Mitofusin 2 in POMC neurons connects ER stress with leptin resistance and energy imbalance.

Mitofusin 2 (MFN2) plays critical roles in both mitochondrial fusion and the establishment of mitochondria-endoplasmic reticulum (ER) interactions. Hypothalamic ER stress has emerged as a causative factor for the development of leptin resistance, but the underlying mechanisms are largely unknown. Here, we show that mitochondria-ER contacts in anorexigenic pro-opiomelanocortin (POMC) neurons in the hypothalamus are decreased in diet-induced obesity. POMC-specific ablation of Mfn2 resulted in loss of mitochondria-ER contacts, defective POMC processing, ER stress-induced leptin resistance, hyperphagia, reduced energy expenditure, and obesity. Pharmacological relieve of hypothalamic ER stress reversed these metabolic alterations. Our data establish MFN2 in POMC neurons as an essential regulator of systemic energy balance by fine-tuning the mitochondrial-ER axis homeostasis and function. This previously unrecognized role for MFN2 argues for a crucial involvement in mediating ER stress-induced leptin resistance.

The Mitochondria-Targeted Antioxidant MitoQ Modulates Mitochondrial Function and Endoplasmic Reticulum Stress in Pancreatic ß Cells Exposed to Hyperglycaemia.

BACKGROUND/AIMS: Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in ß cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to ß cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic ß cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions. METHODS: We incubated the pancreatic ß cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O2 consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting). RESULTS: MitoQ increased insulin secretion and prevented the enhancement of ROS production and O2 consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions. CONCLUSION: Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic ß cell function.

[Spanish validation of Game Addiction Scale for Adolescents (GASA)]. / Validación española de la Escala de Adicción a Videojuegos para Adolescentes (GASA).

OBJECTIVE: The aim of this study is to adapt and validate the Game Addiction Scale for Adolescents (GASA) to the Spanish youth population. DESIGN: Cultural adaptation and validation study. SETTING: Secondary Education centres. PARTICIPANTS: Two independent studies were conducted on a group of 466 young people with a mean age of 15.27 years (13-18, SD: 1.83) and 48.7% ♀ and on another group of 566, with a mean age of 21.24 years (19-26; SD: 1.86) 44.1% ♀. MEASUREMENTS: Addiction to video games (GASA); Game behavior (Game habits usage questionnaire), Impulsiveness (Plutchik Impulsiveness Scale) and Group Pressure (Ad hoc questionnaire). RESULTS: The Spanish version of GASA has shown good reliability and true to the original scale factor structure. As regards criterion validity, GASA scores are significantly different according to four criteria related to problem gambling: Game intensity and frequency, impulsiveness, and peer pressure. CONCLUSIONS: The results show that the adapted version GASA is adequate and a valid tool for assessing problematic gaming behaviour.

Design and validation of a Cannabis Use Intention Questionnaire (CUIQ) for adolescents. / Diseño y validación de una escala de intención de consumo de cannabis (CUIQ) para adolescentes.

In Spain, one in four 14 to 18-year-old adolescents has used cannabis during the last twelve months. Demand for treatment has increased in European countries. These facts have prompted the development of preventive interventions that require screening tools in order to identify the vulnerable population and to properly asses the efficacy of such interventions. The Theory of Planned Behaviour (TPB), widely used to forecast behavioural intention, has also demonstrated a good predictive capacity in addictions. The aim of this study is to design and validate a Cannabis Use Intention Questionnaire (CUIQ) based on TPB. 1,011 teenagers answered a set of tests to assess attitude towards use, subjective norms, self-efficacy towards non-use, and intention to use cannabis. CUIQ had good psychometric properties. Structural Equation Modelling results confirm the predictive model on intention to use cannabis in the Spanish adolescent sample, classified as users and non-users, explaining 40% of variance of intention to consume. CUIQ is aimed at providing a better understanding of the psychological processes that lead to cannabis use and allowing the evaluation of programmes. This can be particularly useful for improving the design and implementation of selective prevention programmes.

En España, uno de cada cuatro jóvenes de 14 a 18 años declara haber consumido cannabis en el último año. La demanda de tratamiento ha aumentado en todos los países europeos. Ello ha motivado el desarrollo de intervenciones preventivas que requieren instrumentos para el cribado de la población en riesgo y la evaluación de la prevención. La Teoría de la Acción Planificada (TAP), ampliamente utilizada para predecir las intenciones conductuales, ha mostrado una buena capacidad predictiva en el campo de las adicciones. El objetivo del presente trabajo es diseñar y validar un Cuestionario de Intención de Consumo de Cannabis (CUIQ, Cannabis Use Intention Questionnaire) basado en la TAP. 1011 adolescentes completaron una batería de cuestionarios que se compone de cuatro subescalas: actitud hacia el consumo, norma subjetiva, autoeficacia hacia la abstinencia e intención de consumo. El Cuestionario CUIQ obtuvo buenas características psicométricas. Las ecuaciones estructurales confirmaron el modelo predictivo sobre la intención de consumo en adolescentes españoles (consumidores y no consumidores), llegando a explicar el 40% de la varianza. El CUIQ tiene como objetivo una mejor comprensión del proceso psicológico que conduce al consumo de cannabis y permitir la evaluación de programas. Esto puede ser especialmente útil para mejorar el diseño e implementación de programas de prevención selectiva.

Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis.

AIMS/HYPOTHESIS: Glycogen accumulation occurs in beta cells of diabetic patients and has been proposed to partly mediate glucotoxicity-induced beta cell dysfunction. However, the role of glycogen metabolism in beta cell function and its contribution to diabetes pathophysiology remain poorly understood. We investigated the function of beta cell glycogen by studying glucose homeostasis in mice with (1) defective glycogen synthesis in the pancreas; and (2) excessive glycogen accumulation in beta cells. METHODS: Conditional deletion of the Gys1 gene and overexpression of protein targeting to glycogen (PTG) was accomplished by Cre-lox recombination using pancreas-specific Cre lines. Glucose homeostasis was assessed by determining fasting glycaemia, insulinaemia and glucose tolerance. Beta cell mass was determined by morphometry. Glycogen was detected histologically by periodic acid-Schiff's reagent staining. Isolated islets were used for the determination of glycogen and insulin content, insulin secretion, immunoblots and gene expression assays. RESULTS: Gys1 knockout (Gys1 (KO)) mice did not exhibit differences in glucose tolerance or basal glycaemia and insulinaemia relative to controls. Insulin secretion and gene expression in isolated islets was also indistinguishable between Gys1 (KO) and controls. Conversely, despite effective glycogen overaccumulation in islets, mice with PTG overexpression (PTG(OE)) presented similar glucose tolerance to controls. However, under fasting conditions they exhibited lower glycaemia and higher insulinaemia. Importantly, neither young nor aged PTG(OE) mice showed differences in beta cell mass relative to age-matched controls. Finally, a high-fat diet did not reveal a beta cell-autonomous phenotype in either model. CONCLUSIONS/INTERPRETATION: Glycogen metabolism is not required for the maintenance of beta cell function. Glycogen accumulation in beta cells alone is not sufficient to trigger the dysfunction or loss of these cells, or progression to diabetes.

Effects of sardine-enriched diet on metabolic control, inflammation and gut microbiota in drug-naïve patients with type 2 diabetes: a pilot randomized trial.

BACKGROUND: Nutrition therapy is the cornerstone of treating diabetes mellitus. The inclusion of fish (particularly oily fish) at least two times per week is recommended by current international dietary guidelines for type 2 diabetes. In contrast to a large number of human studies examining the effects of oily fish on different cardiovascular risk factors, little research on this topic is available in patients with type 2 diabetes. The aims of this pilot study were to investigate the effects of a sardine-enriched diet on metabolic control, adiponectin, inflammatory markers, erythrocyte membrane fatty acid (EMFA) composition, and gut microbiota in drug-naïve patients with type 2 diabetes. METHODS: 35 drug-naïve patients with type 2 diabetes were randomized to follow either a type 2 diabetes standard diet (control group: CG), or a standard diet enriched with 100 g of sardines 5 days a week (sardine group: SG) for 6 months. Anthropometric, dietary information, fasting glycated hemoglobin, glucose, insulin, adiponectin, inflammatory markers, EMFA and specific bacterial strains were determined before and after intervention. RESULTS: There were no significant differences in glycemic control between groups at the end of the study. Both groups decreased plasma insulin (SG: -35.3%, P = 0.01, CG: -22.6%, P = 0.02) and homeostasis model of assessment--insulin resistance (HOMA-IR) (SG: -39.2%, P = 0.007, CG: -21.8%, P = 0.04) at 6-months from baseline. However only SG increased adiponectin in plasma compared to baseline level (+40.7%, P = 0.04). The omega-3 index increased 2.6% in the SG compared to 0.6% in the CG (P = 0.001). Both dietary interventions decreased phylum Firmicutes (SG and CG: P = 0.04) and increased E. coli concentrations (SG: P = 0.01, CG: P = 0.03) at the end of the study from baseline, whereas SG decreased Firmicutes/Bacteroidetes ratio (P = 0.04) and increased Bacteroides-Prevotella (P = 0.004) compared to baseline. CONCLUSIONS: Although enriching diet with 100 g of sardines 5 days a week during 6 months to a type 2 diabetes standard diet seems to have neutral effects on glycemic control in drug-naïve patients with type 2 diabetes, this nutritional intervention could have beneficial effects on cardiovascular risk. Furthermore, both dietary interventions decreased HOMA-IR and altered gut microbiota composition of drug-naïve patients with type 2 diabetes. TRIAL REGISTRATION: Trial number and name of the registry: NCT02294526, ClinicalTrials.gov.

Diversity of Y-chromosomal and mtDNA Markers Included in Mediscope Chip within Two Albanian Subpopulations from Croatia and Kosovo: Preliminary Data.

The aim of this preliminary study is to analyze genetic specificity of Kosovo Albanians comparing with neighboring populations using new genetic tool - MEDISCOPE gene chip, to investigate the feasibility of this approach. We collected 37 DNA samples (9 Croats, 17 Albanians from Croatia and 11 Albanians from Kosovo) from unrelated males born in Croatia and Kosovo. Additionally, samples were expanded with female individuals and mtDNA analysis included a total of 61 samples (15 Croats, 23 Albanians from Croatia and 23 Albanians from Kosovo). This pilot study suggests that the usage of the MEDISCOPE chip could be recognized as an efficient tool within recognition of the population genetic specificity even within extremely small sample size.

Circulating SFRP5 levels are elevated in drug-naïve recently diagnosed type 2 diabetic patients as compared with prediabetic subjects and controls.

BACKGROUND: Secreted frizzled-related protein 5 (SFRP5) has been linked to obesity. Results are conflicting regarding its association with type 2 diabetes (T2D) in humans. We aimed to investigate circulating SFRP5 in prediabetes and T2D and its potential association with parameters of insulin resistance and beta-cell function. METHODS: We studied 70 drug-naïve T2D patients, 70 prediabetic subjects and 70 controls. All subjects were body mass index matched to the T2D patients and overweight or obese. SFRP5, hormones and cytokines levels were measured by ELISA. RESULTS: Serum SFRP5 levels were elevated in T2D patients as compared with prediabetic subjects (median 15.6, interquartile range [9-24.5] ng/mL vs 9.8 [5-14.2] ng/mL, p < 0.001, respectively) and controls (15.6 [9-24.5] ng/mL vs 10.4 [6.7-16.6] ng/mL, P < 0.001, respectively). No differences were found in serum SFRP5 levels between prediabetic subjects and controls (9.8 [5-14.2] ng/mL vs 10.4 [6.7-16.6] ng/mL, p = 0.472, respectively). After adjusting for potential confounders (age, gender, body mass index, triglycerides, high-density lipoprotein cholesterol and blood pressure), T2D was still associated with higher values of SFRP5 as compared with prediabetes in multinomial logistic regression analysis (fully adjusted odds ratio 3.50, 95% confidence interval 1.40-8.79, p = 0.008). The association was more subtle when comparing T2D with normal glucose tolerance state (fully adjusted odds ratio 2.18, 95% confidence interval 0.91-5.21, p = 0.078). CONCLUSIONS: Circulating SFRP5 levels were independently associated with T2D as compared with prediabetes and normal glucose tolerance state.

Integrative analysis reveals novel pathways mediating the interaction between adipose tissue and pancreatic islets in obesity in rats.

AIMS/HYPOTHESIS: Comprehensive characterisation of the interrelation between the peripancreatic adipose tissue and the pancreatic islets promises novel insights into the mechanisms that regulate beta cell adaptation to obesity. Here, we sought to determine the main pathways and key molecules mediating the crosstalk between these two tissues during adaptation to obesity by the way of an integrated inter-tissue, multi-platform analysis. METHODS: Wistar rats were fed a standard or cafeteria diet for 30 days. Transcriptomic variations by diet in islets and peripancreatic adipose tissue were examined through microarray analysis. The secretome from peripancreatic adipose tissue was subjected to a non-targeted metabolomic and proteomic analysis. Gene expression variations in islets were integrated with changes in peripancreatic adipose tissue gene expression and protein and metabolite secretion using an integrated inter-tissue pathway and network analysis. RESULTS: The highest level of data integration, linking genes differentially expressed in both tissues with secretome variations, allowed the identification of significantly enriched canonical pathways, such as the activation of liver/retinoid X receptors, triacylglycerol degradation, and regulation of inflammatory and immune responses, and underscored interaction network hubs, such as cholesterol and the fatty acid binding protein 4, which were unpredicted through single-tissue analysis and have not been previously implicated in the peripancreatic adipose tissue crosstalk with beta cells. CONCLUSIONS/INTERPRETATION: The integrated analysis reported here allowed the identification of novel mechanisms and key molecules involved in peripancreatic adipose tissue interrelation with beta cells during the development of obesity; this might help the development of novel strategies to prevent type 2 diabetes.

Characterization of the transcriptional activity of the basic helix-loop-helix (bHLH) transcription factor Atoh8.

The atonal-related Neurogenin/NeuroD family of basic helix-loop-helix (bHLH) transcription factors comprises potent inducers of neuronal and endocrine differentiation programs in the nervous and digestive system. Atonal homolog 8 (Atoh8) displays high similarity in the bHLH domain with NeuroD proteins. Yet, available evidences indicate that Atoh8 has distinctive features including a ubiquitous expression pattern in embryonic tissues and the ability to inhibit differentiation. To gain insights into Atoh8 function, we aimed at identifying Atoh8 targets and investigated the effects of Atoh8 on global gene expression patterns in pancreatic mPAC cells, a model of bHLH-dependent endocrine differentiation. Our data reveal that Atoh8 is a weak transcriptional activator and does not exhibit proendocrine activity. Conversely, it blocks the induction of a reduced group of gene targets of the atonal-related proendocrine factor Neurogenin3. We show that Atoh8 lacks a transactivation domain and possesses intrinsic repressor activity that depends on a conserved Proline-rich domain. Atoh8 binds the ubiquitous E protein E47 and its ability to repress transcription may partly result from its ability to inhibit E47/E47 and Neurogenin3/E47 dimer activities. These results reveal distinctive transcriptional properties of Atoh8 within the atonal-related bHLH family that may be associated with the acquisition of new biological functions.

Tungstate promotes ß-cell survival in Irs2-/- mice.

Pancreatic ß-cells play a central role in type 2 diabetes (T2D) development, which is characterized by the progressive decline of the functional ß-cell mass that is associated mainly with increased ß-cell apoptosis. Thus, understanding how to enhance survival of ß-cells is key for the management of T2D. The insulin receptor substrate-2 (IRS-2) protein is pivotal in mediating the insulin/IGF signaling pathway in ß-cells. In fact, IRS-2 is critically required for ß-cell compensation in conditions of increased insulin demand and for ß-cell survival. Tungstate is a powerful antidiabetic agent that has been shown to promote ß-cell recovery in toxin-induced diabetic rodent models. In this study, we investigated whether tungstate could prevent the onset of diabetes in a scenario of dysregulated insulin/IGF signaling and massive ß-cell death. To this end, we treated mice deficient in IRS2 (Irs2(-/-)), which exhibit severe ß-cell loss, with tungstate for 3 wk. Tungstate normalized glucose tolerance in Irs2(-/-) mice in correlation with increased ß-cell mass, increased ß-cell replication, and a striking threefold reduction in ß-cell apoptosis. Islets from treated Irs2(-/-) exhibited increased phosphorylated Erk1/2. Interestingly, tungstate repressed apoptosis-related genes in Irs2(-/-) islets in vitro, and ERK1/2 blockade abolished some of these effects. Gene expression profiling showed evidence of a broad impact of tungstate on cell death pathways in islets from Irs2(-/-) mice, consistent with reduced apoptotic rates. Our results support the finding that ß-cell death can be arrested in the absence of IRS2 and that therapies aimed at reversing ß-cell mass decline are potential strategies to prevent the progression to T2D.

Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle.

Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin-stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin-resistant cells. Melatonin treatment increases CAMKII and p-CREB but had no effect on p-AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin-induced increase in p-AKT in palmitic acid-treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p-AKT, mitochondrial respiration, and p-CREB and PGC-1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB-PGC-1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.

Variable patterns of obesity and cardiometabolic phenotypes and their association with lifestyle factors in the Di@bet.es study.

BACKGROUND AND AIM: Prevalence rates of "metabolically healthy obese" (MHO) subjects vary depending on the criteria used. This study examined the prevalence and characteristics of MHO subjects and metabolically abnormal normal-weight subjects and compared the findings with the NHANES 1999-2004 study. The aims of the present study were, first, to determine the prevalence rates of MHO and MNHNO subjects using the same criteria as those of the National Health and Nutrition Examination Survey (NHANES) (1999-2004) study, and second to compare the prevalence and correlates of obese subjects who are resistant to the development of adiposity-associated cardiometabolic abnormalities (CA) and normal-weight individuals who display cardiometabolic risk factor clustering between the Spanish and the US populations. METHODS AND RESULTS: Di@bet.es study is a national, cross-sectional population-based survey of 5728 adults conducted in 2009-2010. Clinical, metabolic, sociodemographic, and anthropometric data and information about lifestyle habits, such as physical activity, smoking habit, alcohol intake and food consumption, were collected. Subjects were classified according to their body mass index (BMI) (normal-weight, <25 kg/m(2); overweight, 25-29.9 kg/m(2); and obese, >30 kg/m(2)). CA included elevated blood pressure; elevated levels of triglycerides, fasting glucose, and high-sensitivity C-reactive protein (hs-CRP); and elevated homeostasis model assessment of insulin resistance (HOMA-IR) value and low high-density lipoprotein cholesterol (HDL-c) level. Two phenotypes were defined: metabolically healthy phenotype (0-1 CA) and metabolically abnormal phenotype (≥2 CA). The prevalence of metabolically abnormal normal-weight phenotype was slightly lower in the Spanish population (6.5% vs. 8.1%). The prevalence of metabolically healthy overweight and MHO subjects was 20.9% and 7.0%, respectively, while in NHANES study it was 17.9% and 9.7%, respectively. Cigarette smoking was associated with CA in each phenotype, while moderate physical activity and moderate alcohol intake were associated with being metabolically healthy. Olive oil intake was negatively associated with the prevalence of CA. CONCLUSIONS: Smoking, physical activity level, and alcohol intake contribute to the explanation of the prevalence of CA in the Spanish population, as in the US population. However in Spain, olive oil intake contributes significantly to the explanation of the variance in the prevalence of CA.

Targeting type 2 diabetes: lessons from a knockout model of insulin receptor substrate 2.

Insulin receptor substrate 2 (IRS2) is a widely expressed protein that regulates crucial biological processes including glucose metabolism, protein synthesis, and cell survival. IRS2 is part of the insulin - insulin-like growth factor (IGF) signaling pathway and mediates the activation of the phosphotidylinositol 3-kinase (PI3K)-Akt and the Ras-mitogen-activated protein kinase (MAPK) cascades in insulin target tissues and in the pancreas. The best evidence of this is that systemic elimination of the Irs2 in mice (Irs2(-/-)) recapitulates the pathogenesis of type 2 diabetes (T2D), in that diabetes arises as a consequence of combined insulin resistance and beta-cell failure. Indeed, work using this knockout mouse has confirmed the importance of IRS2 in the control of glucose homeostasis and especially in the survival and function of pancreatic beta-cells. These studies have shown that IRS2 is critically required for beta-cell compensation in conditions of increased insulin demand. Importantly, islets isolated from T2D patients exhibit reduced IRS2 expression, which supports the likely contribution of altered IRS2-dependent signaling to beta-cell failure in human T2D. For all these reasons, the Irs2(-/-) mouse has been and will be essential for elucidating the inter-relationship between beta-cell function and insulin resistance, as well as to delineate therapeutic strategies to protect beta-cells during T2D progression.

Downregulation of Sfrp5 promotes beta cell proliferation during obesity in the rat.

AIMS/HYPOTHESIS: During obesity, the increment in beta cell mass in response to the rising demand for insulin is essential to maintain normal glucose homeostasis. However, the precise cellular and molecular mechanisms involved in beta cell mass plasticity remain poorly understood. The Wnt signalling pathway has been suggested as one possible modulator of beta cell proliferation, which represents the principal process involved in beta cell mass expansion. Here, we sought to determine the mechanisms involved in beta cell mass proliferation using diet-induced obese rats. METHODS: Wistar rats aged 8 weeks old were fed a standard or cafeteria diet. Global transcriptomic analysis of pancreatic rat islets was performed using microarray analysis. Genetic loss-of-function approaches were performed in dispersed primary rat islets and the beta cell line INS1E. Gene expression was measured by real-time PCR, protein levels by immunoblot analysis, proliferation rates by ELISA and apoptosis by flow cytometry. RESULTS: Sfrp5, coding for secreted frizzled-related protein 5, is downregulated in the pancreatic islets of cafeteria-diet-fed rats as well as in the pancreatic islets of human obese patients. We demonstrate that silencing Sfrp5 increases beta cell proliferation, which correlates with activation of Wnt signalling and enhanced levels of proliferation markers. In addition, we show that expression of Sfrp5 in beta cells is modulated by IGF binding protein 3 (IGFBP3) secreted from visceral adipose tissue. CONCLUSIONS/INTERPRETATION: Together, these findings reveal an important role for SFRP5 and Wnt signalling in the regulation of beta cell proliferation in obesity.

Factors affecting levels of urinary albumin excretion in the general population of Spain: the Di@bet.es study.

The present study was undertaken to examine the prevalence of urinary ACR (albumin/creatinine ratio) >30 mg/g and the associated clinical and environmental factors in a representative sample of the population of Spain. Di@bet.es study is a national, cross-sectional population-based survey conducted in 2009-2010. Clinical, metabolic, socio-demographic, anthropometric data and information about lifestyle habit were collected. Those subjects without KDM (known diabetes mellitus) were given an OGTT (oral glucose tolerance test). Albumin and creatinine were measured in a urinary sample and ACR was calculated. The population prevalence of ACR >30 mg/g was 7.65% (adjusted for sex and age). The prevalence of ACR >30 mg/g increased with age (P<0.001). Subjects with carbohydrate metabolism disorders had a greater prevalence of ACR >30 mg/g but after being adjusted for age, sex and hypertension, was significant only in those subjects with UKDM (unknown diabetes mellitus) {OR (odd ratio), 2.07 [95% CI (confidence interval), 1.38-3.09]; P<0.001] and KDM [OR, 3.55 (95% CI, 2.63-4.80); P<0.001]. Prevalence of ACR >30 mg/g was associated with hypertension [OR, 1.48 (95% CI, 1.12-1.95); P=0.001], HOMA-IR (homoeostasis model assessment of insulin resistance) [OR, 1.47 (95% CI, 1.13-1.92); P≤0.01], metabolic syndrome [OR, 2.17 (95% CI, 1.72-2.72); P<0.001], smoking [OR, 1.40 (95% CI, 1.06-1.83); P≤0.05], physical activity [OR, 0.68 (95% CI, 0.54-0.88); P≤0.01] and consumption of fish [OR, 0.38 (95% CI, 0.18-0.78); P≤0.01]. This is the first study that reports the prevalence of ACR >30 mg/g in the Spanish population. The association between clinical variables and other potentially modifiable environmental variables contribute jointly, and sometimes interactively, to the explanation of prevalence of ACR >30 mg/g. Many of these risk factors are susceptible to intervention.

Direct reprogramming of human fibroblasts into insulin-producing cells using transcription factors.

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing ß cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create ß-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into ß-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a ß-cell fate. Reprogrammed cells exhibit ß-cell features including ß-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.

Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion.

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

In vivo diabetogenic action of CD4+ T lymphocytes requires Fas expression and is independent of IL-1 and IL-18.

CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic ß cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1ß has been described as a key cytokine involved in Fas upregulation on mouse ß cells. We addressed whether CD4(+) T cells require IL-1ß to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1ß-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1ß-deficient mice. Neither IL-1ß nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated ß-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1ß unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD ß cells in vivo.

Expression of TMEM16A and SLC4A4 in human pancreatic islets.

BACKGROUND/AIMS: Stimulation of insulin release by D-glucose is accompanied by Cl(-) and HCO(3)(-) efflux from pancreatic islet cells. The efflux of these anions may involve volume-regulated anion channels, including possibly TMEM16A, and the Na(+)-HCO(3)(-)-cotransporter SLC4A4. The present study was designed to explore the expression of both TMEM16A and SLC4A4 in human pancreatic islets. METHODS: Pancreases were obtained from human cadaveric donors. Immunodetection of TMEM16A and SLC4A4 was performed by immunohistochemistry on sections of fixed pancreas, while real-time PCR for the study of corresponding gene expression was performed on RNA extracted from both total pancreatic pieces and isolated pancreatic islets. RESULTS: RT-PCR yielded lower levels of SLC4A4 in isolated islets than in the total pancreas, whilst a mirror image prevailed for TMEM16A mRNA. Immunohistochemistry of human pancreas, however, indicated comparable immunostaining of SLC4A4 in insulin-producing cells and exocrine pancreatic cells, whilst that of TMEM16A appeared less pronounced in insulin-producing cells than in exocrine cells. CONCLUSION: The present findings support the view that, in humans like in rodent, the regulation of anion fluxes in insulin-producing cells may involve both SLC4A4 and TMEM16A.