Globular adiponectin improves high glucose-suppressed endothelial progenitor cell function through endothelial nitric oxide synthase dependent mechanisms

Plasma levels of adiponectin, an adipose-specific protein with putative anti-atherogenic properties, could be down-regulated in obese and diabetic subjects. Recent insights suggest that the injured endothelial monolayer is regenerated by circulating endothelial progenitor cells (EPCs), but high glucose reduces number and functions of EPCs. Here, we tested the hypothesis that globular adiponectin can improve high glucose-suppressed EPC functions by restoration of endothelial nitric oxide synthase (eNOS) activity. Late EPCs isolated from healthy subjects appeared with cobblestone shape at 2-4 weeks. EPCs were incubated with high glucose (25 mM) and treatment with globular adiponectin for functional study. Migration and tube formation assays were used to evaluate the vasculogenetic capacity of EPCs. The activities of eNOS, Akt and concentrations of nitric oxide (NO) were also determined. Administration of globular adiponectin at physiological concentrations promoted EPC migration and tube formation, and dose-dependently upregulated phosphorylation of eNOS, Akt and augmented NO production. Chronic incubation of EPCs in high-glucose medium significantly impaired EPC function and induced cellular senescence, but these suppression effects were reversed by treatment with globular adiponectin. Globular adiponectin reversed high glucose-impaired EPC functions through NO- and p38 MAPK-related mechanisms. In addition, nude mice that received EPCs treated with adiponectin in high glucose medium showed a significant improvement in blood flow than those received normal saline and EPCs incubated in high glucose conditions. The administration of globular adiponectin improved high glucose-impaired EPC functions in vasculogenesis by restoration of eNOS activity. These beneficial effects may provide some novel rational to the vascular protective properties of adiponectin.     

Exposure to platelets promotes functional properties of endothelial progenitor cells

Recent evidence indicates that endothelial progenitor cells (EPCs) have an important role in the process of repair following vascular injury, and that platelets mediate their recruitment to sites of injury. Platelets and EPCs can interact and bind directly. However, there is limited information on the effect of platelets on EPC function following this interaction. We, therefore, aimed to assess the in vitro effect of platelets on functional properties of EPCs. Human EPCs were isolated from donated Buffy coats and purified on a magnetic separation column specific for CD133. They were incubated either on fibronectin matrix, or co-incubated with washed platelets (isolated from healthy volunteers), for 7 days. Number of EPC colony forming units (CFU) was quantified, and endothelial cell lineage confirmed by immunostaining. Functional properties of the cultured cells were evaluated by MTT—proliferation assay and migration assay using the Boyden chamber. Co-incubation of EPCs with platelets compared to incubation of EPCs alone (on fibronectin matrix) resulted in higher number of CFUs after 7 days (6.5 ± 1.3 vs. 3.5 ± 0.5 CFUs/well, respectively, P = 0.005). In addition, co-incubation of EPCs with platelets versus EPCs alone was associated with higher proportion of living cells, by the MTT assay (0.2 ± 0.01 vs. 0.12 ± 0.04 MTT 570 nm respectively, P = 0.003), and higher number of migrated EPCs, assessed by the migration assay (1400 ± 212 vs. 580 ± 180 migrated cells/2000 cells, respectively, P < 0.0001). In vitro exposure to platelets promotes the capacity of EPCs to form colonies, proliferate and migrate. Therefore, the interaction with platelets appears to augment EPC functional properties.     

Near normalisation of blood glucose improves the potentiating effect of GLP-1 on glucose-induced insulin secretion in patients with type 2 diabetes

Aims/hypothesis The ability of glucagon-like peptide-1 (GLP-1) to enhance beta cell responsiveness to i.v. glucose is impaired in patients with type 2 diabetes mellitus compared with healthy individuals. We investigated whether 4 weeks of near normalisation of blood glucose (BG) improves the potentiation of glucose-stimulated insulin secretion by GLP-1. Methods Nine obese patients with type 2 diabetes and inadequate glycaemic control (HbA_1c 8.0 ± 0.4%) were investigated before and after 4 weeks of near normalisation of BG using insulin treatment (mean diurnal blood glucose 6.4 ± 0.3 mmol/l, HbA_1c 6.6 ± 0.3%). Nine matched healthy participants were also studied. Beta cell function was investigated before and after insulin treatment using stepwise glucose infusions and infusion of saline or GLP-1 (1.0 pmol kg⁻¹ min⁻¹), resulting in supraphysiological total GLP-1 concentrations of approximately 200 pmol/l. The responsiveness to glucose or glucose+GLP-1 was expressed as the slope of the linear regression line relating insulin secretion rate (ISR) and plasma glucose concentration (pmol kg⁻¹ min⁻¹ [mmol/l]⁻¹). Results In the diabetic participants, the slopes during glucose+saline infusion did not differ before and after insulin treatment (0.33 ± 0.07 and 0.39 ± 0.04, respectively; p = NS). In contrast, near normalisation of blood glucose improved beta cell sensitivity to glucose during glucose+GLP-1 infusion (1.27 ± 0.2 before vs 1.73 ± 0.31 after; p < 0.01). In the healthy participants, the slopes during the glucose+saline and glucose+GLP-1 infusions were 1.01 ± 0.14 and 4.79 ± 0.53, respectively. Conclusions/interpretation A supraphysiological dose of GLP-1 enhances beta cell responses to glucose in patients with type 2 diabetes, and 4 weeks of near normalisation of blood glucose further improves this effect. ClinicalTrials.gov ID no.: NCT00612625     

Diesel Exhaust Particles Impair Endothelial Progenitor Cells, Compromise Endothelial Integrity, Reduce Neoangiogenesis, and Increase Atherogenesis in Mice

The mechanisms of the harmful cardiovascular effects of small particulate matter are incompletely understood. Endothelial progenitor cells (EPCs) predict outcome of patients with vascular disease. The aim of our study was to examine the effects of diesel exhaust particles (DEP) on EPC and on the associated vascular damage in mice. C57Bl/6 mice were exposed to DEP. 2 μg DEP/day was applicated intranasally for 3 weeks. Exposure to DEP reduced DiLDL/lectin positive EPC to 58.4 ± 5.6 % (p < 0.005). Migratory capacity was reduced to 65.8 ± 3.9 % (p < 0.0001). In ApoE^?/? mice, DEP application reduced the number of EPC to 75.6 ± 6.4 % (p < 0.005) and EPC migration to 58.5 ± 6.8 % (p < 0.005). Neoangiogenesis was reduced to 39.5 ± 14.6 % (p < 0.005). Atherogenesis was profoundly increased by DEP treatment (157.7 ± 18.1 % vs. controls, p < 0.05). In cultured human EPC, DEP (0.1–100 μg/mL) reduced migratory capacity to 25 ± 2.6 % (p < 0.001). The number of colony-forming units was reduced to 8.8 ± 0.9 % (p < 0.001) and production of reactive oxygen species was elevated by DEP treatment (p < 0.001). Furthermore, DEP treatment increased apoptosis of EPC (to 266 ± 62 % of control, p < 0.05). In a blood–brain barrier model, DEP treatment impaired endothelial cell integrity during oxygen–glucose deprivation (p < 0.001). Diesel exhaust particles impair endothelial progenitor cell number and function in vivo and in vitro. The reduction in EPC was associated with impaired neoangiogenesis and a marked increase in atherosclerotic lesion formation.     

Bone marrow transplantation improves endothelial function in hypertensive Dahl salt-sensitive rats

Bone marrow-derived endothelial progenitor cells (EPCs) constitute an important endogenous system in the maintenance of endothelial integrity and vascular homeostasis. Cardiovascular risk factors are associated with a reduced number and functional capacity of EPCs. Here we investigated the effect of transplantation of bone marrow–derived cells from Dahl salt-resistant rat into age-matched Dahl salt-sensitive (DS) rat on blood pressure, endothelial function, and circulating EPC number. The recipient DS rats were fed a normal (0.5% NaCl, NS) or high-salt (4% NaCl, HS) diet for 6 weeks after bone marrow transplantation (BMT). DS rats on a NS or a HS diet without BMT were used as controls. Hypertensive DS (HS-DS) rat (systolic blood pressure: 213 ± 4 mm Hg vs. 152 ± 4 mm Hg in NS, P < .05) manifested impaired endothelium-dependent relaxation to acetylcholine (EDR), increased gene expression of vascular oxidative stress and proinflamamtory cytokines, and decreased eNOS expression. BMT on HS-DS rat significantly improved EDR and eNOS expression, reduced oxidative stress without reduction in SBP (206 ± 6 mm Hg). Flow cytometry analysis showed that there was no difference in the number of circulating EPCs, demonstrated by expression of EPC markers CD34, cKit, and vascular endothelial growth factor, between hypertensive and normotensive rats. Surprisingly, BMT resulted in a 5- to 10-fold increase in the previously mentioned EPC markers in hypertensive, but not normotensive rat. These results suggest that DS rat has an impaired ability to increase bone marrow–derived EPCs in response to HS diet challenge, which may contribute to endothelial dysfunction.     

Carnitine revisited: potential use as adjunctive treatment in diabetes

Aims/hypothesis This study examined the efficacy of supplemental l-carnitine as an adjunctive diabetes therapy in mouse models of metabolic disease. We hypothesised that carnitine would facilitate fatty acid export from tissues in the form of acyl-carnitines, thereby alleviating lipid-induced insulin resistance. Materials and methods Obese mice with genetic or diet-induced forms of insulin resistance were fed rodent chow  ± 0.5% l-carnitine for a period of 1–8 weeks. Metabolic outcomes included insulin tolerance tests, indirect calorimetry and mass spectrometry-based profiling of acyl-carnitine esters in tissues and plasma. Results Carnitine supplementation improved insulin-stimulated glucose disposal in genetically diabetic mice and wild-type mice fed a high-fat diet, without altering body weight or food intake. In severely diabetic mice, carnitine supplementation increased average daily respiratory exchange ratio from 0.886 ± 0.01 to 0.914 ± 0.01 (p < 0.01), reflecting a marked increase in systemic carbohydrate oxidation. Similarly, under insulin-stimulated conditions, carbohydrate oxidation was higher and total energy expenditure increased from 172 ± 10 to 210 ± 9 kJ kg fat-free mass⁻¹ h⁻¹ in the carnitine-supplemented compared with control animals. These metabolic improvements corresponded with a 2.3-fold rise in circulating levels of acetyl-carnitine, which accounts for 86 and 88% of the total acyl-carnitine pool in plasma and skeletal muscle, respectively. Carnitine supplementation also increased several medium- and long-chain acyl-carnitine species in both plasma and tissues. Conclusions/interpretation These findings suggest that carnitine supplementation relieves lipid overload and glucose intolerance in obese rodents by enhancing mitochondrial efflux of excess acyl groups from insulin-responsive tissues. Carefully controlled clinical trials should be considered. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Peripheral glucose metabolism is altered by epileptic seizures

The aim of the present study was to investigate the status of jejunal absorption and peripheral metabolism of glucose in Wistar Audiogenic Rats (WAR), a genetic model of epilepsy, after seizures induced by intensive sound exposure. The jejunal loop of rats was isolated and infused (0.5 mL min⁻¹) with Tyrode solution containing twice the normal concentrations of glucose, sodium, and potassium. Samples were taken at 5 or 10-min intervals over a 40-min period. At the end of the experiment, samples of liver and gastrocnemius muscle were taken to measure the levels of glycogen, glucose-6-phosphate, fructose-6-phosphate and glucose transporter-4 (GLUT4). Hepatic glucose-6-phosphate increased in WAR submitted to audiogenic seizure (21.90 ± 3.08) as compared to non-susceptible Wistar rats (8.12 ± 0.87) and to WAR not submitted to audiogenic stimulation (5.17 ± 0.97). In addition, an increase in hepatic fructose-6-phosphate, an intermediate metabolite of the glycolytic pathway, was observed in WAR submitted to audiogenic seizure (5.98 ± 0.99) compared to non-susceptible Wistar rats (2.38 ± 0.53). According to the present results, jejunal absorption of glucose was not changed by seizures. However, generalized tonic–clonic seizures produced by sound stimulation resulted in a decrease in muscle glycogen content. In addition, our results demonstrated that the concentration of GLUT4 in the gastrocnemius muscle of WAR was 1.6-fold higher than that observed in resistant rats and that the audiogenic stimulus led to decreased concentration of this receptor in the muscle of WAR animals.     

Tissue specificity of insulin resistance in humans: fat in the liver rather than muscle is associated with features of the metabolic syndrome

Aims/hypothesis The aim of this study was to investigate whether intrahepatic and intramyocellular fat are related to insulin resistance in these respective tissues or to the metabolic syndrome. Methods Hepatic (insulin 1.8 pmol kg⁻¹ min⁻¹ combined with [3-³H]glucose) and muscle (insulin 6.0 pmol kg⁻¹ min⁻¹) insulin sensitivity were measured on separate occasions in 45 non-diabetic men (age 42 ± 1 years, BMI 26.2 ± 0.6 kg/m²) using the euglycaemic–hyperinsulinaemic clamp. Liver fat and intramyocellular lipid (IMCL) were measured by proton magnetic resonance spectroscopy and body composition by magnetic resonance imaging. We also determined fasting serum insulin and adiponectin concentrations, components of the metabolic syndrome and maximal oxygen consumption. Results In participants with high [median 12.0% (interquartile range 5.7–18.5%)] vs low [2.0% (1.0–2.0%)] liver fat, fasting serum triacylglycerols (1.6 ± 0.2 vs 1.0 ± 0.1 mmol/l, p = 0.002) and fasting serum insulin (55 ± 4 vs 32 ± 2 pmol/l, p < 0.0001) were increased and serum HDL-cholesterol (1.26 ± 0.1 vs 1.48 ± 0.1 mmol/l, p = 0.02) and fasting serum adiponectin (9.5 ± 1.2 vs 12.2 ± 1.2 μg/ml, p = 0.05) decreased. In participants with high [19.5% (16.0–26.0%)] vs low [5.0% (2.3–7.5%)] IMCL, these parameters were comparable. Liver fat was higher in participants with [10.5% (3.0–18.0%)] than in those without [2.0% (1.5–6.0%), p = 0.010] the metabolic syndrome, even independently of obesity, while IMCL was comparable. Insulin suppression of glucose rate of appearance and serum NEFA was significantly impaired in the high liver fat group. Conclusions/interpretation Fat accumulation in the liver rather than in skeletal muscle is associated with features of the metabolic syndrome, i.e. increased fasting serum triacylglycerols and decreased fasting serum HDL-cholesterol, as well as with hyperinsulinaemia and low adiponectin.     

Progesterone impairs cell respiration and suppresses a compensatory increase in glucose transport in isolated rat skeletal muscle: a non-genomic mechanism contributing to metabolic adaptation to late pregnancy?

Aims/hypothesis The aim of the study was to gain better insight into the mechanisms responsible for impaired glucose metabolism during late pregnancy. We explored the direct effects of progesterone on glucose metabolism of skeletal muscle. Methods Specimens of skeletal muscle from untreated rats were incubated with progesterone and rates of substrate fluxes through the various pathways of glucose metabolism were analysed. Results Progesterone dose-dependently reduced the rates of glucose and pyruvate oxidation (insulin-stimulated rates after 5 h of exposure to 1 and 10 μmol/l progesterone: glucose oxidation, −6 ± 4%, NS, and −39 ± 4%, p < 0.001; pyruvate oxidation, −28 ± 2% and −55 ± 4%, p < 0.001 each) and increased lactate release (+28 ± 4% and +58 ± 9%, p < 0.005 each), which indicated inhibition of mitochondrial respiratory function. Impairment of cell respiration, e.g. by the specific inhibitor rotenone, is known to trigger a compensatory increase in glucose transport, but this response was blunted in the case of progesterone (change of glucose transport in response to 10 μmol/l progesterone vs 60 nmol/l rotenone, both causing a reduction in glucose oxidation by −39%: progesterone, +14 ± 8% vs rotenone, +84 ± 23%, p < 0.03). Further experiments dealt with the underlying mechanisms and revealed a rapid mode of action (50 μmol/l progesterone, reduction in insulin-stimulated glucose oxidation after 30 min: −29 ± 7%, p < 0.01) not affected by blockers of gene expression or the nuclear progesterone receptor. Conclusions/interpretation Progesterone inhibits cell respiration and at the same time suppresses a compensatory increase in glucose transport, causing cellular carbohydrate deficiency in isolated rat skeletal muscle. This effect is mediated by a direct, rapid and non-genomic mechanism and could contribute to pregnancy-associated changes in glucose homeostasis. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorised users.     

Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet

Aims/hypothesis Increased glucagon secretion predicts deterioration of glucose tolerance, and high glucagon levels contribute to hyperglycaemia in type 2 diabetes. Inhibition of glucagon action may therefore be a potential novel target to reduce hyperglycaemia. Here, we investigated whether chronic treatment with a glucagon receptor antagonist (GRA) improves islet dysfunction in female mice on a high-fat diet (HFD). Materials and methods After 8 weeks of HFD, mice were treated with a small molecule GRA (300 mg/kg, gavage once daily) for up to 30 days. Insulin secretion was studied after oral and intravenous administration of glucose and glucagon secretion after intravenous arginine. Islet morphology was examined and insulin secretion and glucose oxidation were measured in isolated islets. Results Fasting plasma glucose levels were reduced by GRA (6.0 ± 0.2 vs 7.4 ± 0.5 mmol/l; p = 0.017). The acute insulin response to intravenous glucose was augmented (1,300 ± 110 vs 790 ± 64 pmol/l; p < 0.001). The early insulin response to oral glucose was reduced in mice on HFD + GRA (1,890 ± 160 vs 3,040 ± 420 pmol/l; p = 0.012), but glucose excursions were improved. Intravenous arginine significantly increased the acute glucagon response (129 ± 12 vs 36 ± 6 ng/l in controls; p < 0.01), notably without affecting plasma glucose. GRA caused a modest increase in alpha cell mass, while beta cell mass was similar to that in mice on HFD + vehicle. Isolated islets displayed improved glucose-stimulated insulin secretion after GRA treatment (0.061 ± 0.007 vs 0.030 ± 0.004 pmol islet⁻¹ h⁻¹ at 16.7 mmol/l glucose; p < 0.001), without affecting islet glucose oxidation. Conclusions/interpretation Chronic glucagon receptor antagonism in HFD-fed mice improves islet sensitivity to glucose and increases insulin secretion, suggesting improvement of key defects underlying impaired glucose tolerance and type 2 diabetes.     

Glucose increases hepatic lipase expression in HepG2 liver cells through upregulation of upstream stimulatory factors 1 and 2

Aims/hypothesis  Elevated hepatic lipase (HL, also known as LIPC) expression is a key factor in the development of the atherogenic lipid profile in type 2 diabetes and insulin resistance. Recently, genetic screens revealed a possible association of type 2 diabetes and familial combined hyperlipidaemia with the USF1 gene. Therefore, we investigated the role of upstream stimulatory factors (USFs) in the regulation of HL. Methods  Levels of USF1, USF2 and HL were measured in HepG2 cells cultured in normal- or high-glucose medium (4.5 and 22.5 mmol/l, respectively) and in livers of streptozotocin-treated rats. Results  Nuclear extracts of cells cultured in high glucose contained 2.5 ± 0.5-fold more USF1 and 1.4 ± 0.2-fold more USF2 protein than cells cultured in normal glucose (mean ± SD, n = 3). This coincided with higher DNA binding of nuclear proteins to the USF consensus DNA binding site. Secretion of HL (2.9 ± 0.5-fold), abundance of HL mRNA (1.5 ± 0.2-fold) and HL (–685/+13) promoter activity (1.8 ± 0.3-fold) increased in parallel. In chromatin immunoprecipitation assays, the proximal HL promoter region was immunoprecipitated with anti-USF1 and anti-USF2 antibodies. Co-transfection with USF1 or USF2 cDNA stimulated HL promoter activity 6- to 16-fold. USF and glucose responsiveness were significantly reduced by removal of the −310E-box from the HL promoter. Silencing of the USF1 gene by RNA interference reduced glucose responsiveness of the HL (−685/+13) promoter region by 50%. The hyperglycaemia in streptozotocin-treated rats was associated with similar increases in USF abundance in rat liver nuclei, but not with increased binding of USF to the rat Hl promoter region. Conclusions/interpretation  Glucose increases HL expression in HepG2 cells via elevation of USF1 and USF2. This mechanism may contribute to the development of the dyslipidaemia that is typical of type 2 diabetes.     

Lowering serum urate does not improve endothelial function in patients with type 2 diabetes

Aims/hypothesis Endothelial dysfunction contributes to excess cardiovascular risk in patients with type 2 diabetes. There is strong evidence of an association between high serum uric acid concentrations and endothelial dysfunction, and uric acid has been proposed as an independent cardiovascular risk factor in type 2 diabetes. We hypothesised that lowering of uric acid concentrations might allow restoration of endothelial function in this high-risk group. Methods Intravenous urate oxidase (1.5 mg) was administered to ten patients with type 2 diabetes and ten healthy participants in a two-way, randomised, placebo-controlled, crossover study. Forearm blood flow responses to intra-brachial acetylcholine, sodium nitroprusside and N ^G-monomethyl-l-arginine (L-NMMA) were measured using venous occlusion plethysmography. The augmentation index (AIx) was determined by pulse wave analysis as a measure of large arterial stiffness. Results Acetylcholine and L-NMMA evoked lesser responses in patients with type 2 diabetes than in healthy participants. Baseline AIx was higher in patients with type 2 diabetes (mean ± SD: 13.1 ± 6.9%) than in healthy participants (2.0 ± 5.1%; p = 0.006). Urate oxidase lowered serum uric acid concentrations by 64 ± 11% (p < 0.001), but this had no effect on forearm blood flow responses or AIx in either group. Conclusions/interpretation Substantial short-term lowering of uric acid did not have a direct vascular effect, suggesting that, on its own, this might not be an effective strategy for restoring endothelial function in patients with type 2 diabetes.     

Hyperinsulinaemia during exercise does not suppress hepatic glycogen concentrations in patients with type 1 diabetes: a magnetic resonance spectroscopy study

Aims/hypothesis We compared in vivo changes in liver glycogen concentration during exercise between patients with type 1 diabetes and healthy volunteers. Methods We studied seven men with type 1 diabetes (mean ± SEM diabetes duration 10 ± 2 years, age 33 ± 3 years, BMI 24 ± 1 kg/m², HbA_1c 8.1 ± 0.2% and VO₂ peak 43 ± 2 ml [kg lean body mass]⁻¹ min⁻¹) and five non-diabetic controls (mean ± SEM age 30 ± 3 years, BMI 22 ± 1 kg/m², HbA_1c 5.4 ± 0.1% and VO₂ peak 52 ± 4 ml [kg lean body mass]⁻¹ min⁻¹, before and after a standardised breakfast and after three bouts (EX1, EX2, EX3) of 40 min of cycling at 60% VO₂ peak. ¹³C Magnetic resonance spectroscopy of liver glycogen was acquired in a 3.0 T magnet using a surface coil. Whole-body substrate oxidation was determined using indirect calorimetry. Results Blood glucose and serum insulin concentrations were significantly higher (p < 0.05) in the fasting state, during the postprandial period and during EX1 and EX2 in subjects with type 1 diabetes compared with controls. Serum insulin concentration was still different between groups during EX3 (p < 0.05), but blood glucose concentration was similar. There was no difference between groups in liver glycogen concentration before or after the three bouts of exercise, despite the relative hyperinsulinaemia in type 1 diabetes. There were also no differences in substrate oxidation rates between groups. Conclusions/interpretation In patients with type 1 diabetes, hyperinsulinaemic and hyperglycaemic conditions during moderate exercise did not suppress hepatic glycogen concentrations. These findings do not support the hypothesis that exercise-induced hypoglycaemia in patients with type 1 diabetes is due to suppression of hepatic glycogen mobilisation. K. Chokkalingam and K. Tsintzas contributed equally to this study.     

Proliferation of sorted human and rat beta cells

Aims/hypothesis The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors. Methods Human beta cells were purified by FACS by virtue of their high zinc content using Newport Green, and excluding ductal and dead cells. Rat beta cells were sorted by autofluorescence or using the same method developed for human cells. Cells were plated on poly-l-lysine or ECMs from rat or human bladder carcinoma cells or bovine corneal ECM and incubated in the presence of BrdU with or without growth factors. Results The newly developed method for sorting human beta cells yields a population containing 91.4 ± 2.8% insulin-positive cells with a low level of spontaneous apoptosis and a robust secretory response to glucose. Beta cells from 8-week-old rats proliferated in culture and this was increased by ECM. Among growth factors, only human growth hormone (hGH) and the glucagon-like peptide-1 analogue liraglutide enhanced proliferation of rat beta cells, with a significant increase on both poly-l-lysine and ECM. By contrast, sorted adult human beta cells from 16 donors aged 48.9 ± 14.3 years (range 16–64 years) failed to replicate demonstrably in vitro regardless of the substratum or growth factors used. Conclusions/interpretation These findings indicate that, in our conditions, the fully differentiated human adult insulin-producing beta cell was unable to proliferate in vitro. This has important implications for any attempt to expand cells from pancreases of donors of this age group. By contrast, the rat beta cells used here were able to divide in vitro, and this was enhanced by ECM, hGH and liraglutide. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Very low levels of vitamin D in systemic sclerosis patients

Vitamin D displays many extraosseous immunomodulatory effects. The aim of the study was to evaluate the level of vitamin D in patients with systemic sclerosis (SSc) and to analyze the associations between the concentration of the vitamin and clinical manifestations. In March-April 2009, 65 consecutive SSc patients underwent evaluation of vitamin D concentrations by the LIAISON immunoassay (normal 30-100 ng/ml). Serum levels between 10 and 30 ng/ml were classified as vitamin D insufficiency, while concentrations <10 ng/ml as vitamin D deficiency. None of the patients were receiving vitamin D supplementation at the time of or during the year prior to study entry. The mean level of vitamin D was 15.8 ± 9.1 ng/ml. Only three cases showed normal values; vitamin D insufficiency and deficiency were found in 43 and 19 cases, respectively. Patients with vitamin D deficiency showed longer disease duration (13.1 ± 6.8 versus 9.4 ± 5.5 years, P = 0.026), lower diffusing lung capacity for carbon monoxide (63.7 ± 12.4 versus 76.4 ± 20.2, P = 0.014), higher estimated pulmonary artery pressure (28.9 ± 9.9 versus 22.8 ± 10.4, P = 0.037) and higher values of ESR (40 ± 25 versus 23 ± 13 mm/h, P = 0.001) and of CRP (7 ± 7 and 4 ± 2 mg/l, P = 0.004) in comparison with patients with vitamin D insufficiency; moreover, late nailfold videocapillaroscopic pattern was more frequently found (52.6% versus 18.6%, P = 0.013). None of the patients showed evidence of overt mal-absorption. Low levels of vitamin D are very frequent in patients with SSc. Intestinal involvement is not likely the cause of vitamin D deficit; other factors such as skin hyperpigmentation and reduced sun exposition for psychological and social reasons may be implicated. Patients with vitamin D deficiency showed more severe disease in comparison with patients with vitamin D insufficiency, above all concerning lung involvement. Further trials are awaited to determine whether vitamin D could represent a modifiable factor able to interfere with SSc evolution.     

Substrate source utilisation in long-term diagnosed type 2 diabetes patients at rest,and during exercise and subsequent recovery

Aims/hypothesis Disturbances in substrate source metabolism and, more particularly, in fatty acid metabolism, play an important role in the aetiology and progression of type 2 diabetes. However, data on substrate source utilisation in type 2 diabetes are inconclusive. Methods [U-¹³C]palmitate and [6,6-²H₂]glucose tracers were used to assess plasma NEFA and glucose oxidation rates and to estimate the use of muscle- and/or lipoprotein-derived triacylglycerol and muscle glycogen. Subjects were ten male patients who had a long-term (7 ± 1 years) diagnosis of type 2 diabetes and were overweight, and ten matched healthy, male control subjects. Muscle biopsy samples were collected before and after exercise to assess muscle fibre type-specific intramyocellular lipid and glycogen content. Results At rest and during exercise, the diabetes patients had greater values than the controls for palmitate rate of appearance (Ra) (rest, 2.46 ± 0.18 and 1.85 ± 0.20 respectively; exercise, 3.71 ± 0.36 and 2.84 ± 0.20 μmol kg⁻¹ min⁻¹) and rate of disappearance (Rd) (rest, 2.45 ± 0.18 and 1.83 ± 0.20; exercise, 3.64 ± 0.35 and 2.80 ± 0.20 μmol kg⁻¹ min⁻¹ respectively). This was accompanied by significantly higher fat oxidation rates at rest and during recovery in the diabetes patients (rest, 0.11 ± 0.01 in diabetes patients and 0.09 ± 0.01 in controls; recovery, 0.13 ± 0.01 and 0.11 ± 0.01 g/min respectively), despite significantly greater plasma glucose Ra, Rd and circulating plasma glucose concentrations. Furthermore, exercise significantly lowered plasma glucose concentrations in the diabetes patients, as a result of increased blood glucose disposal. Conclusion This study demonstrates that substrate source utilisation in long-term-diagnosed type 2 diabetes patients, in whom compensatory hyperinsulinaemia is no longer present, shifts towards an increase in whole-body fat oxidation rate and is accompanied by disturbances in fat and carbohydrate handling.     

Evidence for abnormal glucose uptake or metabolism in thalamus during acute hyperglycaemia in type 1 diabetes—a <Superscript>1</Superscript>H MRS study

Acute hyperglycaemia impairs cognitive function. It is however not known, whether different brain regions are equally exposed to glucose during acute hyperglycemia or whether the brain is able to adjust its glucose uptake or metabolism in response to blood glucose fluctuation. We studied the effect of acute hyperglycaemia on the brain glucose concentration in seven men with type 1 diabetes with daily glucose fluctuations of 11 ± 3 mmol/l, and in eleven age-matched non-diabetic men. Glucose was quantified with proton magnetic resonance spectroscopy in three different brain regions at baseline (fasting glycaemia) and twice during a 2 h hyperglycaemic clamp with plasma glucose increase of 12 mmol/l. The increase in brain glucose during acute hyperglycaemia in the non-diabetic group was: cortex (2.7 ± 0.9 mmol/l) > thalamus (2.3 ± 0.7 mmol/l) > white matter (1.7 ± 0.7 mmol/l, P = 0.021 vs. cortex) and in the diabetic group: cortex (2.0 ± 0.7 mmol/l) > white matter (1.3 ± 0.7 mmol/l) > thalamus (1.1 ± 0.4 mmol/l, P = 0.010 vs. cortex). In the diabetic group, the glucose increase in the thalamus was attenuated compared to the non-diabetic participants (P = 0.011). In conclusion, the increase of glucose during acute hyperglycaemia seems to be dependent on the brain tissue type. The high exposure of cortex to excess glucose and the altered glucose uptake or metabolism in the thalamus may thus contribute to hyperglycaemia related cognitive dysfunction.     

Evidence that autonomic mechanisms contribute to the adaptive increase in insulin secretion during dexamethasone-induced insulin resistance in humans

Aims/hypothesis This study examined whether autonomic mechanisms contribute to adaptively increased insulin secretion in insulin-resistant humans, as has been proposed from studies in animals. Methods Insulin secretion was evaluated before and after induction of insulin resistance with or without interruption of neural transmission. Insulin resistance was induced by dexamethasone (15 mg given over 3 days) in nine healthy women (age 67 years, BMI 25.2 ± 3.4 kg/m², fasting glucose 5.1 ± 0.4 mmol/l, fasting insulin 46 ± 6 pmol/l). Insulin secretion was evaluated as the insulin response to intravenous arginine (5 g) injected at fasting glucose and after raising glucose to 13 to15 mmol/l or to >28 mmol/l. Neural transmission across the ganglia was interrupted by infusion of trimethaphan (0.3–0.6 mg kg⁻¹ min⁻¹). Results As an indication of insulin resistance, dexamethasone increased fasting insulin (to 75 ± 8 pmol/l, p < 0.001) without significantly affecting fasting glucose. Arginine-induced insulin secretion was increased by dexamethasone at all glucose levels (by 64 ± 12% at fasting glucose, by 80 ± 19% at 13–15 mmol glucose and by 43 ± 12% at >28 mmol glucose; p <0.001 for all). During dexamethasone-induced insulin resistance, trimethaphan reduced the insulin response to arginine at all three glucose levels. The augmentation of the arginine-induced insulin responses by dexamethasone-induced insulin resistance was reduced by trimethaphan by 48 ± 6% at fasting glucose, 61 ± 8% at 13–15 mmol/l glucose and 62 ± 8% at >28 mmol/l glucose (p < 0.001 for all). In contrast, trimethaphan did not affect insulin secretion before dexamethasone was given. Conclusions/interpretations Autonomic mechanisms contribute to the adaptative increase in insulin secretion in dexamethasone-induced insulin resistance in healthy participants.     

Docosahexaenoic acid status in females of reproductive age with maple syrup urine disease

Individuals with maple syrup urine disease (MSUD) have impaired metabolism of branched-chain amino acids (BCAA) valine, isoleucine, and leucine. Life-long dietary therapy is recommended to restrict BCAA intake and thus prevent poor neurological outcomes and death. To maintain adequate nutritional status, the majority of protein and nutrients are derived from synthetic BCAA-free medical foods with variable fatty acid content. Given the restrictive diet and the importance of omega-3 fatty acids, particularly docosahexaenoic acid (DHA), in neurological development, this study evaluated the dietary and fatty acid status of females of reproductive age with MSUD attending a metabolic camp. Healthy controls of similar age and sex were selected from existing normal laboratory data. Total lipid fatty acid concentration in plasma and erythrocytes was analyzed using gas chromatography–mass spectroscopy. Participants with MSUD had normal to increased concentrations of plasma and erythrocyte alpha linolenic acid (ALA) but significantly lower concentrations of plasma and erythrocyte docosahexaenoic acid (DHA) as percent of total lipid fatty acids compared with controls (plasma DHA: MSUD 1.03 ± 0.35, controls 2.87 ± 1.08; P = 0.001; erythrocyte DHA: MSUD 2.58 ± 0.58, controls 3.66 ± 0.80; P = 0.011). Dietary records reflected negligible or no DHA intake over the 3-day period prior to the blood draw (range 0–2 mg). These results suggest females of reproductive age with MSUD have lower blood DHA concentrations than age-matched controls. In addition, the presence of ALA in medical foods and the background diet may not counter the lack of preformed DHA in the diet. The implications of these results warrant further investigation.