Tight spatial regulation of extracellular morphogen signaling within the close confines of a developing embryo is critical for proper organogenesis. Given the complexity of extracellular signaling in developing organs, together with the proximity of adjacent organs that use disparate signaling pathways, we postulated that a physical barrier to signaling may exist between organs in the embryo. Here we describe a previously unrecognized role for the embryonic coelomic epithelium in providing a physical barrier to contain morphogenic signaling in the developing mouse pancreas. This layer of cells appears to function both to contain key factors required for pancreatic epithelial differentiation, and to prevent fusion of adjacent organs during critical developmental windows. During early foregut development, this barrier appears to play a role in preventing splenic anlage-derived activin signaling from inducing intestinalization of the pancreas-specified epithelium.

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.

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

Protection and restoration of a functional β-cell mass are fundamental strategies for prevention and treatment of diabetes. Consequently, knowledge of signals that determine the functional β-cell mass is of immense clinical relevance. Transforming growth factor β (TGFβ) superfamily signaling pathways play a critical role in development and tissue specification. Nevertheless, the role of these pathways in adult β-cell homeostasis is not well defined. Here, we ablated TGFβ receptor I and II genes in mice undergoing two surgical β-cell replication models (partial pancreatectomy or partial duct ligation), representing two triggers for β-cell proliferation, increased β-cell workload and local inflammation, respectively. Our data suggest that TGFβ receptor signaling is necessary for baseline β-cell proliferation. By either provision of excess glucose or treatment with exogenous insulin, we further demonstrated that inflammation and increased β-cell workload are both stimulants for β-cell proliferation but are TGFβ receptor signaling dependent and independent, respectively. Collectively, by using a pancreas-specific TGFβ receptor-deleted mouse model, we have identified two distinct pathways that regulate adult β-cell proliferation. Our study thus provides important information for understanding β-cell proliferation during normal growth and in pancreatic diseases.

Pancreatic alpha-cells, like beta-cells, express ATP-sensitive K(+) (K(ATP)) channels. To determine the physiological role of K(ATP) channels in alpha-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic alpha-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null alpha-cells showed continuous oscillations in [Ca(2+)](i) regardless of the glucose concentration. These findings indicate that K(ATP) channels in alpha-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack K(ATP) channels maintain euglycemia.

Early diagnosis of liver injuries caused by drugs or occupational exposures is necessary to enable effective treatments and prevent liver failure. Whereas histopathology remains the gold standard for assessing hepatotoxicity in animals, plasma aminotransferase levels are the primary measures for monitoring liver dysfunction in humans. In this study, using Sprague Dawley rats, we investigated whether integrated analyses of transcriptomic and metabolomic data with genome-scale metabolic models (GSMs) could identify early indicators of injury and provide new insights into the mechanisms of hepatotoxicity. We obtained concurrent measurements of gene-expression changes in the liver and kidneys, and expression changes along with metabolic profiles in the plasma and urine, from rats 5 or 10 h after exposing them to one of two classical hepatotoxicants, acetaminophen (2 g/kg) or bromobenzene (0.4 g/kg). Global multivariate analyses revealed that gene-expression changes in the liver and metabolic profiles in the plasma and urine of toxicant-treated animals differed from those of controls, even at time points much earlier than changes detected by conventional markers of liver injury. Furthermore, clustering analysis revealed that both the gene-expression changes in the liver and the metabolic profiles in the plasma induced by the two hepatotoxicants were highly correlated, indicating commonalities in the liver toxicity response. Systematic GSM-based analyses yielded metabolites associated with the mechanisms of toxicity and identified several lipid and amino acid metabolism pathways that were activated by both toxicants and those uniquely activated by each. Our findings suggest that several metabolite alterations, which are strongly associated with the mechanisms of toxicity and occur within injury-specific pathways (e.g., of bile acid and fatty acid metabolism), could be targeted and clinically assessed for their potential as early indicators of liver damage.

A key question in diabetes research is whether new β-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into β-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-β receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new β-cells in normal young developing mice as well as in adult TGF-β signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to β-cells in the postnatal pancreas but also importantly implicates TGF-β signaling in this process.

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.

Glucose-dependent insulinotropic polypeptide (GIP) is a member of a structurally related group of hormones that also includes glucagon, glucagon-like peptides, and secretin. GIP is an incretin, known to modulate glucose-induced insulin secretion. Recent studies have shown that glucagon is necessary for early insulin-positive differentiation, and a similar role for incretins in regulating embryonic insulin-positive differentiation seems probable. Here we studied the role of GIP signaling in insulin-positive differentiation in the embryonic mouse pancreas.

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.

Autophagy is known to play a pivotal role in intracellular quality control through the degradation of subcellular damaged organelles and components. Whereas autophagy is essential for maintaining β-cell function in pancreatic islets, it remains unclear as to how the cellular autophagy affects the homeostasis and function of glucagon-secreting α cells. To investigate the role of autophagy in α cells, we generated a mutant mouse model lacking Atg7, a key molecule for autophagosome formation, specifically in α cells. Histological analysis demonstrated more glucagon-positive cells, with a multilayered structure, in the islets under Atg7 deficiency, although metabolic profiles, such as body weight, blood glucose, and plasma glucagon levels were comparable between Atg7-deficient mice and control littermates. Consistent with our previous findings that Atg7 deficiency suppressed β-cell proliferation, cellular proliferation was suppressed in Atg7-deficient α cells. These findings suggest that α-cell autophagy plays a role in maintaining α-cell area and normal islet architecture but appears to be dispensable for metabolic homeostasis.

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.

Pancreatic β-cell loss and dysfunction are critical components of all types of diabetes. Human and rodent β-cells are able to proliferate, and this proliferation is an important defense against the evolution and progression of diabetes. Transforming growth factor-β (TGF-β) signaling has been shown to affect β-cell development, proliferation, and function, but β-cell proliferation is thought to be the only source of new β-cells in the adult. Recently, β-cell dedifferentiation has been shown to be an important contributory mechanism to β-cell failure. In this study, we tie together these two pathways by showing that a network of intracellular TGF-β regulators, smads 7, 2, and 3, control β-cell proliferation after β-cell loss, and specifically, smad7 is necessary for that β-cell proliferation. Importantly, this smad7-mediated proliferation appears to entail passing through a transient, nonpathologic dedifferentiation of β-cells to a pancreatic polypeptide-fold hormone-positive state. TGF-β receptor II appears to be a receptor important for controlling the status of the smad network in β-cells. These studies should help our understanding of properly regulated β-cell replication.

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

Islet α-cell dysfunction has been shown to contribute to type 2 diabetes; however, whether islet α-cell inflammation is involved in the occurrence of pancreatitis is largely unknown. The aims of this study were to investigate how NF-κB inducing kinase (NIK) regulates pancreatic α-cell function, both in vitro and in vivo, and to assess how islet α-cell inflammation induced by NIK affects the development of pancreatitis. Methods: We utilized adenovirus-mediated NIK overexpression, ELISA, qPCR, RNA-seq, and Western blot analyses to study the role of NIK in islet α cells in vitro. Islet α-cell-specific NIK overexpressing (α-NIK-OE) mice were generated, and pancreatic α/β-cell function and the occurrence of pancreatitis in these mice were assessed via ELISA, qPCR, and immunohistochemical analyses. Results: The LTβR/noncanonical NF-κB signaling pathway is present in islet α cells. Overexpression of NIK in αTC1-6 cells induces inflammation and cell death, contributing to a decrease in the expression and secretion of glucagon. Additionally, α-cell specific overexpression of NIK (α-NIK-OE) results in α-cell death, lower serum glucagon levels, and hypoglycemia in mice. Strikingly, α-NIK-OE mice also display a reduced β-cell mass, growth retardation, pancreatitis, and postnatal death. Conclusions: Islet α-cell specific overexpression of NIK results in islet α-cell dysfunction and causes islet β-cell death and pancreatitis, which are most likely due to paracrine secretion of cytokines and chemokines from islet α cells, thus leading to hypoglycemia, growth retardation, and postnatal death in mice.

Kidney injury caused by disease, trauma, environmental exposures, or drugs may result in decreased renal function, chronic kidney disease, or acute kidney failure. Diagnosis of kidney injury using serum creatinine levels, a common clinical test, only identifies renal dysfunction after the kidneys have undergone severe damage. Other indicators sensitive to kidney injury, such as the level of urine kidney injury molecule-1 (KIM-1), lack the ability to differentiate between injury phenotypes. To address early detection as well as detailed categorization of kidney-injury phenotypes in preclinical animal or cellular studies, we previously identified eight sets (modules) of co-expressed genes uniquely associated with kidney histopathology. Here, we used mercuric chloride (HgCl2)-a model nephrotoxicant-to chemically induce kidney injuries as monitored by KIM-1 levels in Sprague Dawley rats at two doses (0.25 or 0.50 mg/kg) and two exposure lengths (10 or 34 h). We collected whole transcriptome RNA-seq data derived from five animals at each dose and time point to perform a toxicogenomics analysis. Consistent with documented injury phenotypes for HgCl2 toxicity, our kidney-injury-module approach identified the onset of necrosis and dilation as early as 10 h after a dose of 0.50 mg/kg that produced only mild injury as judged by urinary KIM-1 excretion. The results of these animal studies highlight the potential of the kidney-injury-module approach to provide a sensitive and histopathology-specific readout of renal toxicity.