The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity.

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

Protection from ß-cell apoptosis by inhibition of TGF-ß/Smad3 signaling.

Prevailing insulin resistance and the resultant hyperglycemia elicits a compensatory response from pancreatic islet beta cells (ß-cells) that involves increases in ß-cell function and ß-cell mass. However, the sustained metabolic stress eventually leads to ß-cell failure characterized by severe ß-cell dysfunction and progressive loss of ß-cell mass. Whereas, ß-cell dysfunction is relatively well understood at the mechanistic level, the avenues leading to loss of ß-cell mass are less clear with reduced proliferation, dedifferentiation, and apoptosis all potential mechanisms. Butler and colleagues documented increased ß-cell apoptosis in pancreas from lean and obese human Type 2 diabetes (T2D) subjects, with no changes in rates of ß-cell replication or neogenesis, strongly suggesting a role for apoptosis in ß-cell failure. Here, we describe a permissive role for TGF-ß/Smad3 in ß-cell apoptosis. Human islets undergoing ß-cell apoptosis release increased levels of TGF-ß1 ligand and phosphorylation levels of TGF-ß's chief transcription factor, Smad3, are increased in human T2D islets suggestive of an autocrine role for TGF-ß/Smad3 signaling in ß-cell apoptosis. Smad3 phosphorylation is similarly increased in diabetic mouse islets undergoing ß-cell apoptosis. In mice, ß-cell-specific activation of Smad3 promotes apoptosis and loss of ß-cell mass in association with ß-cell dysfunction, glucose intolerance, and diabetes. In contrast, inactive Smad3 protects from apoptosis and preserves ß-cell mass while improving ß-cell function and glucose tolerance. At the molecular level, Smad3 associates with Foxo1 to propagate TGF-ß-dependent ß-cell apoptosis. Indeed, genetic or pharmacologic inhibition of TGF-ß/Smad3 signals or knocking down Foxo1 protects from ß-cell apoptosis. These findings reveal the importance of TGF-ß/Smad3 in promoting ß-cell apoptosis and demonstrate the therapeutic potential of TGF-ß/Smad3 antagonism to restore ß-cell mass lost in diabetes.

Corrigendum to "Islet ß-Cell Mass Preservation and Regeneration in Diabetes Mellitus: Four Factors with Potential Therapeutic Interest".

[This corrects the article DOI: 10.1155/2012/230870.].

Dissecting the Brain/Islet Axis in Metabesity.

The high prevalence of type 2 diabetes mellitus (T2DM), together with the fact that current treatments are only palliative and do not avoid major secondary complications, reveals the need for novel approaches to treat the cause of this disease. Efforts are currently underway to identify therapeutic targets implicated in either the regeneration or re-differentiation of a functional pancreatic islet ß-cell mass to restore insulin levels and normoglycemia. However, T2DM is not only caused by failures in ß-cells but also by dysfunctions in the central nervous system (CNS), especially in the hypothalamus and brainstem. Herein, we review the physiological contribution of hypothalamic neuronal and glial populations, particularly astrocytes, in the control of the systemic response that regulates blood glucose levels. The glucosensing capacity of hypothalamic astrocytes, together with their regulation by metabolic hormones, highlights the relevance of these cells in the control of glucose homeostasis. Moreover, the critical role of astrocytes in the response to inflammation, a process associated with obesity and T2DM, further emphasizes the importance of these cells as novel targets to stimulate the CNS in response to metabesity (over-nutrition-derived metabolic dysfunctions). We suggest that novel T2DM therapies should aim at stimulating the CNS astrocytic response, as well as recovering the functional pancreatic ß-cell mass. Whether or not a common factor expressed in both cell types can be feasibly targeted is also discussed.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

The type 2 diabetes-associated HMG20A gene is mandatory for islet beta cell functional maturity.

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.

GATA6 Controls Insulin Biosynthesis and Secretion in Adult ß-Cells.

GATA4 and GATA6 play essential, but redundant, roles in pancreas formation in mice, and GATA6 mutations cause pancreatic agenesis in humans. GATA6 mutations have also recently been linked to adult-onset diabetes, with subclinical or no exocrine insufficiency, suggesting an important role for GATA6 in human ß-cell physiology. To investigate the role of GATA6 in the adult endocrine pancreas, we generated mice in which Gata6 is specifically inactivated in the pancreas. These mice develop glucose intolerance. Islets deficient in GATA6 activity display decreased insulin content and impaired insulin secretion. Gata6-deficient ß-cells exhibit ultrastructural abnormalities, including increased immature insulin granules, swollen mitochondria, and disorganized endoplasmic reticulum. We also demonstrate that Pdx1 expression in adult ß-cells depends on GATA sites in transgenic reporter mice and that loss of GATA6 greatly affects ß-cell-specific gene expression. These findings demonstrate the essential role of GATA6 in ß-cell function.

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice.

BACKGROUND AND PURPOSE: Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with levothyroxine from birth to 24 weeks of age, when mice were killed. The effects of levothyroxine supplementation on metabolic health were determined. C57BL/6 mice treated with levothyroxine for 2 weeks and then challenged with streptozotocin to monitor survival. Mechanistic experiments were conducted in the pancreas, liver and skeletal muscle. RIP-B7.1 mice were treated with levothyroxine for 2 weeks and were subsequently immunized to trigger experimental autoimmune diabetes (EAD). Metabolic tests were performed. Mice were killed and metabolic tissues were extracted for immunohistological analyses. KEY RESULTS: Long-term levothyroxine supplementation enhanced glucose clearance and reduced circulating glucose in C57BL/6 mice. Levothyroxine increased simultaneously the proliferation and apoptosis of pancreatic beta cells, promoting the maintenance of a highly insulin-expressing beta cell population. Levothyroxine increased circulating insulin levels, inducing sustained activation of IRS1-AKT signalling in insulin-target tissues. Levothyroxine-treated C57BL/6 mice challenged with streptozotocin exhibited extended survival. Levothyroxine blunted the onset of EAD in RIP-B7.1 mice by inducing beta cell proliferation and preservation of insulin-expressing cells. CONCLUSIONS AND IMPLICATIONS: Interventions based on the use of thyroid hormones or thyromimetics could be explored to provide therapeutic benefit in patients with type 1 diabetes mellitus.

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.

AIMS/HYPOTHESIS: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. METHODS: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. RESULTS: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. CONCLUSIONS/INTERPRETATION: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.

PAX4 Defines an Expandable ß-Cell Subpopulation in the Adult Pancreatic Islet.

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects ß-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes ß-cell dedifferentiation and hyperglycemia, mimicking ß-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable ß-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched ß-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4(+) progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP(+) subpopulation expanded during pregnancy, a state of active ß-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP(+) cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP(+) cells were more resistant to apoptosis than their GFP(-) counterparts. Our data suggest PAX4 defines an expandable ß-cell sub population within adult islets.

A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional ß-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

Hepatocyte growth factor/c-Met signaling is required for ß-cell regeneration.

Hepatocyte growth factor (HGF) is a mitogen required for ß-cell replication during pregnancy. To determine whether HGF/c-Met signaling is required for ß-cell regeneration, we characterized mice with pancreatic deletion of the HGF receptor, c-Met (PancMet KO mice), in two models of reduced ß-cell mass and regeneration: multiple low-dose streptozotocin (MLDS) and partial pancreatectomy (Ppx). We also analyzed whether HGF administration could accelerate ß-cell regeneration in wild-type (WT) mice after Ppx. Mouse islets obtained 7 days post-Ppx displayed significantly increased c-Met, suggesting a potential role for HGF/c-Met in ß-cell proliferation in situations of reduced ß-cell mass. Indeed, adult PancMet KO mice displayed markedly reduced ß-cell replication compared with WT mice 7 days post-Ppx. Similarly, ß-cell proliferation was decreased in PancMet KO mice in the MLDS mouse model. The decrease in ß-cell proliferation post-Ppx correlated with a striking decrease in D-cyclin levels. Importantly, PancMet KO mice showed significantly diminished ß-cell mass, decreased glucose tolerance, and impaired insulin secretion compared with WT mice 28 days post-Ppx. Conversely, HGF administration in WT Ppx mice further accelerated ß-cell regeneration. These results indicate that HGF/c-Met signaling is critical for ß-cell proliferation in situations of diminished ß-cell mass and suggest that activation of this pathway can enhance ß-cell regeneration.

Islet ß-Cell Mass Preservation and Regeneration in Diabetes Mellitus: Four Factors with Potential Therapeutic Interest.

Islet ß-cell replacement and regeneration are two promising approaches for the treatment of Type 1 Diabetes Mellitus. Indeed, the success of islet transplantation in normalizing blood glucose in diabetic patients has provided the proof of principle that cell replacement can be employed as a safe and efficacious treatment. Nonetheless, shortage of organ donors has hampered expansion of this approach. Alternative sources of insulin-producing cells are mandatory to fill this gap. Although great advances have been achieved in generating surrogate ß-cells from stem cells, current protocols have yet to produce functionally mature insulin-secreting cells. Recently, the concept of islet regeneration in which new ß-cells are formed from either residual ß-cell proliferation or transdifferentiation of other endocrine islet cells has gained much interest as an attractive therapeutic alternative to restore ß-cell mass. Complementary approaches to cell replacement and regeneration could aim at enhancing ß-cell survival and function. Herein, we discuss the value of Hepatocyte Growth Factor (HGF), Glucose-Dependent Insulinotropic Peptide (GIP), Paired box gene 4 (Pax4) and Liver Receptor Homolog-1 (LRH-1) as key players for ß-cell replacement and regeneration therapies. These factors convey ß-cell protection and enhanced function as well as facilitating proliferation and transdifferentiation of other pancreatic cell types to ß-cells, under stressful conditions.

Activation of protein kinase C-ζ in pancreatic ß-cells in vivo improves glucose tolerance and induces ß-cell expansion via mTOR activation.

OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor-induced ß-cell replication in vitro. However, the effect of direct PKC-ζ activation in the ß-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in ß-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and ß-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the ß-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and ß-cell functional markers in TG and wild-type mouse islets. RESULTS: TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased ß-cell proliferation, size, and mass compared with wild-type littermates. The increase in ß-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3ß and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and ß-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in ß-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance-identifying mTOR as a novel downstream mediator of PKC-ζ-induced ß-cell replication and expansion in vivo. CONCLUSIONS PKC:-ζ, through mTOR activation, modifies the expression pattern of ß-cell cycle molecules leading to increased ß-cell replication and mass with a concomitant enhancement in ß-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.

RB regulates pancreas development by stabilizing Pdx1.

RB is a key substrate of Cdks and an important regulator of the mammalian cell cycle. RB either represses E2Fs that promote cell proliferation or enhances the activity of cell-specific factors that promote differentiation, although the mechanism that facilitates this dual interaction is unclear. Here, we demonstrate that RB associates with and stabilizes pancreatic duodenal homeobox-1 (Pdx-1) that is essential for embryonic pancreas development and adult ß-cell function. Interestingly, Pdx-1 utilizes a conserved RB-interaction motif (RIM) that is also present in E2Fs. Point mutations within the RIM reduce RB-Pdx-1 complex formation, destabilize Pdx-1 and promote its proteasomal degradation. Glucose regulates RB and Pdx-1 levels, RB/Pdx-1 complex formation and Pdx-1 degradation. RB occupies the promoters of ß-cell-specific genes, and knockdown of RB results in reduced expression of Pdx-1 and its target genes. Further, RB-deficiency in vivo results in reduced pancreas size due to decreased proliferation of Pdx-1(+) pancreatic progenitors, increased apoptosis and aberrant expression of regulators of pancreatic development. These results demonstrate an unanticipated regulatory mechanism for pancreatic development and ß-cell function, which involves RB-mediated stabilization of the pancreas-specific transcription factor Pdx-1.

Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes.

OBJECTIVE: To determine the role of hepatocyte growth factor (HGF)/c-Met on ß-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS: We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced ß-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS: Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in ß-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and ß-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for ß-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced ß-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and ß-cell apoptosis. c-Met-null ß-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human ß-cells against cytokines. CONCLUSIONS: These results show that HGF/c-Met is critical for ß-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing ß-cell protection.

Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis.

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment. However, whether HGF is a beta-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the beta-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased beta-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and beta-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse beta-cells and normal beta-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-alpha and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased beta-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates beta-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for beta-cell survival in an environment with excessive fatty acid supply.

Protein kinase C-zeta activation markedly enhances beta-cell proliferation: an essential role in growth factor mediated beta-cell mitogenesis.

OBJECTIVE: Diabetes results from a deficiency of functional beta-cells. Previous studies have identified hepatocyte growth factor (HGF) and parathyroid hormone-related protein (PTHrP) as two potent beta-cell mitogens. The objective of this study is to determine 1) whether HGF and PTHrP have additive/synergistic effects on beta-cell growth and proliferation; 2) the signaling pathways through which these growth factors mediate beta-cell mitogenesis; and 3) whether activation of this/these signaling pathway(s) enhances human beta-cell replication. RESEARCH DESIGN AND METHODS: We generated and phenotypically analyzed doubly transgenic mice overexpressing PTHrP and HGF in the beta-cell. INS-1 and primary mouse and human islet cells were used to identify mitogenic signaling pathways activated by HGF and/or PTHrP. RESULTS: Combined overexpression of HGF and PTHrP in the beta-cell of doubly transgenic mice did not result in additive/synergistic effects on beta-cell growth and proliferation, suggesting potential cross-talk between signaling pathways activated by both growth factors. Examination of these signaling pathways in INS-1 cells revealed atypical protein kinase C (PKC) as a novel intracellular target activated by both HGF and PTHrP in beta-cells. Knockdown of PKC zeta, but not PKC iota/lambda, expression using specific small-interfering RNAs blocked growth factor-induced INS-1 cell proliferation. Furthermore, adenovirus-mediated delivery of kinase-dead PKC zeta completely inhibited beta-cell proliferation in primary islet cells overexpressing PTHrP and/or HGF. Finally, adenovirus-mediated delivery of constitutively active PKC zeta in mouse and human primary islet cells significantly enhanced beta-cell proliferation. CONCLUSIONS: PKC zeta is essential for PTHrP- and HGF-induced beta-cell proliferation. PKC zeta activation could be useful in therapeutic strategies for expanding beta-cell mass in vitro and in vivo.

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines.

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).