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Review
. 2016 Aug 2;16(3):162-79.
doi: 10.17305/bjbms.2016.919. Epub 2016 May 22.

Apoptosis in pancreatic β-islet cells in Type 2 diabetes

Tatsuo Tomita  1
Affiliations
Review

Apoptosis in pancreatic β-islet cells in Type 2 diabetes

Tatsuo Tomita. Bosn J Basic Med Sci. .

Abstract

Apoptosis plays important roles in the pathophysiology of Type 2 diabetes mellitus (T2DM). The etiology of T2DM is multifactorial, including obesity-associated insulin resistance, defective insulin secretion, and loss of β-cell mass through β-cell apoptosis. β-cell apoptosis is mediated through a milliard of caspase family cascade machinery in T2DM. The glucose-induced insulin secretion is the principle pathophysiology of diabetes and insufficient insulin secretion results in chronic hyperglycemia, diabetes. Recently, hyperglycemia-induced β-cell apoptosis has been extensively studied on the balance of pro-apoptotic Bcl-2 proteins (Bad, Bid, Bik, and Bax) and anti-apoptotic Bcl family (Bcl-2 and Bcl-xL) toward apoptosis in vitro isolated islets and insulinoma cell culture. Apoptosis can only occur when the concentration of pro-apoptotic Bcl-2 exceeds that of anti-apoptotic proteins at the mitochondrial membrane of the intrinsic pathway. A bulk of recent research on hyperglycemia-induced apoptosis on β-cells unveiled complex details on glucose toxicity on β-cells in molecular levels coupled with cell membrane potential by adenosine triphosphate generation through K+ channel closure, opening Ca2+ channel and plasma membrane depolarization. Furthermore, animal models using knockout mice will shed light on the basic understanding of the pathophysiology of diabetes as a glucose metabolic disease complex, on the balance of anti-apoptotic Bcl family and pro-apoptotic genes. The cumulative knowledge will provide a better understanding of glucose metabolism at a molecular level and will lead to eventual prevention and therapeutic application for T2DM with improving medications.

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Figures

FIGURE 1
FIGURE 1
There are the extrinsic (receptor-mediated, red) and intrinsic (mitochondria-driven, blue) apoptosis pathways as opposed to the survival proteins such as the P13/Akt signaling circuity (yellow). From Lee SC, et al., Int J Biochem 2007;39:497-504.
FIGURE 2
FIGURE 2
Extrinsic and intrinsic pathways leads to apoptosis via cytochrome c and “apoptosome.” Extrinsic pathway: Fas-Fas L binding leads to the death-inducing signaling complex (DISC) where DISC-caspase-8 complex is activated, leading to caspase-3 activation. Intrinsic pathway: Pro-apoptotic Bcl proteins (Bad, Bid, Bik, Bim) become activated and translocate to the mitochondria, where they bind or inactivate Bcl proteins or form pores in the mitochondrial membrane, which facilitates the release of cytochrome c into cytosol. Once cytochrome c accumulates in the cytosol, it complexes with apocaspase-9 and Apaf-1 to form the “apoptosome,” which, in turn, activates caspase-3. From Emamaulee and Shapiro, Diabetes 2006;55:1907-14.
FIGURE 3
FIGURE 3
Amyloid deposited Type 2 diabetes mellitus islets (A-H). These amyloid deposited islets have fewer islet cells, consisting of major β-cells (A, 80%), α-cells (B, 10%) and δ-cells (C, 10%) and less densely stained cleaved caspase-3 positive cells (D,* at about 2%) as compared with about 2% cleaved caspase-3 positive cells in the control islets (Table 1). The nuclear positive staining for cleaved caspase-3 (E) is smaller than that of cytoplasmic positive staining of insulin, glucagon, and somatostatin (A-C). The stroma of amyloid deposited islets is Congo red positive (F) and is birefringent under polarized light (G). The recently amyloid deposited islet is also positive for islet amyloid polypeptide (IAPP) (H). The less amyloid deposited islets from the same subject reveal more cleaved caspase-3 positive islet cells at about 15% (E) than trabecular more amyloid deposited islets at about 2% (D). Immunostained for insulin (A), glucagon (B), somatostatin (C), cleaved caspase-3 (D, E), Congo red (F, G), and IAPP (H). A: Insulin, B: Glucagon, C: SRIF, D and E: Cleaved caspase -3 and H: IAPP immunostained. F and G: Congo red. From Tomita T. Pathology 2010;42:432-7, with permission.
FIGURE 4
FIGURE 4
Amyloid deposits in Type 2 diabetes mellitus diabetic islets: Case 10 (A-C) with amyloid deposits in 95% of the islets and Case 14 (D-F) with amyloid deposits >99% of the islets. In the Case 10 islets, the residual β-cells with plump cytoplasm (A) are minor cells and σ-cells with small and compact cytoplasm (C) are major islet cells. Islet amyloid polypeptide (IAPP) immunostaining with 1:400 diluted IAPP antibody reveals weak staining in the islets occupied by amyloid at 95%. (B) In the islets occupied by amyloid >99% reveal a few β-cells (D) of which large cytoplasm is immunostained strongly for insulin whereas amyloid deposits are moderately immunostained for IAPP. (E) In the Case 14, lesser amyloid deposited islets reveal strong immunostaining for the β-cell cytoplasm without nucleus with continuous moderate positive immunostaining to the fibrous amyloid stroma using 1:400 dilution of anti-IAPP antibody. (F) A and D: Insulin, C: Glucagon, B, E, and F: IAPP antibody at 1:400 immunostained for IAPP. From Tomita T. Islets, 2012;4:223-32, with permission.
FIGURE 5
FIGURE 5
Islet amyloid polypeptide (IAPP) immunocytochemical staining for control (A) and Type 2 diabetes mellitus (T2DM) islets, Case 10 (B-D) and Case 14 (E) IAPP immunostaining was performed using 1:400 for control islets (A) and using 1:200 dilution for T2DM islets (B-D). Control islets were strongly immunostained for the majority of islet cell cytoplasm (A). Both a few control and T2DM islet cytoplasm without nucleus was strongly immunopostive for IAPP (*) (A and B). T2DM islets occupying 95% amyloid deposits were immunostained moderately for IAPP in trabecular amyloid deposits whereas viable islet cells surrounded by amyloid deposits were negative for IAPP (B). In the islets occupied by >99% massive amyloid deposits, which were strongly immunostained for IAPP containing no IAPP-positive β-cells, instead strongly IAPP-positive, scattered single islet cells (s) were noted (C and D). By amyloid p immunostaining, stromal amyloid deposits were moderately positive and perivascular amyloid deposits were strongly positive for amyloid p (E). A-D: IAPP, E: Amyloid P immunostained. From Tomita T. Islets 2012;4:423-232, with permission.
FIGURE 6
FIGURE 6
A-E: Bcl family gene regulation in human islets cultured in high versus normal glucose. Expression of Bcl-2, Bcl-xl, Bad, Bid, and Bik mRNA was detected by RT-PCR and quantified by FluorImager analysis of ethidium signal. In each experiment, band densities were normalized against cyclophyllin, and the result are expressed as mRNA level to NGI control islets (NGI = 100%). A: Bcl-2. Bcl-x (HG5 vs. NG5, **P <0.01). C: Bad (HG5 vs. NG5, **P <0.01). D: Bid (HG5 vs. NG5, ***P <0.01) E: Bik (HG5 vs. NG5, **P <0.01). One representative gel is also shown. Islets from six donors were analysed. Means ± SD of relative expression of the genes are shown in bar graph. Statistical analysis was performed by ANOVA
FIGURE 7
FIGURE 7
Effects of high glucose on apoptosis in MIN6NB cells. The MIN6N8 cells were treated with different glucose concentrations (5.5-45 mM) for the indicated times. (A) DNA fragmentation (upper left) and TUNEL assay (lower left). The cleavage of caspase-3 (upper right) and poly(ADP-ribose) polymerase (PARP) (lower left) was analyzed. (B) Expression on apoptotic proteins. (C) Release of cytochrome C and Bax translocation. The blots were reprobed with antibodies to cytochrome C oxidase and voltage-dependent anion channels (VDAC). (D) Bax immunocytochemistry. Fluorescent microscopic images for Bax (green), Mito Tracker CMXRos (red) and final merged images (localization of Bax at mitochondria) are shown (upper). Fold of cells exhibiting punctuate ABax and percentage of Bax colocalization with mitochondria was determined by counting ~20-100 cells for each condition (lower). Results represent the average ± SE from three independent experiments (*p < 0.05, **p < 0.01) > (E): Interaction of Bax with VDAC, (F) Bax oligomerization. From Kim et al., Diabetes 2005;54:2601-11.
FIGURE 8
FIGURE 8
Characterization of the insulin secretion defect in Bad−/− islets. (A) Perifused islets from Bad−/− mice (red) with 25 mM glucose secreted significantly less insulin compared with that of Bad+/+ islets (black). (B) Insulin secretion throughout the perifusion (min 0-40), first phase (min 8-15) and second phase (min 15-40). (C) Glucose-induced changes in ATP/ADP ratio in Bad+/+ and Bad−/− islets - 5.5 mM (black), 25 mM (blue). (D) Insulin secretion in response to glucose 5.5 mM and 25 mM, 10 mM σ-ketoisocaproate (KIC), 0.25 mM tolbutamide and carbachol. (E) GCK activity in homogenates of primary islets isolated from Bad+/+ (black) and Bad−/− mice (red). (F) Insulin secretion by Bad+/+ (black) and Bad−/− (red) islets perifused with incrementally increasing concentration of glucose. From Daniel NN, et al. Nature 2003;424:952-6.
FIGURE 9
FIGURE 9
Loss of Bcl-2 enhances β-cell glucose responses. (A) Quantitative PCR quantification of Bcl-2 and Bcl-xL mRNA levels in islets from Bcl-2+/- and Bcl-2−/− islets compared to Bcl-2+/+ islets. (B) Average cytosolic Ca2+ levels of dispersed islet cells from Bcl-2+/+, Bcl-2+/−, and Bcl-2−/− islets. (C) Incremental area under the curve of Ca2+ responses by Bcl-2+/+, Bcl-2+/−, and Bcl-2−/− islets. (D) Integrated cytosolic Ca2+ responses of Bcl-2−/− and Bcl-2+/+ β-cells depolarized with 30 mM KCl, (E and F) integrated Ca2+ and NAD(P)H autofluorescence increases of intact islet cells, normalized Bcl-2+/+ control islet cells. (G) Insulin secretion profiles of perifused islets from Bcl-2+/+ and Bcl-2−/− islets. (H) Quantified area under the curve of insulin secretion profiles by Bcl-2+/+ and Bcl-2−/− islets. From Luciani DS, et al. Diabetes 2013;62:170-82.
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