Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) can possibly serve as a cellular metabolic read-out for lifestyle factors and lifestyle interventions. However, the impact of PBMC composition on PBMC metabolism is not yet clear, neither is the differential impact of a longer-term lifestyle factor versus a short-term lifestyle intervention. We investigated the effect of aerobic fitness level and a recent exercise bout on PBMC metabolism in females. PBMCs from 31 young female adults divided into a high-fit (V̇O2peak ≥ 47 mL/kg/min, N = 15) and low-fit (V̇O2peak ≤ 37 mL/kg/min, N = 16) group were isolated at baseline and overnight after a single bout of exercise (60 minutes, 70% V̇O2peak). Oxygen consumption rate (OCR) and glycolytic rate (GR) were measured using extracellular flux (XF) assays and PBMC subsets were characterized using fluorescence-activated cell sorting (FACS). Basal OCR, FCCP-induced OCR, spare respiratory capacity, ATP-linked OCR, and proton leak were significantly higher in high-fit compared to low-fit females (all P < 0.01), while no significant differences in glycolytic rate (GR) were found (all P > 0.05). A recent exercise bout did not significantly affect GR or OCR parameters (all P > 0.05). The overall PBMC composition was similar between high-fit and low-fit females. Mitochondrial PBMC function was significantly higher in PBMCs from high-fit compared to low-fit females, which was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health.

Pituitary adenylate cyclase-activating polypeptide receptor activation in the hypothalamus recruits unique signaling pathways involved in energy homeostasis

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts pleiotropic effects on ventromedial nuclei (VMN) of the hypothalamus and its control of feeding and energy expenditure through the Type I PAC1 receptor. However, the endogenous role of PAC1R's in the VMN and the downstream signaling responsible for PACAP's effects on energy balance are unknown. Numerous studies have revealed that PAC1Rs are coupled to both Gas/ adenylate cyclase/protein kinase A (Gas/AC/PKA) and Gaq/phospholipase C/protein kinase C (Gaq/PLC/PKC), while also undergoing trafficking following stimulation. To determine the endogenous role PAC1R's and downstream signaling that may explain PACAP's pleiotropic effects, we used RNA interference to knockdown VMN PAC1Rs and pharmacologically inhibited PKA, PKC and PAC1R trafficking. Knocking down PAC1Rs increased meal sizes, reduced total number of meals, and induced body weight gain. Inhibition of either PKA or PKC alone in awake male Sprague Dawley rats, attenuated PACAP's hypophagic and anorectic effects during the dark phase. However, PKA or PKC inhibition potentiated PACAP's thermogenic effects during the light phase. Analysis of locomotor activity revealed that PKA inhibition augmented PACAP's locomotor effects, however, PKC inhibition had no effect. Finally, PACAP infusion in the VMN induces surface PAC1R trafficking into the cytosol which was blocked by endocytosis inhibitors. Subsequently, inhibition of PAC1R trafficking into the cytosol attenuated PACAP-induced hypophagia. These results revealed that endogenous PAC1Rs uniquely engage PKA, PKC and receptor trafficking to mediate PACAP's pleiotropic effects in VMN control of feeding and metabolism.

Long-term light and moderate exercise intervention similarly prevent both hippocampal and glycemic dysfunction in pre-symptomatic type 2 diabetic rats

A pre-diabetic population has an increased risk of cognitive decline as well as type 2 diabetes mellitus (T2DM). The present study investigated whether the progression of memory dysfunction and dysregulated brain glycogen metabolism is prevented with four months of exercise intervention from the pre-symptomatic stage in T2DM rat model. Memory function and biochemical and molecular profiles were assessed in the pre-symptomatic stage of OLETF rats, a T2DM model, with LETO rats as genetic control. These rats were subjected to light- or moderate-intensity treadmill running for four months with repetition of the same experiments. Significant hippocampal-dependent memory dysfunction was observed in the pre-symptomatic stage of OLETF rats, accompanied by downregulated levels of hippocampal monocarboxylate transporter 2 (MCT2), a neuronal lactate-transporter, without alteration in hippocampal glycogen levels. Four months of light or moderate exercise from the pre-symptomatic stage of T2DM normalized glycemic parameters and also hippocampal molecular normalization through MCT2, glycogen, and brain-derived neurotrophic factor (BDNF) levels with the improvement of memory dysfunction in OLETF rats. A four-month exercise regimen from the pre-symptomatic stage of T2DM at light and moderate intensities contributed to the prevention of the development of T2DM and the progression of cognitive decline with hippocampal lactate-transport and BDNF improvement.

Duodenal mucosal resurfacing with GLP-1 receptor agonism increases postprandial unconjugated bile acid in patients with insulin-dependent type 2 diabetes

Introduction: Duodenal Mucosal Resurfacing (DMR) is a new endoscopic ablation technique aimed at improving glycemia and metabolic control in patients with type 2 diabetes mellitus (T2DM). DMR appears to improve insulin resistance, which is the root cause of T2DM, but its mechanism of action is largely unknown. Bile acids function as intestinal signalling molecules in glucose and energy metabolism via the activation of farnesoid X receptor and secondary signalling (e.g. via fibroblast growth factor 19[FGF19]), and are linked to metabolic health. Methods: We investigated the effect of DMR and GLP-1 on postprandial bile acid responses in 16 patients with insulin-dependent T2DM, using mixed meal tests performed at baseline and six months after the DMR procedure. Results: The combination treatment allowed discontinuation of insulin treatment in 11/16 (69%) of patients while improving glycaemic and metabolic health. We found increased postprandial unconjugated bile acid responses (all p<0.05), an overall increased secondary bile acid response (p=0.036) and a higher 12α-hydroxylated:non12α-hydroxylated ratio (p<0.001). Total bile acid concentrations were unaffected by the intervention. Postprandial FGF19 and 7-alpha-hydroxy-4-cholesten-3-one (C4) concentrations decreased post-intervention (both p<0.01). Conclusion and discussion: Our study demonstrates that DMR with GLP-1 modulates the postprandial bile acid response. The alterations in postprandial bile acid responses may be the result of changes in the microbiome, ileal bile acid uptake and improved insulin sensitivity. Controlled studies are needed to elucidate the mechanism linking the combination treatment to metabolic health and bile acids.

Metformin Improves Skeletal Muscle Microvascular Insulin Resistance in Metabolic Syndrome

Aims: Microvascular insulin resistance is present in metabolic syndrome and may contribute to increased cardiovascular disease risk and the impaired metabolic response to insulin observed. Metformin improves metabolic insulin resistance in humans. Its effects on macro and microvascular insulin resistance has not been defined. Methods: Eleven non-diabetic, metabolic syndrome subjects were studied four times (before and after 12 weeks treatment with placebo or metformin) using a crossover design, with an eight week washout interval between treatments. On each occasion, we measured three indices of large artery function (pulse wave velocity-PWV, radial pulse wave separation analysis (PWSA), brachial artery endothelial function (flow-mediated dilation-FMD) as well as muscle microvascular perfusion (contrast-enhanced ultrasound-CEU) before and 120 min into a 150 min, 1 mU/min/kg euglycemic insulin clamp. RESULTS: Metformin decreased body mass index (BMI), fat weight, and % body fat (P<0.05, each), placebo had no effect. Metformin (not placebo) improved metabolic insulin sensitivity, (clamp glucose infusion rate, P<0.01). PWV, and FMD after insulin were unaffected by metformin treatment. PWSA improved with insulin only after metformin P<0.01). Insulin decreased muscle microvascular blood volume measured by contrast ultrasound both before and after placebo and before metformin (P<0.02 for each) but not after metformin. CONCLUSIONS: Short-term metformin treatment improves both metabolic and muscle microvascular response to insulin. Metformin's effect on microvascular insulin responsiveness may contribute to its beneficial metabolic effects. Metformin did not improve aortic stiffness or brachial artery endothelial function, but enhaced radial pulse wave properties consistent with relaxation of smaller arterioles.

Tissue specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole-body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole-body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, beta cell area, or genes regulating beta cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.

Plasma FGF21 concentrations and spontaneous self-selection of protein suggest that 15% protein in the diet may not be enough for male adult rats

Protein requirement has been determined at 10-15% energy. Under dietary self-selection, rats ingest 25-30% energy as protein and regulate FGF21 (a hormone signaling protein deficiency) to levels lower than those measured with a 15% protein (15P) diet. Our hypothesis is that if a 15P diet was indeed sufficient to ensure protein homeostasis, it is probably a too low protein level to ensure optimal energy homeostasis. Adult male Wistar rats were used in this study. The first objective was to determine the changes in food intake, body composition and plasma FGF21, IGF-1 and PYY concentrations in rats fed 8P, 15P, 30P, 40P or 50P diets. The second was to determine whether the FGF21 levels measured in the rats were related to spontaneous protein intake. Rats were fed a 15P diet and then allowed to choose between a protein diet and a protein-free diet. Food intake and body weight were measured throughout the experiments. Body composition was determined at different experimental stages. Plasma samples were collected to measure FGF21, IGF-1 and PYY concentrations. A 15P diet appears to result in higher growth than that observed with the 30P, 40P and 50P diets. However, the 15P diet probably does not provide optimal progression of body composition owing to a tendency of 15P rats to fix more fat and energy in the body. The variable and higher concentrations of FGF21 in the 15P diet suggest a deficit in protein intake, but this does not appear to be a parameter reflecting the adequacy of protein intake relative to individual protein requirements.

Susceptibility to diet-induced obesity at thermoneutral conditions is independent of UCP1

Objective Activation of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) upon cold stimulation leads to substantial increase in energy expenditure to defend body temperature. Increases in energy expenditure after a high caloric food intake, termed diet-induced thermogenesis, are also attributed to BAT. These properties render BAT a potential target to combat diet-induced obesity. However, studies investigating the role of UCP1 to protect against diet-induced obesity are controversial and rely on the phenotyping of a single constitutive UCP1-knockout model. To address this issue, we generated a novel UCP1-knockout model by Cre-mediated deletion of Exon 2 in the UCP1 gene. We studied the effect of constitutive UCP1 knockout on metabolism and the development of diet-induced obesity. Methods UCP1 knockout and wildtype mice were housed at 30°C and fed a control diet for 4-weeks followed by 8-weeks of high-fat diet. Body weight and food intake were monitored continuously over the course of the study and indirect calorimetry was used to determine energy expenditure during both feeding periods. Results Based on Western blot analysis, thermal imaging and noradrenaline test, we confirmed the lack of functional UCP1 in knockout mice. However, body weight gain, food intake and energy expenditure were not affected by deletion of UCP1 gene function during both feeding periods. Conclusion We introduce a novel UCP1-KO mouse enabling the generation of conditional UCP1-knockout mice to scrutinize the contribution of UCP1 to energy metabolism in different cell types or life stages. Our results demonstrate that UCP1 does not protect against diet-induced obesity at thermoneutrality.

Considerations and Challenges of Islet Transplantation and Future Therapies on the Horizon

Islet transplantation is a treatment for selected adults with Type 1 diabetes and severe hypoglycemia. Islets from two or more donor pancreases, a scarce resource, are usually required to impact on glycemic control but the treatment falls short of a cure. Islets are avascular when transplanted into the hypoxic liver environment and subjected to inflammatory insults, immune attack and toxicity from systemic immunosuppression. The Collaborative Islet Transplant Registry with outcome data on over 1000 islet transplant recipients has demonstrated that larger islet numbers transplanted and older age of recipient are associated with better outcomes. Induction with T cell depleting agents and the TNF-α inhibitor Etanercept and maintenance systemic immunosuppression with mTOR inhibitors in combination with calcineurin inhibitors also appear advantageous, but concerns remain over immunosuppressive toxicity. We discuss strategies and therapeutics which address specific challenges of islet transplantation, many of which are at the pre-clinical stage of development. On the horizon are adjuvant cell therapies with mesenchymal stromal cells and regulatory T cells that have been used in preclinical models and in humans in other contexts; such a strategy may enable reductions in immunosuppression in the early peri-transplant period when the islets are vulnerable to apoptosis. Human embryonic stem-cell derived islets are in early phase clinical trials and hold the promise of an inexhaustible supply of insulin producing cells; effective encapsulation of such cells or, silencing of the HLA complex would eliminate the need for immunosuppression, enabling this therapy to be used in all those with Type 1 diabetes.

Nitric Oxide-Dependent Micro-and Macro-Vascular Dysfunction Occurs Early in Adolescents with Type 1 Diabetes

Background: Arterial stiffness and endothelial dysfunction are both reported in children with type 1 diabetes (DM1) and may predict future cardiovascular events. In health, nitric-oxide (NO) relaxes arteries and increases microvascular perfusion. The relationships between NO-dependent macro- and microvascular functional responses and arterial stiffness have not been studied in adolescents with DM1. Here we assessed macro- and microvascular function in DM1 adolescents and aged-matched controls at baseline and during an oral glucose challenge (OGTT). Methods: DM1 adolescents (n=16) and controls (n=14) were studied before and during an OGTT. At baseline we measured: A) large artery stiffness using both aortic augmentation index (AI) and carotid-femoral pulse wave velocity (cfPWV); B) brachial flow-mediated dilation (FMD) and forearm endothelial function using post-ischemic flow velocity (PIFV); and C) forearm muscle microvascular blood volume (MBV) using contrast-enhanced ultrasound. Following OGTT, AI, cfPWV and MBV were reassessed at 60 min and MBV again at 120 min. Within individual and between-group comparisons were made by paired and unpaired t-tests or repeated measures ANOVA. Results: Baseline FMD was lower (p=0.02) in DM1. PWV at 0 and 60 min did not differ between groups. Baseline AI did not differ between groups but declined with OGTT only in controls (p=0.02) and was lower than DM1 at 60 min (p<0.03). Baseline MBV was comparable in DM1 and control groups, but declined in DM1 at 120 min (p=0.01) and was lower than the control group (p<0.03). There was an inverse correlation between plasma glucose and MBV at 120 min (r= -0.523, p<0.01). No differences were noted between groups for VO2max (ml/min/kg), body fat (%), or BMI. Conclusions: NO-dependent macro- and microvascular function, including FMD and AI, and microvascular perfusion respectively are impaired early in the course of DM1, precede increases of arterial stiffness, and may provide an early indicator of vascular risk.