Research Interests and Techniques

The development of type-2 diabetes occurs when pancreatic β-cells fail to compensate for insulin resistance in peripheral tissues due to defects in insulin secretion or loss of β-cell mass. Importantly, both pancreatic beta cell function and mass can be modulated by nutrients and hormones, which are mediated, at least in part, through changes in the rate of protein synthesis and/or the activity of key components of the translational machinery. My work is primarily aimed at understanding the molecular mechanisms by which nutrients and hormones, such as glucagon-like peptide-1 (GLP1), regulate protein synthesis/the activity of key components of the translational machinery in pancreatic β-cells. This is important in understanding the pathophysiology of type-2 diabetes and identifying new rational strategies for the treatment and possible prevention of this disease.

1. Nutrient and Hormonal Regulation of Protein Synthesis in Pancreatic β-cells

The pancreatic ß-cell rapidly releases insulin in response to an influx of nutrients, such as amino acids or glucose. In order to maintain insulin stores within the ß-cell, there is a rapid increase in proinsulin synthesis (10-20 fold within 40min). Additionally, there is a co-ordinated increase in the synthesis of a subset of proteins and a two-fold increase in total protein synthesis. These rapid increases in proinsulin and total protein synthesis are mediated entirely at the translational level.

In a number of other cell types, the signalling pathways that regulate protein synthesis through the phosphorylation of translation factors and regulatory proteins have been partially characterised (see figure 1). Key steps in the regulation of protein synthesis include: i.) the assembly of the initiation complex, eIF4F, containing the initiation factors eIF4G, a large scaffolding protein, eIF4E, the protein which binds to the 5’-cap structure (7mGTP) and eIF4A, a RNA helicase and ii.) the binding of the initiator tRNA to the 40S ribosomal subunit mediated by eIF2.

However, the regulation of ß-cell protein synthesis and the mechanism by which protein synthesis is regulated in ß-cells is poorly understood. Therefore, one of our aims is to understand the molecular mechanisms of action of nutrients in the translational regulation of both total protein and proinsulin synthesis in isolated islets of Langerhans and the pancreatic ß-cell line, MIN6, particularly focusing the regulatory components of translation and the signalling pathways leading to their control. Our work is particularly relevant to type II diabetes where increased demand of both the synthesis and secretion of insulin ultimately leads to ß-cell dysfunction.

2. PERK and the unfolded protein response in pancreatic β-cells

Most transmembrane, ER-resident and secretory proteins are co-translationally translocated into the lumen of the ER where they are post-translationally modified, folded and trafficked to specific compartments of the endomembrane system. The accumulation of improperly folded or processed proteins in the ER, which can occur in response to specific pathophysiological states, leads to ER stress and the induction of an adaptive response known as the unfolded protein response (UPR) transduced by two related ER transmembrane serine-threonine kinases, pancreatic ER kinase (PERK) and inositol requiring enzyme-1 (IRE1).

PERK phosphorylates the a-subunit of eukaryotic initiation factor 2 (eIF2a) and the transcription factor NF-E2-related factor-2 (Nrf2) resulting in the repression of protein synthesis, and hence a decrease in ER protein folding load, and an increase in the expression of mRNAs encoding anti-apoptotic proteins involved in maintaining redox homeostasis and combating oxidative stress. Importantly, PERK activity is essential for pancreatic b-cell viability as pancreatic b-cells from PERK knock-out mice die within weeks of birth as do the pancreatic b-cells from infants with loss of function mutations within PERK.

These effects are likely mediated through the phosphorylation of PERK’s substrate eIF2a, as homozygous serine 51 to alanine (Ser51Ala) ‘knock-in' mice (therefore creating a non-phosphorylatable eIF2a) also show severe b-cell deficiency. IRE1 activation leads to the activation of the X-box transcription factor XBP1 resulting in an increase in the expression of mRNAs encoding proteins that are important in increasing the folding capacity of the ER and clearing the ER of misfolded proteins. Collectively, the activation of both IRE1 and PERK result in the alleviation of ER stress and hence promote cell survival.  However, the prolonged activation of the UPR ultimately leads to the expression of mRNAs encoding pro-apoptotic proteins leading to cell death.

The development of type 2 diabetes occurs when pancreatic b-cells fail to compensate for insulin resistance in peripheral tissues due to defects in insulin secretion or loss of b-cell mass. Elevated levels of non-esterified free fatty acids (FFA) have been shown to be cytotoxic to b-cells in obesity-associated diabetes models as well as in normal b-cells.

Interestingly, these effects may be mediated through the activation of UPR as incubation of pancreatic b-cells with palmitate results in the phosphorylation of eIF2a and XBP1 splicing. Moreover, heterozygous eIF2a Ser51Ala 'knock-in' mice, fed on a high fat diet, show abnormal b-cell ER morphology, defective proinsulin trafficking and a reduced number of secretory granules. Chronic elevated levels of circulating glucose, hallmarks of impaired fasting glucose and type 2 diabetes, have also been implicated in pancreatic b-cell dysfunction and death and we have preliminary data indicating that chronic exposure of pancreatic b-cells to high glucose also activates PERK and the UPR.

However, the patho/physiological roles of the activation of PERK and IRE1 in response to lipids or elevated levels of glucose in pancreatic b-cells are poorly understood. Therefore, an important aim of the lab is to determine the patho/physiological roles of the activation of the UPR in response to glucose and lipids in the pancreatic b-cell line MIN6 and rodent islets of Langerhans.

3. L-type VGCC signalling to Erk in Pancreatic β-cells

Glucagon like peptide-1 (GLP1) is a Gs-coupled receptor agonist released from intestinal L-cells in response to nutrient ingestion. GLP1 acts upon pancreatic ß-cells stimulating both insulin gene expression and secretion. These effects are mediated through the activation of multiple signal transduction pathways including the extra-cellular regulated kinase (Erk) pathway. Work in our laboratory has demonstrated that GLP1 activates Erk through a mechanism dependent on the influx of extracellular calcium through L-type voltage gated calcium channels. Interestingly, we have also shown that GLP1 activation of Erk is independent of Raf yet dependent of MEK. My group is currently further investigating the molecular mechanisms of calcium dependent Erk pathway signalling in pancreatic ß-cells.

Fig1.  Model for glucose dependent GLP1 activation of Erk.

Research Group and Funding

Present group members

Norhan Mohammed El-Sayed
Dr Edith Gomez
Harry Holkham
Omotola Omikorede
Jianling Xie Zhang
Xue Chan Zhao

Current funding

Diabetes UK studentship
AstraZENECA BBSRC CASE studentship
MRC PhD studentship
Wellcome Trust Project Grant

Recent Publications

Peer Reviewed Articles

Gomez E, Powell ML, Bevington A, Herbert TP (2008). A decrease in cellular energy status stimulates PERK-dependent eIF2alpha phosphorylation and regulates protein synthesis in pancreatic beta-cells. Biochem J. Mar 15;410(3):485-93

Evans K, Nasim Z, Brown J, Butler H, Kauser S, Varoqui H, Erickson JD, Herbert TP, Bevington A. (2007) Acidosis-Sensing Glutamine Pump SNAT2 Determines Amino Acid Levels and Mammalian Target of Rapamycin Signalling to Protein Synthesis in L6 Muscle Cells. J Am Soc Nephrol. 2007 May;18(5):1426-36. Epub 2007 Apr 11.

Greenman IC, Gomez E, Moore CE, Herbert TP. Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic beta-cells (2007). J Endocrinol. 2007 Jan;192(1):179-87.

Greenman IC, Gomez E, Moore CE, Herbert TP. (2005). The selective recruitment of mRNA to the ER and an increase in initiation are important for glucose-stimulated proinsulin synthesis in pancreatic beta-cells. Biochemical Journal. Oct 15;391(Pt 2):291-300.

Gomez E, Powell ML, Greenman IC, Herbert TP. (2004). Glucose-stimulated protein synthesis in pancreatic beta-cells parallels an increase in the availability of the translational ternary complex (eIF2-GTP.Met-tRNAi) and the dephosphorylation of eIF2 alpha. Journal of  Biological Chemistry. Dec 24;279(52):53937-46.

Gomez E, Pritchard C and Herbert TP. (2002). cAMP dependent protein kinase and Ca++ influx through L-type voltage gated calcium channels mediate Raf independent activation of extracellular regulated kinase in response to glucagon like peptide-1 in pancreatic ß-cells. Journal of  Biological Chemistry. Dec 13;277(50):48146-51.

Herbert TP, Tee AR and Proud CG (2002). The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. Journal of Biological Chemistry. Mar 29;277(13):11591-6.

Wicksteed B, Herbert TP, Alarcon C, Lingohr MK, Moss LG, Rhodes CJ. (2001). Cooperativity between the preproinsulin mRNA untranslated regions is necessary for glucose-stimulated translation. Journal of  Biological Chemistry. Jun 22;276(25):22553-8.

Herbert TP, Fåhraeus R, Prescott A, Lane DP, Proud CG. (2000). Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E. Current Biology. 10:793-796.  

Herbert TP, Kilhams GR, Batty I and Proud CG. (2000). Distinct signalling pathways mediate insulin and phorbol ester stimulated eIF4F assembly and protein synthesis in HEK 293 cells. Journal of Biological Chemistry. 275(15):11249-56.

Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, Yamauchi T, Feener EP, Herbert TP, Rhodes CJ, King GL. (2000). Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo: a specific vascular action of insulin. Circulation. 101(6):676-81.

Book Chapters

Herbert TP and Proud CG. (2006). Regulation of translation elongation and co-translational protein targeting. In Sonenberg N, Hershey JWB and Mathews MB. (ed.), Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Herbert TP and Rhodes CJ. (1997). Regulation of prohormone conversion by co-ordinated control of processing endopeptidase biosynthesis with that of the prohormone substrate. Book chapter in Proteolytic and cellular mechanisms in prohormone processing, Springer-verlag, Heildleberg, Germany Ed: Vivian YH Hook.