Overexpression of Bcl-xL in beta -cells prevents cell death but impairs mitochondrial signal for insulin secretion

Yun-Ping Zhou1, John C. Pena2, Michael W. Roe1, Anshu Mittal1, Matteo Levisetti1, Aaron C. Baldwin1, William Pugh1, Diane Ostrega1, Noreen Ahmed1, Vytautas P. Bindokas3, Louis H. Philipson1, Douglas Hanahan4, Craig B. Thompson2, and Kenneth S. Polonsky1

1 Department of Medicine, Section of Endocrinology; 2 Howard Hughes Medical Institute and Departments of Medicine, Molecular Genetics and Cell Biology, 3 Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637; and 4 Department of Biochemistry, University of California at San Francisco, San Francisco, California 94143


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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To study effects of Bcl-xL in the pancreatic beta -cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-xL expression in beta -cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 µM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-xL transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-xL mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-xL islets had impaired insulin secretory and intracellular free Ca2+ ([Ca2+]i) responses to glucose and KCl. Furthermore, insulin and [Ca2+]i responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-xL islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-xL islets than in wild-type islets. Glucose-, PME-, and alpha -ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-xL islets. Thus, although Bcl-xL promotes beta -cell survival, high levels of expression of Bcl-xL result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.

apoptosis; calcium; islets of Langerhans; mitochondria


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APOPTOSIS OR PROGRAMMED cell death is an important biological process essential for the development and maintenance of multicellular organisms (36, 42). Changes in the rate of apoptosis may underlie a variety of common diseases (38). Members of the bcl-2 protooncogene family encode proteins that function either to promote or to inhibit apoptosis (3, 12, 43). Anti-apoptotic products of this gene family, such as Bcl-2 and Bcl-xL, prevent programmed cell death in response to a wide variety of stimuli (3). Bcl-xL is present in mitochondrial, endoplasmic reticulum, and nuclear membranes (38, 29). Recent studies have demonstrated that Bcl-xL or Bcl-2 inhibits apoptosis via multiple effects in mitochondria, such as inhibiting release of mitochondrial cytochrome c (11), increasing mitochondrial Ca2+ uptake (22), facilitating mitochondrial ATP/ADP exchange (40), and maintaining normal mitochondrial volume (41). Because mitochondria play an important role in the stimulus-secretion coupling of glucose-induced insulin secretion in pancreatic beta -cells (19, 24), whether Bcl-xL changes mitochondrial function in beta -cells, thereby affecting insulin secretion, is an important issue to address.

Apoptosis is known to mediate autoimmune destruction of beta -cells in type 1 diabetes (13) and postpartum involution of beta -cell mass in the rat (34). We and others have recently suggested that subtle increases in the rate of beta -cell apoptosis may contribute to loss of beta -cell mass in some animal models of type 2 diabetes, such as the Zucker fatty diabetic rat (26) and Psammomys obesus (1). In light of these observations, interventions that could reduce the rate of beta -cell apoptosis may have substantial potential in the prevention and treatment of diabetes. One approach is to overexpress the anti-apoptotic protein Bcl-xL in pancreatic beta -cells. The present study was undertaken to test the feasibility of such an approach by producing transgenic mice that overexpress Bcl-xL in the pancreatic beta -cell. The results demonstrate that Bcl-xL overexpression does partially protect beta -cells from death induced by thapsigargin, an agent that induces apoptosis in insulin-secreting beta -cells by inhibiting the sarcoendoplasmic reticulum Ca2+-ATPase and depleting intracellular Ca2+ stores (46). However, marked overexpression of Bcl-xL resulted in a severe defect in insulin secretion and hyperglycemia in the Bcl-xL transgenic mice. Further studies revealed that the impairment in pancreatic beta -cell function can largely be accounted for by alteration in mitochondrial nutrient metabolism and the consequent generation of ATP or other signaling molecules for insulin secretion. These unexpected phenotypic and functional changes associated with the overexpression of Bcl-xL in beta -cells should be taken into account in efforts that target Bcl-xL or other proteins to the pancreatic beta -cell for therapeutic purposes.


    MATERIALS AND METHODS
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Generation of the transgenic mice. Two Bcl-xL rat insulin promoter (RIP) constructs were employed to express Bcl-xL in pancreatic beta -cells (Fig. 1A). Both vectors contained the same blunt-ended 0.82-kb human bcl-xL cDNA (25). To induce specific expression in beta -cells, the bcl-xL cDNA was ligated to the 3' ends of either RIP1 or RIP7. RIP1, a 0.50-kb Sac I/BstB I fragment of the RIP (9) was used to generate Fd 1 Bcl-xL trangenic mice. RIP7, used in the Fd 2 transgenic line, was composed of a much longer (12.9-kb) promoter/enhancer intron region from the rat insulin II gene (23). Original founders were screened by PCR analysis using the primers 5'-CCGAATTCTTG GACAATGGAC-TGGTTGA-3' and 3'-CCGAATTCGTAGAG-TGGATGATCAGTG-5' and were confirmed by Southern blot using a 32P-labeled full-length bcl-xL cDNA probe. Once founder lines were established, PCR was used exclusively to identify transgenic progeny.


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Fig. 1.   Transgene construct and expression of Bcl-xL gene in pancreatic islets. A: founder (Fd) 1 transgenic line [rat insulin promoter (RIP) 1 construct] had the bcl-xL cDNA cloned downstream of RIP1 and was immediately followed by an intronic sequence with a polyadenylation sequence. Isolation of the construct was completed using the displayed restriction sites. The Fd 2 transgenic line (RIP7 construct) used the enhancer region upstream of RIP2. The bcl-xL cDNA was cloned directly downstream of the intronic sequence followed by a polyadenylation sequence. I, intron; P(A), polyadenylation signal. B: Western blot analysis of wild-type vs. transgenic islets from both Fd 1 and 2 mice.

Immunoblotting. Islets from wild-type and Bcl-xL transgenic mice were washed in PBS and lysed by passing them through a 25-gauge needle five times in RIPA buffer (1% Nonidet P-40, 1% deoxycholate, and 0.1% SDS) containing aprotinin, phenylmethylsulfonyl fluoride, and leupeptin. Protein samples (50 µg, quantified by bicinchoninic acid; Pierce, Rockford, IL) were separated in a 15% polyacrylamide-SDS gel. Proteins were transferred to nitrocellulose and blotted with a monoclonal antibody to Bcl-xL (18). Reactive proteins were detected using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL), and the intensities of the bands were quantified with a densitometer (Molecular Dynamics, Sunnyvale, CA).

Immunohistochemistry. Pancreatic tissue was fixed in 10% neutral buffered Formalin and paraffin embedded. Sections were deparaffinized and rehydrated by standard methods before immunohistochemistry. An antigen retrieval protocol (boiling the slides in citrate buffer for 10 min) was employed for Bcl-xL staining in sections of Fd 1 Bcl-xL mice. The primary antibodies used were a polyclonal guinea pig anti-bovine insulin antibody (1:1,000); a cocktail of polyclonal antibodies for glucagon, somatostatin, and pancreatic polypeptide (1:1,000; Linco Research, St. Charles, MO); and a monoclonal anti-Bcl-xL antibody (1:200). All blocking and developing reagents were from a Histomouse-SP Bulk Kit (Zymed Laboratories, San Francisco, CA) and were used according to the manufacturer's instructions. Sections treated with the same protocol in the absence of the primary antibodies served as negative controls.

Isolation of pancreatic islets and tests of in vitro cell viability. Islets of Langerhans were isolated from the pancreata of 8- to 12-wk-old Bcl-xL transgenic mice and their wild-type littermates by collagenase digestion and discontinuous Ficoll gradient separation, a modification of the original method of Lacy and Kostianovsky (14). Islets were cultured in RPMI 1640 medium supplemented with 11 mM glucose, 5% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

Islet cell viability after 48 h exposure to thapsigargin was determined by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as previously described (20, 46).

Assessment of glucose tolerance. Intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 4-h fast in Bcl-xL transgenic and wild-type control mice. Blood was sampled from the tail vein before and 30, 60, and 120 min after injection of 2 g/kg dextrose intraperitoneally.

Insulin secretion from isolated perifused pancreatic islets. Insulin secretion from isolated islets was measured in a temperature-controlled multichamber perifusion system (ACUSYST-S; Cellex Biosciences, Minneapolis, MN), as previously described (37), after an overnight culture in RPMI 1640 medium (with 11 mM glucose).

Insulin secretion from the in situ-perfused pancreas. The pancreas was perfused in situ in a humidified, temperature-controlled chamber using a modification of a protocol previously described in detail (27).

Intracellular free Ca2+ measurements. Islets attached to glass coverslips were loaded with fura 2-AM (5 µM; Molecular Probes, Eugene, OR) for 30 min at 37°C in the Krebs-Ringer bicarbonate (KRB) medium (no BSA). The coverslip was mounted in a microperifusion chamber on the specimen stage of a microscope equipped for epifluorescence and was perifused with KRB at a rate of 2.5 ml/min at 37°C as described in detail elsewhere (31). Intracellular free Ca2+ concentration ([Ca2+]i) was measured using dual-wavelength fluorescence video microscopy and was expressed as the ratio of the fluorescence intensity (detected at 510 nm) of fura 2 stimulated with light at excitation wavelengths of 340 and 380 nm (ratio 340/380) as described (31).

Glucose utilization and mitochondrial glucose oxidation in islets. These parameters were determined simultaneously by measuring the formation of 3H2O from D-[5-3H]glucose (45) and 14CO2 from [6-14C]glucose (35) in the same test tube. Duplicate batches of 10 islets each were incubated in 100 µl of KRB containing 2 or 24 mM glucose together with 0.35 µCi D-[6-14C]glucose and 1 µCi D-[5-3H]glucose.

NAD(P)H autofluorescence measurement. The reduced forms of NAD and NADP, referred to as NAD(P)H, were measured using the same imaging system described for the measurement of [Ca2+]i. Autofluorescence derived from NAD(P)H was excited at 365 nm and was measured at 495 nm in dye-free islets (28).

Mitochondrial membrane potential measurement. Rhodamine 123 (Rh123) was used as an indicator of mitochondrial membrane potential (Delta Psi m; see Ref. 4). This fluorescent lipophilic cationic dye is known to be specifically partitioned into negatively charged mitochondrial membranes. In cells preloaded with Rh123, when Delta Psi m increases (hyperpolarization) as seen after nutrient stimulation, more Rh123 is concentrated into the mitochondrial membrane, leading to an aggregation of dye molecules and quenching of the fluorescence signal. Depolarization of Delta Psi m, on the other hand, allows dye to redistribute from mitochondria into the cytosol, resulting in an increase of the Rh123 signal. In the present study, intact islets were loaded by incubation with 10 µg/ml Rh123 in KRB (with 2 mM glucose) for 20 min at 37°C. Rh123 fluorescence was excited at 490 nm and was measured at 530 nm with a FITC cube.

ATP response to nutrient stimulation. Dynamic changes in the intracellular ATP-to-ADP ratio were measured indirectly by monitoring free Mg2+ levels with the fluorescent dye MgGreen (16), since Mg2+ has much greater affinity for ATP than ADP. Thus a rise in ATP or the ATP-to-ADP ratio decreases free Mg2+ levels, and this reduces the MgGreen signal. Islets were loaded with 5 µM of the dye in KRB with zero glucose for 30 min and rinsed for 15 min before data acquisition. MgGreen signal was detected at 535 nm after excitation at 495 nm. To document the ability of this system to detect a change in ATP, at the end of most experimental protocols, islets were exposed to 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler that leads to ATP consumption and a 21 ± 4% rise of MgGreen signal over the basal intensity.

Assay methods. Insulin concentrations were measured by a double-antibody RIA using a rat insulin standard. Pancreatic insulin content was measured after acid ethanol (0.18 M HCl in 75% ethanol) extraction of the whole pancreas (8) and was quantified by RIA after dilution. To quantify DNA fragmentation, lysates from 200 mouse islets were centrifuged at 15,000 g for 15 min to separate fragmented DNA from intact DNA, and the amount of DNA in the two fractions was determined spectrofluorometrically with Hoechst 33258 after phenol-chloroform extraction (46).

Data presentation and statistics. Intensity data of the NAD(P)H, Delta Psi m, and ATP measurements were normalized using their own basal levels at 2 mM glucose [designated as 1 for NAD(P)H and Delta Psi m and 100 for ATP] to correct the variation that resulted from different islet sizes. All data are presented as means ± SE, and unpaired Student's t-test or ANOVA was used for comparison between different groups.


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Islet morphology and Bcl-xL expression in pancreatic islets. There were no significant differences in litter size and body weight between the two transgenic founder lines and their wild type littermates throughout their lifetime.

Immunohistochemical staining with anti-insulin antibody (Fig. 2, A-C) or a cocktail of anti-glucagon/somatostatin/pancreatic peptide sera (Fig. 2, D-F) revealed normal islet architecture in both Fd 1 and Fd 2 Bcl-xL mice. As expected, insulin-positive beta -cells were located in the center of the islet, and other endocrine cells were present in the surrounding mantle. Pancreatic insulin content was not different in the Fd 1, Fd 2, and wild-type mice (26.5 ± 2.3, 29.8 ± 4.4, and 34 ± 2.8 µmol/mg pancreas, respectively, n = 7 for each group). Therefore, overexpression of Bcl-xL in beta -cells did not affect either islet morphology or insulin content.


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Fig. 2.   Islet morphology and Bcl-xL expression in wild-type (A, D, and G), Fd 1 (B, E, and H), and Fd 2 (C, F, and I) Bcl-xL transgenic mice. Pancreatic sections (5 µm) were immunochemically stained for insulin (A-C), glucagon/somatostatin/pancreatic polypeptide (D-F), and Bcl-xL (G-I) with hematoxylin as counterstain. In islets from all 3 groups of animals, insulin-positive beta -cells were distributed in the center of islets and were surrounded by non-beta -cell hormone-positive cells. Bcl-xL-positive cells were only found in islets from the 2 founder lines of Bcl-xL transgenic mice. Aggregates of Bcl-xL staining were found in most of the Fd 2 islets (I).

The specific expression of Bcl-xL protein in islet cells was documented by immunohistochemistry using anti-Bcl-xL serum. As shown in Fig. 2, G-I, Bcl-xL-positive cells were seen in the center of the islets from both founders of Bcl-xL transgenic mice but not in the non-beta -cells, exocrine tissue or liver, small intestine, and lymph nodes (data not shown). The expression level of Bcl-xL in Fd 2 Bcl-xL islets was markedly higher than in Fd 1 mice, as evidenced by a significantly stronger signal and greater percentage of Bcl-xL-positive beta -cells. The distribution of Bcl-xL staining was also dramatically different between Fd 2 and Fd 1 mice. Large Bcl-xL-positive aggregates were found in most of the beta -cells of the Fd 2 mice, whereas in beta -cells of the Fd 1 mice, Bcl-xL staining was evenly distributed in the cytosol.

The difference in expression levels of Bcl-xL between Fd 1 and Fd 2 transgenic mice was further evidenced by Western blot analysis (Fig. 1B). On the basis of densitometry, Bcl-xL expression in beta -cells was increased 2- to 3-fold in Fd 1 and at least 10-fold in Fd 2 transgenic mice over littermate controls.

Effects of Bcl-xL on thapsigargin-induced cell death and DNA fragmentation in islets. In our previous study, we have demonstrated that thapsigargin consistently induces a sizable degree of apoptosis in islet beta -cells (46). Therefore, we used it as a convenient apoptosis inducer to test whether overexpression of Bcl-xL confers protection against apoptosis in islet beta -cells. As shown in Fig. 3A, thapsigargin treatment dramatically reduced the viability of wild-type islets, whereas Bcl-xL- overexpressing transgenic islets had significantly higher cell viability. After exposure to 10 µM thapsigargin for 48 h, 64 ± 4.6% of wild-type islets remained viable compared with 81 ± 1.6% in Fd 1 and 86 ± 1.0% in Fd 2 Bcl-xL islets (P < 0.05 vs. wild type for both). Protection against apoptosis induced by high concentrations of thapsigargin (20 µM) was greater in Fd 2 than in Fd 1 Bcl-xL transgenic islets, as indicated by a greater degree of viability (65 ± 4 vs. 27 ± 2%, P < 0.01, Fig. 3A). DNA fragmentation measurement was used as an additional indicator of thapsigargin-induced apoptosis in Bcl-xL and wild-type islets. Thapsigargin (10 µM) caused less DNA fragmentation in Bcl-xL islets (Fig. 3B) than in wild-type islets, which is consistent with resistance to apoptosis.


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Fig. 3.   Thapsigargin-induced cell death and DNA fragmentation in islets from Bcl-xL transgenic and wild-type mice. A: islets from both Fd 1 and Fd 2 Bcl-xL transgenic and wild-type (WT) mice were cultured in RPMI 1640 medium with varying concentrations of thapsigargin for 48 h, and cell death was determined as the decrease in cell viability monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. B: islets from the Fd 2 Bcl-xL mice (Bcl-x) and their wild-type controls were exposed to 10 µM of thapsigargin for 48 h. Fragmented DNA and intact DNA in islet lysates were then quantified spectrofluorometrically with the specific dye Hoechst 33258. Data represent means ± SE of 3 (Fd 2) or 5 (Fd 1) separate experiments. * P < 0.05 and ** P < 0.01 compared with wild type.

Plasma glucose and insulin levels during IPGTT. In the course of studies on the in vivo effects of overexpression of Bcl-xL in pancreatic islets, unanticipated hyperglycemia was found in random specimens from 85% of male and 60% of the female Fd 2 Bcl-xL mice. To determine whether the overexpression of Bcl-xL is associated with a systematic reduction in glucose tolerance, IPGTTs were performed on the transgenic and wild-type animals. As illustrated in Fig. 4, the Fd 1 Bcl-xL mice and wild-type controls had comparable blood glucose profiles after intraperitoneal glucose administration. In contrast, Fd 2 Bcl-xL mice had fasting hyperglycemia (12 ± 2 vs. 9 ± 1 mmol/l in control, P < 0.01) and significantly higher glucose levels after the glucose load (19 ± 2 vs. 9 ± 1 mmol/l at 2 h, P < 0.001). Despite the presence of hyperglycemia, serum insulin levels of Fd 2 Bcl-xL mice 30 min after glucose administration were >50% lower than the wild type (4.3 ± 1.0 vs. 9.5 ± 1.7 pmol/l, P < 0.01). The basal serum insulin concentrations of the two groups were similar (3.7 ± 0.5 in control and 3.0 ± 1.0 pmol/l in Bcl-xL mice).


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Fig. 4.   Blood glucose levels during ip glucose tolerance tests in Bcl-xL Fd 1 (A) and Fd 2 (B) transgenic mice and their wild-type littermates. Glucose (2 g/kg body wt) was administrated ip after a 4-h fast. Data represent means ± SE of 10 mice in each group.

To determine the age of diabetes onset in Fd 2 mice, IPGTTs were performed in 4-, 8-, and 12-wk-old animals. Blood glucose levels obtained after fasting (16 ± 0.9 vs. 10 ± 0.5 mmol/l, P < 0.01) and 2 h after intraperitoneal glucose administration (18 ± 0.6 vs. 9.7 ± 0.4 mmol/l, P < 0.001) were already markedly elevated at 4 wk of age in Fd 2 Bcl-xL mice.

Insulin secretory and [Ca2+]i responses to glucose and KCl in isolated perifused islets. To identify the cause of hyperglycemia in Fd 2 Bcl-xL transgenic mice, insulin secretory responses to glucose and KCl were measured in perifused islets. As shown in Table 1, insulin secretion in response to 16.7 mM glucose was reduced by 60% in islets from the Fd 2 Bcl-xL (5.5 ± 0.6 vs. 13 ± 1.9 pmol/l, P < 0.001) compared with wild-type islets but was normal in Fd 1 Bcl-xL islets (15 ± 1.9 pmol/l). KCl-stimulated insulin secretion in islets of Fd 2 mice was also significantly attenuated (by 34%).

                              
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Table 1.   Insulin secretion in isolated islets from Bcl-xL transgenic and wild-type mice

The intracellular signaling pathway of stimulus-secretion coupling in the pancreatic beta -cell involves an increase in [Ca2+]i, which triggers the exocytosis of insulin. We therefore estimated changes in [Ca2+]i in response to secretagogues. [Ca2+]i responses to 12 mM glucose and 30 mM KCl, as measured by the area under the curve (AUC), were decreased by 62 ± 7 and 46 ± 8%, respectively, in islets from Bcl-xL mice (Fig. 5A).


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Fig. 5.   Intracellular free Ca2+ concentration ([Ca2+]i) responses to glucose, KCl, and carbachol in islets from Bcl-xL transgenic and wild-type mice. Islets from Fd 2 Bcl-xL and wild-type mice were loaded with 5 µM fura 2-AM for 30 min and then were perifused with Krebs-Ringer bicarbonate (KRB) buffer with 12 mM glucose or 30 mM KCl. Changes in [Ca2+]i, as reflected by the ratio absorbance at 340 to 380 nm, were monitored with a computerized digital imaging system. Representative records of [Ca2+]i responses to glucose and KCl (A) and carbachol (CCh) before and after exposure to EGTA (B) in islets from Fd 2 Bcl-xL transgenic and wild-type mice are shown.

Long-term hyperglycemia per se is known to cause a wide range of disturbances of islet function, including a reduction in the [Ca2+]i response to glucose (15). Therefore, additional [Ca2+]i experiments were performed in islets from insulin-treated Bcl-xL mice that had been normoglycemic for 1 wk. The responses to glucose, pyruvate methylester (PME), and KCl were reduced to a similar extent in islets from insulin-treated and untreated Bcl-xL mice. The integrated [Ca2+]i responses also remained reduced after insulin treatment and represented only 68 ± 7, 61 ± 2 (P < 0.01), and 86 ± 10% (P < 0.05) of the responses in wild-type islets, respectively, indicating that defects of the [Ca2+]i response in Bcl-xL mice are not secondary to hyperglycemia.

Insulin secretory and [Ca2+]i responses to carbachol. Carbachol, a muscarinic agonist, stimulates insulin secretion by releasing Ca2+ from intracellular stores, a process that is mediated by inositol 1,4,5-trisphosphate (IP3; see Ref. 7). Addition of EGTA (2.5 mM) to the incubation medium, by preventing entry of extracellular Ca2+ into islets, isolated the effects of carbachol on release of Ca2+ from intracellular stores. Addition of 250 µM carbachol in the presence or absence of extracellular Ca2+ triggered similar [Ca2+]i responses in Bcl-xL and wild-type islets (Fig. 5B), suggesting that the intracellular Ca2+ stores and their release mechanisms are intact in Fd 2 Bcl-xL islets. Consistent with this observation, insulin responses to carbachol from perifused Fd 2 Bcl-xL islets in the presence of 2 mM glucose were not impaired (Table 1).

Insulin secretory and [Ca2+]i responses to mitochondrial substrates and tolbutamide. To test mitochondrial function in Bcl-xL islets, two agents that are metabolized in mitochondria [PME, a cell-permeable derivative of pyruvate (44), and alpha -ketoisocaproic acid (KIC)] were used as secretagogues as well. As shown in Fig. 6, insulin responses to glucose and PME in pancreata from Bcl-xL mice were reduced by 79 ± 10 and 76 ± 13%, respectively (P < 0.01 for both, see Fig. 6B). [Ca2+]i responses to 24 mM PME and 10 mM KIC were reduced by 58 ± 3 and 58 ± 5%, respectively (P < 0.01, n = 5) in Fd 2 Bcl-xL islets compared with a 52 ± 7% reduction in the [Ca2+]i response to glucose (comparisons were made based on AUC, data not shown).


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Fig. 6.   A: insulin secretory responses to glucose, pyruvate methyl ester (PME), and tolbutamide (Tolb) in perfused pancreas. Isolated pancreata from Bcl-xL transgenic and wild-type mice were sequentially stimulated by 20 mM glucose, 24 mM PME, and 250 µM tolbutamide for 10 min each. Each agent was washed out for 15 min with KRB medium containing 5 mM glucose. B: insulin secretion from pancreata of Fd 2 Bcl-xL mice as percentage of that from pancreata of wild-type mice. Data represent means ± SE of 3 mice in each group. * P < 0.05 and ** P < 0.01 compared with wild-type mice.

Insulin secretion in response to tolbutamide, a sulfonylurea that closes the ATP-sensitive K+ channel directly by binding to the sulfonylurea receptor subunit of the channel (32), was also significantly reduced (by ~50%, P < 0.05, Fig. 6).

Glucose metabolism in islets from Bcl-xL transgenic mice. The rates of glycolytic flux (3H2O production from [5-3H]glucose) and glucose oxidation within mitochondria (14CO2 production from [6-14C]glucose) were measured simultaneously. As illustrated in Fig. 7A, the basal glucose metabolic rates at 2 mM glucose were not different in the two groups of islets. At 24 mM glucose, glycolytic flux in Bcl-xL islets was slightly higher (statistically nonsignificant) than in wild-type islets, whereas the rate of glucose oxidation in mitochondria was significantly lower in Fd 2 Bcl-xL than in wild-type islets (P < 0.01 at 24 mM glucose, Fig. 7A). Hence, the ratio of glucose oxidation to utilization in Bcl-xL islets was significantly reduced at 24 mM glucose (Fig. 7B).


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Fig. 7.   Glucose utilization and oxidation in islets from Fd 2 Bcl-xL and wild-type mice. 3H2O production from D-[5-3H]glucose and 14CO2 from D-[6-14C]glucose were determined simultaneously in batches of 10 islets incubated in duplicate for 2 h in 100 µl KRB medium. A: rates of glycolytic flux (D-[5-3H]glucose right-arrow 3H2O) and mitochondrial glucose oxidation (D-[6-14C]glucose right-arrow 14CO2) at different concentrations of glucose in Fd 2 Bcl-xL and wild-type islets. B: ratios of glucose oxidation to utilization. Data represent means ± SE of 4 mice in each group. ** P < 0.01 compared with wild-type mice.

Consistent with defective mitochondrial nutrient metabolism, the NAD(P)H responses to 10 mM KIC or 20 mM PME in Fd 2 Bcl-xL islets were significantly lower than the responses in wild-type islets (Fig. 8). However, the NAD(P)H response to glucose was not different between Fd 2 Bcl-xL and wild-type islets.


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Fig. 8.   NAD(P)H responses to glucose, PME, and alpha -ketoisocaproic acid (KIC). Autofluorescence of NAD(P)H in perifused islets was measured with same imaging system used for measurement of [Ca2+]i. Intensity of the fluorescent signal was normalized with its own basal level (at 2 mM glucose) to correct for variation in intensity resulting from the differences in islet size. A: typical NAD(P)H responses to 12 mM glucose, 10 mM KIC, and 20 mM PME in wild-type (top) and Bcl-xL (bottom) islets. B: integrated NAD(P)H responses [calculated from the area under the curve (AUC)] in Bcl-xL islets expressed as %response in wild-type islets. Data represent means ± SE of 6 experiments performed in islets from different animals. * P < 0.05 compared with wild-type mice.

Responses of Delta Psi m to glucose and PME. Nutrient metabolism and NAD(P)H generation in beta -cells normally increases the rate of electron transport and proton efflux across the innermitochondrial membrane, leading to the hyperpolarization of Delta Psi m and ATP production. In the present study, increasing glucose concentration from 2 to 12 mM induced a two-phase decrease of the Rh123 signal in wild-type islets: a rapid drop within the first 120 s, followed by a much slower second-phase decrease lasting for another 120 s before maintaining a hyperpolarized state (Fig. 9A). After withdrawal of 12 mM glucose, Delta Psi m returned almost completely to the prestimulation level. Stimulation of wild-type islets with 24 mM PME caused a steep drop of Delta Psi m that reached the nadir in <1 min. The response of Delta Psi m to glucose in Fd 2 Bcl-xL islets was markedly different from that of control islets. The normal biphasic decrease in Delta Psi m was replaced by a single, slow decrease of the Rh123 signal in Bcl-xL islets, and no rapid drop at the beginning of stimulation was observed. The degree of hyperpolarization of Delta Psi m, as measured by the maximum decrease in Rh123 signal, was decreased by 50% in Fd 2 Bcl-xL islets relative to that of wild-type controls (Fig. 9B).


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Fig. 9.   Alteration in mitochondrial membrane potential (Delta Psi m) induced by glucose, PME, and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Islets were loaded with rhodamine 123 (Rh123) in KRB for 30 min, and the fluorescence intensity was monitored as in Fig. 8. Data were normalized for each experiment to the fluorescent intensity obtained at 2 mM glucose. A: representative Delta Psi m responses to 12 mM glucose, 20 mM PME, and 10 µM CCCP in wild-type (left) and Bcl-xL (right) islets. B: summary of Delta Psi m changes over the basal level in Bcl-xL and wild-type islets. Data represent means ± SE of 8 experiments performed in islets from different animals. * P < 0.05 and ** P < 0.01 compared with wild-type mice.

The pattern of the Delta Psi m response to PME in Fd 2 Bcl-xL islets was similar to that of wild-type controls but was attenuated by 60 ± 5% (P < 0.01). No such abnormality was observed in Fd 1 Bcl-xL islets.

In an effort to compare the dynamics of Delta Psi m in Bcl-xL and wild-type islets, we treated the islets with 10 µM CCCP, an uncoupler of oxidative phosphorylation in mitochondria that disrupts Delta Psi m (33). As expected, addition of CCCP immediately resulted in a robust increase in Rh123 signal (Fig. 9), reflecting a release of Rh123 from the mitochondrial membrane into the cytosol after the depolarization of Delta Psi m. The rise of the Rh123 signal induced by CCCP stimulation over the basal level in Fd 2 Bcl-xL islets was significantly lower than in wild-type islets (220 ± 34 vs. 305 ± 33%, P < 0.01), indicating the preservation of Delta Psi m, even in the presence of CCCP in the transgenic islets.

Intracellular ATP response to glucose and KIC. Changes in ATP level or the ATP-to-ADP ratio after glucose and KIC stimulation were measured indirectly by monitoring changes in the intracellular free Mg2+ level with MgGreen (16). As shown in Fig. 10, stimulation of wild-type islets with 16 mM glucose or 10 mM KIC elicited a 20-25% decrease in free Mg2+, reflecting an increase in ATP levels. A decrease of only 8-11% was obtained in Fd 2 Bcl-xL islets with the same concentration of glucose and KIC (P < 0.01 vs. wild-type controls), again suggesting that a defect in metabolism was primarily at the level of the Bcl-xL mitochondria.


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Fig. 10.   Response of ATP or ATP-to-ADP ratio to glucose and KIC in islets from Bcl-xL mice. After loading islets with 10 µM MgGreen-AM in KRB without glucose for 30 min, the signal was monitored at 535 nm after excitation at 495 nm. A: representative changes after stimulation by glucose and KIC. B: AUC of the responses to glucose and KIC in islets from Fd 2 Bcl-xL and wild-type mice. * P < 0.01 compared with wild-type mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In view of the mounting evidence that apoptosis mediates destruction of beta -cells in type 1 and possibly type 2 diabetes, interventions that reduce the rate of beta -cell apoptosis could potentially offer therapeutic benefit in the prevention and treatment of these conditions. The present study was undertaken as a prelude to the development of such a therapeutic approach. Transgenic mice were produced in which the anti-apoptotic protein Bcl-xL was expressed in the pancreatic beta -cells. Tissue-specific expression of Bcl-xL was obtained by using rat insulin II promoter constructs, and the level of Bcl-xL expression was determined by the length of the promoters. The longer promoter construct, RIP7, resulted in levels of Bcl-xL that were ~10-fold those seen in wild-type islets, whereas a shorter promoter, RIP1 led to an ~2- to 3-fold increase in the level of expression. This experimental design allowed us to study the effects of two levels of overexpression of Bcl-xL on the function of pancreatic islets as well as the ability of islets to resist apoptosis induced by thapsigargin.

Bcl-xL overexpression in pancreatic beta -cells did not alter islet development or morphology in either founder line of transgenic mice, as evidenced by the normal islet architecture and insulin staining. Normal pancreatic insulin content, in conjunction with normal pancreas weight, excludes the possibility of major changes in beta -cell mass in the transgenic animals. Additionally, increased expression of Bcl-xL protein in transgenic islets afforded protection against thapsigargin-induced cell death when compared with wild-type islets, and the level of protection appeared to correlate with the quantity of Bcl-xL expressed in islets. These results are consistent with previous findings in other cell types and insulinoma cell lines transfected with Bcl-2 (10, 17). This is the first demonstration that Bcl-xL promotes the survival of primary beta -cells.

Thapsigargin is known to induce apoptosis in many cell types, including insulin-secreting beta -cells, by depleting intracellular Ca2+ stores (46). Cytokines such as interleukin-1beta , interferon-gamma , and tumor necrosis factor-alpha have been widely implicated in autoimmune destruction of beta -cells associated with type 1 diabetes. However, the potency of the cytokines to induce cell death in intact islets was much lower than thapsigargin in our hands, and others have shown that cytokines also induce necrosis in pancreatic beta -cells. Because thapsigargin is a more potent inducer of beta -cell death, we would expect that islets overexpressing Bcl-xL are protected against cytokine-induced cell death.

The high level of Bcl-xL expression achieved in the Fd 2 animals was associated with significant impairment of glucose tolerance manifested by increases in the blood glucose concentration both in the fasting state and 2 h after the intraperitoneal administration of glucose. The elevation in blood glucose was present at an early age (at least 4 wk) in both male and female animals and was associated with defective insulin secretion in response to glucose and other secretagogues in the isolated perfused pancreas and isolated perifused islets. The reduction in insulin secretion was paralleled by a similar reduction in [Ca2+]i responses to glucose and KCl. However, the pathways that stimulate insulin secretion after release of Ca2+ from intracellular stores appear to be intact, since the insulin secretory responses to carbachol, an agent that activates phospholipase C and generates IP3 (7), were normal. Normal insulin secretory responses to carbachol indicate that the exocytosis process distal to the rise in [Ca2+]i is intact in Bcl-xL transgenic islets.

Taken together with the normal islet morphology and insulin content, these results indicate that the high level of Bcl-xL protein expression in the Fd 2 transgenic mice interferes with the beta -cell signaling pathways activated by glucose. The precise mechanisms responsible for the anti-apoptotic effects of Bcl-xL have not been fully defined. Its subcellular localization to the mitochondrial membrane (12) and the recent demonstration of the effects of Bcl-xL on mitochondrial function (11, 22, 29, 40, 41) raised the possibility that the impairment of beta -cell function associated with Bcl-xL overexpression may be related to disturbances in mitochondrial signaling mediating glucose-induced insulin secretion. We therefore tested the hypothesis that beta -cell dysfunction in Fd 2 Bcl-xL transgenic mice may be related to important mitochondrial effects of Bcl-xL overexpression.

These studies uncovered considerable evidence for mitochondrial dysfunction. In Fd 2 Bcl-xL pancreas or islets, the insulin secretory responses to the mitochondrial nutrients PME (Fig. 1) and KIC (data not shown) were reduced to a similar extent as the response to glucose. In addition, glucose oxidation was significantly reduced at 24 mM glucose in the face of a normal glycolytic flux. Reduced NAD(P)H responses to PME or KIC stimulation in Fd 2 Bcl-xL islets provided further evidence for reduced metabolism of these two nutrients within the mitochondria. Bcl-xL islets also demonstrated altered regulation of Delta Psi m, as evidenced by reduced hyperpolarization responses after nutrient stimulation. The maintenance of a normal Delta Psi m is crucial to a number of mitochondrial functions, such as ATP production and homeostasis of intramitochondrial Ca2+ levels, which are important for the regulation of metabolism (30). In the present study, we found that the ATP production in response to glucose and KIC stimulation was indeed reduced in Bcl-xL islets compared with wild-type islets.

The normal NAD(P)H response to glucose in Fd 2 Bcl-xL islets is surprising in light of the alteration in glucose oxidation. The reason for this apparent discrepancy is not known. It may be explained by the difference in the length of time for each experiment. The glucose oxidation is the total amount of oxidation over 120 min, whereas the change in NAD(P)H is the maximal amplitude over 10 min. Over the 120-min period, glucose would continue to be oxidized, and this would occur in all of the cells of the wild-type islets but may not happen in cells overexpressing Bcl-xL. Also, because the change in fluorescence is a relative value, it is difficult to quantitatively compare the two experiments.

ATP plays an important role in the regulation of exocytosis of insulin (24, 32), and depletion of ATP impairs tolbutamide-induced insulin secretion (21). The reduction in KCl and tolbutamide-induced [Ca2+]i and insulin responses seen in islets of Fd 2 Bcl-xL mice are likely due to mitochondrial dysfunction in the beta -cell, since similar results have recently been observed in a pancreatic beta -cell line lacking mitochondrial DNA (39). Because mitochondrial DNA encodes proteins responsible for ATP production through oxidative phosphorylation, depletion of mitochondrial DNA is expected to abolish mitochondrial ATP generation. In Fd 2 Bcl-xL islets, there is indeed a dramatic reduction in nutrient metabolism and ATP production. These observations indicate that ATP or other factors generated from mitochondrial nutrient metabolism play an important role in KCl- and tolbutamide-induced [Ca2+]i responses and insulin secretion and in glucose-induced insulin secretion.

Increased expression of Bcl-xL seen in islets from Fd 1 and Fd 2 mice was associated with protection from thapsigargin-induced apoptosis. These results are consistent with those of Biroccio et al. (2) and others, who have suggested that a decrease in mitochondrial nutrient metabolism may help to restrain the release of cytochrome c and other death-promoting molecules from the space between the inner and outer mitochondrial membranes to the cytosol. However, although even a modest overexpression of Bcl-xL, as seen in the Fd 1 islets, was sufficient to protect the islets from thapsigargin-induced cell death, only marked overexpression of the protein, as seen in the islets from the Fd 2 animals, resulted in sufficient disturbance of mitochondrial distribution and metabolism to reduce glucose-induced insulin secretion.

Although it is possible that the diabetic phenotype in the Fd 2 line could also be linked to a very rare insertional disruption of the host gene, we believe that the chance of this mechanism being responsible for the observed changes is very small. First, we always performed wild-type × transgenic breeding to avoid the disruption of both alleles of a particular gene. Second, the phenotype of the Fd 2 Bcl-xL mice is unique; the animals were viable, reproductively competent, and had a glucose-signaling defect consistent with possible effects of Bcl-xL. It is also unlikely that we introduced a gene disruption to a position that happened to be more important for beta -cell function then elsewhere. However, because we did not get the chance to study more positive founders generated with the RIP7 construct, we can not totally rule out insertional disruption of a host gene as a mechanism for the phenotype of the Fd 2 animals.

In summary, the two- to threefold increase in Bcl-xL expression seen in islets from the Fd 1 animals afforded protection against thapsigargin-induced apoptosis and did not interfere with insulin secretion. The ~10-fold increase in expression of Bcl-xL found in the Fd 2 animals afforded greater protection against thapsigargin-induced apoptosis but was associated with a severe defect in insulin secretion that caused diabetes. This defect in insulin secretion is due to a novel mitochondrial defect resulting in reduced responses to mitochondrial substrates, decreased mitochondrial glucose oxidation, and abnormal control of Delta /Psi m. These results underscore the complexity of developing therapeutic approaches by transferring novel proteins into beta -cells to modify their underlying function or to enhance their ability to survive. In designing such therapeutic strategies, not only must the desired therapeutic effect of the protein under study be considered but unexpected adverse influences of the particular protein on islet function need to be considered also. On the other hand, the Fd 2 Bcl-xL mice represent a unique model of beta -cell-specific mitochondrial impairment that underscores the critical role of mitochondrial metabolism in insulin secretion (5, 6).


    ACKNOWLEDGEMENTS

We thank Kimberly Fox for technical assistance.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-31842, DK-44840, DK-20595, and DK-49799, the Blum-Kovler Foundation, and the Jack and Dollie Galter Center of Excellence of the Juvenile Diabetes Foundation International. M. Levisetti was supported by a mentor-based postdoctoral fellowship award of American Diabetes Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: K. S. Polonsky, Section of Endocrinology, Univ. of Chicago, MC 1027, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: polonsky{at}medicine.bsd.uchicago.edu).

Received 10 May 1999; accepted in final form 12 October 1999.


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