Andrew T. Templin

Islet β-Cell Endoplasmic Reticulum Stress Precedes the Onset of Type 1 Diabetes in the Nonobese Diabetic Mouse Model

Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.

Palmitate Induces mRNA Translation and Increases ER Protein Load in Islet β Cells via Activation of the Mammalian Target of Rapamycin Pathway

Saturated free fatty acids (FFAs) have complex effects on the islet β-cell, acutely promoting adaptive hyperplasia but chronically impairing insulin release. The acute effects of FFAs remain incompletely defined. To elucidate these early molecular events, we incubated mouse β-cells and islets with palmitate and then studied mRNA translation by polyribosomal profiling and analyzed signaling pathways by immunoblot analysis. We found that palmitate acutely increases polyribosome occupancy of total RNA, consistent with an increase in mRNA translation. This effect on translation was attributable to activation of mammalian target of rapamycin (mTOR) pathways via L-type Ca2+ channels but was independent of insulin signaling. Longer incubations led to depletion of polyribosome-associated RNA, consistent with activation of the unfolded protein response (UPR). Pharmacologic inhibition of mTOR suppressed both the acute effects of palmitate on mRNA translation and the chronic effects on the UPR. Islets from mice fed a high-fat diet for 7 days showed increases in polyribosome-associated RNA and phosphorylation of S6K, both consistent with activation of mTOR. Our results suggest that palmitate acutely activates mRNA translation and that this increase in protein load contributes to the later UPR.

Translational Control of Inducible Nitric Oxide Synthase by p38 MAPK in Islet β-Cells

The MAPKs are transducers of extracellular signals such as proinflammatory cytokines. In islet β-cells, cytokines acutely activate expression of the Nos2 gene encoding inducible nitric oxide synthase (iNOS), which ultimately impairs insulin release. Because iNOS production can also be regulated posttranscriptionally, we asked whether MAPKs participate in posttranscriptional regulatory events in β-cells and primary islets in response to cytokine signaling. We show that cytokines acutely reduce cellular oxygen consumption rate and impair aconitase activity. Inhibition of iNOS with l-NMMA or inhibition of Nos2 mRNA translation with GC7 [an inhibitor of eukaryotic translation initiation factor 5A (eIF5A) activity] reversed these defects, as did inhibition of p38 MAPK by PD169316. Although inhibition of p38 had no effect on the nuclear translocation of nuclear factor κB or the abundance of Nos2 transcripts during the immediate period after cytokine exposure, its inhibition or knockdown resulted in significant reduction in iNOS protein, a finding suggestive of a permissive role for p38 in Nos2 translation. Polyribosomal profiling experiments using INS-1 β-cells revealed that Nos2 mRNA remained associated with polyribosomes in the setting of p38 inhibition, in a manner similar to that seen with blockade of translational elongation by cycloheximide. Consistent with a role in translational elongation, p38 activity is required in part for the activation of the translational factor eIF5A by promoting its hypusination. Our results suggest a novel signaling pathway in β-cells in which p38 MAPK promotes translation elongation of Nos2 mRNA via regulation of eIF5A hypusination.

Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells

Significance Elevated circulating proinsulin and a poor biological response to insulin are observed early in individuals with type 2 diabetes. Genome-wide association studies have recently identified genes associated with proinsulin processing, and clinical observations suggest that elevated proinsulin and its intermediates are markers of dysfunctional insulin-secreting β cells. Here, we propose a previously unidentified mechanism in the regulation of an enzyme that is involved in proinsulin processing called carboxypeptidase E (CPE). Disruption of insulin signaling in β cells reduces expression of a scaffolding protein, eukaryotic translation initiation factor 4 gamma 1, that is required for the initiation of translation and occurs via regulation of two transcription factors, namely, pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1. Together, these effects lead to reduced levels of CPE protein and poor proinsulin processing in β cells. Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of β-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying β-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in β cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.

Deoxyhypusine synthase haploinsufficiency attenuates acute cytokine signaling

Deoxyhypusine synthase (DHS) catalyzes the post-translational formation of the amino acid hypusine. Hypusine is unique to the eukaryotic translational initiation factor 5A (eIF5A), and is required for its functions in mRNA shuttling, translational elongation, and stress granule formation. In recent studies, we showed that DHS promotes cytokine and ER stress signaling in the islet β cell and thereby contributes to its dysfunction in the setting of diabetes mellitus. Here, we review the evidence supporting a role for DHS (and hypusinated eIF5A) in cellular stress responses, and provide new data on the phenotype of DHS knockout mice. We show that homozygous knockout mice are embryonic lethal, but heterozygous knockout mice appear normal with no evidence of growth or metabolic deficiencies. Mouse embryonic fibroblasts from heterozygous knockout mice attenuate acute cytokine signaling, as evidenced by reduced production of inducible nitric oxide synthase, but show no statistically significant defects in proliferation or cell cycle progression. Our data are discussed with respect to the utility of sub-maximal inhibition of DHS in the setting of inflammatory states, such as diabetes mellitus.