Larry G. Moss

Identification of β-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA

Of the three critical enhancer elements that mediate β-cell-specific and glucose-responsive expression of the insulin gene, only the identity of the transcription factor binding to the RIPE3b element (RIPE3b1) has remained elusive. Using a biochemical purification approach, we have identified the RIPE3b1 factor as a mammalian homologue of avian MafA/L-Maf (mMafA). The avian MafA is a cell-type determination factor that expressed ectopically can trigger lens differentiation program, but no mammalian homologue of avian MafA has previously been identified. Here, we report cloning of the human mafA (hMafA) and demonstrate that it can specifically bind the insulin enhancer element RIPE3b and activate insulin-gene expression. In addition, mMafA has a very restrictive cellular distribution and is selectively expressed in pancreatic β but not in α cells. We suggest that mMafA has an essential role in the function and differentiation of β-cells and thus may be associated with the pathophysiological origins of diabetes.

Regeneration of the Pancreas in Adult Zebrafish

OBJECTIVE Regenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian β-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic β-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans. RESEARCH DESIGN AND METHODS Adult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells. RESULTS Zebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively β-cells. CONCLUSIONS The ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases.

INDIVIDUAL BETA CELLS WITHIN THE INTACT ISLET DIFFERENTIALLY RESPOND TO GLUCOSE

Insulin production by the pancreatic islet is tightly coupled to the concentration of blood glucose. The mechanism by which glucose controls proinsulin biosynthesis in β cells is poorly understood. Analysis of insulin gene expression in individual cells within whole, living islets using adenovirus gene transfer and direct observation of insulin promoter-directed green fluorescent protein activity indicates that β cells are functionally heterogeneous. An increase in glucose concentration not only stimulates expression within individual β cells, but unexpectedly acts to increase the total number of positive cells. The net islet response to a given glucose stimulus reflects an integrated action of β cells with individually differing behaviors. This additional level of functional complexity may provide new insights into the pathophysiology and treatment of diabetes mellitus.

Distribution and tissue specificity of 4-aminobutyrate-2-oxoglutarate aminotransferase.

A rapid and specific method for assaying 4-aminobutyrate-2-oxoglutarate aminotransferase was developed. The method was based on the selectivity of ion exchange resin and the speed of vacuum filtration. With this new method, the aminotransferase activity in various tissues has been determined as follows: brain, 10.2; spinal cord, 11.8; liver, 5.7; kidney, 4.6; heart, 0.5; lung, 0.4 nmol glutamate formed/min/mg. No activity could be detected in muscle preparations. When the aminotransferases were tested with the antibody, against the purified 4-aminobutyrate aminotransferase from brain, no difference could be detected among brain, spinal cord, and kidney preparations as judged from the results of immunodiffusion, inhibition of enzyme activity by antibody, and microcomplement fixation. It is concluded that 4-aminobutyrate aminotransferases from various tissues of the mouse are probably identical or closely related.

Nkx6.1 regulates islet β-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors

Significance Loss of pancreatic islet β cells occurs in both major forms of diabetes, and strategies for restoring β cells are needed. The homeobox transcription factor NK6 homeobox 1 (Nkx6.1) activates β-cell proliferation and insulin secretion when overexpressed in pancreatic islets, but the molecular pathway involved in the proliferative response is unknown. We show that Nkx6.1 induces expression of orphan nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3), which stimulate proliferation via two mechanisms: (i) increased expression of the cell cycle inducers E2F transcription factor 1 and cyclin E1; and (ii) induction of anaphase-promoting complex elements, and degradation of the cell cycle inhibitor p21. These studies reveal a new bipartite pathway for activation of β-cell proliferation that could guide development of therapeutic strategies for diabetes. Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

Sulfonylurea signal transduction.

In the pancreatic beta cells the proximal step in sulfonylurea signal transduction is the binding of these clinically important drugs to high-affinity receptors in the beta cell membrane. Using HIT cells as a model system, we have established an extremely close correlation between the affinity of binding of glyburide and its analog, iodoglyburide, and the activation of various steps in stimulus-secretion coupling--inhibition of 86Rb+ efflux, increase in [Ca2+]i resulting from gating of voltage-gated calcium channels by cell depolarization, and the exocytosis of insulin. Two different L-type channel cDNAs have been identified in an HIT cell library, one neuroendocrine in type and one more cardiac-like. A HIT cell membrane protein of Mr 140,000, which we believe to be the high-affinity sulfonylurea receptor, can be covalently linked to 5(125)-iodo-2-hydroxyglyburide by ultraviolet irradiation. The receptor has been solubilized and retains binding activity and the same rank order of displacement of the 5(125)-iodo-2-hydroxyglyburide as observed with the native receptor. The Mr 140,000 protein has been partially purified and the amino acid sequences of three proteolytic fragments have been used to design oligonucleotides to screen HIT cell cDNA libraries. Since the binding constant of glyburide or iodoglyburide is closely correlated with the ability of these compounds to inhibit the ATP-sensitive K+ channel, increase [Ca2+]i, and elicit insulin secretion, we have identified the Mr 140,000 protein as the sulfonylurea receptor. Expression of the cloned cDNA should allow us to test this hypothesis directly.

Pdx-1 Activates Islet α- and β-Cell Proliferation via a Mechanism Regulated by Transient Receptor Potential Cation Channels 3 and 6 and Extracellular Signal-Regulated Kinases 1 and 2

ABSTRACT The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and β-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, cooverexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly β-cell proliferation, whereas Pdx-1 stimulates both α- and β-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1-stimulated but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 but not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and small interfering RNA (siRNA)-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1.

Tissue and regional distribution of cysteic acid decarboxylase. A new assay method.

A sensitive and rapid assay method for cysteic acid decarboxylase was developed which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of35S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.

Genetic risk analysis of a patient with fulminant autoimmune type 1 diabetes mellitus secondary to combination ipilimumab and nivolumab immunotherapy

BackgroundCheckpoint inhibitor immunotherapy is becoming an effective treatment modality for an increasing number of malignancies. As a result, autoinflammatory side-effects are also being observed more commonly in the clinic. We are currently unable to predict which patients will develop more severe toxicities associated with these treatment regimens.Case presentationWe present a patient with stage IV melanoma that developed rapid onset autoimmune type 1 diabetes (T1D) in response to combination ipilimumab and nivolumab immunotherapy. At the time of the patient’s presentation with diabetes ketoacidosis, a confirmed anti-GAD antibody seroconversion was noted. Longer-term follow-up of this patient has demonstrated a durable complete response based on PET CT imaging along with a persistently undetectable C-peptide level. Single nucleotide polymorphism gene sequencing and HLA risk allele analysis has revealed the patient to lack any established genetic predisposition to the development of autoimmune T1D.ConclusionsWhile larger studies are necessary to better understand the role of genetic risk factors for the development of autoimmune toxicities in those patients undergoing checkpoint inhibitor immunotherapy, these results suggest that pre-screening patients for known T1D risk alleles may not be indicated. Additional investigation is needed to determine whether an approach such as T cell receptor clonotypic analysis to identify the presence of autoreactive T cell clones may be an effective approach for predicting which patients are at risk for the development of autoinflammatory toxicities while undergoing checkpoint inhibitor immunotherapy.

Imaging beta cell regeneration and interactions with islet vasculature in transparent adult zebrafish.

Blood vessel networks provide nutrients and gaseous exchange that are essential for functions. Pancreatic islet capillaries deliver oxygen to endocrine cells while transporting hormones to organs and peripheral locations throughout the body. We have developed a zebrafish diabetes model in which adult islets can be followed in vivo during beta cell regeneration while calibrating changes in beta cell mass and fasting blood glucose levels. After genetic ablation, beta cells are initially dysfunctional or dying, and blood glucose levels increase fourfold. During a 2-week period, hyperglycemia eventually normalizes as beta cell mass regenerates. We show that mCherry-fluorescent, insulin-positive beta cells re-emerge in close contact with the vascular endothelium. Alterations in the dense vascular network of zebrafish islets were visualized by the expression of green fluorescent protein (GFP) in endothelial cells derived from the Fli transcription factor promoter. The rapid destruction and regeneration of beta cell mass was evaluated in the same animal over time, providing a functional model for investigating the interactions of islet cell types with vascular cells as well as the consequences of hyperglycemia on other tissues. Regenerating adult zebrafish can be utilized as vertebrate, metabolically active models for generating new insights into treatments for type 2 diabetes.