Mediterranean diet adherence in individuals with prediabetes and unknown diabetes: the Di@bet.es Study.

BACKGROUND AND AIMS: Mediterranean diet (MedDiet) is causally related to diabetes and is a dietary pattern recommended to individuals with diabetes. We investigated MedDiet adherence in individuals with prediabetes and unknown (PREDM/UKDM) or known diabetes (KDM) compared to those with normal glucose metabolism (NORMAL). METHODS: This was a national, population-based, cross-sectional, cluster-sampling study. MedDiet adherence was scored (MedScore, mean ± SD 24 ± 5) using a qualitative food frequency questionnaire. Logistic regression was used to examine the association between MedScore and PREDM/UKDM or KDM versus control subjects. RESULTS: We evaluated 5,076 individuals. Mean age was 50 years, 57% were female, 826 (582/244) were PREDM/UKDM, 478 were KDM and 3,772 were NORMAL. Mean age increased across MedScore tertiles (46, 51 and 56 years, p < 0.0001). Higher age-adjusted adherence to MedDiet (5-unit increment in the MedScore) was associated with lower and nondifferent odds (OR, 95% CI) of prevalent PREDM/UKDM (0.88, 0.81-0.96, p = 0.001) and KDM (0.97, 0.87-1.07, p = 0.279), respectively, compared to individuals in the NORMAL group. CONCLUSIONS: In a representative sample of the whole Spanish population, MedDiet adherence is independently associated with PREDM/UKDM. Therapeutic intervention may be, in part, responsible for the lack of differences in adherence observed between the KDM and NORMAL groups. However, reverse causation bias cannot be ruled out in cross-sectional studies.

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice.

AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function. METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment. RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice. CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Prevalence of diabetes mellitus and impaired glucose regulation in Spain: the Di@bet.es Study.

AIMS/HYPOTHESIS: The Di@bet.es Study is the first national study in Spain to examine the prevalence of diabetes and impaired glucose regulation. METHODS: A population-based, cross-sectional, cluster sampling study was carried out, with target population being the entire Spanish population. Five thousand and seventy-two participants in 100 clusters (health centres or the equivalent in each region) were randomly selected with a probability proportional to population size. Participation rate was 55.8%. Study variables were a clinical and demographic structured survey, lifestyle survey, physical examination (weight, height, BMI, waist and hip circumference, blood pressure) and OGTT (75 g). RESULTS: Almost 30% of the study population had some carbohydrate disturbance. The overall prevalence of diabetes mellitus adjusted for age and sex was 13.8% (95% CI 12.8, 14.7%), of which about half had unknown diabetes: 6.0% (95% CI 5.4, 6.7%). The age- and sex-adjusted prevalence rates of isolated impaired fasting glucose (IFG), isolated impaired glucose tolerance (IGT) and combined IFG-IGT were 3.4% (95% CI 2.9, 4.0%), 9.2% (95% CI 8.2, 10.2%) and 2.2% (95% CI 1.7, 2.7%), respectively. The prevalence of diabetes and impaired glucose regulation increased significantly with age (p < 0.0001), and was higher in men than in women (p < 0.001). CONCLUSIONS/INTERPRETATION: The Di@bet.es Study shows, for the first time, the prevalence rates of diabetes and impaired glucose regulation in a representative sample of the Spanish population.

Long-term safety and efficacy of linagliptin as monotherapy or in combination with other oral glucose-lowering agents in 2121 subjects with type 2 diabetes: up to 2 years exposure in 24-week phase III trials followed by a 78-week open-label extension.

Aim: The aim of this study was to evaluate the long-term safety, tolerability and efficacy of the dipeptidyl peptidase-4 inhibitor linagliptin given either alone or in combination with other oral glucose-lowering agents in persons with type 2 diabetes. Methods: A 78-week open-label extension study evaluated subjects who participated in one of four preceding 24-week, randomised, double-blind, placebo-controlled parent trials and who received linagliptin, linagliptin + metformin, linagliptin + metformin + a sulphonylurea or linagliptin + pioglitazone (all with linagliptin administered orally once daily). Individuals receiving one of these treatments during a previous trial continued the same treatment (n = 1532) for up to a total of 102 weeks, whereas those previously receiving placebo were switched to linagliptin (n = 589). All 2121 participants received at least one dose of the trial medication and were included in the primary safety analysis. Results: In subjects previously receiving active treatment, the glycosylated haemoglobin A(1c) reduction achieved during the 24-week parent trials was sustained through the 78-week extension period (change from baseline to week 102: -0.8%). Drug-related adverse events were experienced by 14.3% of participants. Hypoglycaemia occurred in 13.9% of participants and was similar between those previously receiving treatment (13.6%) and those switching from placebo to linagliptin (14.6%). Hypoglycaemia occurred most frequently with the use of metformin + a sulphonylurea background therapy (11%). Overall, no clinically relevant changes in body weight were observed. Conclusion: Long-term treatment with linagliptin was well tolerated with no change in the safety profile observed during the extension study. Sustained long-term glycaemic control was maintained for up to 102 weeks with either linagliptin monotherapy or linagliptin in combination with other oral glucose-lowering agents.

Paradoxical suboptimal vitamin D levels in a Mediterranean area: a population-based study.

Policies in sunny countries, such as those in the Mediterranean area, do not promote vitamin D supplementation despite some studies might suggest the high prevalence of sub-optimal levels. The objective was to determine the vitamin D levels by 25-hydroxyvitamin D (25(OH)D) of a Mediterranean population and their characteristics. This population-based study included a database of public health system from all individuals living in Catalonia > 18 years who had some measure of 25(OH)D between January 2018 and April 2021. More than half million people were classified based on 25(OH)D measurements to study their characteristics. Three vitamin D categories were created: < 20 ng/ml deficiency, 20-30 ng/ml insufficiency and > 30 ng/ml optimal. Less than 10% of the population residing in Catalonia had recent 25(OH)D determinations and the majority of determinations were in ≥ 45 years and in women. Around 80% of young people with determination had sub-optimal levels but the prevalence of vitamin D supplementation prescription increased with age which was associated with better values of 25(OH)D. In a Mediterranean area 25(OH)D determinations were low despite the high prevalence of suboptimal levels in the population with recent determination. In addition, the measurements were especially concentrated in people ≥ 45 years of age and in women who were, in addition, the groups to whom the most vitamin D supplementation was prescribed. On the contrary, young people presented few determinations of 25(OH)D and, although majority of them showed sub-optimal levels, vitamin D supplementation was not prescribed in most cases.

Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity.

AIMS/HYPOTHESIS: Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans. METHODS: Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy. RESULTS: Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules. CONCLUSIONS/INTERPRETATION: Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.

Efficacy and safety of initial combination therapy with linagliptin and pioglitazone in patients with inadequately controlled type 2 diabetes: a randomized, double-blind, placebo-controlled study.

AIMS: To compare the efficacy, safety and tolerability of linagliptin or placebo administered for 24 weeks in combination with pioglitazone in patients with type 2 diabetes mellitus (T2DM) exhibiting insufficient glycaemic control (HbA1c 7.5-11.0%). METHODS: Patients were randomized to receive the initial combination of 30 mg pioglitazone plus 5 mg linagliptin (n = 259) or pioglitazone plus placebo (n = 130), all once daily. The primary endpoint was change from baseline in HbA1c after 24 weeks of treatment, adjusted for baseline HbA1c and prior antidiabetes medication. RESULTS: After 24 weeks of treatment, the adjusted mean change (±s.e.) in HbA1c with the initial combination of linagliptin plus pioglitazone was -1.06% (±0.06), compared with -0.56% (±0.09) for placebo plus pioglitazone. The difference in adjusted mean HbA1c in the linagliptin group compared with placebo was -0.51% (95% confidence interval [CI] -0.71, -0.30; p < 0.0001). Reductions in fasting plasma glucose (FPG) were significantly greater for linagliptin plus pioglitazone than with placebo plus pioglitazone; -1.8 and -1.0 mmol/l, respectively, equating to a treatment difference of -0.8 mmol/l (95% CI -1.2, -0.4; p < 0.0001). Patients taking linagliptin plus pioglitazone, compared with those receiving placebo plus pioglitazone, were more likely to achieve HbA1c of <7.0% (42.9 vs. 30.5%, respectively; p = 0.0051) and reduction in HbA1c of ≥0.5% (75.0 vs. 50.8%, respectively; p < 0.0001). ß-cell function, exemplified by the ratio of relative change in adjusted mean HOMA-IR and disposition index, improved. The proportion of patients that experienced at least one adverse event was similar for both groups. Hypoglycaemic episodes (all mild) occurred in 1.2% of the linagliptin plus pioglitazone patients and none in the placebo plus pioglitazone group. CONCLUSION: Initial combination therapy with linagliptin plus pioglitazone was well tolerated and produced significant and clinically meaningful improvements in glycaemic control. This combination may offer a valuable additive initial treatment option for T2DM, particularly where metformin either is not well tolerated or is contraindicated, such as in patients with renal impairment.

Sodium tungstate regulates food intake and body weight through activation of the hypothalamic leptin pathway.

AIMS: Sodium tungstate is an anti-obesity drug targeting peripheral tissues. In vivo, sodium tungstate reduces body weight gain and food intake through increasing energy expenditure and lipid oxidation, but it also modulates hypothalamic gene expression when orally administered, raising the possibility of a direct effect of sodium tungstate on the central nervous system. METHODS: Sodium tungstate was administered intraperitoneally (ip) to Wistar rats, and its levels were measured in cerebrospinal fluid through mass spectrometry. Body weight gain and food intake were monitored for 24 h after its administration in the third ventricle. Hypothalamic protein was obtained and subjected to western blot. In vitro, hypothalamic N29/4 cells were treated with 100 µM sodium tungstate or 1 nM leptin, and protein and neural gene expression were analysed. RESULTS: Sodium tungstate crossed the blood-brain barrier, reaching a concentration of 1.31 ± 0.07 mg/l in cerebrospinal fluid 30 min after ip injection. When centrally administered, sodium tungstate decreased body weight gain and food intake and increased the phosphorylation state of the main kinases and proteins involved in leptin signalling. In vitro, sodium tungstate increased the phosphorylation of janus kinase-2 (JAK2) and extracellular signal-regulated kinase-1/2 (ERK1/2), but the activation of each kinase did not depend on each other. It regulated c-myc gene expression through the JAK2/STAT system and c-fos and AgRP (agouti-related peptide) gene expression through the ERK1/2 pathway simultaneously and independently. CONCLUSIONS: Sodium tungstate increased the activity of several kinases involved in the leptin signalling system in an independent way, making it a suitable and promising candidate as a leptin-mimetic compound in order to manage obesity.

Glucose and fructose as functional modulators of overall dog, but not boar sperm function.

The main aim of the present work was to test the effects of glucose and fructose on the phosphorylation levels of proteins linked to the control of overall sperm function in two species with very different metabolic characteristics, dog and boar. Incubation of dog spermatozoa with 10mM glucose increased serine phosphorylation of proteins related to cell cycle and signal transduction including cyclins B and E, Cdk2, Cdk6, Cdc6, PYK2, c-kit, Raf-1, TRK and several protein phosphatases. Incubation of dog spermatozoa with 10mM fructose decreased serine phosphorylation levels of cyclins B and D3, Cdk1/Cdc2, Cdk2, Cdk6, Akt, PI3 kinase, ERK-1 and protein kinase C. Incubation of boar spermatozoa with glucose or fructose did not modify any of the phosphorylation patterns studied. Given that one important difference between dog and boar spermatozoa is the presence of glucokinase (GK) in dog but not in boar, GK-transfected COS7 cells were incubated with either 10mM glucose or 10mM fructose. Incubation of GK-transfected cells with fructose decreased serine phosphorylation of cyclin A, ERK-2 and Hsp-70. In contrast, incubation of control COS7 cells with fructose increased serine phosphorylation of Cdk6, Cdk1/Cdc2, protein kinase C and Hsp-70. Incubation with glucose did not induce any significant effect. Our results indicate that monosaccharides act as signalling compounds in dog spermatozoa after ejaculation through changes in the phosphorylation levels of specific proteins. One of the factors that may be related to the action of sugars is the equilibrium of the total sperm hexokinase activity, in which the presence or absence of GK appears to be relevant.

Type 2 diabetes preventive effects with a 12-months sardine-enriched diet in elderly population with prediabetes: An interventional, randomized and controlled trial.

BACKGROUND: Fish could play a role in preventing type 2 diabetes (T2D) but there has been little specification about the type of fish and the preventive mechanism involved in its health claim. The sardine is a source of omega-3 and taurine that, in isolation or in synergy, would produce T2D-delaying through different molecular mechanism. HYPOTHESIS: The consumption of twice a week of sardine, during one year would reduce T2D-developing risk in a population with prediabetes (preDM) and old age. DESIGN: 152 subjects with fasting glucose between 100-124 mg/dL aged ≥65 yo were recruited from three primary care centers in Barcelona and were randomly distributed among two interventional groups: control group (CG) and sardine group (SG). Both groups received same T2D-prevention nutritional during a year but only SG had to add 200 g of sardine per week. All variables were collected before to start and at the end of the diet. (ClinicalTrials.gov: NCT03557541). RESULTS: 152 people were randomized into CG (n=77) and SG (n=75) with 18 and 12 drop outs respectively. Subjects in SG, significantly compared to CG, decreased percentage classified-individuals in a very high risk group to develop T2D according to FINDRISC (p=0.035). In addition to increasing HDL-cholesterol and adiponectin and decreasing triglycerides (p<0.05) and blood pressure (<0.05), SG showed a lower HOMA-IR (p=0.032). The consumption of sardine characteristics nutrients as omega-3, EPA and DHA, vitamin D, fluorine and taurine were higher for SG (p<0.05). These results agreed with the increased of taurine, fatty acid (FA) omega-3 and bile acids circulating metabolites (p<0.05). Changes erythrocyte membrane FA were detected only in SG with a decrease of 5 omega-6 FA (p<0.001) and an increase of 3 omega-3 FA types (p<0.001). CONCLUSION: We conclude that a year T2D-prevention diet with sardine supplementation has a greater protective effect against developing T2D and CV events.

Fatty liver index as a predictor for type 2 diabetes in subjects with normoglycemia in a nationwide cohort study.

Our aim was to evaluate whether fatty liver index (FLI) is associated with the risk of type 2 diabetes (T2DM) development within the Spanish adult population and according to their prediabetes status; additionally, to examine its incremental predictive value regarding traditional risk factors. A total of 2260 subjects (Prediabetes: 641 subjects, normoglycemia: 1619 subjects) from the Di@bet.es cohort study were studied. Socio-demographic, anthropometric, clinical data and survey on habits were recorded. An oral glucose tolerance test was performed and fasting determinations of glucose, lipids and insulin were made. FLI was calculated and classified into three categories: Low (< 30), intermediate (30-60) and high (> 60). In total, 143 people developed diabetes at follow-up. The presence of a high FLI category was in all cases a significant independent risk factor for the development of diabetes. The inclusion of FLI categories in prediction models based on different conventional T2DM risk factors significantly increase the prediction power of the models when all the population was considered. According to our results, FLI might be considered an early indicator of T2DM development even under normoglycemic condition. The data also suggest that FLI could provide additional information for the prediction of T2DM in models based on conventional risk factors.

The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker.

AIMS/HYPOTHESIS: Transmembrane protein 27 (TMEM27) is a membrane protein cleaved and shed by pancreatic beta cells that has been proposed as a beta cell mass biomarker. Despite reports of its possible role in insulin exocytosis and cell proliferation, its function in beta cells remains controversial. We aimed to characterise the function of TMEM27 in islets and its potential use as a beta cell mass biomarker. METHODS: To determine TMEM27 function, we studied TMEM27 gene expression and localisation in human healthy and diabetic islets, the correlation of its expression with cell cycle and insulin secretion genes in human islets, its expression in tungstate-treated rats, and the effects of its overproduction on insulin secretion and proliferation in a beta cell line and islets. To elucidate its utility as a beta cell mass biomarker, we studied TMEM27 cleavage in a beta cell line, islets and primary proximal tubular cells. RESULTS: TMEM27 mRNA levels in islets are lower in diabetic donors than in controls. Its gene expression correlates with that of insulin and SNAPIN in human islets. TMEM27 expression is downregulated in islets of tungstate-treated rats, which exhibit decreased insulin secretion and increased proliferation. TMEM27 overproduction in a beta cell line and islets significantly enhanced glucose-induced insulin secretion, with modest or no effects on proliferation. Finally, TMEM27 is cleaved and shed by renal proximal tubular cells and pancreatic islets. CONCLUSIONS/INTERPRETATION: Our data support a role for TMEM27 in glucose-induced insulin secretion but not in cell proliferation. The finding that its cleavage is not specific to beta cells challenges the current support for its use as a potential beta cell mass biomarker.

Proof-of-concept trial on the efficacy of sodium tungstate in human obesity.

AIM: Considering the poor long-term success of current dietary and pharmacological interventions, we aimed to evaluate the potential effect of sodium tungstate in the treatment of grade I and II obesity (ClinicalTrials.gov identifier: NCT00555074). METHODS: Prospective, randomized, placebo-controlled, double-blind, proof-of-concept study was carried out. Following a 2-week lead-in period, 30 obese (body mass index, BMI 30.0-39.9 kg/m(2)), non-diabetic subjects were randomized to receive either sodium tungstate (100 mg bid) or placebo for 6 weeks. The primary study endpoint was the absolute change in body weight relative to the time of randomization. RESULTS: Treatment with sodium tungstate [-0.135 ± 0.268 kg (95% CI -0.686 to +0.416 kg)] was not associated with a significant weight loss compared to placebo [-0.063 ± 0.277 kg (95% CI -0.632 to +0.507 kg)] (p = 0.854). Likewise, treatment with sodium tungstate was not associated with significant changes in fat mass (DEXA), resting energy expenditure (indirect calorimetry) or caloric consumption (3-day food records). CONCLUSION: Our data do not support sodium tungstate as a pharmacological agent in the treatment of human obesity.

Healthy dietary pattern and their corresponding gut microbiota profile are linked to a lower risk of type 2 diabetes, independent of the presence of obesity.

BACKGROUND: Prediabetes and old age are both high risk factors for developing Type 2 Diabetes (T2D), while obesity is one of the most important factors triggering the disease. Nutritional interventions are the most effective tool for preventing T2D, as they improve different biochemical and anthropometric outcomes and growth-promoting/inhibiting gut microbiota populations. However, to date there are no specific dietary recommendations to stop the development of T2D in elderly groups, for whom hypocaloric diets and other commonly used weight-loss programs could be considered dangerous. The objective of our study, thus, was to understand the impact of dietary patterns on T2D risk as related to gut microbiota profile in obese and non-obese elderly prediabetic subjects. METHODS: A cross-sectional study was performed in 182 subjects ≥65 years old with prediabetes, divided into obese (OB) or non-obese (NOB) subgroups, and their risk of developing T2D was measured according to FINDRISK score and biochemical parameters. Also, clusters into different dietary patterns in each group by PCA analysis was related with gut microbiota, which was analyzed from stool samples by qPCR. The creation of clusters was used to re-evaluate T2D risk. RESULTS: OB was at higher risk of developing T2D and showed worse metabolic outcomes. Unhealthier and healthier dietary pattern clusters were observed for both OB (OB-6 and OB-5 respectively) and NOB (NOB-2 and NOB-3 respectively) groups. Results obtained from the gut microbiota showed that only Prevotella was higher in NOB, but when comparisons were made between clusters, a clear relation with dietary pattern was observed; showing in healthier dietary clusters a decrease in Prevotella, an increase of Faecalibacterium prausnitzii and an increase in lactic acid bacteria. T2D risk was greater in the obese group between unhealthier dietary clusters. No difference between healthier dietary clusters was observed. CONCLUSION: A healthy dietary pattern and the growth-promoting beneficial and growth-inhibiting disadvantageous gut microbiota populations linked to it provide protection against the development of T2D in an obese population with advanced age and preDM.

Incidence of diabetes mellitus in Spain as results of the nation-wide cohort di@bet.es study.

Our aim was to determine the incidence of type 2 diabetes mellitus in a nation-wide population based cohort from Spain (di@bet.es study). The target was the Spanish population. In total 5072 people older than 18 years,were randomly selected from all over Spain). Socio-demographic and clinical data, survey on habits (physical activity and food consumption) and weight, height, waist, hip and blood pressure were recorder. A fasting blood draw and an oral glucose tolerance test were performed. Determinations of serum glucose were made. In the follow-up the same variables were collected and HbA1c was determined. A total of 2408 subjects participated in the follow-up. In total, 154 people developed diabetes (6.4% cumulative incidence in 7.5 years of follow-up). The incidence of diabetes adjusted for the structure of age and sex of the Spanish population was 11.6 cases/1000 person-years (IC95% = 11.1-12.1). The incidence of known diabetes was 3.7 cases/1000 person-years (IC95% = 2.8-4.6). The main risk factors for developing diabetes were the presence of prediabetes in cross-sectional study, age, male sex, obesity, central obesity, increase in weight, and family history of diabetes. This work provides data about population-based incidence rates of diabetes and associated risk factors in a nation-wide cohort of Spanish population.

Role of iduronate-2-sulfatase in glucose-stimulated insulin secretion by activation of exocytosis.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme expressed in pancreatic islets responsible for the degradation of proteoglycans such as perlecan and dermatan sulfate. Previous findings of our group demonstrated the involvement of IDS in the normal pathway of lysosomal degradation of secretory peptides, suggesting a role of this enzyme in beta-cell secretory functionality. The present study was undertaken to characterize the effect of IDS overexpression on insulin release. INS1E cells were transiently transfected with a construct encoding human IDS (hIDS). hIDS overexpression was associated with a gain of function detected by a reduction in heparan sulfate content. hIDS potentiated the glucose-stimulated insulin secretory response compared with controls (61%) with no changes in insulin mRNA levels or insulin peptide content. Results on quantification of the exocytotic process showed a significant increase in hIDS-transfected cells compared with controls. Furthermore, ultramorphological analysis demonstrated an increase in the number of granules in the immediate vicinity of the plasma membrane in hIDS-transfected cells and a decrease in total vesicles per square micrometer. hIDS overexpression induced phosphorylation of protein kinase C (PKC) alpha and its newly myristoylated alanine-rich C kinase substrate, MARCKS. We conclude that IDS has a role in glucose-stimulated insulin secretion via a mechanism that involves the activation of exocytosis through phosphorylation of PKCalpha and MARCKS.

Dual effects of sodium tungstate on adipocyte biology: inhibition of adipogenesis and stimulation of cellular oxygen consumption.

OBJECTIVE: We have recently shown the in vivo anti-obesity effects of sodium tungstate. In this study, we investigate the in vitro effects of sodium tungstate on adipocyte differentiation and function. METHODS: 3T3-F442A cells were allowed to differentiate in the presence of sodium tungstate, and were analyzed for triglyceride (TG) accumulation, adipocyte differentiation and mitochondrial oxygen consumption. RESULTS: Sodium tungstate treatment of adipose cells decreased TG accumulation and adipocyte differentiation. Expression of key genes for adipocyte function (aP2, ACC, fatty acid synthase (FAS) and lipoprotein lipase (LPL)) and differentiation (CCAAT enhancer-binding protein (C/EBP)alpha and peroxisome proliferator-activated receptor gamma (PPARgamma)) was reduced by sodium tungstate, whereas C/EBPbeta isoform LIP expression level was increased. TG accumulation and changes in C/EBPbeta expression were partially recovered by inactivating the erk1/2 pathway. Finally, tungstate treatment increased the oxygen consumption of adipose cells without changes in the expression of oxidative genes. CONCLUSIONS: Sodium tungstate inhibits adipocyte differentiation by promoting the translation of LIP, a master dominant-negative regulator of this process, and regulates the mitochondrial oxygen consumption of adipose cells. These effects contribute to the anti-obesity activity of sodium tungstate and confirm its potential as a powerful alternative for the treatment of obesity.

Correction: The anti-metastatic activity of collagenase-2 in breast cancer cells is mediated by a signaling pathway involving decorin and miR-21.

The original microRNA hybridization data for this article, which has been available for the scientific community upon request, has now been deposited in the GEO repository under accession number GSE124432.

Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4.

AIMS/HYPOTHESIS: To investigate the mechanism by which human islet amyloid polypeptide (hIAPP) fibril formation results in calcium influx across the plasma membrane of pancreatic beta cells, and its association with apoptosis. METHODS: Cytoplasmic intracellular calcium concentrations ([Ca(2+)](i)) were monitored for 2 h as the 340/380 nm fluorescence ratio in fura-2 loaded cells of the MIN6 mouse pancreatic beta cell line. Cell morphology was evaluated by transmission electron microscopy, and viability by FACS. RESULTS: hIAPP (10 micromol/l) increased [Ca(2+)](i) in 21% of MIN6 cells in standard buffer, and in 8% of cells in Na(+)-free buffer. Transient receptor potential (TRP) channel inhibitors (gadolinium and ruthenium red) prevented the [Ca(2+)](i) rise under both conditions, whilst nifedipine was only effective in the presence of Na(+). hIAPP increased apoptosis in both insulinoma cells and islets in primary culture, and cell viability was partially rescued by ruthenium red (p < 0.001). By RT-PCR, we detected expression of the mechanosensitive TRP cation channel subfamily V member 4 (Trpv4) in MIN6 cells and mouse pancreas. Small interference RNA against Trpv4 prevented hIAPP-induced [Ca(2+)](i) rises, decreased hIAPP-triggered expression of the endoplasmic reticulum (ER) stress response, and reduced hIAPP-triggered cell death by 50% (p < 0.05). CONCLUSIONS/INTERPRETATION: Alterations in [Ca(2+)](i) play a key role in hIAPP-induced beta cell cytotoxicity. By electron microscopy, we detected extracellular hIAPP aggregates adjacent to irregular invaginated regions of the plasma membrane. We propose that TRPV4 channels may sense physical changes in the plasma membrane induced by hIAPP aggregation, enabling Ca(2+) entry, membrane depolarisation and activation of L-type Ca(2+) channels. Decreasing the activity of TRPV4 prevented hIAPP-induced [Ca(2+)](i) changes, reduced hIAPP-triggered ER stress and improved cell viability.

The effect of the availability of inhaled insulin on glycaemic control in patients with Type 2 diabetes failing on oral therapy: the evaluation of Exubera as a therapeutic option on insulin initiation and improvement in glycaemic control in clinical practice (EXPERIENCE) trial.

AIM: To examine the impact of inhaled human insulin (Exubera, EXU) on patient or physician willingness to adopt insulin after oral glucose-lowering agent failure. METHODS: During a randomized controlled trial in primary, secondary and tertiary care in Europe and North America, 739 patients using >or= 2 oral glucose-lowering agents with glycated haemoglobin (HbA(1c)) >or= 8.0% were assigned to two treatment groups: Group 1 (standard care with the option of EXU) or Group 2 (standard care only). Standard care included adjusting oral therapy (optimizing current regimen or adding/omitting agents) and/or initiating subcutaneous (s.c.) insulin. The primary endpoint was difference in HbA(1c) between randomized groups at 26 weeks. Secondary outcomes included differences in the rate of uptake of insulin therapy, proportion achieving satisfactory glycaemic control, treatment satisfaction and safety outcomes. RESULTS: At baseline, insulin was initiated by more [odds ratio 6.0; 95% confidence interval (CI) 4.2 to 8.8; P < 0.0001] patients in Group 1 (86.2%; 76.7% EXU plus 9.5% s.c.) than Group 2 (50.7%; s.c. insulin only). At 26 weeks, mean (sd) changes in HbA(1c) from baseline were -2.0% (1.2%) and -1.7% (1.3%) in Groups 1 and 2, respectively, a difference of -0.2% (95% CI: -0.1% to -0.4%; P = 0.004). In Group 1, 45% of patients achieved an HbA(1c)