Instruct: Given a scientific claim, retrieve passages that support or refute the claim.
Query: TMEM27 is a marker for beta cells.
The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.
Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. IL-1beta and/or TNF-alpha plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.
Thiazolidinediones (TZDs) improve glycemic control and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). There is growing evidence from in vivo and in vitro studies that TZDs improve pancreatic beta-cell function. The aim of this study was to determine whether TZD-induced improvement in glycemic control is associated with improved beta-cell function. We studied 11 normal glucose-tolerant and 53 T2DM subjects [age 53+/-2 yr; BMI 29.4+/-0.8 kg/m2; fasting plasma glucose (FPG) 10.3+/-0.4 mM; Hb A1c 8.2+/-0.3%]. Diabetic patients were randomized to receive placebo or TZD for 4 mo. Subjects received 1) 2-h OGTT with determination of plasma glucose, insulin, and C-peptide concentrations and 2) two-step euglycemic insulin (40 and 160 mU.m-2.min-1) clamp with [3-(3)H]glucose. T2DM patients were then randomized to receive 4 mo of treatment with pioglitazone (45 mg/day), rosiglitazone (8 mg/day), or placebo. Pioglitazone and rosiglitazone similarly improved FPG, mean plasma glucose during OGTT, Hb A1c, and insulin-mediated total body glucose disposal (Rd) and decreased mean plasma FFA during OGTT (all P<0.01, ANOVA). The insulin secretion/insulin resistance (disposition) index [DeltaISR(AUC)/Deltaglucose(AUC)/IR] was significantly improved in all TZD-treated groups: +1.8+/-0.7 (PIO+drug-naïve diabetics), +0.7+/-0.3 (PIO+sulfonylurea-treated diabetics), and 0.7+/-0.2 (ROSI+sulfonylurea-withdrawn diabetics) vs. -0.2+/-0.3 in the two placebo groups (P<0.01, all TZDs vs. placebo, ANOVA). Improved insulin secretion correlated positively with increased body weight, fat mass, and Rd and inversely with decreased plasma glucose and FFA during the OGTT. In T2DM patients, TZD treatment leads to improved beta-cell function, which correlates strongly with improved glycemic control.
Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3+, including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25+CD4+ and CD25−CD4+ T cell subsets), were as suppressive as the “classic” CD4+CD25hi T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.
Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.
Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.
CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.
The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. Recent studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function. This review addresses the heterogeneity of TCM and TEM, their differentiation stages, and the current models for their generation and maintenance in humans and mice.
EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.
To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.
Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in vitro and in vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.
Notch signals are necessary for the functional outcomes of T cell receptor β-selection, including differentiation, proliferation and rescue from apoptosis. The mechanism underlying this requirement for T cell development is unknown. Here we show that Notch receptor and Delta-like 1 ligand interactions promoted the survival of CD4−CD8− pre–T cells through the maintenance of cell size, glucose uptake and metabolism. Furthermore, the trophic effects of Notch signaling were mediated by the pathway of phosphatidylinositol-3-OH kinase and the kinase Akt, such that expression of active Atk overcame the requirement for Notch in β-selection. Collectively, our results demonstrate involvement of Notch receptor–ligand interactions in the regulation of cellular metabolism, thus enabling the autonomous signaling capacity of the pre–T cell receptor complex.*Note: In the version of this article initially published online, in the fourth sentence of the abstract, the term "Atk" was a misspelling; this should be "Akt. " In the fourth sentence of the second paragraph of the introduction, the name of the second kinase mentioned, "PI(3)K-dependent kinase 1," was incorrect; this should read "phosphoinositide-dependent kinase 1. " These errors have been corrected for the HTML and print versions of the article.
Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.
TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation.
Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.
Class B GPCRs can activate multiple signalling effectors with the potential to exhibit biased agonism in response to ligand stimulation. Previously, we highlighted key TM domain polar amino acids that were crucial for the function of the GLP-1 receptor, a key therapeutic target for diabetes and obesity. Using a combination of mutagenesis, pharmacological characterisation, mathematical and computational molecular modelling, this study identifies additional highly conserved polar residues located towards the TM helical boundaries of Class B GPCRs that are important for GLP-1 receptor stability and/or controlling signalling specificity and biased agonism. This includes (i) three positively charged residues (R3.30227, K4.64288, R5.40310) located at the extracellular boundaries of TMs 3, 4 and 5 that are predicted in molecular models to stabilise extracellular loop 2, a crucial domain for ligand affinity and receptor activation; (ii) a predicted hydrogen bond network between residues located in TMs 2 (R2.46176), 6 (R6.37348) and 7 (N7.61406 and E7.63408) at the cytoplasmic face of the receptor that is important for stabilising the inactive receptor and directing signalling specificity, (iii) residues at the bottom of TM 5 (R5.56326) and TM6 (K6.35346 and K6.40351) that are crucial for receptor activation and downstream signalling; (iv) residues predicted to be involved in stabilisation of TM4 (N2.52182 and Y3.52250) that also influence cell signalling. Collectively, this work expands our understanding of peptide-mediated signalling by the GLP-1 receptor.
In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.
We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.
Thymic epithelial cells (TEC) form the structural and functional microenvironment necessary for the establishment and quality control of the T cell repertoire. In addition, they provide an ectopic source of numerous tissue-restricted antigens (TRA), a feature called promiscuous gene expression (pGE). How the regulation of pGE is related to the cell biology of TEC subset(s), e.g. their turnover and developmental interrelationship is still poorly understood. The observation that pGE is foremost a property of phenotypically and functionally mature medullary TEC (mTEC) implies that the full implementation of pGE is contingent on mTEC differentiation. Here, we show that the emergence of TEC subsets and pGE is tightly correlated during ontogeny and we provide evidence that mature CD80pos mTEC develop from an immature CD80neg subset. This differentiation step proceeds continuously in the postnatal thymus. While mature mTEC turnover in 2 to 3 weeks, immature mTEC encompass a smaller cycling and a larger non-cycling pool. The latter might serve as a reservoir of committed precursors, which sustain this renewal process. Our data document that mTEC represent a highly dynamic cell population, and they imply that the availability and display of TRA in the thymus undergoes a perpetual temporal and spatial reorganization.
Proper regulation of energy storage in adipose tissue is crucial for maintaining insulin sensitivity and molecules contributing to this process have not been fully revealed. Here we show that type II transmembrane protein tenomodulin (TNMD) is upregulated in adipose tissue of insulin-resistant versus insulin-sensitive individuals, who were matched for body mass index (BMI). TNMD expression increases in human preadipocytes during differentiation, whereas silencing TNMD blocks adipogenesis. Upon high-fat diet feeding, transgenic mice overexpressing Tnmd develop increased epididymal white adipose tissue (eWAT) mass, and preadipocytes derived from Tnmd transgenic mice display greater proliferation, consistent with elevated adipogenesis. In Tnmd transgenic mice, lipogenic genes are upregulated in eWAT, as is Ucp1 in brown fat, while liver triglyceride accumulation is attenuated. Despite expanded eWAT, transgenic animals display improved systemic insulin sensitivity, decreased collagen deposition and inflammation in eWAT, and increased insulin stimulation of Akt phosphorylation. Our data suggest that TNMD acts as a protective factor in visceral adipose tissue to alleviate insulin resistance in obesity.
Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.
Endoplasmic reticulum (ER) stress causes pancreatic β-cell dysfunction and contributes to β-cell loss and the progression of type 2 diabetes. Wolfram syndrome 1 (WFS1) has been shown to be an important regulator of the ER stress signalling pathway; however, its role in β-cell function remains unclear. Here we provide evidence that WFS1 is essential for glucose- and glucagon-like peptide 1 (GLP-1)-stimulated cyclic AMP production and regulation of insulin biosynthesis and secretion. Stimulation with glucose causes WFS1 translocation from the ER to the plasma membrane, where it forms a complex with adenylyl cyclase 8 (AC8), an essential cAMP-generating enzyme in the β-cell that integrates glucose and GLP-1 signalling. ER stress and mutant WFS1 inhibit complex formation and activation of AC8, reducing cAMP synthesis and insulin secretion. These findings reveal that an ER-stress-related protein has a distinct role outside the ER regulating both insulin biosynthesis and secretion. The reduction of WFS1 protein on the plasma membrane during ER stress is a contributing factor for β-cell dysfunction and progression of type 2 diabetes.
Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.
Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and alpha-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K(+) channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.
Memory function often declines with age, and is believed to deteriorate initially because of changes in synaptic function rather than loss of neurons. Some individuals then go on to develop Alzheimer's disease with neurodegeneration. Here we use Tg2576 mice, which express a human amyloid-β precursor protein (APP) variant linked to Alzheimer's disease, to investigate the cause of memory decline in the absence of neurodegeneration or amyloid-β protein amyloidosis. Young Tg2576 mice (< 6 months old) have normal memory and lack neuropathology, middle-aged mice (6–14 months old) develop memory deficits without neuronal loss, and old mice (> 14 months old) form abundant neuritic plaques containing amyloid-β (refs 3–6). We found that memory deficits in middle-aged Tg2576 mice are caused by the extracellular accumulation of a 56-kDa soluble amyloid-β assembly, which we term Aβ*56 (Aβ star 56). Aβ*56 purified from the brains of impaired Tg2576 mice disrupts memory when administered to young rats. We propose that Aβ*56 impairs memory independently of plaques or neuronal loss, and may contribute to cognitive deficits associated with Alzheimer's disease.