Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation

Abstract

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Figure 1

The promoter region of insulin gene. The organization of the proximal portion (-340 bp to +1 bp) of the insulin promoter including critical transcription activation elements and their binding transcription factors are shown. The critical transcription activation elements are illustrated as boxes. Above the boxes are shown the names of transcription factors that can bind to corresponding elements. Elements A3, C1, E1 have been implicated in β cell-specific expression of the insulin gene, which is mediated by the restricted cellular distribution of their transcription factors PDX-1, MafA, Beta-2. The synergistic activities of PDX-1, MafA and Beta-2 are illustrated by green arrows. In addition to transcription factors, DNA-binding proteins such as HMG I (Y) can promote the binding of PDX-1 and Beta2/E47 to their corresponding elements.

Figure 2

Insulin synthesis and secretion process. After preproinsulin mRNA transcription, preproinsulin is synthesized in the ER and converted into proinsulin, proinsulin is transported through the Golgi apparatus and packaged into immature clathrin-coated granules, where proinsulin is processed into insulin and c-peptide. The immature granules can then become mature granules containing cystalized insulin. After glucose stimulation insulin granules exhibit two characteristic phases that consist of a rapidly initiated but transient first phase and a sustained second phase, because the granules are divided into two different pools. (1) A limited pool of granules (< 5%) ready for immediate release and is referred to as the "readily releasable pool" (RRP), which account for the first release phase. (2) Most of the granules (> 95%) belong to a reserve pool responsible for the second-phase of insulin secretion, granules in this pool must undergo mobilization before they can gain release competence.

Figure 3

Glucose control of insulin secretion. Glucose-stimulates insulin secretion via two mechanisms: the triggering pathway and the amplifying pathway. (1) Glucose enters beta cells through GLUT-2 and undergoes glycolysis. This metabolism increases the ratio of ATP to ADP, which inhibits the ATP-sensitive K_ATP-channels, leading to membrane depolarization and opening of voltage-dependent calcium channels (VDCC), with a resultant major increase in cytosolic calcium, which, in turn, triggers exocytosis. SNARE proteins play a critical role in insulin granule secretion. The linking of the plasma membrane proteins syntaxin and SNAP-25 to vesicle protein VAMP-2/synaptobrevin-2 cause the docking of the vesicle, bringing insulin granules in close contact with the plasma membrane and calcium channels, after opening of calcium channels, the readily releasable pool (RRP) insulin granules located nearby are exposed to high level of Ca2+, resulting in RRP granule exocytosis. CDK5 can inhibit VDCC activity by phosphorylating its subunit, thus inhibiting insulin secretion; however, high glucose concentrations inhibit the Cdk5 kinase activity which in turn increases Ca²⁺ influx, leading to enhanced insulin secretion. (2) Insulin granules in reserve pool must undergo acidification to gain secretion competence. This mobilization or priming process is dependent on the simultaneous operation of a V-type H⁺-ATPase and ClC-3 Cl^- channels. Cl^- uptake determines the extent of granular acidification by providing a counter-ion required to allow continuous H⁺ pumping. ADP can inhibit Cl- channel activity, however, glucose metabolism reduces the ADP level, leading to the loss of inhibition to Cl- channels, so insulin secretory granules undergo acidification and the secretion process is augmented.

Figure 4

Glucose control of insulin gene transcription. Glucose metabolism in beta cells generates upstream signals that are responsible for the activation of factors involved in insulin transcription. (1) Glucose metabolism causes a shift of transcription factor PDX-1 from the cytoplasm to the nucleus, increases its activation domain and binding to A3 element. The effects are in part, due to the activity of phosphatidylinositol 3-kinase (PI3-K); another kinase stress-activated protein kinase 2 (SAPK2/P38) might be involved in this process. An alternative pathway involves histone and PDX-1. When glucose levels are low, PDX-1 interacts with histone deacetylases Hdac-1 and Hdac-2 and recruits them to insulin gene promoter, which causes the deacetylation of histone H4 and results in down-regulation of insulin gene expression. High concentrations of glucose diminish this inhibiting activity. (2) Stimulatory concentrations of glucose can activate ERK1/2, which promotes BETA2 and E47 heterodimerization and binding to E-box sites. (3) Glucose affects MafA at the mRNA level. Stimulatory glucose levels increase MafA transcription and result in increased MafA protein.

Figure 5

Glucose control of insulin translation. Glucose stimulates insulin synthesis largely by promoting insulin translation initiation. (1) Glucose promotes the phosphorylation of eIF-4EBP and activates eIF-4E; eIF-4E, eIF-4A and eIF-4G form eIF-4F, a complex whose functions include recognition of preproinsulin mRNA and recruiting 40S ribosome to mRNA. (2) eIF2 is a critical factor regulating protein biosynthesis. It is active only in GTP-bound state. A factor named eIF-2B functions to convert GDP-bound eIF-2 to GTP-bound eIF-2. The activity of eIF-2B is transiently up-regulated after glucose stimulation. Additionally, phosphorylation of alpha subunit of eIF2 (eIF2α) inhibits the formation of eIF2-GTP·Met-tRNAi translational ternary complex, which binds 40S ribosome and is indispensable for protein translation. Glucose causes the dephosphorylation of eIF2α, and induces an increase in the availability of the translational ternary complex.