Reduced Β-cell Mass Research Articles

Loss of glucose-stimulated β-cell Nr4a1 expression impairs insulin secretion and glucose homeostasis.

A central aspect of type 2 diabetes is decreased functional β-cell mass. The orphan nuclear receptor Nr4a1 is critical for fuel utilization, but little is known regarding its regulation and function in the β-cell. Nr4a1 expression is decreased in type 2 diabetes rodent β-cells and type 2 diabetes patient islets. We have shown that Nr4a1-deficient mice have reduced β-cell mass and that Nr4a1 knockdown impairs glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 β-cells. Here, we demonstrate that glucose concentration directly regulates β-cell Nr4a1 expression. We show that 11 mM glucose increases Nr4a1 expression in INS-1 832/13 β-cells and primary mouse islets. We show that glucose functions through the cAMP/PKA/CREB pathway to regulate Nr4a1 mRNA and protein expression. Using Nr4a1-/- animals, we show that Nr4a1 is necessary for GSIS and systemic glucose handling. Using RNA-seq, we define Nr4a1-regulated pathways in response to glucose in the mouse islet, including Glut2 expression. Our data suggest that Nr4a1 plays a critical role in the β-cells response to the fed state.NEW & NOTEWORTHY Nr4a1 has a key role in fuel metabolism and β-cell function, but its exact role is unclear. Nr4a1 expression is regulated by glucose concentration using cAMP/PKA/CREB pathway. Nr4a1 regulates Glut2, Ndufa4, Ins1, In2, Sdhb, and Idh3g expression in response to glucose treatment. These results suggest that Nr4a1 is necessary for proper insulin secretion both through glucose uptake and metabolism machinery.

Talin-1 inhibits Smurf1-mediated Stat3 degradation to modulate β-cell proliferation and mass in mice

Insufficient pancreatic β-cell mass and reduced insulin expression are key events in the pathogenesis of diabetes mellitus (DM). Here we demonstrate the high expression of Talin-1 in β-cells and that deficiency of Talin-1 reduces β-cell proliferation, which leads to reduced β-cell mass and insulin expression, thus causing glucose intolerance without affecting peripheral insulin sensitivity in mice. High-fat diet fed exerbates these phenotypes. Mechanistically, Talin-1 interacts with the E3 ligase smad ubiquitination regulatory factor 1 (Smurf1), which prohibits ubiquitination of the signal transducer and activator of transcription 3 (Stat3) mediated by Smurf1, and ablation of Talin-1 enhances Smurf1-mediated ubiquitination of Stat3, leading to decreased β-cell proliferation and mass. Furthermore, haploinsufficiency of Talin-1 and Stat3 genes, but not that of either gene, in β-cell in mice significantly impairs glucose tolerance and insulin expression, indicating that both factors indeed function in the same genetic pathway. Finally, inducible deletion Talin-1 in β-cell causes glucose intolerance in adult mice. Collectively, our findings reveal that Talin-1 functions as a crucial regulator of β-cell mass, and highlight its potential as a therapeutic target for DM patients.

Slc12a2 loss in insulin-secreting β-cells links development of overweight and metabolic dysregulation to impaired satiation control of feeding.

Male mice lacking the Na+-K+-2Cl- cotransporter Slc12a2 (Nkcc1) specifically in insulin-secreting β-cells (Slc12a2βKO) have reduced β-cell mass and mild β-cell secretory dysfunction associated with overweight, glucose intolerance, insulin resistance, and metabolic abnormalities. Here, we confirmed and extended previous results to female Slc12a2βKO mice, which developed a similar metabolic syndrome-like phenotype as males, albeit milder. Notably, male and female Slc12a2βKO mice developed overweight without consuming excess calories. Analysis of the feeding microstructure revealed that young lean Slc12a2βKO male mice ate meals of higher caloric content and at a relatively lower frequency than normal mice, particularly during the night. In addition, overweight Slc12a2βKO mice consumed significantly larger meals than lean mice. Therefore, the reduced satiation control of feeding precedes the onset of overweight and is worsened in older Slc12a2βKO mice. However, the time spent between meals remained intact in lean and overweight Slc12a2βKO mice, indicating conserved satiety responses to ad libitum feeding. Nevertheless, satiety was intensified during and after refeeding only in overweight males. In lean females, satiety responses to refeeding were delayed relative to age- and body weight-matched control mice but normalized in overweight mice. Since meal size did not change during refeeding, these data suggested that the satiety control of eating after fasting is impaired in lean Slc12a2βKO mice before the onset of overweight and independently of their reduced satiation responses. Therefore, our results support the novel hypothesis that reduced satiation precedes the onset of overweight and the development of metabolic dysregulation.NEW & NOTEWORTHY Obesity, defined as excess fat accumulation, increases the absolute risk for metabolic diseases. Although obesity is usually attributed to increased food intake, we demonstrate that body weight gain can be hastened without consuming excess calories. In fact, impaired meal termination control, i.e., satiation, is detectable before the development of overweight in an animal model that develops a metabolic syndrome-like phenotype.

Defining the ferroptotic phenotype of beta cells in type 1 diabetes and its inhibition as a potential antidiabetic strategy.

Recently, the involvement of ferroptotic cell death in the reduction of β-cell mass in diabetes has been demonstrated. To elucidate the mechanisms of β-cell ferroptosis and potential antidiabetic effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in vivo, a mouse model of type 1 diabetes (T1D) was used. Animals were divided into three groups: control (vehicle-treated), diabetic (streptozotocin-treated, 40 mg/kg, from days 1-5), and diabetic treated with Fer-1 (1 mg/kg, from days 1-21). On day 22, glycemia and insulinemia were measured and pancreases were isolated for microscopic analyses. Diabetes disturbed general parameters of β-cell mass (islet size, β-cell abundance and distribution) and health (insulin and PDX-1 expression), increased lipid peroxidation in islet cells, and phagocytic removal of iron-containing material. It also downregulated the main players of the antiferroptotic pathway - Nrf2, GPX4, and xCT. In contrast, Fer-1 ameliorated the signs of deterioration of β-cell/islets, decreased lipid peroxidation, and reduced phagocytic activity, while upregulated expression of Nrf2 (and its nuclear translocation), GPX4, and xCT in β-cell/islets. Overall, our study confirms ferroptosis as an important mode of β-cell death in T1D and suggests antiferroptotic agents as a promising strategy for the prevention and treatment of diabetes.

1754-P: Reduced Incretin Effect Predicts Diabetes Appearance in a Cohort of Nondiabetic Subjects after Acute Beta-Cell Mass Reduction

In type 2 diabetes incretin effect (IE) is reduced but still contributes to 20-35% of insulin response to oral glucose. One reason is the impairment of GIP and GLP1 effects. The role of IE in nondiabetic people is still under debate. In this study we evaluated changes in GLP1 levels, differential β-cell responses and IE during oral vs intravenous stimuli in nondiabetic humans before and after pancreatoduodenectomy (PD). We enrolled 37 nondiabetic patients affected by periampullary nonmalignant neoplasms scheduled for PD. Subjects underwent a 2-h hyperglycemic clamp (HC), a 830 kcal mixed meal test (MMT), and an oral glucose tolerance test (OGTT) before and after PD. GLP1 secretion was calculated during MMT stimulation. β-cell glucose sensitivity (βCGS, pmol·min−1m−2·mM−1) was calculated as the ratio of insulin secretion and glucose increments during both HC and MMT. IE was calculated as the ratio of βCGS obtained during MMT and HC. Based on post-PD OGTT, we divided nondiabetic subjects into 3 groups according to glucose tolerance after PD: normal glucose tolerance (postNGT, n=11), impaired glucose tolerance (postIGT, n=15), or diabetes (postDM, n=11). Before surgery, postIGT subjects exhibited increased GLP1 secretion during MMT compared to the other 2 groups (GLP1 AUC: postIGT 12638±688 vs postNGT 6994±1773 vs postDM 6443±1687 pmol/L·min, P<0.05). Further, a similar βCGS was observed in the 3 groups following HC stimulation (postNGT 95.2±15.5 vs postIGT 64.3±8.72 vs postDM 73.8±11.8, P=0.2), while a scaled reduction in βCGS was observed in postIGT and post-DM following MMT (postNGT 206±50.0 vs postIGT 111±19.9 vs postDM 88.2±8.45, P<0.05). Accordingly, we observed a scaled reduction of IE in postIGT and postDM groups (postNGT 2.35±0.52 vs post-IGT 1.37±0.13 vs post-DM 0.86±0.18, P<0.05). Our data show that GLP1 secretion increases in people at risk of IGT, possibly to compensate reduced IE, which seems to predict DM appearance after β-cell mass reduction. Disclosure G.Di giuseppe: None. J.J.Holst: Advisory Panel; Novo Nordisk A/S, Eli Lilly and Company, Board Member; Bainan Biotech, Antag Therapeutics, Consultant; Alphasights, Eli Lilly and Company, ShouTi Pharma Inc., Zealand Pharma A/S, Other Relationship; Novo Nordisk, Novo Nordisk Pharma, Mayo Clinic, Boehringer-Ingelheim, Scohia Pharma Inc., Research Support; ARLA, European Union, Novo Nordisk Foundation. A.Giaccari: Speaker's Bureau; Abbott, AstraZeneca, Boehringer-Ingelheim, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk, Sanofi. T.Mezza: None. C.Cefalo: None. S.Moffa: None. G.Ciccarelli: None. L.Soldovieri: None. F.Cinti: None. U.Capece: None. F.Impronta: None. A.Mari: Consultant; Eli Lilly and Company. Funding Università Cattolica del Sacro Cuore; European Foundation for the Study of Diabetes; Ministero della Salute

82-OR: NRF2 Regulates Neonatal ß-Cell Mass Expansion

The pediatric population suffers from increased incidence of both major types of diabetes in recent decades, highlighting the urgent need for discovering mechanisms that regulate functional β-cell mass. We found that the master antioxidant regulator, Nrf2, is required for adaptive adult β-cell expansion under hypercaloric conditions via regulation of β-cell proliferation, β-cell survival, and maintenance of β-cell identity. Additionally, we found that Nrf2 expression in β-cells of 7-day-old mice is 15-fold higher of that in adult β-cells (p<0.0001, n=6). Based on these observations, we hypothesized that Nrf2 regulates β-cell proliferation and survival at early stages of life and contributes to the maintenance of normal β-cell mass later in life. To test this, we deleted Nrf2 in β-cells by crossing InsCre with Nrf2lox/lox mice (βNrf2KO). βNrf2KO mice at 28 days of age exhibited a 65% reduction of β-cell mass compared to control mice (p<0.0005, n=6). Importantly, at 7 days of age, βNrf2KO mice displayed a 65% reduction of β-cell proliferation (p<0.0001, n=6), a 22% reduction of nuclear Pdx1 levels (p<0.0001, n=6), a 5.84-fold increase in β-cell death (±0.04, p<0.001, n=6) and a 2.93-fold increase in β-cell oxidative stress (p<0.001, n=6) compared to control mice. Surprisingly, daily administration of antioxidant NAC to 7-day-old βNrf2KO mice only partially restored β-cell proliferation but completely blunted oxidative stress compared to control mice. This suggests that neonatal β-cell proliferation induced by Nrf2 relies on additional mechanisms beyond oxidative stress. Interestingly, RNAseq of isolated islets from 7-day-old βNrf2KO mice showed decreased expression of mitochondrial encoded genes and upregulation of stress-responsive genes. This suggests that Nrf2 controls β-cell proliferation by regulating mitochondrial bioenergetics. We conclude that Nrf2 is required for β-cell mass expansion in neonatal ages by controlling β-cell proliferation, identity, and survival. Disclosure S.Baumel-alterzon: None. L.S.Katz: None. L.Lambertini: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D.Scott: None. Funding National Institutes of Health (DK128387, DK114338)

Glucose-Lowering Effects of Imeglimin and Its Possible Beneficial Effects on Diabetic Complications.

Mitochondrial dysfunction is a prominent pathological feature of type 2 diabetes, which contributes to β-cell mass reduction and insulin resistance. Imeglimin is a novel oral hypoglycemic agent with a unique mechanism of action targeting mitochondrial bioenergetics. Imeglimin reduces reactive oxygen species production, improves mitochondrial function and integrity, and also improves the structure and function of endoplasmic reticulum (ER), changes which enhance glucose-stimulated insulin secretion and inhibit the apoptosis of β-cells, leading to β-cell mass preservation. Further, imeglimin inhibits hepatic glucose production and ameliorates insulin sensitivity. Clinical trials into the effects of imeglimin monotherapy and combination therapy exhibited an excellent hypoglycemic efficacy and safety profile in type 2 diabetic patients. Mitochondrial impairment is closely associated with endothelial dysfunction, which is a very early event in atherosclerosis. Imeglimin improved endothelial dysfunction in patients with type 2 diabetes via both glycemic control-dependent and -independent mechanisms. In experimental animals, imeglimin improved cardiac and kidney function via an improvement in mitochondrial and ER function or/and an improvement in endothelial function. Furthermore, imeglimin reduced ischemia-induced brain damage. In addition to glucose-lowering effects, imeglimin can be a useful therapeutic option for diabetic complications in type 2 diabetic patients.

Zhx2 maintains islet β-cell mass and function by transcriptionally regulating Pax6

Emerging evidence shows that pancreatic β-cell function and quality are key determinants in the progression of type 2 diabetes (T2D). The transcription factor zinc finger homeobox 2 (Zhx2) is involved in proliferation and development of multiple cells. However, the exact role of Zhx2 in β-cells and T2D remains completely unknown. Here, we report that Zhx2 orchestrates β-cell mass and function by regulating paired box protein pax-6 (Pax6). We found that β-cell-specific knockout Zhx2 (Zhx2BKO) mice showed a decrease in β-cell proliferation and glucose homeostasis. Under prediabetic and diabetic conditions, we discovered glucose intolerance in both Zhx2BKO-HFD mice and Zhx2BKO-db/db mice, with reduced β-cell mass and insulin secretion. Mechanistically, we demonstrated that Zhx2 targeted the Pax6 promoter region (-1740∼-1563; -862∼-559; -251∼+75), enhanced promoter activity. Overall, Zhx2 maintains β-cell function by transcriptionally regulating Pax6, which provides a therapeutic target for diabetes intervention.

Metformin and Gegen Qinlian Decoction boost islet α-cell proliferation of the STZ induced diabetic rats

BackgroundThe traditional Chinese medicine Gegen Qinlian Decoction (GQD), as well as metformin, had been reported with anti-diabetic effects in clinical practice.ObjectiveTo verify whether these two medicines effectively ameliorate hyperglycemia caused by deficiency of islet β-cell mass which occurs in both type 1 and type 2 diabetes.MethodsSD rats were injected with a single dose of STZ (55 mg/kg) to induce β-cell destruction. The rats were then divided into control, diabetes, GQD and metformin group. GQD and metformin groups were administered with GQD extract or metformin for 6 weeks. The islet α-cell or β-cell mass changes were tested by immunohistochemical and immunofluorescent staining. The potential targets and mechanisms of GQD and metformin on cell proliferation were tested using in silico network pharmacology. Real-time PCR was performed to test the expression of islet cells related genes and targets related genes.ResultsBoth GQD and metformin did not significantly reduce the FBG level caused by β-cell mass reduction, but alleviated liver and pancreas histopathology. Both GQD and metformin did not change the insulin positive cell mass but increased α-cell proliferation of the diabetic rats. Gene expression analysis showed that GQD and metformin significantly increased the targets gene cyclin-dependent kinase 4 (Cdk4) and insulin receptor substrate (Irs1) level.ConclusionThis research indicates that GQD and metformin significantly increased the α-cell proliferation of β-cell deficiency induced diabetic rats by restoring Cdk4 and Irs1 gene expression.

XAF1 overexpression exacerbates diabetes by promoting pancreatic β-cell apoptosis

AimsPancreatic β-cell apoptosis may be involved in the onset and progression of type 2 diabetes mellitus, although its mechanism remains unclear. We previously demonstrated that macrophage-derived interferon (IFN) β induced X-linked inhibitor of apoptosis–associated factor 1 (XAF1) expression in β-cells and accelerated β-cell apoptosis in vitro. Here, we explored the effects of XAF1 on β-cell function and progression of diabetes in vivo.MethodsPancreatic β-cell-selective XAF1 overexpressing (Xaf1 Tg) mice were generated. Xaf1 Tg mice and their wild-type (WT) littermates were fed either a normal diet or a 40% or 60% high-fat diet (HFD). The effects of β-cell XAF1 on β-cell apoptosis and exacerbation of diabetes were investigated.ResultsPalmitic acid induced IFNβ expression in macrophages, and HFD intake promoted macrophage infiltration in pancreatic islets, both of which cooperatively upregulated XAF1 expression in mouse islets. Furthermore, HFD-fed Xaf1 Tg mice demonstrated increased β-cell apoptosis, lowered insulin expression, and impaired glucose tolerance compared with WT mice fed the same diet. These effects were more pronounced in the 60%HFD group than in the 40%HFD group.ConclusionsPancreatic β-cell XAF1 expression was enhanced via HFD-induced, macrophage-derived IFNβ, which promoted β-cell apoptosis and led to a reduction in insulin secretion and progression of diabetes. To our knowledge, this is the first report to demonstrate an association between pancreatic β-cell XAF1 overexpression and exacerbation of diabetes, thus providing insight into the mechanism of β-cell mass reduction in diabetes.

248-LB: Nrf2 Is Essential for Expanding Neonatal Pancreatic Beta-Cell Mass

Patients with both major forms of diabetes suffer from insufficient functional β-cell mass. Moreover, the sharp increase in the annual incidence of both type 1 (1.8%) and type 2 (4.8%) diabetes in the pediatric population in the last decades highlights the urgent need for discovering the mechanisms that regulate functional β-cell mass. β-cell proliferation, which reaches its maximal peak during postnatal stages is the major mechanism for increasing β-cell mass in early postnatal ages. We previously found that induction of the antioxidant regulator, Nrf2, is required for adaptive β-cell expansion under hypercaloric conditions via stimulation of β-cell proliferation, increased β-cell survival, and maintenance of β-cell identity in adult mice. Based on these observations, we hypothesize that Nrf2 regulates β-cell proliferation and survival at early stages of life and contribute to maintain normal β-cell mass later in life. Nrf2 expression levels were found to correlate with β-cell proliferation in wild type neonatal mice. Importantly, 28 day-old β-cell specific Nrf2 knockout mice (Ins1Cre-Nrf2lox/lox) exhibited 50% reduction in β-cell mass, 58% reduction in total islet number, 43% decrease in extra-large islets, 46% decrease in large islets and 53% decrease in extra small islets, compared to control mice. No changes in pancreas weight were observed. Additionally, depletion of Nrf2 in β-cells increased β-cell death in 7 (5.8-fold) and 14 (12.5-fold) day-old mice and attenuated β-cell proliferation in 1- and 7-day-old mice. Surprisingly, we also found a marked decrease in islet size in 1- and 7-day-old mice, suggesting that Nrf2 may affect β-cell proliferation, survival and/or development during embryonic stages. We conclude that Nrf2 is required for β-cell mass expansion in postnatal ages by controlling β-cell proliferation and survival. Disclosure S. Baumel-alterzon: None. A. Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D. Scott: None.

Prenatal exposure to a mixture of PAHs causes the dysfunction of islet cells in adult male mice: Association with type 1 diabetes mellitus

Polycyclic aromatic hydrocarbons (PAHs) have been detected throughout the human body. Whether exposure to PAHs is associated with the incidence of type 1 diabetes mellitus should be investigated. To this end, pregnant mice were exposed to mixed PAHs (5, 50, or 500 μg/kg) once every other day during gestation. The adult male offspring displayed impaired glucose tolerance and reduced serum levels of glucagon and insulin. Immunohistochemical staining revealed increased numbers of apoptotic β-cells and a reduced β-cell mass in these males. The downregulated expression of pancreatic estrogen receptor α, androgen receptor, and transcription factor PDX1 was responsible for impacting β-cell development. The relatively reduced α-cell area was associated with downregulated ARX expression. The transcription of Isn2 and Gcg in pancreatic tissue was downregulated, which indicated that the function of β-cells and α-cells was impaired. Methylation levels in the Isn2 promotor were significantly elevated in mice prenatally exposed to 500 µg/kg PAHs, which was consistent with the change in its mRNA levels. The number of macrophages infiltrating islets was significantly increased, indicating that prenatal PAH exposure might reduce islet cell numbers in an autoimmune manner. This study shows that prenatal exposure to PAHs may promote the pathogenesis of type 1 diabetes mellitus.

Exosomal miR-140–3p and miR-143–3p from TGF-β1-treated pancreatic stellate cells target BCL2 mRNA to increase β-cell apoptosis

BackgroundPeople with chronic pancreatitis (CP) normally develop a fibrotic pancreas with reduced β-cell mass. Limited studies have focused on the development and pathogenesis of CP-related diabetes. MiRNAs packaged as exosomes are the key regulators of β-cell dysfunction. This study aimed to define the effect of exosomal miRNA from activated pancreatic stellate cells (PSCs) on β-cells. MethodsExosomes in the supernatants of mouse PSCs lines were extracted via ultracentrifugation and then identified. The role of exosomes secreted by transforming growth factor-β1 (TGF-β1)-treated PSCs in β-cell function was assessed. MiRNAs were prepared from exosomes extracted from TGF-β1-treated and untreated PSCs (T-Exo or C-Exo), and the miRNA expression profiles were compared by microarray. Then, miR-140–3p and miR-143–3p were overexpressed or inhibited in MIN6 cells and islets to determine their molecular and functional effects. ResultsExosomes were the predominant extracellular vesicles secreted by PSCs into the culture medium. The MIN6 cells incubated with T-Exo had less insulin secretion and lower viability than the MIN6 cells incubated with PBS or C-Exo. MiR-140–3p and miR-143–3p were notably upregulated in T-Exo. Enhancing the expression of miR-140–3p and miR-143–3p in β-cells decreased the cell count and viability and increased the cleaved caspase-3 levels. Mechanistically, T-Exo mediated the intercellular transfer of miR-140–3p and miR-143–3p by targeting the B-cell lymphoma 2 gene in recipient β-cells to induce cell death. ConclusionsExosomal miRNA transfer as a communication mode between PSCs and β-cells, which may be explored for its therapeutic utility.

Knockout of Nur77 Leads to Amino Acid, Lipid, and Glucose Metabolism Disorders in Zebrafish.

Orphan nuclear receptor Nur77 has been reported to be implicated in a diverse range of metabolic processes, including carbohydrate metabolism and lipid metabolism. However, the detailed mechanism of Nur77 in the regulation of metabolic pathway still needs to be further investigated. In this study, we created a global nur77 knockout zebrafish model by CRISPR/Cas9 technique, and then performed whole-organism RNA sequencing analysis in wildtype and nur77-deficient zebrafish to dissect the genetic changes in metabolic-related pathways. We found that many genes involved in amino acid, lipid, and carbohydrate metabolism changed by more than twofold. Furthermore, we revealed that nur77−/− mutant displayed increased total cholesterol (TC) and triglyceride (TG), alteration in total amino acids, as well as elevated glucose. We also demonstrated that the elevated glucose was not due to the change of glucose uptake but was likely caused by the disorder of glycolysis/gluconeogenesis and the impaired β-cell function, including downregulated insb expression, reduced β-cell mass, and suppressed insulin secretion. Importantly, we also verified that targeted expression of Nur77 in the β cells is sufficient to rescue the β-cell defects in global nur77−/− larvae zebrafish. These results provide new information about the global metabolic network that Nur77 signaling regulates, as well as the role of Nur77 in β-cell function.

Targeting β-cell dedifferentiation and transdifferentiation: opportunities and challenges.

The most distinctive pathological characteristics of diabetes mellitus induced by various stressors or immune-mediated injuries are reductions of pancreatic islet β-cell populations and activity. Existing treatment strategies cannot slow disease progression; consequently, research to genetically engineer β-cell mimetics through bi-directional plasticity is ongoing. The current consensus implicates β-cell dedifferentiation as the primary etiology of reduced β-cell mass and activity. This review aims to summarize the etiology and proposed mechanisms of β-cell dedifferentiation and to explore the possibility that there might be a time interval from the onset of β-cell dysfunction caused by dedifferentiation to the development of diabetes, which may offer a therapeutic window to reduce β-cell injury and to stabilize functionality. In addition, to investigate β-cell plasticity, we review strategies for β-cell regeneration utilizing genetic programming, small molecules, cytokines, and bioengineering to transdifferentiate other cell types into β-cells; the development of biomimetic acellular constructs to generate fully functional β-cell-mimetics. However, the maturation of regenerated β-cells is currently limited. Further studies are needed to develop simple and efficient reprogramming methods for assembling perfectly functional β-cells. Future investigations are necessary to transform diabetes into a potentially curable disease.

Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass

ObjectiveEarly postnatal life is a critical period for the establishment of the functional β-cell mass that will sustain whole-body glucose homeostasis during the lifetime. β cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult β cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal β-cell development. MethodsTo study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from β-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. β-cell mass was studied using histomorphometric analysis and optical projection tomography. β-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted β cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. ResultsElimination of Gsα in β cells led to reduced β-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key β-cell identity and maturation genes in postnatal β-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. ConclusionWe conclude that Gsα is required for β-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal β cells, which may have implications for using cAMP-raising agents to promote β-cell regeneration and maturation in diabetes.

1254-P: ß-Cell Cdk1 Deletion Reveals a- and ß-Cell Compensatory Mechanisms for Hypoplasia

Diabetes is characterized by a loss of functional β-cell mass. With current rodent models of chemically-induced diabetes such as streptozotocin, it is difficult to distinguish the consequence of β-cell loss from the decline in individual β-cell function. Here, we generated a mouse model of severe β-cell mass loss by deleting β-cell CDK1 within the first week of life (Cdk1Ucn3Cre, Cdk1-floxed:Ucn3-Cre). At 4 weeks of age, islet mass in Cdk1Ucn3Cre islets is comparable to controls, although islet architecture is disrupted with α-cells having formed more heterotypic contacts with β-cells. By 12 weeks of age, Cdk1Ucn3Cre mice exhibit significantly reduced β-cell mass as well as α-cell mass, but maintain glucose tolerance and ex vivo insulin secretion in response to glucose, amino acids, and the gut incretin GIP. We therefore hypothesized that functional compensations in Cdk1Ucn3Cre islets arise from both β- and α-cells, which are insulinotropic in the fed state. Interestingly, α-cell glucagon secretion from Cdk1Ucn3Cre islets exhibits hypersensitivity to suppression by glucose, including when glucagonotropic amino acids are present. In parallel, we observed a reduction in amino acid-stimulated cAMP in β‑cells from Cdk1Ucn3Cre islets. We also found a left-shift in the glucose dependence of β-cell calcium oscillations in Cdk1Ucn3Cre islets, identifying β-cell recruitment as one mechanism offsetting the loss of α-cell paracrine signaling. Importantly, GIP elicited a much larger glucagon response from glucose- and amino-acid stimulated Cdk1Ucn3Cre islets, implying that α-cell compensation contributes significantly to the maintained insulin secretory output in vivo. In sum, the Cdk1Ucn3Cre model reveals the interdependence of α- and β-cell mass, and function, and will be useful to define the temporal sequence of islet compensation for chronic loss of β-cell mass under stressed conditions. Disclosure S. Sdao: None. M. Adams: None. H. R. Foster: None. B. Blum: None. M. J. Merrins: None.

Conversion of α-Cells to β-Cells in the Postpartum Mouse Pancreas Involves Lgr5 Progeny.

In contrast to the skin and the gut, where somatic stem cells and their niche are well characterized, a definitive pancreatic multipotent cell population in the adult pancreas has yet to be revealed. Of particular interest is whether such cells may be endogenous in patients with diabetes, and if so, can they be used for therapeutic purposes? In the current study, we used two separate reporter lines to target Cre-recombinase expression to the Lgr5- or glucagon-expressing cells in the pancreas. We provide evidence for the existence of a population of cells within and in the proximity of the ducts that transiently express the stem-cell marker Lgr5 during late gestational stages. Careful timing of tamoxifen treatment in Lgr5EGFP-IRES-CreERT2;R26Tomato mice allowed us to show that these Lgr5-expressing progenitor cells can differentiate into α-cells during pregnancy. Furthermore, we report on a spontaneous lineage conversion of α- to β-cells specifically after parturition. The contribution of Lgr5 progeny to the β-cell compartment through an α-cell intermediate phase early after pregnancy appears to be part of a novel mechanism that would counterbalance against excessive β-cell mass reduction during β-cell involution.

Metabolic and Energy Imbalance in Dysglycemia-Based Chronic Disease.

Metabolic flexibility is the ability to efficiently adapt metabolism based on nutrient availability and requirement that is essential to maintain homeostasis in times of either caloric excess or restriction and during the energy-demanding state. This regulation is orchestrated in multiple organ systems by the alliance of numerous metabolic pathways under the master control of the insulin-glucagon-sympathetic neuro-endocrine axis. This, in turn, regulates key metabolic enzymes and transcription factors, many of which interact closely with and culminate in the mitochondrial energy generation machinery. Metabolic flexibility is compromised due to the continuous mismatch between availability and intake of calorie-dense foods and reduced metabolic demand due to sedentary lifestyle and age-related metabolic slowdown. The resultant nutrient overload leads to mitochondrial trafficking of substrates manifesting as mitochondrial dysfunction characterized by ineffective substrate switching and incomplete substrate utilization. At the systemic level, the manifestation of metabolic inflexibility comprises reduced skeletal muscle glucose disposal rate, impaired suppression of hepatic gluconeogenesis and adipose tissue lipolysis manifesting as insulin resistance. This is compounded by impaired β-cell function and progressively reduced β-cell mass. A consequence of insulin resistance is the upregulation of the mitogen-activated protein kinase pathway leading to a pro-hypertensive, atherogenic, and thrombogenic environment. This is further aggravated by oxidative stress, advanced glycation end products, and inflammation, which potentiates the risk of micro- and macro-vascular complications. This review aims to elucidate underlying mechanisms mediating the onset of metabolic inflexibility operating at the main target organs and to understand the progression of metabolic diseases. This could potentially translate into a pharmacological tool that can manage multiple interlinked conditions of dysglycemia, hypertension, and dyslipidemia by restoring metabolic flexibility. We discuss the breadth and depth of metabolic flexibility and its impact on health and disease.

Regulation of Adaptive Cell Proliferation by Vagal Nerve Signals for Maintenance of Whole-Body Homeostasis: Potential Therapeutic Target for Insulin-Deficient Diabetes.

In insulin-resistant states such as obesity, pancreatic β-cells proliferate to prevent blood glucose elevations. Failure of this β-cells proliferative response leads to the development of diabetes. On the other hand, when organs are damaged, cells proliferate to repair the organs. Therefore, these proliferations are compensatory mechanisms aimed at maintaining whole-body homeostasis. We previously discovered vagal signal-mediated systems regulating adaptive proliferation of β-cells and hepatocytes. Neuron-mediated liver-β-cell inter-organ crosstalk is involved in promotion of β-cell proliferation during obesity, and in this system, vagal signals directly stimulate β-cell proliferation. Meanwhile, in the liver, the multi-step mechanisms whereby vagal nerve signals activate hepatic resident macrophages are involved in hepatocyte proliferation after severe injury. Diabetes mellitus develops on the pathological basis of insufficient insulin action. Insulin action insufficiency is attributable to insulin resistance, i.e., the failure of insulin to exert sufficient effects, and/or to impairment of insulin secretion. Impairment of insulin secretion is attributable not only to the β-cell dysfunction but also to functional β-cell mass reduction. In this regard, there are already therapeutic options to increase insulin secretion from residual β-cells, such as sulfonyl urea and incretin-related drugs. In contrast, there are as yet no applicable therapeutic strategies to increase functional β-cell mass in vivo. Therefore, we have conducted the basic investigations to tackle this issue based on the discovery of neuron-mediated liver-β-cell inter-organ crosstalk. This review introduces vagal signal-mediated regulatory systems of adaptive cell proliferation in vivo and efforts to develop cell-increasing therapies based on vagal nerve-mediated cell proliferation.