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Endocrinology Vol. 146, No. 10 4417-4424
Copyright © 2005 by The Endocrine Society

Activation of Caspase 8 in the Pituitaries of Streptozotocin-Induced Diabetic Rats: Implication in Increased Apoptosis of Lactotrophs

Ana I. Arroba, Laura M. Frago, Jesús Argente and Julie A. Chowen

Hospital Infantil Universitario Niño Jesús, Universidad Autónoma, Department of Endocrinology, 28009 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Julie A. Chowen, Department of Endocrinology, Hospital Niño Jesús, Menéndez Pelayo, 65, 28009 Madrid, Spain. E-mail: jachowen{at}telefonica.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lactotroph cell death is increased in streptozotocin-induced diabetic rats. To determine the mechanism involved, cell death proteins were accessed in pituitaries of diabetic (streptozotocin at 65 mg/kg, 2 months evolution) and control male rats by Western blot analysis and double immunohistochemistry. The intact and cleaved forms of caspase 9 were increased in diabetic rat pituitaries compared with controls. Although the proforms of caspases 3, 6, and 7 were increased in diabetic rat pituitaries, their activated forms were either unchanged or decreased. Activation of these effector caspases may be blocked by the increased expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in diabetic rat pituitaries. However, in diabetic rats, XIAP expression in lactotrophs was decreased, suggesting that this cell type is not protected. Caspase 8, p53, and nuclear factor {kappa}B were more highly activated in diabetic rat pituitaries, with caspase 8 colocalization in lactotrophs being increased. These results suggest that, in the pituitaries of diabetic rats, the cascades of normal cell turnover are partially inhibited, possibly via XIAP, and this may be cell specific. Furthermore, activation of the extrinsic cell-death pathway, including activation of caspase 8, may underlie the diabetes-associated increase in lactotroph death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIABETES MELLITUS is a metabolic disease that, if poorly controlled, can have diverse secondary complications, including retinopathies, renal dysfunction, peripheral neuropathies, cardiovascular problems, increased risk of dementia, and hormonal imbalances (1, 2, 3, 4, 5, 6). One common observation among many of these conditions is increased cell death in the affected tissues or organs. Indeed, in addition to the primary loss of pancreatic ß cells in type I diabetes mellitus (7), increased programmed cell death occurs in the retina, kidney, cardiovascular tissue, neurons, oligodendrocytes, epithelial tissue, and pituitary (8, 9, 10, 11, 12, 13).

Cell death can occur by necrosis or apoptosis, with these two mechanisms having distinct histological and biochemical markers (14, 15). In contrast to necrosis, apoptosis involves a cascade of intracellular events that ultimately culminates in cell destruction (14, 15, 16, 17). This process involves caspases, cysteine-dependent, aspartate-specific proteases, that exist in an inactivated state that, when activated, initiate the death program. In what is referred to as the intrinsic cell death pathway, the upstream or initiator caspases, including caspase 9, respond to a death signal and then activate downstream or effector caspases such as caspases 3, 6, and 7. These effector caspases then trigger processes that ultimately result in cell death. The intrinsic apoptotic pathway can be initiated by external signals or internal changes, such as release of cytochrome c from mitochondria; indeed, mitochondria are a very important component of this cascade (14, 15, 16, 17). Release of apoptogenic factors from mitochondria can be induced by distinct factors, including members of the Bcl-2 protein family. The balance between proapoptotic and antiapoptotic members of this family has a crucial role in determining the integrity of the mitochondria and, hence, cell death (14, 15, 16, 17).

The extrinsic cell-death pathway involves activation of extracellular death receptors, which belong to the TNF receptor superfamily (15, 17, 18). Binding of the appropriate ligand to one of these receptors results in receptor aggregation and recruitment of FADD (Fas-associated death domain) and procaspase 8 (also called FLICE or MACH-1). Procaspase 8 can then be activated by self-cleavage or cleavage by another caspase 8 molecule (19, 20). Activated caspase 8, functioning as an initiator caspase, activates downstream executioner caspases that cleave cell death substrates or directly induces apoptosis (21). In distinct paradigms, caspase 8 activation has been implicated in p53-mediated apoptosis (22, 23). The transcription factor p53 modulates various genes involved in cell death (24, 25) and, in some tissues, hyperglycemia has been shown to activate p53, which then results in cell death (26).

It was previously thought that, once activated, the cascade of events leading to cell death could not be stopped. However, it is now known that members of the inhibitor of apoptosis protein (IAP) family block apoptotic pathways at various levels of the intracellular cascade (17, 27, 28). One of the most potent IAPs described to date is X-chromosome-linked IAP (XIAP), which has been shown to inhibit caspases 3, 7, and 9 through domain-specific binding (28). Activation of the extrinsic apoptotic pathway can be inhibited by the presence of FLICE-like inhibitory protein (FLIP). This protein binds to FADD; thus, reducing the availability of FADD for binding to caspase 8, which, as stated above, is essential for its activation (29). Although TNF receptors are involved in activation of the extrinsic cell death pathway, activation of these receptors can also induce IAPs, with this process being mediated through activation of nuclear factor {kappa}B (NF{kappa}B) (30). NF{kappa}B also inhibits apoptosis by activating survival signals such as Bax, Bcl-2, and Bcl-xL, as well as FLIP (31). Indeed, it has been suggested that FLIP inhibition of TNF{alpha}-induced activation of caspase 8 may be mediated by NF{kappa}B (31, 32, 33). Hence, activation of TNF receptors can result in activation of the extrinsic cell death pathway and inhibition of both the intrinsic and extrinsic pathways.

We have reported recently that there is an increase in the number of lactotrophs undergoing cell death in poorly controlled diabetic rats (13). This could underlie, at least in part, the decrease in prolactin secretion observed in poorly controlled diabetic patients and laboratory animals (1, 5). In diabetic animals, the activation of caspase 9 and subsequently caspase 3 (9, 10, 12) or a decrease in Bcl-2 levels (34) have been associated with increased apoptosis in various tissues. Hence, it is conceivable that the intrinsic or mitochondrial pathway is involved in increased death of lactotrophs in poorly controlled diabetes. Indeed, in physiological situations in which lactotroph cell death is increased, such as during weaning, changes in Bcl-2, Bax, and p53 expression have been reported (35). The Bcl-2 family of proteins is also involved in estrogen and thyroid hormone modulation of lactotroph turnover (36). Furthermore, it was reported recently that nitric oxide-induced pituitary cell death involves activation of caspases 3 and 9 (37), whereas bromocryptine-induced cell death involves p53 activation, as well as Bcl-2 inhibition (38).

The aims of this study were to first determine what intracellular signaling pathways associated with programed cell death are modified in the anterior pituitary of poorly controlled diabetic rats. Second, we assessed the changes occurring in lactotrophs of diabetic rats to attempt to explain their increased death rate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Electrophoresis reagents were from Bio-Rad (Hercules, CA). All other reagents were obtained from Sigma (St. Louis, MO) or Merck (Barcelona, Spain) unless otherwise indicated.

Animals and drug administration
Adult male Wistar rats from our in-house breeding colony were used. Animals were kept on a 12-h dark, 12-h light cycle and received food and water ad libitum. Diabetes was induced by injecting streptozotocin (65 mg/kg, ip). Controls received vehicle only. The appearance of diabetes was confirmed by blood glucose measurement via tail puncture and by using an automatic glucose analyzer (Glucocard Memory 2; A. Menarini Diagnostics, Florence, Italy). Blood glucose levels were measured at the beginning of the study, 10 d after streptozotocin injection to ensure the presence of diabetes and at the time the animals were killed. Animals were considered to be diabetic if they maintained a mean glucose level of more than 300 mg/dl. Two months after the induction of diabetes, the animals were killed by decapitation. The pituitary glands were removed and stored at –80 C until processed. Tissue was processed for protein extraction as indicated below, and samples were stored at –80 C. Three to six rats per group were used for each analysis. Animals were handled following the guidelines of the European Union.

ELISA cell death detection
This assay was performed according to the instructions of the manufacturer (Roche Diagnostics, Mannheim, Germany) as briefly described and with the following modifications. Tissue was homogenized in 300 µl of incubation buffer, placed on ice for 1 h, and centrifuged at 1200 x g for 5 min at 4 C, and the supernatant was collected. The microtiter plates were prepared by adding 100 µl of the coating solution (anti-histone antibody) to each well and incubating for 1 h at room temperature. The coating solution was removed, and 200 µl of incubation buffer was added to each well, covered, and incubated for 30 min at room temperature. The wells were then rinsed three times, and the samples (25 µl of sample plus 75 µl of incubation buffer) were added and incubated for 90 min at room temperature. This dilution was chosen after preliminary assays showed it to be the most adequate for detecting changes. After washing, 100 µl of the conjugate solution (anti-DNA-peroxidase) was added. The wells were covered and incubated at room temperature for 90 min. After washing, 100 µl of substrate solution was added, mixed, and incubated for 15 min. The resulting color was then measured at 405 nm on an automatic microplate analyzer (Biotek Instruments, Winooski, VT). Each sample was measured in duplicate in each assay. Background measurements were made, and this value was subtracted from the mean value of each sample.

Immunoblotting
For Western blotting, tissue was homogenized in 300 µl of radioimmunoprecipitation assay lysis buffer with an EDTA-free protease inhibitor cocktail (Roche Diagnostics) on ice. After homogenization, the samples were centrifuged at 12,000 x g for 5 min at 4 C, and the clear supernatants were transferred to a new tube. Protein concentration was estimated by Bio-Rad protein assay. Protein (30–60 µg) was resolved using 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Bio-Rad). Filters were blocked with Tris-buffered saline (TBS) containing 5% (wt/vol) nonfat dried milk and incubated with the appropriate primary antibody (for details, see Table 1Go). Filters were subsequently washed and incubated with the corresponding secondary antibody conjugated with peroxidase or biotin (1:2000; Pierce, Rockford, IL). Bound peroxidase activity was visualized by chemiluminescence (DuPont NEN Life Science Products, Boston, MA) and quantified by densitometry using Bio-1D (Vilber Lourmat, Marne La Vallee, France). All blots were rehybridized with actin to normalize each sample for gel-loading variability.


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  		TABLE 1. Concentrations and sources of the primary antibodies used for Western blot and immunohistochemistry analyses

 
Immunohistochemistry
Immunohistochemistry was performed on frozen 12 µm cryostat sections that were fixed in 4% paraformaldehyde (w/v), washed in TBS plus 0.1% BSA and 0.1% Triton X-100 (this buffer was used for all subsequent washes), and blocked in TBS containing 3% BSA and 1% Triton X-100 for 2 h. Sections were left overnight in a humid chamber at 4 C with primary antibodies for prolactin plus caspase 8, cleaved caspase 3, or XIAP in blocking solution (for details, see Table 1Go). Afterward, sections were washed and incubated with a biotin-conjugated antirabbit or antimouse antibody (1:1000; Pierce) and/or an antiguinea pig antibody conjugated to Alexa Fluor 488 (1:2000; Molecular Probes, Eugene, OR) for 90 min. Sections were then incubated in streptavidin-Alexa Fluor 633 conjugate (1:2000; Molecular Probes) for 1 h. After the addition of the Alexa fluorochromes, all incubations were performed in the dark. The resulting signal was visualized by using a confocal microscope (Leica, Madrid, Spain). The primary antibody was omitted for negative control slides and resulted in very low background labeling (data not shown).

To determine the approximate number of double-labeled cells, images were captured using a 63x objective and stored. In each field, the number of immunopositive prolactin cells and those positive for prolactin plus caspase-8 or prolactin plus XIAP were determined. Six sections per animal were analyzed and approximately four fields per section. The mean percentage of double-labeled lactotrophs was then determined.

Statistics
All results are reported as mean ± SEM. All Western blot results are normalized to actin levels in the same sample and then to mean control levels in each assay (control is 100%). Student’s t test was used for comparison between groups, and significance was chosen as P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell death
Cell death, as determined by ELISA for histones, was significantly increased in the pituitary of diabetic rats compared with controls (controls, 100 ± 5 vs. 190 ± 5 diabetic rats; P < 0.001).

Bcl-2 family
Mean protein concentrations of the antiapoptotic Bcl-2 family members Bcl-2{alpha} and Bcl-XL were significantly higher in diabetic animals compared with control animals (Table 2Go). Mean levels of Bax, a proapoptotic member of this family, were not significantly different between the two groups. Mean levels of BAD, another proapoptotic protein, were slightly but significantly lower in the pituitary of diabetic rats compared with controls. Furthermore, the phosphorylated or inactivated form of this protein was higher in the diabetic pituitaries compared with controls (Table 2Go).


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  		TABLE 2. Relative protein levels of members of the Bcl-2 superfamily in the pituitaries of diabetic and control rats

 
Caspase activation in the anterior pituitary
Concentrations of both the intact (45 kDa) and fragmented forms of caspase 9 (35 kDa) were significantly higher in diabetic rats compared with controls (P < 0.01 and P < 0.001, respectively) (Fig. 1AGo).



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  		FIG. 1. A, The 45- and 35-kDa forms of caspase 9 were higher (P < 0.05 and P < 0.002, respectively) in pituitaries of diabetic rats compared with control rats. B, Levels of the inactive form of caspase 3 (32 kDa) were higher (P < 0.02) in the pituitaries of diabetic rats. However, the activated form (17 kDa) of caspase 3 was higher (P < 0.001) in control compared with diabetic rat pituitary glands. C, Procaspase 6 (34 kDa) levels were higher (P < 0.001) in the pituitaries of diabetic rats, whereas levels of the cleaved form (20 kDa) of caspase 6 were not significantly different. Data are represented as mean ± SE and are normalized to control results for each experiment. *, Significantly different from control group. n = 5.

 
Although the concentration of the intact form of caspase 3 (32 kDa) was higher in pituitaries of diabetic rats (P < 0.01), levels of the fragmented form (17 kDa) were significantly lower (P < 0.001) (Fig. 1BGo).

The concentration of the proform of caspase 6 was 3-fold higher in diabetic rats compared with controls (P < 0.001) (Fig. 1CGo); however, there was no significant difference between these two groups in the concentration of cleaved caspase 6.

Levels of procaspase 7 were 4-fold higher (P < 0.001) (Fig. 2AGo) in the pituitaries of diabetic rats, whereas concentrations of the fragmented form were significantly lower compared with controls (P < 0.01) (Fig. 2BGo).



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  		FIG. 2. A, Procaspase 7 levels were higher (P < 0.001) in pituitaries of diabetic rats compared with control rats. B, The cleaved form of caspase 7 was significantly lower (P < 0.03) in diabetic rats compared with control rats. C, Levels of both procaspase 8 (P < 0.002) and activated caspase 8 (P < 0.005) were higher in pituitaries of diabetic rats compared with controls rats. Data are represented as mean ± SE and are normalized to control results for each experiment. *, Significantly different from control group. n = 5. For caspase 7, distinct antibodies were used to detect the proform and the cleaved from, whereas the same antibody detected the two forms of caspase 8.

 
Both the proform (P < 0.01) and the fragmented form (P < 0.01) of caspase 8 were significantly higher in pituitaries of diabetic rats (Fig. 2CGo).

Apoptosis inhibitors
No significant difference in the mean concentration of the protein FLIP was found between control and diabetic pituitaries (control, 100.0 ± 3.8; diabetic, 96.5 ± 9.5).

The concentration of the apoptosis inhibitor XIAP was significantly higher in the pituitaries of diabetic rats compared with controls (P < 0.05) (Fig. 3Go). In addition, the fragmented form (30 kDa) was only detected in the pituitaries of diabetic rats.



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  		FIG. 3. Mean concentrations of XIAP were higher in diabetic rat pituitaries compared with controls (P < 0.05). In addition, the cleaved form (30 kDa) was only detected in pituitaries of diabetic rats. *, Significantly different from control group. n = 6.

 
NF{kappa}B and p53
As the liberation of inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}) by phosphorylation activates NF{kappa}B (39), levels of this protein were used to determine the activation state of NF{kappa}B. As can be seen in Figure 4AGo, concentrations of phosphorylated I{kappa}ß{alpha} (pI{kappa}b{alpha}) were 4-fold higher in the pituitaries of diabetic rats compared with controls (P < 0.001).



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  		FIG. 4. A, Mean concentrations of pI{kappa}B{alpha} were significantly higher in pituitaries of diabetic rats compared with controls (n = 3; P < 0.001). B, Mean concentrations of p53 were significantly higher in pituitaries of diabetic rats compared with controls (n = 3; P < 0.001). *, Significantly different from control group.

 
Concentrations of the tumor suppressor protein p53 were significantly increased in the pituitaries of diabetic rats compared with control rats (P < 0.001) (Fig. 4BGo).

Immunohistochemistry study of lactotrophs
Cells immunopositive for caspase 8 were found in the pituitaries from both control and diabetic rats (Fig. 5Go, B and E, respectively). Caspase 8 was found to colocalize with prolactin in the pituitaries of both control [10 ± 5% of prolactin-positive cells (Fig. 5CGo)] and diabetic [38 ± 7% of prolactin-positive cells (Fig. 5FGo)] rats.



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  		FIG. 5. Immunohistochemistry for prolactin (PRL; green) and caspase 8 (CASP-8; red) in the pituitary of a control (A–C) and a diabetic (D–F) rat. Prolactin colocalized with caspase 8 in cells of both diabetic and control pituitaries (filled arrows), although the percentage of double-labeled cells was higher in diabetic rats. In both groups, there were caspase 8-positive cells that did not express prolactin (open arrows). In F, the inset shows in increased magnification of the dashed box. Immunohistochemistry for prolactin (green) and XIAP (red) in the pituitary of a control (G–I) and diabetic (J–L) rat. Double-labeled cells could be found in the pituitary of control rats (filled arrows) but not in the diabetic rat pituitary. Cells immunopositive for XIAP (open arrows), but not prolactin, were found in both control and diabetic rat pituitaries; however, the number of XIAP-positive cells was greater in the diabetic rat. In L, the inset shows in increased magnification of the dashed box. Immunohistochemistry for prolactin (PRL; green) and cleaved caspase 3 (CASP-3; red) in the pituitary of a diabetic rat (M–O). Although overall levels of cleaved caspase 3 were decreased in the pituitaries of diabetic rats, cleaved caspase 3 colocalized with prolactin (O; filled arrow).

 
In the pituitaries of both control and diabetic rats, cells immunopositive for XIAP were found (Fig. 5Go, H and K, respectively). In control animals, approximately 12 ± 6% of prolactin-positive cells colocalized XIAP (Fig. 5IGo), whereas in diabetic rats less than 1% of prolactin-positive cells also expressed XIAP (Fig. 5LGo).

Although activation of caspase 3 is decreased in the pituitaries of diabetic rats, cleaved caspase 3 is detected in lactotrophs of diabetic rats (Fig. 5Go, M–O).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The anterior pituitary gland continues to undergo cell death and proliferation throughout life (40), with this process being modulated by the existing hormonal environment or physiological state (13, 40, 41). We have reported previously that there is an increase in cell death in the pituitary of diabetic rats, with lactotrophs being at least one of the affected cell types (13). However, the cause and mechanisms involved in this process remain unknown. Indeed, very little is known about the intracellular mechanisms involved in pituitary cell turnover in the normal animal, let alone in pathological situations. Here we report that caspases 3, 6, 7, 8, and 9 are all expressed and activated to various degrees in the pituitaries of male rats, suggesting that these proteases may be involved in the basal cell turnover that occurs normally in this gland.

Anterior pituitary concentrations of both the 45 and 35 kDa forms of caspase 9 were significantly increased in diabetic rats compared with controls. One mechanism by which caspase 9 can be activated is by cytochrome c release from mitochondria (42). Mitochondria-associated members of the Bcl-2 family of apoptotic proteins regulate cytochrome c release from this organelle and, hence, caspase 9 activation (16, 17, 42). Indeed, the balance and interaction between the proapoptotic and antiapoptotic members of this family are fundamental in determining the final effect on mitochondria and whether this cell death pathway is activated (16, 17). Because changes in Bcl-2 and Bax have been implicated in cell death in the pituitary under both physiological and pathophysiological conditions (35), we suspected that they could play a role in diabetes-induced pituitary cell death. However, we found levels of the antiapoptotic proteins Bcl-2{alpha} and Bcl-XL to be increased in the pituitaries of diabetic rats, whereas no significant change in Bax, a proapoptotic protein, was found. Levels of BAD, which is proapoptotic in its nonphosphorylated form, are decreased, and levels of the inactivated phosphorylated form of BAD are increased in diabetic rats. Because these molecules either homodimerize or heterodimerize with molecules of opposing function, the net influence on apoptosis depends on the ratio between proapoptotic and antiapoptotic members. Hence, the changes in the Bcl-2 family of proteins reported here are balanced toward cell survival and are most likely not involved with the observed increase in cell death in the pituitary gland of diabetic rats.

We previously reported no significant change in Bcl-2 levels in the pituitary of diabetic rats (13). The experiments reported here were performed using an antibody for Bcl-2{alpha} that recognizes amino acids 61–76 of this protein, whereas in the previous experiments, the antibody was directed toward amino acids N1–19, representing part of the BH4 domain of the Bcl-2 proteins. Both antibodies were specific and recognized a single 25–26 kDa protein. The difference in the results could be explained by the possibility that these antibodies recognize different isoforms of Bcl-2 proteins. However, in neither case were Bcl-2 levels decreased, suggesting that changes in this protein are most likely not involved in the increased cell death in the pituitaries of diabetic rats.

Caspase 9 is the initiator of the mitochondrial pathway or intrinsic cell-death pathway and is activated via association of its 45 kDa form with Apaf-1 and cytochrome c (17, 42). Binding of procaspase-9 to Apaf-1 leads to autolytic cleavage of procaspase-9 to generate its 35 kDa form and activation of effector caspases, including caspases 3, 6, and 7, which then execute the cell-death sentence (17). However, although the concentrations of procaspases 3, 6, and 7 are significantly higher in the pituitaries of diabetic rats, the levels of the fragmented forms of caspases 3 and 7 are lower, with no change in cleaved caspase 6 levels. Hence, the increase in cell death in the pituitaries of diabetic rats is not correlated with an increase in activation of the downstream members of the intrinsic cell death pathway but in fact a decrease.

An increase in caspase 9 associated with a decrease or no change in the activation of caspases 3, 6, and 7 suggests that the intracellular cascade may be blocked downstream of activation of the initiator caspase. Of the currently described members of the IAP family, XIAP is reported to be the most potent apoptosis inhibitor, suppressing cell death by BIR domain-specific inhibition (28, 43). Although XIAP may not inhibit the cleavage of caspase 9 in all tissues, it can bind to this protease and block its activity (43), as well as inhibit the activated forms of caspases 3 and 7 (28, 43). Hence, although both forms of caspase 9 are increased in the pituitary of diabetic rats, its activation of downstream caspases could be inhibited by increased expression and fragmentation of XIAP. However, this increase in XIAP does not occur in all pituitary cell types. Although in the diabetic rat the number of XIAP-immunoreactive cells increased, the number of lactotrophs expressing this caspase inhibitor is reduced. This suggests that the increase in XIAP expression is cell type selective and lactotrophs are not protected by this mechanism.

The higher level of cell death in the pituitary of diabetic rats could be explained, at least in part, by the increase in activation of caspase 8. Indeed, the number of lactotrophs expressing caspase 8 is higher in the diabetic rat, suggesting that it may be involved in the increase in death of this cell population. Furthermore, we found no change in the concentration of FLIP, a known inhibitor of this caspase (29). The extrinsic cell death pathway is activated via cell membrane receptors in response to various stimuli, including TNF{alpha} (17), as well as via caspase 9 (44). In turn, caspase 8 may then activate one of the effector caspases (17), although a direct path for caspase 8-elicited apoptosis has also been described (45). Because TNF{alpha} is elevated in the serum of diabetic patients and streptozotocin-induced diabetic rats (46, 47) and has been implicated in apoptosis in diabetes (48), as well as in pituitary cells (49), it could possibly explain the observations reported here. Furthermore, the increased activation of both caspase 8 and p53, which are also modulated by TNF{alpha}, could be interrelated and involved in lactotroph cell death.

The transcription factor p53 is a key protein in tumor suppression, modulating genes involved in apoptosis, cell cycle, DNA repair, and cell fate to facilitate repair of damaged cells or elimination of severely damaged cells (24, 25). In various paradigms, caspase 8 activation has been implicated in p53-mediated apoptosis (22, 23). In addition, transcription of the caspase 8 gene can be induced by p53 (50). Interestingly, hyperglycemia has been shown to activate p53, resulting in cell death in other tissues (26).

The dopamine agonist bromocryptine also stimulates p53 expression in the pituitary, and this has been correlated with increased apoptosis of lactotrophs in male rats (38, 51). Indeed, it has been suggested that changes in prolactin secretion in diabetic rats are due to increased hypothalamic release of dopamine (52). Dopamine not only inhibits prolactin release but decreases the proliferation and stimulates apoptosis of lactotrophs (51, 53). Hence, the increased expression of p53 and lactotroph death seen in diabetic rats could be due to an increase in hypothalamic dopamine release. However, in our studies, Bcl-2 was not inhibited, as occurs with bromocryptine at least in pituitary cell lines (38), and it remains to be determined whether dopamine is capable of activating caspase 8 in lactotrophs.

Another protein that is associated with the extrinsic cell-death pathway is NF{kappa}B, which can be activated by proapoptotic signals, including activation of death receptors (18, 39). However, this protein has a duel role in that it can also participate in the activation of IAPs (27, 30) and other survival signals, such as Bcl-2, Bcl-xL, and FLIP (for review, see Ref.31). Hence, because activation of this protein is increased in the pituitaries of diabetic rats, it could be involved in both the execution of the extrinsic cell-death pathway via caspase 8, as well as stimulation of XIAP, Bcl-2, and Bcl-xL and inhibition of the intrinsic pathway of cell death.

The percentage of cells undergoing cell death in the normal adult male pituitary gland is low. In adult males, it has been estimated at approximately 0.03%, with mitotic figures representing approximately 0.7% (41) or 50–100 cells/mm3 (54) or 190 cells/mm3 (1.7%) in pubertal males (40). The relatively high percentage of cells that express activated effector caspases in the normal pituitary leads one to speculate that these caspases may be involved in processes other than cell death or that they may be chronically inhibited so that apoptosis does not occur at a rapid rate. Indeed, caspases have been implicated in cellular processes other than apoptosis, including proliferation, differentiation, and even receptor internalization (55, 56).

One caveat that must be taken into consideration in these studies is that measurement of overall changes in activation of proteins in the pituitary does not necessarily reflect what occurs in each cell type. However, these studies of the whole anterior pituitary, undertaken to gain insight into what mechanisms may be activated at the level of the lactotroph, yielded surprising and interesting results. It is clear that poorly controlled diabetes results in a complex shift in the activation of specific intracellular mechanisms in the anterior pituitary, most likely corresponding to distinct processes in different cell types. Because each pituitary cell type responds to different stimuli for survival and function, it is possible that they also use specific mechanisms for proliferation, differentiation, and death. Indeed, we have not found increased death of corticotrophs or thyrotrophs (our unpublished observation), suggesting that they may be less susceptible to diabetes-induced cell death and are possibly being protected by the increases in XIAP or antiapoptotic members of the Bcl-2 family reported here. Although the number of somatotrophs decreases in diabetic rats, this change was not significant at 2 months of diabetes, and we did not detect colocalization of growth hormone in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells (13). It is possible that this loss of somatotrophs occurs earlier in the diabetic process or is more gradual, making it more difficult to detect at any one moment. Indeed, after 2 months of diabetes, the majority of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled cells colocalized with prolactin (13), suggesting that, at least at this time point, lactotrophs are the most affected cell type.

Although activation of the effector caspases 3 and 7 was decreased in the diabetic pituitary, there was still fragmentation of these proteins, suggesting that in some cells or cell types these caspases are activated. Indeed, cleaved caspase 3 was detected in lactotrophs of diabetic rats. Hence, it is possible that blockage of effector caspase activation could be occurring in other cell types and not lactotrophs, which is congruent with the lack of expression of XIAP in this cell type.

It is clear that additional investigation is necessary to determine what occurs in each pituitary cell type during poorly controlled diabetes. However, here we demonstrate that increased cell death in the anterior pituitary is associated with increased levels and activation of caspases 9 and 8, as well as the cell death-associated transcription factor p53. Furthermore, lactotrophs, which undergo increased cell death in poorly controlled diabetes, have increased caspase 8 expression, supporting the implication of this in this process. The fact that activation of the effector caspases 3, 6, and 7 is decreased suggests that mechanisms such as XIAP may be activated to hinder diabetes-evoked cell death in this gland. This inhibition could be cell specific, because lactotrophs of diabetic rats do not express XIAP and continue to express cleaved caspase 3. Together, these results indicate that, during poorly controlled diabetes, normal mechanisms of pituitary cell death may be shifted or down-regulated and protective mechanisms increased such that some cell types are less likely to suffer changes in cell number, whereas others such as lactotrophs are more susceptible to the noxious process.


   Footnotes
 
This work was funded by grants from Fondo de Investigación Sanitaria (Grant PI04/0817), Ministerio de Educación (Grant SAF2002-03324), Fundación de Investigación Médica Mutua Madrileña, and Fundación de Endocrinología y Nutrición.

First Published Online June 23, 2005

Abbreviations: IAP, Inhibitor of apoptosis protein; I{kappa}B{alpha}, inhibitor of {kappa}B{alpha}; NF{kappa}B, nuclear factor {kappa}B; TBS, Tris-buffered saline; XIAP, X-chromosome-linked inhibitor of apoptosis protein.

Received April 29, 2005.

Accepted for publication June 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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