA) muscle for 10 days. Comprehensive metabolic and physiological analysis was undertaken to define the influence loss of the VDR on muscle fibre composition, protein synthesis, anabolic and catabolic signalling, mitochondrial phenotype and gene expression. Finally, in vitro lentiviral transfection was used to induce sustained VDR-KD in C2C12 cells to analyse myogenic regulation. Muscle VDR-KD elicited atrophy through a reduction in total protein content, resulting in lower myofibre area. Activation of autophagic processes was observed, with no effect upon muscle protein synthesis or anabolic signalling. Furthermore, RNA-sequencing analysis identified systematic down-regulation of multiple mitochondrial respiration-related protein and genesets. Finally, in vitro VDR-knockdown impaired myogenesis (cell cycling, differentiation and myotube formation). Together, these data indicate a fundamental regulatory role of the VDR in the regulation of myogenesis and muscle mass, whereby it acts to maintain muscle mitochondrial function and limit autophagy.

High Levels of Physical Activity in Later Life Are Associated With Enhanced Markers of Mitochondrial Metabolism.

The age-associated reduction in muscle mass is well characterized; however, less is known regarding the mechanisms responsible for the decline in oxidative capacity also observed with advancing age. The purpose of the current study was therefore to compare mitochondrial gene expression and protein content between young and old recreationally active, and older highly active individuals. Muscle biopsies were obtained from the vastus lateralis of young males (YG: 22 ± 3 years) and older (OG: 67 ± 2 years) males not previously engaged in formal exercise and older male master cyclists (OT: 65 ± 5 years) who had undertaken cycling exercise for 32 ± 17 years. Comparison of gene expression between YG, OG, and OT groups revealed greater expression of mitochondrial-related genes, namely, electron transport chain (ETC) complexes II, III, and IV (p < .05) in OT compared with YG and OG. Gene expression of mitofusion (MFN)-1/2, mitochondrial fusion genes, was greater in OT compared with OG (p < .05). Similarly, protein content of ETC complexes I, II, and IV was significantly greater in OT compared with both YG and OG (p < .001). Protein content of peroxisome proliferator-activated receptor gamma, coactivator 1 α (PGC-1α), was greater in OT compared with YG and OG (p < .001). Our results suggest that the aging process per se is not associated with a decline in gene expression and protein content of ETC complexes. Mitochondrial-related gene expression and protein content are substantially greater in OT, suggesting that exercise-mediated increases in mitochondrial content can be maintained into later life.

The vitamin D receptor regulates mitochondrial function in C2C12 myoblasts.

Vitamin D deficiency has been linked to a reduction in skeletal muscle function and oxidative capacity; however, the mechanistic bases of these impairments are poorly understood. The biological actions of vitamin D are carried out via the binding of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) to the vitamin D receptor (VDR). Recent evidence has linked 1α,25(OH)2D3 to the regulation of skeletal muscle mitochondrial function in vitro; however, little is known with regard to the role of the VDR in this process. To examine the regulatory role of the VDR in skeletal muscle mitochondrial function, we used lentivirus-mediated shRNA silencing of the VDR in C2C12 myoblasts (VDR-KD) and examined mitochondrial respiration and protein content compared with an shRNA scrambled control. VDR protein content was reduced by ~95% in myoblasts and myotubes (P < 0.001). VDR-KD myoblasts displayed a 30%, 30%, and 36% reduction in basal, coupled, and maximal respiration, respectively (P < 0.05). This phenotype was maintained in VDR-KD myotubes, displaying a 34%, 33%, and 48% reduction in basal, coupled, and maximal respiration (P < 0.05). Furthermore, ATP production derived from oxidative phosphorylation (ATPOx) was reduced by 20%, suggesting intrinsic impairments within the mitochondria following VDR-KD. However, despite the observed functional decrements, mitochondrial protein content, as well as markers of mitochondrial fission were unchanged. In summary, we highlight a direct role for the VDR in regulating skeletal muscle mitochondrial respiration in vitro, providing a potential mechanism as to how vitamin D deficiency might impact upon skeletal muscle oxidative capacity.

Lipid remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

Utilizing small nutrient compounds as enhancers of exercise-induced mitochondrial biogenesis.

Endurance exercise, when performed regularly as part of a training program, leads to increases in whole-body and skeletal muscle-specific oxidative capacity. At the cellular level, this adaptive response is manifested by an increased number of oxidative fibers (Type I and IIA myosin heavy chain), an increase in capillarity and an increase in mitochondrial biogenesis. The increase in mitochondrial biogenesis (increased volume and functional capacity) is fundamentally important as it leads to greater rates of oxidative phosphorylation and an improved capacity to utilize fatty acids during sub-maximal exercise. Given the importance of mitochondrial biogenesis for skeletal muscle performance, considerable attention has been given to understanding the molecular cues stimulated by endurance exercise that culminate in this adaptive response. In turn, this research has led to the identification of pharmaceutical compounds and small nutritional bioactive ingredients that appear able to amplify exercise-responsive signaling pathways in skeletal muscle. The aim of this review is to discuss these purported exercise mimetics and bioactive ingredients in the context of mitochondrial biogenesis in skeletal muscle. We will examine proposed modes of action, discuss evidence of application in skeletal muscle in vivo and finally comment on the feasibility of such approaches to support endurance-training applications in humans.

Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells.

How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle selection") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively select the ER Ca2+ store, whereas cholecystokinin additionally recruits a lysosome-related organelle. Similarly, in a pancreatic beta cell line MIN6, acetylcholine selects only the ER, whereas glucose mobilizes Ca2+ from a lysosome-related organelle. We also show that the key to organelle selection is the agonist-specific coupling messenger(s) such that the ER is selected by recruitment of inositol 1,4,5-trisphosphate (or cADP-ribose), whereas lysosome-related organelles are selected by NAADP.

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.

Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans.

Fatty acids have both stimulatory and inhibitory effects on insulin secretion. Long-term exposure to fatty acids results in impaired insulin secretion whilst acute exposure has generally been found to enhance insulin release. However, there are conflicting data in the literature as to the relative efficacy of various fatty acids and on the glucose dependency of the stimulatory effect. Moreover, there is little information on the responses of human islets in vitro to fatty acids. We have therefore studied the acute effects of a range of fatty acids on insulin secretion from rat and human islets of Langerhans at different glucose concentrations. Fatty acids (0.5 mM) acutely stimulated insulin release from rat islets of Langerhans in static incubations in a glucose-dependent manner. The greatest effect was seen at high glucose concentration (16.7 mM) and little or no response was elicited at 3.3 or 8.7 mM glucose. Long-chain fatty acids (palmitate and stearate) were more effective than medium-chain (octanoate). Saturated fatty acids (palmitate, stearate) were more effective than unsaturated (palmitoleate, linoleate, elaidate). Stimulation of insulin secretion by fatty acids was also studied in perifused rat islets. No effects were observed at 3.3 mM glucose but fatty acids markedly potentiated the effect of 16.7 mM glucose. The combination of fatty acid plus glucose was less effective when islets had been first challenged with glucose alone. The insulin secretory responses to fatty acids of human islets in static incubations were similar to those of rat islets. In order to examine whether the responses to glucose and to fatty acids could be varied independently we used an animal model in which lactating rats are fed a low-protein diet during early lactation. Islets from rats whose mothers had been malnourished during lactation were still able to respond effectively to fatty acids despite a lowered secretory response to glucose. These data emphasise the complex interrelationships between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis during undernutrition.