While the mechanism(s) that promotes β cell dysfunction in humans with T2DM is still poorly understood, recent reports indicate the involvement of ER stress (18
) and impaired insulin secretion caused by altered insulin granule exocytosis (27
). Indeed, TRB3, a pseudokinase, has been reported to be induced by ER stress (19
) and upregulated in several pathological conditions (30
). We report here that TRB3 expression levels are upregulated in human and mouse pancreatic islets in multiple pathophysiological conditions characterized by altered glycemia. An increase in TRB3 levels in β cells inhibits insulin signaling and leads to defective insulin exocytosis. Moreover, the polymorphic 84R variant, which is associated with increased risk of T2DM and cardiovascular disease in humans, exhibited a greater inhibition of β cell secretory function. To our knowledge, these findings provide novel insights into molecular mechanisms that directly link TRB3 polymorphisms to altered β cell exocytosis in the development of T2DM.
A polymorphism (rs2295490/Q84R) in exon 2 of TRB3
, which changes glutamine to arginine in codon 84, has been recently reported to be associated with T2DM, insulin resistance, and atherosclerosis in humans (8
). In the present study, in 2 populations of people of mixed European descent (Krakow, Poland, and Boston, Massachusetts, USA), individuals carriers for the minor allele coding for arginine (84R) exhibited an increased risk of T2DM in comparison with homozygotes of the major allele coding for glutamine (QQ84). In another recent study, in an Italian population, the 84R variant also exhibited significant association with early-onset T2DM and relative insulin deficiency (9
) (V. Trischitta and L. Frittitta, personal communication). Unfortunately, the Q84R polymorphism is not in linkage disequilibrium with any of the other known HapMap SNPs, and, therefore, its effect on T2DM risk could not be examined in the publicly available genome-wide association studies (33
In addition to being expressed in islets, the TRB3
gene is reported to be expressed in the liver in humans; however, its expression levels in adipose, skeletal muscle, and brain are either low or undetectable (35
). We pursued functional studies in β cells, based on our findings of reduced C-peptide levels in carriers of the polymorphism and on the report by Prudente et al. (9
) of insulin deficiency, which is likely due to defects in islet secretory function from individuals expressing the polymorphism in an independent cohort. Furthermore, the lack of specific phenotypic data in these individuals that directly point to functional defects in the liver also suggested that we should focus our studies on islets. Nevertheless, we plan to undertake functional studies in hepatocytes in a separate study.
Consistent with the reports that TRB3 expression levels are regulated by insulin signaling and stress (19
), we detected an increase in expression of TRB3 protein in islets from patients with T2DM, islets from obese and insulin resistant mouse models, and observed that insulin signaling negatively regulates TRB3 expression in pancreatic β cells. These data, along with the observation that Q84R polymorphisms are associated with T2DM, implicate an important role for TRB3 in β cell biology.
To study the role of TRB3 and the potential differential effect of the 84R polymorphism on islet/β cell function, we expressed the Q84 major variant or 84R minor variant of TRB3 in dispersed human islet cells or MIN6 cells to mimic the levels of TRB3 observed in diabetic islets (approximately 4-fold). Consistent with the effects observed in hepatocytes (7
) and HUVECs, (37
) both Q84 and 84R TRB3 blunted basal and insulin-stimulated Akt and ERK phosphorylation, with the latter variant having stronger inhibitory effects on Akt activation. Thus, the presence of a polymorphism (e.g., 84R) in TRB3 or a mere increase in expression of TRB3 can both induce an insulin resistant state and lead to poor proliferation and enhanced apoptosis of β cells. Given that TRB3 expression is regulated by insulin signaling, a balance between insulin mediated-suppression of TRB3 and TRB3-mediated suppression of Akt might play a critical role in the regulation of β cell function.
Expression of the TRB3 variants in β cells resulted in a marked inhibition of glucose and non-glucose secretagogue-stimulated insulin secretion. In addition, β cell–specific TRB3 overexpressing mice displayed impaired insulin secretion both in vivo and ex vivo. These data are consistent with significantly lower C-peptide levels in homozygous carriers of the 84R variant in humans in our studies and are in agreement with the relative insulin deficiency during an OGTT in humans carrying the 84R variant reported by Prudente et al. (10
) and V. Trischitta and L. Frittitta (personal communication). Although the defects in GSIS observed in vivo in RIP-TRB3F1 mice occur when first-phase insulin secretion is expected, it would be desirable to confirm the defect using an islet perfusion model.
An alternative mechanism that determines the greater inhibitory effects of 84R TRB3 compared with Q84 TRB3 is the relatively greater stability of 84R TRB3, which potentially increased TRB3 expression and, together with the enhanced affinity of 84R TRB3 with Akt and ATF4, was able to alter islet secretory function and β cell survival. The functional variation between Q84 and 84R TRB3 could also be due to differences in their conformational structures secondary to substitution of uncharged glutamine with charged arginine (38
Consistent with a dominant effect of the 84R variant, the expression of Snap25 and Rab3d was inhibited in 84R TRB3-expressing β cells and RIP-TRB3 mouse islets. Electron microscopic analysis of the plasma membrane of β cells from the transgenic mice localized the site of defect to a significant decrease in the number of docked insulin granules. In addition, we identified the transcription factor CREB as an indirect target of TRB3 that potentially mediates the downregulation of Snap25 and Rab3d expression. We also observed that CREB binding on the Snap25 promoter is regulated by CREB activation and ER stress. ATF4, a member of the CREB family of transcription factors, consistently coimmunoprecipitated with TRB3 in β cells, and the binding of TRB3 to ATF4 inhibited ATF4 transcription activity, providing evidence, for the first time to our knowledge, that ATF4 participates in the regulation of exocytosis in β cells. Indeed, we observed that ATF4 competitively inhibited CREB activation of the Snap25 promoter. These data are supported by our observation that upregulation of TRB3 and ATF4 by ER stress induction downregulates Snap25 and point to ATF4 as a key player in the TRB3-CREB regulation of insulin exocytosis. The reversal in insulin secretion and expression of exocytosis genes in a loss-of-function model (knockdown) strengthens the argument for a role for TRB3 in β cell exocytosis. Further studies are warranted to delineate the molecular pathway(s) and identify the proteins that link the TRB3 variants with Akt and modulate mitochondrial Bax to promote apoptosis in β cells.
We propose that under normal conditions CREB regulates a subset of exocytosis genes by acting via CRE. However, in pathological states, such as obesity and diabetes, and/or when there is an increased demand for protein synthesis, the ER stress/unfolded protein response upregulates ATF4 and its downstream target, TRB3. Subsequently, TRB3 binds to ATF4 and promotes access to CRE sites, resulting in reduced SNAP25 promoter activity and a decrease in insulin exocytosis. Our findings demonstrate what we believe to be a novel regulation of a subset of exocytosis genes, whereby a transcriptionally inactive TRB3/ATF4 complex competitively inhibits CREB binding to CRE-binding sites, leading to downregulation of SNAP25 (Figure F).
In summary, the inhibitory role of TRB3 in β cell function, via impairment of insulin exocytosis and proliferation, and enhancement of apoptosis indicates the significance of targeting the tribbles protein for the development of new therapeutic approaches to prevent progressive β cell dysfunction and improve β cell survival in T2DM.