Samuel B. Stephens

A VGF-Derived Peptide Attenuates Development of Type 2 Diabetes via Enhancement of Islet β-Cell Survival and Function

Deterioration of functional islet β-cell mass is the final step in progression to Type 2 diabetes. We previously reported that overexpression of Nkx6.1 in rat islets has the dual effects of enhancing glucose-stimulated insulin secretion (GSIS) and increasing β-cell replication. Here we show that Nkx6.1 strongly upregulates the prohormone VGF in rat islets and that VGF is both necessary and sufficient for Nkx6.1-mediated enhancement of GSIS. Moreover, the VGF-derived peptide TLQP-21 potentiates GSIS in rat and human islets and improves glucose tolerance in vivo. Chronic injection of TLQP-21 in prediabetic ZDF rats preserves islet mass and slows diabetes onset. TLQP-21 prevents islet cell apoptosis by a pathway similar to that used by GLP-1, but independent of the GLP-1, GIP, or VIP receptors. Unlike GLP-1, TLQP-21 does not inhibit gastric emptying or increase heart rate. We conclude that TLQP-21 is a targeted agent for enhancing islet β-cell survival and function.

Analysis of mRNA Partitioning Between the Cytosol and Endoplasmic Reticulum Compartments of Mammalian Cells

All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments-mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasmic proteins are enriched on cytosolic ribosomes. In current views, this partitioning phenomenon occurs through positive selection-mRNAs encoding signal sequence-bearing proteins are directed into the signal recognition particle pathway early in translation and trafficked as mRNA/ribosome/nascent polypeptide chain complexes to the ER. In the absence of an encoded signal sequence, mRNAs undergo continued translation on cytosolic ribosomes. Recent genome-wide analyses of mRNA partitioning between the cytosol and the ER compartments have identified subsets of mRNAs that are non-canonically partitioned to the ER-although lacking an encoded signal sequence, they are translated on ER-bound ribosomes. These findings suggest that multiple, and as yet unidentified, pathways exist for directing mRNA partitioning in the cell. In this contribution, we briefly review the literature describing the subcellular partitioning patterns of mRNAs and present a detailed methodology for studying this fundamental, yet poorly understood process.

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.

In Vitro and Tissue Culture Methods for Analysis of Translation Initiation on the Endoplasmic Reticulum

For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting channel via the large ribosomal subunit and protein translocation ensues through coupling of the ribosomal nascent protein exit site with the protein-conducting channel. At the termination of translation, ribosomal subunits are thought to dissociate from the ER to return to a common cytoplasmic pool and participate in additional cycles of initiation, translation, targeting, termination, and ER membrane release. Experimental evidence has demonstrated that ER-membrane ribosomes are capable of de novo initiation, that mRNA partitioning to the ER membrane does not, per se, require translation of an encoded signal sequence, and that ribosomal subunit dissociation from the ER membrane is not obligatorily coupled to protein synthesis termination. These findings suggest that the cycle of protein synthesis-initiation, elongation, and termination-can occur on the two-dimensional plane of the ER membrane and challenge current views on the subcellular restriction of translation initiation to the cytosol, the role of the ribosome cycle in partitioning mRNA between the cytosol and ER, and the in vivo basis for termination-induced ribosomal subunit dissociation. In the following chapter, we provide detailed experimental methods to study protein synthesis initiation on the ER membrane.

Delayed apoptosis allows islet β-cells to implement an autophagic mechanism to promote cell survival

Increased β-cell death coupled with the inability to replicate existing β-cells drives the decline in β-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit β-cell loss and thereby promote β-cell recovery. Insulinoma cells have provided useful insight into β-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet β-cells highly vulnerable to ER stress-induced cell death. We propose that islet β-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.

SWELL1 is a glucose sensor regulating β-cell excitability and systemic glycaemia

Insulin secretion is initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC requires β-cell membrane depolarization, which is regulated by a balance of depolarizing and hyperpolarizing ionic currents. Here, we show that SWELL1 mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in both murine and human β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this contributes to membrane depolarization and activation of VGCC-dependent intracellular calcium signaling. SWELL1 depletion in MIN6 cells and islets significantly impairs glucose-stimulated insulin secretion. Tamoxifen-inducible β-cell-targeted Swell1 KO mice have normal fasting serum glucose and insulin levels but impaired glucose-stimulated insulin secretion and glucose tolerance; and this is further exacerbated in mild obesity. Our results reveal that β-cell SWELL1 modulates insulin secretion and systemic glycaemia by linking glucose-mediated β-cell swelling to membrane depolarization and activation of VGCC-triggered calcium signaling.Insulin secretion by β-cells is stimulated by glucose and is dependent on the induction of β-cell membrane depolarization, mainly driven by the closure of KATP channels, which in turn promotes voltage-gated Ca2+ channel opening. Here Kang et al. show that the volume-regulated anion channel, SWELL1, is involved in glucose-stimulated calcium increase and insulin secretion.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

ABSTRACT The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis

The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity.