A novel 3D imaging approach for quantification of GLUT4 levels across the intact myocardium.

Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm×5 mm×3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail.

Impact of physical fitness and exercise training on subcutaneous adipose tissue beiging markers in humans with and without diabetes and a high-fat diet-fed mouse model.

AIMS: Exercise training induces white adipose tissue (WAT) beiging and improves glucose homeostasis and mitochondrial function in rodents. This could be relevant for type 2 diabetes in humans, but the effect of physical fitness on beiging of subcutaneous WAT (scWAT) remains unclear. This translational study investigates if beiging of scWAT associates with physical fitness in healthy humans and recent-onset type 2 diabetes and if a voluntary running wheel intervention is sufficient to induce beiging in mice. MATERIALS AND METHODS: Gene expression levels of established beiging markers were measured in scWAT biopsies of humans with (n = 28) or without type 2 diabetes (n = 28), stratified by spiroergometry into low (L-FIT; n = 14 each) and high physical fitness (H-FIT; n = 14 each). High-fat diet-fed FVB/N mice underwent voluntary wheel running, treadmill training or no training (n = 8 each group). Following the training intervention, mitochondrial respiration and content of scWAT were assessed by high-resolution respirometry and citrate synthase activity, respectively. RESULTS: Secreted CD137 antigen (Tnfrsf9/Cd137) expression was three-fold higher in glucose-tolerant H-FIT than in L-FIT, but not different between H-FIT and L-FIT with type 2 diabetes. In mice, both training modalities increased Cd137 expression and enhanced mitochondrial content without changing respiration in scWAT. Treadmill but not voluntary wheel running led to improved whole-body insulin sensitivity. CONCLUSIONS: Higher physical fitness and different exercise interventions associated with higher gene expression levels of the beiging marker CD137 in healthy humans and mice on a high-fat diet. Humans with recent-onset type 2 diabetes show an impaired adipose tissue-specific response to physical activity.

AMPK and Beyond: The Signaling Network Controlling RabGAPs and Contraction-Mediated Glucose Uptake in Skeletal Muscle.

Impaired skeletal muscle glucose uptake is a key feature in the development of insulin resistance and type 2 diabetes. Skeletal muscle glucose uptake can be enhanced by a variety of different stimuli, including insulin and contraction as the most prominent. In contrast to the clearance of glucose from the bloodstream in response to insulin stimulation, exercise-induced glucose uptake into skeletal muscle is unaffected during the progression of insulin resistance, placing physical activity at the center of prevention and treatment of metabolic diseases. The two Rab GTPase-activating proteins (RabGAPs), TBC1D1 and TBC1D4, represent critical nodes at the convergence of insulin- and exercise-stimulated signaling pathways, as phosphorylation of the two closely related signaling factors leads to enhanced translocation of glucose transporter 4 (GLUT4) to the plasma membrane, resulting in increased cellular glucose uptake. However, the full network of intracellular signaling pathways that control exercise-induced glucose uptake and that overlap with the insulin-stimulated pathway upstream of the RabGAPs is not fully understood. In this review, we discuss the current state of knowledge on exercise- and insulin-regulated kinases as well as hypoxia as stimulus that may be involved in the regulation of skeletal muscle glucose uptake.

Chronic stress alters hepatic metabolism and thermodynamic respiratory efficiency affecting epigenetics in C57BL/6 mice.

Chronic stress episodes increase metabolic disease risk even after recovery. We propose that persistent stress detrimentally impacts hepatic metabolic reprogramming, particularly mitochondrial function. In male C57BL/6 mice chronic variable stress (Cvs) reduced energy expenditure (EE) and body mass despite increased energy intake versus controls. This coincided with decreased glucose metabolism and increased lipid ß-oxidation, correlating with EE. After Cvs, mitochondrial function revealed increased thermodynamic efficiency (ƞ-opt) of complex CI, positively correlating with blood glucose and NEFA and inversely with EE. After Cvs recovery, the metabolic flexibility of hepatocytes was lost. Reduced CI-driving NAD+/NADH ratio, and diminished methylation-related one-carbon cycle components hinted at epigenetic regulation. Although initial DNA methylation differences were minimal after Cvs, they diverged during the recovery phase. Here, the altered enrichment of mitochondrial DNA methylation and linked transcriptional networks were observed. In conclusion, Cvs rapidly initiates the reprogramming of hepatic energy metabolism, supported by lasting epigenetic modifications.

Inhibition of proline-rich tyrosine kinase 2 restores cardioprotection by remote ischaemic preconditioning in type 2 diabetes.

BACKGROUND AND PURPOSE: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline-rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. EXPERIMENTAL APPROACH: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. KEY RESULTS: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. CONCLUSION AND IMPLICATIONS: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes.

Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes.

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes.

Dysfunction of the adhesion G protein-coupled receptor latrophilin 1 (ADGRL1/LPHN1) increases the risk of obesity.

Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.

Skeletal muscle p53-depletion uncovers a mechanism of fuel usage suppression that enables efficient energy conservation.

BACKGROUND: The ability of skeletal muscle to respond adequately to changes in nutrient availability, known as metabolic flexibility, is essential for the maintenance of metabolic health and loss of flexibility contributes to the development of diabetes and obesity. The tumour suppressor protein, p53, has been linked to the control of energy metabolism. We assessed its role in the acute control of nutrient allocation in skeletal muscle in the context of limited nutrient availability. METHODS: A mouse model with inducible deletion of the p53-encoding gene, Trp53, in skeletal muscle was generated using the Cre-loxP-system. A detailed analysis of nutrient metabolism in mice with control and knockout genotypes was performed under ad libitum fed and fasting conditions and in exercised mice. RESULTS: Acute deletion of p53 in myofibres of mice activated catabolic nutrient usage pathways even under ad libitum fed conditions, resulting in significantly increased overall energy expenditure (+10.6%; P = 0.0385) and a severe nutrient deficit in muscle characterized by depleted intramuscular glucose and glycogen levels (-62,0%; P < 0.0001 and -52.7%; P < 0.0001, respectively). This was accompanied by changes in marker gene expression patterns of circadian rhythmicity and hyperactivity (+57.4%; P = 0.0068). These metabolic changes occurred acutely, within 2-3 days after deletion of Trp53 was initiated, suggesting a rapid adaptive response to loss of p53, which resulted in a transient increase in lactate release to the circulation (+46.6%; P = 0.0115) from non-exercised muscle as a result of elevated carbohydrate mobilization. Conversely, an impairment of proteostasis and amino acid metabolism was observed in knockout mice during fasting. During endurance exercise testing, mice with acute, muscle-specific Trp53 inactivation displayed an early exhaustion phenotype with a premature shift in fuel usage and reductions in multiple performance parameters, including a significantly reduced running time and distance (-13.8%; P = 0.049 and -22.2%; P = 0.0384, respectively). CONCLUSIONS: These findings suggest that efficient nutrient conservation is a key element of normal metabolic homeostasis that is sustained by p53. The homeostatic state in metabolic tissues is actively maintained to coordinate efficient energy conservation and metabolic flexibility towards nutrient stress. The acute deletion of Trp53 unlocks mechanisms that suppress the activity of nutrient catabolic pathways, causing substantial loss of intramuscular energy stores, which contributes to a fasting-like state in muscle tissue. Altogether, these findings uncover a novel function of p53 in the short-term regulation of nutrient metabolism in skeletal muscle and show that p53 serves to maintain metabolic homeostasis and efficient energy conservation.

Depletion of TBC1D4 Improves the Metabolic Exercise Response by Overcoming Genetically Induced Peripheral Insulin Resistance.

The Rab-GTPase-activating protein (RabGAP) TBC1D4 (AS160) represents a key component in the regulation of glucose transport into skeletal muscle and white adipose tissue (WAT) and is therefore crucial during the development of insulin resistance and type 2 diabetes. Increased daily activity has been shown to be associated with improved postprandial hyperglycemia in allele carriers of a loss-of-function variant in the human TBC1D4 gene. Using conventional Tbc1d4-deficient mice (D4KO) fed a high-fat diet, we show that moderate endurance exercise training leads to substantially improved glucose and insulin tolerance and enhanced expression levels of markers for mitochondrial activity and browning in WAT from D4KO animals. Importantly, in vivo and ex vivo analyses of glucose uptake revealed increased glucose clearance in interscapular brown adipose tissue and WAT from trained D4KO mice. Thus, chronic exercise is able to overcome the genetically induced insulin resistance caused by Tbc1d4 depletion. Gene variants in TBC1D4 may be relevant in future precision medicine as determinants of exercise response.