The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

Assessing Kidney Injury Induced by Mercuric Chloride in Guinea Pigs with In Vivo and In Vitro Experiments.

Acute kidney injury, which is associated with high levels of morbidity and mortality, affects a significant number of individuals, and can be triggered by multiple factors, such as medications, exposure to toxic chemicals or other substances, disease, and trauma. Because the kidney is a critical organ, understanding and identifying early cellular or gene-level changes can provide a foundation for designing medical interventions. In our earlier work, we identified gene modules anchored to histopathology phenotypes associated with toxicant-induced liver and kidney injuries. Here, using in vivo and in vitro experiments, we assessed and validated these kidney injury-associated modules by analyzing gene expression data from the kidneys of male Hartley guinea pigs exposed to mercuric chloride. Using plasma creatinine levels and cell-viability assays as measures of the extent of renal dysfunction under in vivo and in vitro conditions, we performed an initial range-finding study to identify the appropriate doses and exposure times associated with mild and severe kidney injuries. We then monitored changes in kidney gene expression at the selected doses and time points post-toxicant exposure to characterize the mechanisms of kidney injury. Our injury module-based analysis revealed a dose-dependent activation of several phenotypic cellular processes associated with dilatation, necrosis, and fibrogenesis that were common across the experimental platforms and indicative of processes that initiate kidney damage. Furthermore, a comparison of activated injury modules between guinea pigs and rats indicated a strong correlation between the modules, highlighting their potential for cross-species translational studies.

Evidence of a developmental origin for ß-cell heterogeneity using a dual lineage-tracing technology.

The Cre/loxP system has been used extensively in mouse models with a limitation of one lineage at a time. Differences in function and other properties among populations of adult ß-cells is termed ß-cell heterogeneity, which was recently associated with diabetic phenotypes. Nevertheless, the presence of a developmentally derived ß-cell heterogeneity is unclear. Here, we have developed a novel dual lineage-tracing technology, using a combination of two recombinase systems, Dre/RoxP and Cre/LoxP, to independently trace green fluorescent Pdx1-lineage cells and red fluorescent Ptf1a-lineage cells in the developing and adult mouse pancreas. We detected a few Pdx1+/Ptf1a- lineage cells in addition to the vast majority of Pdx1+/Ptf1a+ lineage cells in the pancreas. Moreover, Pdx1+/Ptf1a+ lineage ß-cells had fewer Ki-67+ proliferating ß-cells, and expressed higher mRNA levels of insulin, Glut2, Pdx1, MafA and Nkx6.1, but lower CCND1 and CDK4 levels, compared with Pdx1+/Ptf1a- lineage ß-cells. Furthermore, more TSQ-high, SSC-high cells were detected in the Pdx1+Ptf1a+ lineage population than in the Pdx1+Ptf1a- lineage population. Together, these data suggest that differential activation of Ptf1a in the developing pancreas may correlate with this ß-cell heterogeneity.

SMAD7 enhances adult ß-cell proliferation without significantly affecting ß-cell function in mice.

The interplay between the transforming growth factor ß (TGF-ß) signaling proteins, SMAD family member 2 (SMAD2) and 3 (SMAD3), and the TGF-ß-inhibiting SMAD, SMAD7, seems to play a vital role in proper pancreatic endocrine development and also in normal ß-cell function in adult pancreatic islets. Here, we generated conditional SMAD7 knockout mice by crossing insulin1Cre mice with SMAD7fx/fx mice. We also created a ß cell-specific SMAD7-overexpressing mouse line by crossing insulin1Dre mice with HPRT-SMAD7/RosaGFP mice. We analyzed ß-cell function in adult islets when SMAD7 was either absent or overexpressed in ß cells. Loss of SMAD7 in ß cells inhibited proliferation, and SMAD7 overexpression enhanced cell proliferation. However, alterations in basic glucose homeostasis were not detectable following either SMAD7 deletion or overexpression in ß cells. Our results show that both the absence and overexpression of SMAD7 affect TGF-ß signaling and modulates ß-cell proliferation but does not appear to alter ß-cell function. Reversible SMAD7 overexpression may represent an attractive therapeutic option to enhance ß-cell proliferation without negative effects on ß-cell function.

Genomics and metabolomics of early-stage thioacetamide-induced liver injury: An interspecies study between guinea pig and rat.

To study the complex processes involved in liver injuries, researchers rely on animal investigations, using chemically or surgically induced liver injuries, to extrapolate findings and infer human health risks. However, this presents obvious challenges in performing a detailed comparison and validation between the highly controlled animal models and development of liver injuries in humans. Furthermore, it is not clear whether there are species-dependent and -independent molecular initiating events or processes that cause liver injury before they eventually lead to end-stage liver disease. Here, we present a side-by-side study of rats and guinea pigs using thioacetamide to examine the similarities between early molecular initiating events during an acute-phase liver injury. We exposed Sprague Dawley rats and Hartley guinea pigs to a single dose of 25 or 100 mg/kg thioacetamide and collected blood plasma for metabolomic analysis and liver tissue for RNA-sequencing. The subsequent toxicogenomic analysis identified consistent liver injury trends in both genomic and metabolomic data within 24 and 33 h after thioacetamide exposure in rats and guinea pigs, respectively. In particular, we found species similarities in the key injury phenotypes of inflammation and fibrogenesis in our gene module analysis for liver injury phenotypes. We identified expression of several common genes (e.g., SPP1, TNSF18, SERPINE1, CLDN4, TIMP1, CD44, and LGALS3), activation of injury-specific KEGG pathways, and alteration of plasma metabolites involved in amino acid and bile acid metabolism as some of the key molecular processes that changed early upon thioacetamide exposure and could play a major role in the initiation of acute liver injury.

Toxicant-Induced Metabolic Alterations in Lipid and Amino Acid Pathways Are Predictive of Acute Liver Toxicity in Rats.

Liver disease and disorders associated with aberrant hepatocyte metabolism can be initiated via drug and environmental toxicant exposures. In this study, we tested the hypothesis that gene and metabolic profiling can reveal commonalities in liver response to different toxicants and provide the capability to identify early signatures of acute liver toxicity. We used Sprague Dawley rats and three classical hepatotoxicants: acetaminophen (2 g/kg), bromobenzene (0.4 g/kg), and carbon tetrachloride (0.3 g/kg), to identify early perturbations in liver metabolism after a single acute exposure dose. We measured changes in liver genes and plasma metabolites at two time points (5 and 10 h) and used genome-scale metabolic models to identify commonalities in liver responses across the three toxicants. We found strong correlations for gene and metabolic profiles between the toxicants, indicative of similarities in the liver response to toxicity. We identified several injury-specific pathways in lipid and amino acid metabolism that changed similarly across the three toxicants. Our findings suggest that several plasma metabolites in lipid and amino acid metabolism are strongly associated with the progression of liver toxicity, and as such, could be targeted and clinically assessed for their potential as early predictors of acute liver toxicity.

Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated ß Cell Aging in Pancreas-specific SMAD7 Mutant Mice.

The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in ß cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated ß cell dysfunction and loss of proliferation capacity, two features of ß cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in ß cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in ß cells prevented ß cell dysfunction and loss in this model. Thus, we present a model of accelerated ß cell aging that may be useful for studying the mechanisms underlying ß cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining ß cell identity in the context of SMAD7 failure.

Gcg CreERT2 knockin mice as a tool for genetic manipulation in pancreatic alpha cells.

AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS: Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.

Epidermal Growth Factor Receptor Signaling Regulates ß Cell Proliferation in Adult Mice.

A thorough understanding of the signaling pathways involved in the regulation of ß cell proliferation is an important initial step in restoring ß cell mass in the diabetic patient. Here, we show that epidermal growth factor receptor 1 (EGFR) was significantly up-regulated in the islets of C57BL/6 mice after 50% partial pancreatectomy (PPx), a model for workload-induced ß cell proliferation. Specific deletion of EGFR in the ß cells of adult mice impaired ß cell proliferation at baseline and after 50% PPx, suggesting that the EGFR signaling pathway plays an essential role in adult ß cell proliferation. Further analyses showed that ß cell-specific depletion of EGFR resulted in impaired expression of cyclin D1 and impaired suppression of p27 after PPx, both of which enhance ß cell proliferation. These data highlight the importance of EGFR signaling and its downstream signaling cascade in postnatal ß cell growth.

A synopsis of factors regulating beta cell development and beta cell mass.

The insulin-secreting beta cells in the endocrine pancreas regulate blood glucose levels, and loss of functional beta cells leads to insulin deficiency, hyperglycemia (high blood glucose) and diabetes mellitus. Current treatment strategies for type-1 (autoimmune) diabetes are islet transplantation, which has significant risks and limitations, or normalization of blood glucose with insulin injections, which is clearly not ideal. The type-1 patients can lack insulin counter-regulatory mechanism; therefore, hypoglycemia is a potential risk. Hence, a cell-based therapy offers a better alternative for the treatment of diabetes. Past research was focused on attempting to generate replacement beta cells from stem cells; however, recently there has been an increasing interest in identifying mechanisms that will lead to the conversion of pre-existing differentiated endocrine cells into beta cells. The goal of this review is to provide an overview of several of the key factors that regulate new beta cell formation (neogenesis) and beta cell proliferation.

M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7.

Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFß superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFß1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFß1-activated SMAD2 nuclear translocation, resulting in TGFß signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Pancreatic duct cells as a source of VEGF in mice.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is essential for proper pancreatic development, islet vascularisation and insulin secretion. In the adult pancreas, VEGF is thought to be predominantly secreted by beta cells. Although human duct cells have previously been shown to secrete VEGF at angiogenic levels in culture, an analysis of the kinetics of VEGF synthesis and secretion, as well as elucidation of an in vivo role for this ductal VEGF in affecting islet function and physiology, has been lacking. METHODS: We analysed purified duct cells independently prepared by flow cytometry, surgical isolation or laser-capture microdissection. We infected duct cells in vivo with Vegf (also known as Vegfa) short hairpin RNA (shRNA) in an intrapancreatic ductal infusion system and examined the effect of VEGF knockdown in duct cells in vitro and in vivo. RESULTS: Pancreatic duct cells express high levels of Vegf mRNA. Compared with beta cells, duct cells had a much higher ratio of secreted to intracellular VEGF. As a bioassay, formation of tubular structures by human umbilical vein endothelial cells was essentially undetectable when cultured alone and was substantially increased when co-cultured with pancreatic duct cells but significantly reduced when co-cultured with duct cells pretreated with Vegf shRNA. Compared with islets transplanted alone, improved vascularisation and function was detected in the islets co-transplanted with duct cells but not in islets co-transplanted with duct cells pretreated with Vegf shRNA. CONCLUSIONS/INTERPRETATION: Human islet preparations for transplantation typically contain some contaminating duct cells and our findings suggest that the presence of duct cells in the islet preparation may improve transplantation outcomes.

Smad signaling pathways regulate pancreatic endocrine development.

Expansion of the pancreatic endocrine cell population occurs during both embryonic development and during post-natal pancreatic growth and regeneration. Mechanisms of the expansion of endocrine cells during embryonic development are not completely understood, and no clear mechanistic link has been established between growth of the embryonic endocrine pancreas and the islet cell replication that occurs in an adult animal. We found that transforming growth factor-beta (TGF-ß) superfamily signaling, which has been implicated in many developmental processes, plays a key role in regulating pancreatic endocrine maturation and development. Specifically, the intracellular mediators of TGF-ß signaling, smad2 and smad3, along with their inhibitor smad7, appear to mediate this process. Smad2, smad3 and smad7 were all broadly expressed throughout the early embryonic pancreatic epithelium. However, during later stages of development, smad2 and smad3 became strongly localized to the nuclei of the endocrine positive cells, whereas the inhibitory smad7 became absent in the endocrine component. Genetic inactivation of smad2 and smad3 led to a significant expansion of the embryonic endocrine compartment, whereas genetic inactivation of smad7 led to a significant decrease in the endocrine compartment. In vitro antisense studies further corroborated these results and supported the possibility that interplay between the inhibitory smad7 and the intracellular mediators smad2/3 is a control point for pancreatic endocrine development. These results should provide a better understanding of the key control mechanisms for ß-cell development.

Hypoglycemia reduces vascular endothelial growth factor A production by pancreatic beta cells as a regulator of beta cell mass.

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.

Neurogenin3 activation is not sufficient to direct duct-to-beta cell transdifferentiation in the adult pancreas.

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.

α-Cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets.

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistent depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of ß-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of ß-cells.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing ß-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

Dysregulation of the norepinephrine transporter sustains cortical hypodopaminergia and schizophrenia-like behaviors in neuronal rictor null mice.

The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.

Specific reprogramming of alpha cells to insulin-producing cells by short glucagon promoter-driven Pdx1 and MafA.

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we used an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 (AAV8) can be used to successfully deliver Pdx1 and MafA to pancreatic alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells were also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.