Cystic fibrosis-related diabetes (CFRD) is a common complication for patients with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The cause of CFRD is unclear, but a commonly observed reduction in first-phase insulin secretion suggests defects at the beta cell level. Here we aimed to examine alpha and beta cell function in the Cftr tm1 EUR/F508del mouse model (C57BL/6J), which carries the most common human mutation in CFTR, the F508del mutation. CFTR expression, beta cell mass, insulin granule distribution, hormone secretion and single cell capacitance changes were evaluated using islets (or beta cells) from F508del mice and age-matched wild type (WT) mice aged 7–10 weeks. Granular pH was measured with DND-189 fluorescence. Serum glucose, insulin and glucagon levels were measured in vivo, and glucose tolerance was assessed using IPGTT. We show increased secretion of proinsulin and concomitant reduced secretion of C-peptide in islets from F508del mice compared to WT mice. Exocytosis and number of docked granules was reduced. We confirmed reduced granular pH by CFTR stimulation. We detected decreased pancreatic beta cell area, but unchanged beta cell number. Moreover, the F508del mutation caused failure to suppress glucagon secretion leading to hyperglucagonemia. In conclusion, F508del mice have beta cell defects resulting in (1) reduced number of docked insulin granules and reduced exocytosis and (2) potential defective proinsulin cleavage and secretion of immature insulin. These observations provide insight into the functional role of CFTR in pancreatic islets and contribute to increased understanding of the pathogenesis of CFRD.
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- Abstract: Diabetes x
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A Edlund, M Barghouth, M Hühn, M Abels, J S E Esguerra, I G Mollet, E Svedin, A Wendt, E Renström, E Zhang, N Wierup, B J Scholte, M Flodström-Tullberg and L Eliasson
S Schmidt, A Hommel, V Gawlik, R Augustin, N Junicke, S Florian, M Richter, D J Walther, D Montag, H-G Joost and A Schürmann
Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3 −/− morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells of Slc2a3 −/− embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.
Esther Nuñez-Durán, Belén Chanclón, Silva Sütt, Joana Real, Hanns-Ulrich Marschall, Ingrid Wernstedt Asterholm, Emmelie Cansby and Margit Mahlapuu
Characterising the molecular networks that negatively regulate pancreatic β-cell function is essential for understanding the underlying pathogenesis and developing new treatment strategies for type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of ectopic fat storage, meta-inflammation, and fibrosis in liver and skeletal muscle. Here, we assessed the role of STK25 in control of progression of non-alcoholic fatty pancreas disease in the context of chronic exposure to dietary lipids in mice. We found that overexpression of STK25 in high-fat-fed transgenic mice aggravated diet-induced lipid storage in the pancreas compared with that of wild-type controls, which was accompanied by exacerbated pancreatic inflammatory cell infiltration, stellate cell activation, fibrosis and apoptosis. Pancreas of Stk25 transgenic mice also displayed a marked decrease in islet β/α-cell ratio and alteration in the islet architecture with an increased presence of α-cells within the islet core, whereas islet size remained similar between genotypes. After a continued challenge with a high-fat diet, lower levels of fasting plasma insulin and C-peptide, and higher levels of plasma leptin, were detected in Stk25 transgenic vs wild-type mice. Furthermore, the glucose-stimulated insulin secretion was impaired in high-fat-fed Stk25 transgenic mice during glucose tolerance test, in spite of higher net change in blood glucose concentrations compared with wild-type controls, suggesting islet β-cell dysfunction. In summary, this study unravels a role for STK25 in determining the susceptibility to diet-induced non-alcoholic fatty pancreas disease in mice in connection to obesity. Our findings highlight STK25 as a potential drug target for metabolic disease.
Michael Rouse, Antoine Younès and Josephine M Egan
Resveratrol (RES) and curcumin (CUR) are polyphenols that are found in fruits and turmeric, and possess medicinal properties that are beneficial in various diseases, such as heart disease, cancer, and type 2 diabetes mellitus (T2DM). Results from recent studies have indicated that their therapeutic properties can be attributed to their anti-inflammatory effects. Owing to reports stating that they protect against β-cell dysfunction, we studied their mechanism(s) of action in β-cells. In T2DM, cAMP plays a critical role in glucose- and incretin-stimulated insulin secretion as well as overall pancreatic β-cell health. A potential therapeutic target in the management of T2DM lies in regulating the activity of phosphodiesterases (PDEs), which degrade cAMP. Both RES and CUR have been reported to act as PDE inhibitors in various cell types, but it remains unknown if they do so in pancreatic β-cells. In our current study, we found that both RES (0.1–10 μmol/l) and CUR (1–100 pmol/l)-regulated insulin secretion under glucose-stimulated conditions. Additionally, treating β-cell lines and human islets with these polyphenols led to increased intracellular cAMP levels in a manner similar to 3-isobutyl-1-methylxanthine, a classic PDE inhibitor. When we investigated the effects of RES and CUR on PDEs, we found that treatment significantly downregulated the mRNA expression of most of the 11 PDE isozymes, including PDE3B, PDE8A, and PDE10A, which have been linked previously to regulation of insulin secretion in islets. Furthermore, RES and CUR inhibited PDE activity in a dose-dependent manner in β-cell lines and human islets. Collectively, we demonstrate a novel role for natural-occurring polyphenols as PDE inhibitors that enhance pancreatic β-cell function.
Xuefeng Yang, Shuang Mei, Haihua Gu, Huailan Guo, Longying Zha, Junwei Cai, Xuefeng Li, Zhenqi Liu and Wenhong Cao
We have previously shown that insulin plays an important role in the nutrient-induced insulin resistance. In this study, we tested the hypothesis that chronic exposure to excess long-acting insulin (glargine) can cause typical type 2 diabetes mellitus (T2DM) in normal mice fed on a chow diet. C57BL/6 mice were treated with glargine once a day for 8 weeks, followed by evaluations of food intake, body weight, blood levels of glucose, insulin, lipids, and cytokines, insulin signaling, histology of pancreas, ectopic fat accumulation, oxidative stress level, and cholesterol content in mitochondria in tissues. Cholesterol content in mitochondria and its association with oxidative stress in cultured hepatocytes and β-cells were also examined. Results show that chronic exposure to glargine caused insulin resistance, hyperinsulinemia, and relative insulin deficiency (T2DM). Treatment with excess glargine led to loss of pancreatic islets, ectopic fat accumulation in liver, oxidative stress in liver and pancreas, and increased cholesterol content in mitochondria of liver and pancreas. Prolonged exposure of cultured primary hepatocytes and HIT-TI5 β-cells to insulin induced oxidative stress in a cholesterol synthesis-dependent manner. Together, our results show that chronic exposure to excess insulin can induce typical T2DM in normal mice fed on a chow diet.
Manon M Roustit, Joan M Vaughan, Pauline M Jamieson and Mark E Cleasby
Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity.
Tingting Yang, Min He, Hailiang Zhang, Paula Q Barrett and Changlong Hu
Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenal adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type CaV3.2/3.3 and L-type CaV1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K+. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effect on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.
K E Lines, P J Newey, C J Yates, M Stevenson, R Dyar, G V Walls, M R Bowl and R V Thakker
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1 +/ − mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1 +/ − mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.
Anne-Marie O'Carroll, Gillian M Howell, Emma M Roberts and Stephen J Lolait
Arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) have both been implicated in modulating insulin secretion from pancreatic β-cells. In the present study, we investigated the insulin-secreting activities of AVP and CRH in wild-type and AVP VIb receptor knockout mice. Both neuropeptides stimulated insulin secretion from isolated mouse pancreatic islets. The response of islets to CRH was increased fourfold by concomitant incubation with a subthreshold dose of AVP that alone did not stimulate insulin secretion. Activation of the endogenously expressed M3 receptor by the cholinergic agonist carbachol also potentiated CRH-induced insulin secretion, indicating that the phenomenon may be pathway specific (i.e. Ca2 +-phospholipase C) rather than agonist specific. The protein kinase C (PKC) inhibitors Ro-31-8425 and bisindolylmaleimide I attenuated the potentiating effect of AVP on CRH-stimulated insulin secretion and blocked AVP-stimulated insulin secretion. A possible interaction between the PKC and protein kinase A pathways was also investigated. The phorbol ester phorbol myristate acetate (PMA) stimulated insulin secretion, while the addition of both PMA and CRH enhanced insulin secretion over that measured with either PMA or CRH alone. Additionally, no AVP potentiation of CRH-stimulated insulin secretion was observed upon incubation in Ca2 +-free Krebs–Ringer buffer. Taken together, the present study suggests a possible synergism between AVP and CRH to release insulin from pancreatic β-cells that relies at least in part on activation of the PKC signaling pathway and is dependent on extracellular Ca2 +. This is the first example of a possible interplay between the AVP and CRH systems outside of the hypothalamic–pituitary–adrenal axis.
Thomas Nicholson, Chris Church, Kostas Tsintzas, Robert Jones, Leigh Breen, Edward T Davis, David J Baker and Simon W Jones
Adipokines have emerged as central mediators of insulin sensitivity and metabolism, in part due to the known association of obesity with metabolic syndrome disorders such as type 2 diabetes. Recent studies in rodents have identified the novel adipokine vaspin as playing a protective role in inflammatory metabolic diseases by functioning as a promoter of insulin sensitivity during metabolic stress. However, at present the skeletal muscle and adipose tissue expression of vaspin in humans is poorly characterised. Furthermore, the functional role of vaspin in skeletal muscle insulin sensitivity has not been studied. Since skeletal muscle is the major tissue for insulin-stimulated glucose uptake, understanding the functional role of vaspin in human muscle insulin signalling is critical in determining its role in glucose homeostasis. The objective of this study was to profile the skeletal muscle and subcutaneous adipose tissue expression of vaspin in humans of varying adiposity, and to determine the functional role of vaspin in mediating insulin signalling and glucose uptake in human skeletal muscle. Our data shows that vaspin is secreted from both human subcutaneous adipose tissue and skeletal muscle, and is more highly expressed in obese older individuals compared to lean older individuals. Furthermore, we demonstrate that vaspin induces activation of the PI3K/AKT axis, independent of insulin receptor activation, promotes GLUT4 expression and translocation and sensitises older obese human skeletal muscle to insulin-mediated glucose uptake.