(-)-Epicatechin Reverses Glucose Intolerance in Rats Housed at Thermoneutrality.

Diabetes is a life-threatening and debilitating disease with pathological hallmarks, including glucose intolerance and insulin resistance. Plant compounds are a source of novel and effective therapeutics, and the flavonoid (-)-epicatechin, common to popular foods worldwide, has been shown to improve carbohydrate metabolism in both clinical studies and preclinical models. We hypothesized that (-)-epicatechin would alleviate thermoneutral housing-induced glucose intolerance. Male rats were housed at either thermoneutral (30 °C) or room temperature (24 °C) for 16 weeks and gavaged with either 1 mg/kg body weight or vehicle for the last 15 days before sacrifice. Rats housed at thermoneutrality had a significantly elevated serum glucose area under the curve (p < 0.05) and reduced glucose-mediated insulin secretion. In contrast, rats at thermoneutrality treated with (-)-epicatechin had improved glucose tolerance and increased insulin secretion (p < 0.05). Insulin tolerance tests revealed no differences in insulin sensitivity in any of the four groups. Pancreatic immunohistochemistry staining showed significantly greater islet insulin positive cells in animals housed at thermoneutrality. In conclusion, (-)-epicatechin improved carbohydrate tolerance via increased insulin secretion in response to glucose challenge without a change in insulin sensitivity.

Acute effects of sedentary breaks on vascular health in adults at risk for type 2 diabetes: A systematic review.

The aim of this systematic review was to evaluate the available evidence regarding the acute effects of interrupting/breaking up prolonged sedentary behavior (SB) on vascular health among individuals at elevated risk for type 2 diabetes (T2D). Searches of MEDLINE, Embase, Web of Science, and Cochrane Library databases were conducted on April 7, 2020. Included studies: (1) examined the effect of breaking up prolonged SB in adults with or at elevated risk for T2D and (2) assessed a vascular health outcome, such as blood pressure (BP), flow-mediated dilation (FMD), pulse-wave velocity, or endothelin-1. A total of 20 articles (17 unique studies) were included. Only three studies reported adequate statistical power for the specified vascular outcome. The available evidence suggests that light and moderate intensity activity breaks are effective in acutely lowering BP when compared to prolonged sitting. The small number of studies that included FMD or other vascular outcomes prohibits conclusions regarding the impact of SB breaks on these outcomes. Few studies evaluating the impact of breaking up SB among adults at risk for T2D have included and been adequately powered to examine vascular outcomes, but our preliminary finding, that certain SB breaks improve BP, provides proof-of-concept for this line of inquiry. Future studies should examine both the acute and chronic vascular effects of breaking up SB among individuals most vulnerable to the effects of SB (e.g. older adults, those with T2D), as these individuals are both highly sedentary and at greatest risk of poor health outcomes. PROSPERO ID: CRD42020183423.

Achieving ADA/ISPAD clinical guideline goals is associated with higher insulin sensitivity and cardiopulmonary fitness in adolescents with type 1 diabetes: Results from RESistance to InSulin in Type 1 ANd Type 2 diabetes (RESISTANT) and Effects of MEtformin on CardiovasculaR Function in AdoLescents with Type 1 Diabetes (EMERALD) Studies.

OBJECTIVE: Most youth with type 1 diabetes do not meet the American Diabetes Association (ADA) and International Society for Pediatric and Adolescent Diabetes (ISPAD) targets for hemoglobin A1c (HbA1c), blood pressure (BP), lipids, and body mass index (BMI). We hypothesized that ISPAD/ADA goal achievement would be associated with better insulin sensitivity (IS) and cardiopulmonary fitness. METHODS: IS was quantified as glucose infusion rate (GIR) from a hyperinsulinemic-euglycemic clamp in youth with type 1 diabetes from the RESistance to InSulin in Type 1 ANd Type 2 diabetes (RESISTANT) (n = 86) and Effects of MEtformin on CardiovasculaR Function in AdoLescents with Type 1 Diabetes (EMERALD) (n = 41) cohorts (n = 127; age 15.7 ± 2.2 years, 52% girls). Cardiopulmonary fitness was measured as peak oxygen consumption (VO2 peak/kg) during upright (RESISTANT) or supine (EMERALD) cycle ergometry and were stratified by cycle type. Goal achievement was defined as HbA1c < 7.5%, BP < 90th percentile, LDL-cholesterol < 100 mg/dL, HDL-cholesterol > 35 mg/dL, triglycerides < 150 mg/dL and BMI < 85th percentile. Participants were stratified into 3 groups: achieving 0-3 goals (n = 52), 4 goals (n = 48), and 5-6 goals (n = 27). Differences between groups were examined with generalized linear models. RESULTS: IS was lower in youth who met 0-3 goals (5.2 ± 3.4 mg/kg/min) vs those who met 4 goals (7.4 ± 4.1 mg/kg/min, P = .04) and those who met 5-6 goals (8.5 ± 4.3 mg/kg/min, P = .003), and remained significant after adjustments for sex and diabetes duration. Upright VO2 peak was lower in youth who met 0-3 goals (25.8 ± 4.6 mL/kg/min) vs those who met 4 goals (33.0 ± 7.8 mL/kg/min, P = .01) and those who met 5-6 goals (33.2 ± 4.4 mL/kg/min, P = .004). Similar and significant relationships were observed in EMERALD participants for supine VO2 peak. CONCLUSIONS: ADA/ISPAD goal achievement was associated with greater IS and cardiopulmonary fitness.

Perivascular adipose tissue remodeling impairs vasoreactivity in thermoneutral-housed rats.

Objective: Vascular pathology, characterized by impaired vasoreactivity and mitochondrial respiration, differs between the sexes. Housing rats under thermoneutral (TN) conditions causes vascular dysfunction and perturbed metabolism. We hypothesized that perivascular adipose tissue (PVAT), a vasoregulatory adipose depot with brown adipose tissue (BAT) phenotype, remodels to a white adipose (WAT) phenotype in rats housed at TN, driving diminished vasoreactivity in a sex-dependent manner. Methods: Male and female Wistar rats were housed at either room temperature (RT) or TN. Endpoints included changes in PVAT morphology, vasoreactivity in vessels with intact PVAT or transferred to PVAT of the oppositely-housed animal, vessel stiffness, vessel mitochondrial respiration and cellular signaling. Results: Remodeling of PVAT was observed in rats housed at TN; animals in this environment showed PVAT whitening and displayed diminished aortae vasodilation (p<0.05), different between the sexes. Juxtaposing PVAT from RT rats onto aortae from TN rats in females corrected vasodilation (p<0.05); this did not occur in males. In aortae of all animals housed at TN, mitochondrial respiration was significantly diminished in lipid substrate experiments (p<0.05), and there was significantly less expression of peNOS (p<0.001). Conclusions: These data are consistent with TN-induced remodeling of PVAT, notably associated with sex-specific blunting of vasoreactivity, diminished mitochondrial respiration, and altered cellular signaling.

Rosiglitazone improves insulin resistance but does not improve exercise capacity in individuals with impaired glucose tolerance: A randomized clinical study.

Dysmetabolic states, such as type 2 diabetes (T2D), characterized by insulin resistance (IR), are associated with fatty liver, increased cardiovascular disease (CVD) risk, and decreased functional exercise capacity (FEC). Rosiglitazone (RO) improves exercise capacity and IR in T2D. However, the effects of RO on FEC and other markers of CVD risk in prediabetes are unknown. We hypothesized that insulin sensitization with RO would improve exercise capacity and markers of CVD risk in participants with impaired glucose tolerance (IGT). Exercise performance (peak oxygen consumption and oxygen uptake kinetics), IR (homeostasis model assessment of IR and quantitative insulin sensitivity check index), and surrogate cardiovascular endpoints (coronary artery calcium (CAC) volume and density and C-reactive protein (CRP)) were measured in participants with IGT after 12 and 18 months of RO or placebo (PL). RO did not significantly improve exercise capacity. Glycemic measures and IR were significantly lower in people on RO compared to PL at 18 months. CAC volume progression was not different between PL and RO groups. RO did not improve exercise capacity during an 18-month intervention despite improved IR and glycemia in people with IGT. Future studies should explore why effects on FEC with RO occur in T2D but not IGT. Understanding these questions may help in targeting therapeutic approaches in T2D and IGT.

(-)-Epicatechin Improves Vasoreactivity and Mitochondrial Respiration in Thermoneutral-Housed Wistar Rat Vasculature.

Cardiovascular disease (CVD) is a global health concern. Vascular dysfunction is an aspect of CVD, and novel treatments targeting vascular physiology are necessary. In the endothelium, eNOS regulates vasodilation and mitochondrial function; both are disrupted in CVD. (−)-Epicatechin, a botanical compound known for its vasodilatory, eNOS, and mitochondrial-stimulating properties, is a potential therapy in those with CVD. We hypothesized that (−)-epicatechin would support eNOS activity and mitochondrial respiration, leading to improved vasoreactivity in a thermoneutral-derived rat model of vascular dysfunction. We housed Wistar rats at room temperature or in thermoneutral conditions for a total of 16 week and treated them with 1mg/kg body weight (−)-epicatechin for 15 day. Vasoreactivity, eNOS activity, and mitochondrial respiration were measured, in addition to the protein expression of upstream cellular signaling molecules including AMPK and CaMKII. We observed a significant improvement of vasodilation in those housed in thermoneutrality and treated with (−)-epicatechin (p < 0.05), as well as dampened mitochondrial respiration (p < 0.05). AMPK and CaMKIIα and ß expression were lessened with (−)-epicatechin treatment in those housed at thermoneutrality (p < 0.05). The opposite was observed with animals housed at room temperature supplemented with (−)-epicatechin. These data illustrate a context-dependent vascular response to (−)-epicatechin, a candidate for CVD therapeutic development.

Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells.

Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle cells (SMCs) in remodeled pulmonary arteries from animals with pulmonary hypertension and in the SMC layers of atherogenic systemic arteries and cardiomyocytes from hypertensive individuals. Loss of CREB can be induced in cultured SMCs by chronic exposure to hypoxia or platelet-derived growth factor BB (PDGF-BB). Here we investigated the signaling pathways and mechanisms by which PDGF elicits depletion of SMC CREB. Chronic PDGF treatment increased CREB ubiquitination in SMCs, while treatment of SMCs with the proteasome inhibitor lactacystin prevented decreases in CREB content. The nuclear export inhibitor leptomycin B also prevented depletion of SMC CREB alone or in combination with lactacystin. Subsequent studies showed that PDGF activated extracellular signal-regulated kinase, Jun N-terminal protein kinase, and phosphatidylinositol 3 (PI3)-kinase pathways in SMCs. Inhibition of these pathways blocked SMC proliferation in response to PDGF, but only inhibition of PI3-kinase or its effector, Akt, blocked PDGF-induced CREB loss. Finally, chimeric proteins containing enhanced cyan fluorescent protein linked to wild-type CREB or CREB molecules with mutations in several recognized phosphorylation sites were introduced into SMCs. PDGF treatment reduced the levels of each of these chimeric proteins except for one containing mutations in adjacent serine residues (serines 103 and 107), suggesting that CREB loss was dependent on CREB phosphorylation at these sites. We conclude that PDGF stimulates nuclear export and proteasomal degradation of CREB in SMCs via PI3-kinase/Akt signaling. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.

ß Cell dysfunction during progression of metabolic syndrome to type 2 diabetes.

In a society where physical activity is limited and food supply is abundant, metabolic diseases are becoming a serious epidemic. Metabolic syndrome (MetS) represents a cluster of metabolically related symptoms such as obesity, hypertension, dyslipidemia, and carbohydrate intolerance, and significantly increases type 2 diabetes mellitus risk. Insulin resistance and hyperinsulinemia are consistent characteristics of MetS, but which of these features is the initiating insult is still widely debated. Regardless, both of these conditions trigger adverse responses from the pancreatic ß cell, which is responsible for producing, storing, and releasing insulin to maintain glucose homeostasis. The observation that the degree of ß cell dysfunction correlates with the severity of MetS highlights the need to better understand ß cell dysfunction in the development of MetS. This Review focuses on the current understanding from rodent and human studies of the progression of ß cell responses during the development of MetS, as well as recent findings addressing the complexity of ß cell identity and heterogeneity within the islet during disease progression. The differential responses observed in ß cells together with the heterogeneity in disease phenotypes within the patient population emphasize the need to better understand the mechanisms behind ß cell adaptation, identity, and dysfunction in MetS.

Content and activity of cAMP response element-binding protein regulate platelet-derived growth factor receptor-alpha content in vascular smooth muscles.

Experiments in vascular smooth muscle cells (SMCs) indicate that the transcription factor cAMP response element-binding protein (CREB), the cyclic nucleotide response element-binding protein, suppresses expression of the platelet-derived growth factor-alpha receptor gene (PDGFRalpha). Adenovirus-mediated expression of constitutively active CREB mutants decreases PDGFRalpha mRNA, PDGFRalpha protein, and PDGFRalpha promoter-luciferase reporter activity in cultured SMCs. Expression of dominant negative CREB protein, A-CREB, increases PDGFRalpha protein content and the PDGFRalpha-promoter activity in SMCs. Active CREB prevents activation of PDGFRalpha promoter-luciferase reporter activity by CCAAT/enhancer-binding protein-delta (C/EBPdelta), shown to mediate IL-1beta stimulation of PDGFRalpha expression. Exposure of cultured SMCs to high glucose or reactive oxidant stress, which decrease CREB protein content and activity, increases PDGFRalpha protein content and promoter activity. Expression of active CREB blunts reactive oxidant stress-induced PDGFRalpha accumulation in SMCs. Loss of CREB protein in aortic walls of rats with streptozotocin-induced diabetes is accompanied by an increase in PDGFRalpha content. In Ob/Ob mice (which demonstrate reduced aortic wall CREB content vs. Ob/- controls), treatment with the peroxisomal proliferator-activated receptor gamma rosiglitazone increases CREB content and decreases PDGFRalpha content in the aortic wall. Thus, both in vitro and in vivo loss of CREB content and activity and subsequent accumulation of PDGFRalpha may contribute to SMC activation during diabetes.

Thiazolidinedione therapy: the benefits of aggressive and early use in type 2 diabetes.

Type 2 diabetes is now a global epidemic, with the number of people affected worldwide predicted to more than double to 300 million by the year 2025. While the importance of good glycemic control in countering the microvascular and macrovascular complications of diabetes is widely recognized, monotherapy with sulfonylureas or metformin achieves target blood glucose levels in only a minority of patients. Consequently, there is a pressing need for new treatment strategies that are more effective in providing sustained glycemic control and so reducing the burden of morbidity and mortality associated with diabetes and its complications. There is growing evidence of the benefits of early intervention with aggressive treatment strategies in improving glycemic control and reducing diabetic complications. To provide sustained control, such strategies need to address the combination of insulin resistance and beta-cell dysfunction that underlies most cases of type 2 diabetes. At the same time, treatment needs to address not only glycemic control but also the range of cardiovascular risk factors that are often found clustered together in patients with type 2 diabetes. This paper reviews the rationale and evidence for early combination therapy including a thiazolidinedione in improving glycemic control, and considers the potential for such aggressive therapy in reducing diabetic complications.

Nonesterified fatty acid exposure activates protective and mitogenic pathways in vascular smooth muscle cells by alternate signaling pathways.

Vascular smooth muscle cells (VSMC) are dynamic cells exposed to fluctuating concentrations of nutrients on a daily basis. Nonesterified fatty acids (NEFA) have been indicted as potential mediators of atherosclerosis and exaggerated VSMC remodeling observed in diabetes, and in vitro data support a model of VSMC activation by NEFA. However, recent observations suggest that metabolic stressors such as oxidants and NEFA may also simultaneously induce cytoprotective events as part of a homeostatic "off switch." Our group has established that the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is important for maintenance of VSMC quiescence, differentiation, and survival. We therefore examined whether acute physiologic NEFA exposure would regulate CREB in primary cultures of bovine aortic VSMC and explored the relationship between signaling to the cytoprotective CREB and the activating mitogen-activated protein kinase pathways. In vitro exposure of VSMC to 3 classes of unsaturated NEFA leads to significant acute, transient, dose-dependent, and repeatedly inducible CREB activation. As expected, extracellular signal-regulated kinase, P38 mitogen-activated protein kinase, Akt, Jun N-terminal kinase, and protein kinase C (PKC) pathways are also activated by NEFA. Using a battery of pharmacologic inhibitors and antioxidants, we demonstrate that CREB activation is mediated by a novel PKC isoform and is reactive oxygen species independent, whereas extracellular signal-regulated kinase activation, in contrast, is mediated by reactive oxygen species and is PKC independent. These data suggest parallel and mechanistically distinct stimulation of separate stabilizing and activating pathways in VSMC response to acute NEFA-mediated stress. Furthermore, the down-regulation of CREB in models of chronic metabolic stress reported in the literature would be expected to disrupt this homeostasis and shift the balance toward VSMC activation, consistent with emerging models of atherosclerosis.

Atherosclerosis in diabetes and insulin resistance.

Atherosclerosis and cardiovascular disease are the major causes of morbidity and mortality in patients with diabetes and those with insulin resistance and the metabolic syndrome. Both conditions profoundly accelerate the development of atherosclerosis and increase the morbidity and mortality of cardiovascular events. The question, therefore, is what are the molecular/biochemical mechanisms that underlie the potentiating influence of diabetes, the metabolic syndrome and/or insulin resistance on the development and progression of atherosclerosis? The following review will focus on the molecular mechanism whereby hyperglycaemia and/or hyperinsulinemia either directly or indirectly promote atherosclerosis.

SP600125, an inhibitor of c-jun N-terminal kinase, activates CREB by a p38 MAPK-mediated pathway.

SP600125, an anthrapyrazolone inhibitor of c-jun N-terminal kinase (JNK), has been used to characterize the role of JNK in apoptotic pathways. In this study, we have demonstrated an additional novel anti-apoptotic action of this inhibitor in MIN6 cells, a mouse beta cell line. SP600125 induced CREB-dependent promoter activation by 2.8-fold at 20 microM, the concentration at which it inhibited c-jun-dependent promoter activation by 51%. There was a significant (P<0.01) increase in CREB phosphorylation (serine 133) at 5 min, which persisted for a period of 2h. Examination of signaling pathways upstream of CREB showed a 2.5-fold increase in the active phospho form of p38 MAPK. This finding was further confirmed by an in vitro kinase assay using ATF-2 as substrate. SB203580, an inhibitor of p38 MAPK, partially blocked SP600125-mediated activation of CREB. These observations suggest that SP600125 could be used as a small molecular weight activator of CREB.

Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons.

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

Cytokine-mediated down-regulation of the transcription factor cAMP-response element-binding protein in pancreatic beta-cells.

Cytokines are known to induce apoptosis of pancreatic beta-cells. Impaired expression of the anti-apoptotic gene bcl-2 is one of the mechanisms involved. In this study, we identified a defect involving transcription factor cAMP-response element-binding protein (CREB) in the expression of bcl-2. Exposure of mouse pancreatic beta-cell line, MIN6 cells, to cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) led to a significant (p < 0.01) decrease in Bcl-2 protein and mRNA levels. Cytokines decreased (56%) the activity of the bcl-2 promoter that contains a cAMP-response element (CRE) site. Similar decreases were seen with a luciferase reporter gene driven by tandem repeats of CRE and a CREB-specific Gal4-luciferase reporter, suggesting a defect at the level of CREB. The active phospho form (serine 133) of CREB diminished significantly (p < 0.01) in cells exposed to cytokines. Examination of signaling pathways upstream of CREB revealed a reduction in the active form of Akt. Cytokine-induced decrease of bcl-2 promoter activity was partially restored when cells were cotransfected with a constitutively active form of Akt. Several end points of cytokine action including decreases in phospho-CREB, phospho-Akt, and BCl-2 levels and activation of caspase-9 were observed in isolated mouse islets. Overexpression of wild-type CREB in MIN6 cells by plasmid transfection and adenoviral infection led to protection against cytokine-induced apoptosis. Adenoviral transfer of dominant-negative forms of CREB, on the other hand, resulted in activation of caspase-9 and exaggeration of cytokine-induced beta-cell apoptosis. Together, these results point to CREB as a novel target for strategies aimed at improving the survival of beta-cells.