PERK in beta cell biology and insulin biogenesis

Abstract

PERK (EIF2AK3) was originally discovered as a major component of the unfolded protein response (UPR). PERK deficiency results in permanent neonatal diabetes, which was initially thought to be caused by a failure to regulate ER stress in insulin-secreting beta cells, culminating in beta cell death. However, subsequent studies found that low beta cell mass was a result of reduced cell proliferation, rather than increased apoptosis. Genetic and cellular studies of Perk-deficient beta cells showed that PERK was crucially required for ER functions including proinsulin trafficking and quality control, unrelated to the ER stress pathway. Under normal physiological conditions, changes in ER calcium levels, mediated by glucose and other insulin secretagogues, regulate PERK activity for the purpose of controlling insulin biogenesis.

Figure 1

The contrasting functions of PERK in the canonical UPR and in the regulation of the insulin secreting beta cell. (a) The UPR-ER stress response is initiated by the accumulation of unfolded proteins in the endoplasmic reticulum that activates the three major arms of the UPR including PERK, IRE1, and ATF6. PERK phosphorylates eIF2α at amino acid residue Serine-51. Phosphorylation at this site results in attenuation of protein synthesis but also in activation of translation of a small number of genes including ATF4. ATF4 increases the transcription of GADD34, which acts as a feedback control to restore normal protein synthesis. ATF4 also activates the transcription of CHOP, which if produced at high levels in conjunction with other factors, may lead to cell death. IRE1 catalyzes the splicing of the transcription factor XBP1, which in turn activates the transcription of genes encoding ER chaperones and other proteins facilitating ERAD function. The activation of ATF6 results in its translocation to the Golgi and generation of the active nuclear form. After trafficking to the nucleus, ATF6 acts in concert with other transcription factors to activate the expression of ER chaperone and ERAD genes. In some cases ATF6 and XBP1 co-activate specific genes. (b) Under normal physiological conditions, PERK activity is dynamically controlled by changes in ER calcium levels. ER calcium levels are heavily influenced by glucose and other insulin secretagogues including those that activate the IP3 receptor localized in the ER membrane. Depending on other factors, glucose can either stimulate calcium uptake through SERCA pump which requires ATP or it can stimulate calcium release from the IP3 receptor via calcium-induced calcium release (CICR). PERK in turn regulates proinsulin trafficking and ERAD. The activity of IRE1α is also regulated by glucose, but in an opposite manner to PERK. In turn, IRE1a regulates proinsulin biogenesis. PERK and IRE1α are likely to activate changes in gene expression as part of their regulation of proinsulin trafficking and biogenesis, but the relevant downstream target genes have not been identified. GLUT2 = glucose transporter-2, KATP = ATP-sensitive potassium channel, VDCC = voltage-dependent calcium channel, PLC = phospholipase C, IP3R = inositol triphosphate receptor, ERAD = ER associated protein degradation, SERCA = Sarcoplasmic-endoplasmic reticulum calcium ATPase - calcium pump, BiP = immunoglobulin binding protein/glucose regulated protein-78.

Figure 1

The contrasting functions of PERK in the canonical UPR and in the regulation of the insulin secreting beta cell. (a) The UPR-ER stress response is initiated by the accumulation of unfolded proteins in the endoplasmic reticulum that activates the three major arms of the UPR including PERK, IRE1, and ATF6. PERK phosphorylates eIF2α at amino acid residue Serine-51. Phosphorylation at this site results in attenuation of protein synthesis but also in activation of translation of a small number of genes including ATF4. ATF4 increases the transcription of GADD34, which acts as a feedback control to restore normal protein synthesis. ATF4 also activates the transcription of CHOP, which if produced at high levels in conjunction with other factors, may lead to cell death. IRE1 catalyzes the splicing of the transcription factor XBP1, which in turn activates the transcription of genes encoding ER chaperones and other proteins facilitating ERAD function. The activation of ATF6 results in its translocation to the Golgi and generation of the active nuclear form. After trafficking to the nucleus, ATF6 acts in concert with other transcription factors to activate the expression of ER chaperone and ERAD genes. In some cases ATF6 and XBP1 co-activate specific genes. (b) Under normal physiological conditions, PERK activity is dynamically controlled by changes in ER calcium levels. ER calcium levels are heavily influenced by glucose and other insulin secretagogues including those that activate the IP3 receptor localized in the ER membrane. Depending on other factors, glucose can either stimulate calcium uptake through SERCA pump which requires ATP or it can stimulate calcium release from the IP3 receptor via calcium-induced calcium release (CICR). PERK in turn regulates proinsulin trafficking and ERAD. The activity of IRE1α is also regulated by glucose, but in an opposite manner to PERK. In turn, IRE1a regulates proinsulin biogenesis. PERK and IRE1α are likely to activate changes in gene expression as part of their regulation of proinsulin trafficking and biogenesis, but the relevant downstream target genes have not been identified. GLUT2 = glucose transporter-2, KATP = ATP-sensitive potassium channel, VDCC = voltage-dependent calcium channel, PLC = phospholipase C, IP3R = inositol triphosphate receptor, ERAD = ER associated protein degradation, SERCA = Sarcoplasmic-endoplasmic reticulum calcium ATPase - calcium pump, BiP = immunoglobulin binding protein/glucose regulated protein-78.

Figure 2

PERK deficiency results in diminished proliferation of beta cells and a severe defect in proinsulin trafficking. (a) Beta cell mass normally increases 20–40 fold during the first four postnatal weeks in mice (24). In contrast, beta cell mass increases only about 2-fold in Perk KO mice. (b) Images of wildtype and Perk KO islets at the 3^rd postnatal week are shown. (c) Proinsulin (red) normally accumulates in the ER-Golgi Intermediate Compartment and Golgi adjacent to the nucleus (blue) whereas insulin (green) is dispersed through the cytoplasm. In a large fraction of beta cells in Perk KO (PKO) mice, proinsulin accumulates in the ER and is not trafficked to the Golgi (24). (c) The ER in wildtype beta cells exhibits an elongated, tubular structure dotted with ribosomes, whereas the ER in Perk KO mice is highly distended with high electron density due to the accumulation of a large amount of proinsulin and other client proteins.

Figure 3

Diabetic progression in Akita insulin mutant mouse is ameliorated by reducing Perk gene dosage. The effect of three Perk genotypes – Perk +/−, Perk +/+, and βPerk; Perk +/+ on the diabetic progression of the insulin Akita mouse (Ins2+/Akita) was investigated (22). At postnatal day 23 the serum glucose level of Akita mice begins to increase and five days later (P28) all mice exceed 200 mg/dl. However, the diabetic progression during this period is inversely related to Perk gene dosage. The Perk dependent progression of diabetes in the Akita mouse is correlated with differences in pancreatic insulin content.

Figure 4

Glucose dynamically regulates PERK activity in the insulin secreting beta cell. Within the normal physiological range of serum glucose, PERK activity is relatively high after an overnight fast but is rapidly reduced by the administration of high glucose (19). We hypothesize that increased PERK activity during fasting (low glucose) increases quality control that serves to limit the anterograde trafficking proinsulin and insulin secretion. However, sustained high levels of circulating glucose negatively impact the function of beta cells, and a high level of PERK activity is seen. We speculate that one of the consequences of glucotoxicity is the accumulation of unfolded proteins in the ER that activate PERK and the other arms of the ER stress pathway.