RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding.

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

Molecular Mechanisms of Glucocorticoid-Induced Insulin Resistance.

Glucocorticoids (GCs) are steroids secreted by the adrenal cortex under the hypothalamic-pituitary-adrenal axis control, one of the major neuro-endocrine systems of the organism. These hormones are involved in tissue repair, immune stability, and metabolic processes, such as the regulation of carbohydrate, lipid, and protein metabolism. Globally, GCs are presented as 'flight and fight' hormones and, in that purpose, they are catabolic hormones required to mobilize storage to provide energy for the organism. If acute GC secretion allows fast metabolic adaptations to respond to danger, stress, or metabolic imbalance, long-term GC exposure arising from treatment or Cushing's syndrome, progressively leads to insulin resistance and, in fine, cardiometabolic disorders. In this review, we briefly summarize the pharmacological actions of GC and metabolic dysregulations observed in patients exposed to an excess of GCs. Next, we describe in detail the molecular mechanisms underlying GC-induced insulin resistance in adipose tissue, liver, muscle, and to a lesser extent in gut, bone, and brain, mainly identified by numerous studies performed in animal models. Finally, we present the paradoxical effects of GCs on beta cell mass and insulin secretion by the pancreas with a specific focus on the direct and indirect (through insulin-sensitive organs) effects of GCs. Overall, a better knowledge of the specific action of GCs on several organs and their molecular targets may help foster the understanding of GCs' side effects and design new drugs that possess therapeutic benefits without metabolic adverse effects.

Slug, a Cancer-Related Transcription Factor, is Involved in Vascular Smooth Muscle Cell Transdifferentiation Induced by Platelet-Derived Growth Factor-BB During Atherosclerosis.

Background Heart attacks and stroke often result from occlusive thrombi following the rupture of vulnerable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) play a pivotal role in plaque vulnerability because of their switch towards a proinflammatory/macrophage-like phenotype when in the context of atherosclerosis. The prometastatic transcription factor Slug/Snail2 is a critical regulator of cell phenotypic transition. Here, we aimed to investigate the role of Slug in the transdifferentiation process of VSMCs occurring during atherogenesis. Methods and Results In rat and human primary aortic smooth muscle cells, Slug protein expression is strongly and rapidly increased by platelet-derived growth factor-BB (PDGF-BB). PDGF-BB increases Slug protein without affecting mRNA levels indicating that this growth factor stabilizes Slug protein. Immunocytochemistry and subcellular fractionation experiments reveal that PDGF-BB triggers a rapid accumulation of Slug in VSMC nuclei. Using pharmacological tools, we show that the PDGF-BB-dependent mechanism of Slug stabilization in VSMCs involves the extracellular signal-regulated kinase 1/2 pathway. Immunohistochemistry experiments on type V and type VI atherosclerotic lesions of human carotids show smooth muscle-specific myosin heavy chain-/Slug-positive cells surrounding the prothrombotic lipid core. In VSMCs, Slug siRNAs inhibit prostaglandin E2 secretion and prevent the inhibition of cholesterol efflux gene expression mediated by PDGF-BB, known to be involved in plaque vulnerability and/or thrombogenicity. Conclusions Our results highlight, for the first time, a role of Slug in aortic smooth muscle cell transdifferentiation and enable us to consider Slug as an actor playing a role in the atherosclerotic plaque progression towards a life-threatening phenotype. This also argues for common features between acute cardiovascular events and cancer.

The flanking peptides issue from the maturation of the human islet amyloid polypeptide (hIAPP) slightly modulate hIAPP-fibril formation but not hIAPP-induced cell death.

Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic ß cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic ß-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.

Exposure to Glucocorticoids in the First Part of Fetal Life is Associated with Insulin Secretory Defect in Adult Humans.

OBJECTIVE: High glucocorticoid levels in rodents inhibit development of beta cells during fetal life and lead to insulin deficiency in adulthood. To test whether similar phenomena occur in humans, we compared beta-cell function in adults who were exposed to glucocorticoids during the first part of fetal life with that of nonexposed subjects. RESEARCH DESIGN AND METHODS: The study was conducted in 16 adult participants exposed to glucocorticoids during the first part of fetal life and in 16 nonexposed healthy participants with normal glucose tolerance who were matched for age, sex, and body mass index (BMI). Exposed participants had been born to mothers who were treated with dexamethasone 1 to 1.5 mg/day from the sixth gestational week (GW) to prevent genital virilization in children at risk of 21-hydroxylase deficiency. We selected offspring of mothers who stopped dexamethasone before the 18th GW following negative genotyping of the fetus. Insulin and glucagon secretion were measured during an oral glucose tolerance test (OGTT) and graded intravenous (IV) glucose and arginine tests. Insulin sensitivity was measured by hyperinsulinemic-euglycemic-clamp. RESULTS: Age, BMI, and anthropometric characteristics were similar in the 2 groups. Insulinogenic index during OGTT and insulin sensitivity during the clamp were similar in the 2 groups. In exposed subjects, insulin secretion during graded IV glucose infusion and after arginine administration decreased by 17% (P = 0.02) and 22% (P = 0.002), respectively, while glucagon secretion after arginine increased. CONCLUSION: Overexposure to glucocorticoids during the first part of fetal life is associated with lower insulin secretion at adult age, which may lead to abnormal glucose tolerance later in life.

Adaptive ß-Cell Neogenesis in the Adult Mouse in Response to Glucocorticoid-Induced Insulin Resistance.

Both type 1 and type 2 diabetes are characterized by deficient insulin secretion and decreased ß-cell mass. Thus, regenerative strategies to increase ß-cell mass need to be developed. To characterize mechanisms of ß-cell plasticity, we studied a model of severe insulin resistance in the adult mouse and defined how ß-cells adapt. Chronic corticosterone (CORT) treatment was given to adult mice and led to rapid insulin resistance and adaptive increased insulin secretion. Adaptive and massive increase of ß-cell mass was observed during treatment up to 8 weeks. ß-Cell mass increase was partially reversible upon treatment cessation and reinduced upon subsequent treatment. ß-Cell neogenesis was suggested by an increased number of islets, mainly close to ducts, and increased Sox9 and Ngn3 mRNA levels in islets, but lineage-tracing experiments revealed that neoformed ß-cells did not derive from Sox9- or Ngn3-expressing cells. CORT treatment after ß-cell depletion partially restored ß-cells. Finally, ß-cell neogenesis was shown to be indirectly stimulated by CORT because serum from CORT-treated mice increased ß-cell differentiation in in vitro cultures of pancreatic buds. Altogether, the results present a novel model of ß-cell neogenesis in the adult mouse and identify the presence of neogenic factors in the serum of CORT-treated mice.

Humanized Mouse Model to Study Type 1 Diabetes.

Key requirements in type 1 diabetes (T1D) are in setting up new assays as diagnostic biomarkers that will apply to prediabetes, likely T-cell assays, and in designing antigen-specific therapies to prevent T1D development. New preclinical models of T1D will be required to help with advancing both aims. By crossing mouse strains that lack either murine MHC class I and class II genes and insulin genes, we developed YES mice that instead express human HLA-A*02:01, HLA-DQ8, and insulin genes as transgenes. The metabolic and immune phenotype of YES mice is basically identical to that of the parental strains. YES mice remain insulitis and diabetes free up to 1 year of follow-up, maintain normoglycemia to an intraperitoneal glucose challenge in the long-term range, have a normal ß-cell mass, and show normal immune responses to conventional antigens. This new model has been designed to evaluate adaptive immune responses to human insulin on a genetic background that recapitulates a human high-susceptibility HLA-DQ8 genetic background. Although insulitis free, YES mice develop T1D when challenged with polyinosinic-polycytidylic acid. They allow the characterization of preproinsulin epitopes recognized by CD8+ and CD4+ T cells upon immunization against human preproinsulin or during diabetes development.

Microvascular vasodilator properties of the angiotensin II type 2 receptor in a mouse model of type 1 diabetes.

Diabetes Mellitus is associated with severe cardiovascular disorders involving the renin-angiotensin system, mainly through activation of the angiotensin II type 1 receptor (AT1R). Although the type 2 receptor (AT2R) opposes the effects of AT1R, with vasodilator and anti-trophic properties, its role in diabetes is debatable. Thus we investigated AT2R-mediated dilatation in a model of type 1 diabetes induced by streptozotocin in 5-month-old male mice lacking AT2R (AT2R-/y). Glucose tolerance was reduced and markers of inflammation and oxidative stress (cyclooxygenase-2, gp91phox p22phox and p67phox) were increased in AT2R-/y mice compared to wild-type (WT) animals. Streptozotocin-induced hyperglycaemia was higher in AT2R-/y than in WT mice. Arterial gp91phox and MnSOD expression levels in addition to blood 8-isoprostane and creatinine were further increased in diabetic AT2R-/y mice compared to diabetic WT mice. AT2R-dependent dilatation in both isolated mesenteric resistance arteries and perfused kidneys was greater in diabetic mice than in non-diabetic animals. Thus, in type 1 diabetes, AT2R may reduce glycaemia and display anti-oxidant and/or anti-inflammatory properties in association with greater vasodilatation in mesenteric arteries and in the renal vasculature, a major target of diabetes. Therefore AT2R might represent a new therapeutic target in diabetes.

NOV/CCN3: A New Adipocytokine Involved in Obesity-Associated Insulin Resistance.

Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV(-/-) mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV(-/-) mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV(-/-) mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance.

Correction: Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

[This corrects the article DOI: 10.1371/journal.pone.0149343.].

Glucagon actions on the kidney revisited: possible role in potassium homeostasis.

It is now recognized that the metabolic disorders observed in diabetes are not, or not only due to the lack of insulin or insulin resistance, but also to elevated glucagon secretion. Accordingly, selective glucagon receptor antagonists are now proposed as a novel strategy for the treatment of diabetes. However, besides its metabolic actions, glucagon also influences kidney function. The glucagon receptor is expressed in the thick ascending limb, distal tubule, and collecting duct, and glucagon regulates the transepithelial transport of several solutes in these nephron segments. Moreover, it also influences solute transport in the proximal tubule, possibly by an indirect mechanism. This review summarizes the knowledge accumulated over the last 30 years about the influence of glucagon on the renal handling of electrolytes and urea. It also describes a possible novel role of glucagon in the short-term regulation of potassium homeostasis. Several original findings suggest that pancreatic α-cells may express a "potassium sensor" sensitive to changes in plasma K concentration and could respond by adapting glucagon secretion that, in turn, would regulate urinary K excretion. By their combined actions, glucagon and insulin, working in a combinatory mode, could ensure an independent regulation of both plasma glucose and plasma K concentrations. The results and hypotheses reviewed here suggest that the use of glucagon receptor antagonists for the treatment of diabetes should take into account their potential consequences on electrolyte handling by the kidney.

Inhibition of the Inflammasome NLRP3 by Arglabin Attenuates Inflammation, Protects Pancreatic ß-Cells from Apoptosis, and Prevents Type 2 Diabetes Mellitus Development in ApoE2Ki Mice on a Chronic High-Fat Diet.

Intraperitoneal injection of arglabin (2.5 ng/g of body weight, twice daily, 13 weeks) into female human apolipoprotein E2 gene knock-in (ApoE2Ki) mice fed a high-fat Western-type diet (HFD) reduced plasma levels of glucose and insulin by ∼20.0% ± 3.5% and by 50.0% ± 2.0%, respectively, in comparison with vehicle-treated mice. Immunohistochemical analysis revealed the absence of active caspase-3 in islet sections from ApoE2Ki mice fed a HFD and treated with arglabin. In addition, arglabin reduced interleukin-1ß (IL-1ß) production in a concentration-dependent manner in Langerhans islets isolated from ApoE2Ki mice treated with lipopolysaccharide (LPS) and with cholesterol crystals. This inhibitory effect is specific for the inflammasome NOD-like receptor family, pyrin domain-containing 3 (NLRP3) because IL-1ß production was abolished in Langerhans islets isolated from Nlrp3(-/-) mice. In the insulin-secreting INS-1 cells, arglabin inhibited, in a concentration-dependent manner, the maturation of pro-IL-1ß into biologically active IL-1ß probably through the inhibition of the maturation of procaspase-1 into active capsase-1. Moreover, arglabin reduced the susceptibility of INS-1 cells to apoptosis by increasing Bcl-2 levels. Similarly, autophagy activation by rapamycin decreased apoptosis susceptibility while autophagy inhibition by 3-methyladenin treatment promoted apoptosis. Arglabin further increased the expression of the autophagic markers Bcl2-interacting protein (Beclin-1) and microtubule-associated protein 1 light chain 3 II (LC3-II) in a concentration-dependent manner. Thus, arglabin reduces NLRP3-dependent inflammation as well as apoptosis in pancreatic ß-cells in vivo and in the INS-1 cell line in vitro, whereas it increases autophagy in cultured INS-1 cells, indicating survival-promoting properties of the compound in these cells. Hence, arglabin may represent a new promising compound to treat inflammation and type 2 diabetes mellitus development.

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation.

BACKGROUND: Fetal exposure to hyperglycemia impacts negatively kidney development and function. OBJECTIVE: Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. DESIGN: Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. RESULTS: Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)--a key enzyme involved in gene expression during early development--was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. CONCLUSION: Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.

Glucose Tolerance Is Improved in Mice Invalidated for the Nuclear Receptor HNF-4γ: A Critical Role for Enteroendocrine Cell Lineage.

Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1-positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic ß-cells, and we report an increase of the ß-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage.

Fetal PGC-1α overexpression programs adult pancreatic ß-cell dysfunction.

Adult ß-cell dysfunction, a hallmark of type 2 diabetes, can be programmed by adverse fetal environment. We have shown that fetal glucocorticoids (GCs) participate in this programming through inhibition of ß-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1α represses genes important for ß-cell development and function. More precisely, PGC-1α inhibited the expression of the key ß-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1α complex to the Pdx1 promoter. To explore PGC-1α function, we generated mice with inducible ß-cell PGC-1α overexpression. Mice overexpressing PGC-1α exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased ß-cell mass, and ß-cell hypotrophy. Interestingly, PGC-1α expression in fetal life only was sufficient to impair adult ß-cell function whereas ß-cell PGC-1α overexpression from adult age had no consequence on ß-cell function. Altogether, our results demonstrate that the GR and PGC-1α participate in the fetal programming of adult ß-cell function through inhibition of Pdx1 expression.

ß- and α-cell dysfunctions in africans with ketosis-prone atypical diabetes during near-normoglycemic remission.

OBJECTIVE: Ketosis-prone atypical diabetes (KPD) is a subtype of diabetes in which the pathophysiology is yet to be unraveled. The aim of this study was to characterize ß- and α-cell functions in Africans with KPD during remission. RESEARCH DESIGN AND METHODS: We characterized ß- and α-cell functions in Africans with KPD during remission. The cohort comprised 15 sub-Saharan Africans who had been insulin-free for a median of 6 months. Patients in remission were in good glycemic control (near-normoglycemic) and compared with 15 nondiabetic control subjects matched for age, sex, ethnicity, and BMI. Plasma insulin, C-peptide, and glucagon concentrations were measured in response to oral and intravenous glucose and to combined intravenous arginine and glucose. Early insulin secretion was measured during a 75-g oral glucose tolerance test. Insulin secretion rate and glucagon were assessed in response to intravenous glucose ramping. RESULTS: Early insulin secretion and maximal insulin secretion rate were lower in patients compared with control participants. In response to combined arginine and glucose stimulation, maximal insulin response was reduced. Glucagon suppression was also decreased in response to oral and intravenous glucose but not in response to arginine and insulin. CONCLUSIONS: Patients with KPD in protracted near-normoglycemic remission have impaired insulin response to oral and intravenous glucose and to arginine, as well as impaired glucagon suppression. Our results suggest that ß- and α-cell dysfunctions both contribute to the pathophysiology of KPD.

Developmental programming of neonatal pancreatic ß-cells by a maternal low-protein diet in rats involves a switch from proliferation to differentiation.

Maternal low-protein diets (LP) impair pancreatic ß-cell development, resulting in later-life failure and susceptibility to type 2 diabetes (T2D). We hypothesized that intrauterine and/or postnatal developmental programming seen in this situation involve altered ß-cell structure and relative time course of expression of genes critical to ß-cell differentiation and growth. Pregnant Wistar rats were fed either control (C) 20% or restricted (R) 6% protein diets during pregnancy (1st letter) and/or lactation (2nd letter) in four groups: CC, RR, RC, and CR. At postnatal days 7 and 21, we measured male offspring ß-cell fraction, mass, proliferation, aggregate number, and size as well as mRNA level for 13 key genes regulating ß-cell development and function in isolated islets. Compared with CC, pre- and postnatal LP (RR) decreased ß-cell fraction, mass, proliferation, aggregate size, and number and increased Hnf1a, Hnf4a, Pdx1, Isl1, Rfx6, and Slc2a2 mRNA levels. LP only in pregnancy (RC) also decreased ß-cell fraction, mass, proliferation, aggregate size, and number and increased Hnf1a, Hnf4a, Pdx1, Rfx6, and Ins mRNA levels. Postnatal LP offspring (CR) showed decreased ß-cell mass but increased ß-cell fraction, aggregate number, and Hnf1a, Hnf4a, Rfx6, and Slc2a2 mRNA levels. We conclude that LP in pregnancy sets the trajectory of postnatal ß-cell growth and differentiation, whereas LP in lactation has smaller effects. We propose that LP promotes differentiation through upregulation of transcription factors that stimulate differentiation at the expense of proliferation. This results in a decreased ß-cell reserve, which can contribute to later-life predisposition to T2D.

Novel transgenic mice for inducible gene overexpression in pancreatic cells define glucocorticoid receptor-mediated regulations of beta cells.

Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology.

Specific control of pancreatic endocrine ß- and δ-cell mass by class IIa histone deacetylases HDAC4, HDAC5, and HDAC9.

OBJECTIVE: Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development. RESEARCH DESIGN AND METHODS: We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments. RESULTS: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine ß- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing ß-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing ß-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper ß-and δ-cell differentiation and amplifies endocrine ß- and δ-cells. CONCLUSIONS: We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic ß/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for ß-cell differentiation-based therapies.