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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:1873-1878, August 2005

Nutrient-Gene Interactions

Low-Protein Diets Reduce PKA{alpha} Expression in Islets from Pregnant Rats1,2

Marciane Milanski, Vanessa Cristina Arantes*, Fabiano Ferreira{dagger}, Marise Auxiliadora de Barros Reis*, Everardo Magalhães Carneiro**, Antonio Carlos Boschero**, Carla Beatriz Collares-Buzato{ddagger} and Márcia Queiroz Latorraca*,3

Secretaria de Estado de Saúde, Mato Grosso, MT, Brazil; * Departamento de Alimentos e Nutrição, Faculdade de Nutrição, Universidade Federal de Mato Grosso, Cuiabá, MT, Brazil; {dagger} Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Recife, PE, Brazil; ** Departamento de Fisiologia e Biofísica and {ddagger} Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil

3To whom correspondence should be addressed. E-mail: mqlator{at}terra.com.br.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We investigated the effect of protein restriction on insulin secretion and the expression of protein kinase (PK)A{alpha} and PKC{alpha} in islets from control and pregnant rats. Adult control nonpregnant (CN) and control pregnant (CP) rats were fed a normal-protein diet (17%), whereas low-protein nonpregnant (LPN) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) for 15 d. In the presence of 2.8 and 8.3 mmol glucose/L, insulin secretion by islets of CP rats was higher than that by islets of CN rats. Compared with the CN groups, insulin secretion by islets of LPN rats was lower with 8.3 but not with 2.8 mmol glucose/L. The insulin secretion by islets of LPP rats was higher than by LPN rats at both glucose concentrations. IBMX (1 mmol/L), a phosphodiesterase inhibitor, increased insulin secretion by islets from pregnant rats, and this effect was greater in islets of CP rats than in LPP rats. Forskolin (0.01–100 µmol/L), a stimulator of adenylyl cyclase, increased insulin secretion only in islets of CN and CP rats, with a higher 50% effective concentration in islets of CP rats compared with CN rats. The insulin secretion induced by phorbol 12-myristate 13-acetate (a stimulator of PKC) was higher in islets of LPN and LPP rats than in the respective controls, especially at 8.3 mmol glucose/L. PKA{alpha}, but not PKC{alpha}, expression was lower in islets of rats fed low protein than in the controls, regardless of the physiological status of the rats. All endocrine cells of the islets, including ß-cells, expressed the PKA{alpha} isoform. The cytoplasmic distribution of this enzyme in ß-cells was not modified by pregnancy and/or protein restriction. In conclusion, our results indicate that the response of islets from rats fed low protein during pregnancy is similar to that of control rats, at least for physiologic glucose concentration. However, the decreased response to IBMX and forskolin indicates decreased production and/or sensitivity to cAMP; this was associated with a decrease in PKA expression, which may result in lower PKA activity.

KEY WORDS: • low-protein diet • insulin secretion • PKA{alpha} • PKC{alpha} • pregnancy • rats

Malnutrition and pregnancy are associated with functional changes in pancreatic islets. A low-protein diet impairs insulin secretion in response to glucose (1–3) as a consequence of several alterations, including a smaller size and/or cell volume of ß-cells (4); an inappropriate recognition of glucose as a stimulus due to low glucoreceptor expression and/or decreased metabolism of glucose (5); reduced mitochondrial substrate oxidation caused by impaired activity of the ß-cell mitochondrial glycerophosphate dehydrogenase, possibly associated with other enzymatic anomalies (6); a diminished capacity of glucose to increase the Ca2+ uptake and/or to reduce Ca2+ efflux from ß-cells (1,7); an alteration in the cAMP and phospholipase C (PLC)4 pathways; and diminished amounts of protein kinase (PK)C{alpha} and PKA{alpha} (2,3). The reduced amount of these kinases was attributed to hormonal changes (8,9) during undernutrition (10) or to a direct effect of a low-protein diet on the expression of these proteins (2,3).

In contrast, the adaptation of islets to pregnancy involves increased glucose-stimulated insulin secretion with a reduction in the threshold for stimulation (11). These adaptative changes are controlled by lactogenic hormones (placental lactogen and/or prolactin) that increase ß-cell proliferation and islet volume (12), insulin synthesis (13), and gap-junctional coupling among ß-cells (14); enhance glucose metabolism through greater glucose transporter (GLUT) 2 expression (15) and glucokinase activity (16); and increase the cAMP and PLC metabolism (17,18).

Diacylglycerol (DAG), derived from hydrolysis of phospholipids by PLC, and cAMP enhance insulin release by increasing the sensitivity to Ca2+ by the secretory machinery (19,20). DAG and cAMP activate PKC and PKA, respectively, which in turn phosphorylate proteins that act directly on intracellular Ca2+ levels (21,22). In addition, PKA also increases the integrated Ca2+ current in ß-cells (21).

We observed recently that islets from pregnant rats fed a low-protein diet exhibited higher sensitivity to glucose than islets from nonpregnant rats fed a normal-protein diet (data not shown). Because cAMP/PKA and PLC/PKC systems are involved in the reduction of stimulatory threshold glucose concentration and in the potentiation of insulin secretion during pregnancy (11), we investigated the effect of protein restriction on insulin secretion as well as the expression of PKA{alpha} and PKC{alpha} in pancreatic islets from pregnant rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diet. The animal experiments were approved by the institutional Committee for Ethics in Animal Experimentation (Instituto de Biologia, Universidade Estadual de Campinas). Virgin female Wistar rats (90 d old) were obtained from the university’s breeding colony. Mating was achieved by housing males with females overnight, and pregnancy was confirmed by the examination of vaginal smears for the presence of sperm. Pregnant and nonpregnant females were each randomly assigned to 2 diet groups, control and low protein. The control nonpregnant (CN) and pregnant (CP) groups were fed a 17% protein diet, and the low-protein nonpregnant (LPN) and pregnant (LPP) groups were fed a 6% protein diet from d 1 to 15 of pregnancy. The diets were isocaloric, as described in Table 1. During the experimental period, rats had free access to food and water and were housed at 22°C with a 12-h light:dark cycle. At the end of this experimental period, the rats were weighed and killed by decapitation. Blood samples were collected and allowed to clot; sera were stored at –20°C for the subsequent measurement of insulin. Serum glucose (23), serum albumin (24), and serum total protein (25) levels were determined immediately after decapitation.


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  		TABLE 1 Composition of the control and low-protein diets1

 
    Islet isolation and insulin secretion. The pancreas was removed from rats and digested with collagenase (Boehringer Mannheim), as described elsewhere (26). In the first series of experiments, groups of 5 islets were incubated for 90 min at 37°C in Krebs-bicarbonate buffer containing glucose (2.8 and 8.3 mmol/L) and equilibrated with a mixture of 95% O2 and 5% CO2 to give a pH of 7.4. The incubation medium contained (mmol/L): NaCl, 115; KCl, 5; CaCl2, 2.56; MgCl2, 1; NaHCO3, 24; and bovine serum albumin (3 g/L; Sigma). In the second series of experiments, insulin secretion was measured in response to: 1) glucose (2.8 mmol/L) in the absence and presence of isobutylmethylxanthine (IBMX; 1 mmol/L; Sigma), 2) glucose (2.8 mmol/L) associated with increasing concentrations of forskolin (0.01–100 µmol/L), and 3) glucose (2.8 or 8.3 mmol/L) in the absence or presence of phorbol 12-myristate 13-acetate (PMA; 100 nmol/L; Sigma). The insulin released was measured by RIA using rat insulin as the standard. The forskolin concentration that produced a response that was 50% of the maximum (50% effective concentration; EC50) was expressed as the mean negative logarithm (pD2).

    Immunohistochemistry. Pancreata from all experimental groups were dissected, fixed in 10% formaldehyde (v:v, in 100 mmol/L PBS) for at least 12 h and embedded in paraffin. Pairs of serial sections (5 µm) of paraffin-embedded tissue were placed on silane-treated glass slides, dewaxed, hydrated with 50 mmol/L Trizma-buffered saline (TBS), and incubated for 30 min with undiluted hydrogen peroxide solution (Sigma) to block endogenous peroxidase. After a 1-h incubation with TBS containing 5% dry skimmed milk and 0.1% Tween 20, each pair of sections was incubated overnight with primary antibodies for PKA{alpha} (Santa Cruz Biotechnology) or insulin (Dako) (diluted 1:50 in TBS containing 3% dry skimmed milk). The PKA{alpha} tissue distribution was detected using the rabbit biotin-avidin-peroxidase ImmunoCruz Staining System. For the insulin immunoreaction, the sections were incubated for 90 min with anti-guinea pig IgG conjugated with peroxidase (diluted 1:1500 in TBS containing 1% dry skimmed milk) and developed with 3,3'diaminobenzidine (Sigma). All sections were counterstained with Ehrlich’s hematoxylin and photographed using a digital camera (CoolSNAP-Pro, Media Cybernetics) coupled to a Nikon Eclipse E800 microscope. The negative controls for the immunoreaction were obtained by omitting the primary antibodies from the incubation.

    Western blotting. After isolation, groups of islets were pelleted by centrifugation (15,000 x g) and then resuspended in 50–100 µL of homogenization buffer containing protease inhibitors (27,28). The islets were sonicated and the total protein content was determined (29). Samples containing 70 µg of protein from each experimental group were incubated for 5 min at 80°C with 4X concentrated Laemmli sample buffer (1 mmol sodium phosphate/L, pH 7.8; 0.1% bromophenol blue; 50% glycerol; 10% SDS; 2% mercaptoethanol) (4:1, v:v) and assayed on 8% polyacrylamide gels at 120 V for 30 min. Electrotransfer of proteins to nitrocellulose membranes (Bio-Rad) was performed for 1 h at 120 V in buffer containing methanol and SDS. After checking the transfer efficiency by Ponceau S staining, the membranes were blocked with 5% skimmed milk in Tween-Tris-buffered saline (TTBS) (10 mmol Tris/L, 150 mmol NaCl/L, 0.5% Tween 20) overnight at 4°C. PKC{alpha} and PKA{alpha} were detected in the membranes after a 2-h incubation at room temperature with anti-PKA{alpha} rabbit polyclonal IgG and anti-cPKC{alpha} mouse monoclonal IgG (Santa Cruz; diluted 1:500 in TTBS containing 3% dry skimmed milk). Enhanced chemiluminescence (SuperSignal West Pico, Pierce) after incubation with a horseradish peroxidase-conjugated secondary antibody was used for detection.

    Statistical analysis. The results were expressed as the means ± SEM for the number of rats (n) indicated. For islets, n refers to the number of experiments performed. Bartlett’s test for the homogeneity of variances was used initially to check the fit of the data to the parametric ANOVA assumptions. To correct for variance heterogeneity or normality (30), serum insulin and insulin secretion in response to glucose were log-transformed, and EC50 was ln-transformed. Serum metabolites, body weight, insulin secretion in response to glucose (2.8 and 8.3 mmol/L), EC50, and expression of PKC{alpha} and PKA{alpha} were analyzed by 2-way ANOVA (nutritional status and physiological status). Insulin secretion stimulated by glucose (2.8 mmol/L) plus IBMX and glucose (2.8 or 8.3 mmol/L) plus PMA was evaluated by 3-way ANOVA (nutritional status, physiological status, and absence or presence of IBMX or PMA). When necessary, these analyses were followed by Tukey’s honestly significant difference test to determine the significance of individual differences. The level of significance was set at P < 0.05. The data were analyzed using the Statistic Software package (Statsoft).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Characteristics of the rats. Regardless of nutritional status, pregnant rats had higher body weights and lower serum glucose concentrations than nonpregnant rats. At the end of the experimental period, serum total protein, albumin, and insulin levels did not differ among the groups (Table 2).


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  		TABLE 2 Final body weight and serum total protein, albumin, insulin, and glucose concentrations in pregnant and nonpregnant rats that consumed control or low-protein diets1

 
    Insulin secretion. Insulin secretion at the low glucose concentration (2.8 mmol/L) was significantly higher for islets from CP and LPP rats compared with their respective CN and LPN controls (Fig. 1A). With high glucose (8.3 mmol/L), the insulin secretion by islets from LPN rats was lower than for CN rats (P < 0.05). Pregnancy increased the secretory response in both groups. Thus, islets from LPP and CP rats had similar insulin secretion (Fig. 1B).



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  		FIGURE 1 Glucose stimulation of insulin secretion by islets from CN, CP, LPN, and LPP rats. Groups of 5 islets were incubated for 90 min in Krebs-bicarbonate medium containing 2.8 (A) and 8.3 (B) mmol glucose/L. The columns represent the cumulative 90-min insulin secretion and are the means ± SEM of 5–9 independent experiments. Means without a common letter differ, P < 0.05.

 
At nonstimulatory concentrations of glucose (2.8 mmol/L) IBMX did not alter the insulin secretion by islets from CN rats, but increased the insulin release by islets of LPN rats (P < 0.05). In islets from pregnant rats, the potentiation of insulin secretion by IBMX was lower in the LPP group than in the CP group (Fig. 2).



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  		FIGURE 2 IBMX-induced insulin secretion by islets from CN, CP, LPN, and LPP rats in medium containing 2.8 mmol glucose/L. The columns represent the cumulative 90-min insulin secretion and are the means ± SEM of 5–6 independent experiments. Means without a common letter differ, P < 0.05.

 
At all forskolin concentrations (0.01–100 µmol/L), the insulin secretion by islets from the low-protein groups was lower than that from the control groups, regardless of the physiological status. In islets from LPN and LPP rats, the increase in insulin secretion, even at high concentrations of forskolin, was only marginal. In islets from CN rats, a significant increase in insulin secretion was observed only at higher concentrations of forskolin (1–100 µmol/L), whereas in islets from CP rats, forskolin increased insulin secretion at a concentration as low as 0.1 µmol/L. The half-maximal release concentration of forskolin was affected by nutritional (P < 0.001) and physiological (P < 0.001) status, as well as by the interaction between them (P < 0.001). Thus, pregnancy reduced the EC50 value in the control and low-protein groups. However, the EC50 for the LPP group was higher than that for the CP group (LPP = 0.026 ± 0.004 µmol/L, n = 4; LPN = 0.461 ± 0.210 µmol/L, n = 4; CP = 0.002 ± 0.001 µmol/L, n = 4; CN = 0.240 ± 0.022 µmol/L, n = 4).

At 2.8 and 8.3 mmol glucose/L, PMA increased insulin secretion more markedly in islets from LPP rats compared with islets from CP rats (Fig. 3A and B). At 2.8 mmol glucose/L (Fig. 3A), insulin secretion induced by PMA was 329 and 700% higher in islets from the LPN and LPP groups, respectively, than from their respective controls (LPN and LPP without PMA). In islets from CN and CP rats, PMA increased insulin secretion by 526 and 315%, respectively (P < 0.05). At 8.3 mmol glucose/L, (Fig. 3B) the increase in insulin secretion provoked by PMA in islets from LPN and CN rats was 369 and 549%, respectively (P < 0.05). In islets from LPP and CP rats, the increase was 436 and 259%, respectively, relative to the respective controls (no PMA) (P < 0.05).



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  		FIGURE 3 PMA stimulation of insulin secretion in islets from CN, CP, LPN, and LPP rats in medium containing 2.8 (A) and 8.3 (B) mmol glucose/L. The columns represent the cumulative 90-min insulin secretion and are the means ± SEM of 5–7 independent experiments. Means without a common letter differ, P < 0.05.

 
The PKA{alpha} expression in islets from rats fed a low-protein diet (LPN and LPP) was 33% lower than in control rats (CN and CP). A significant effect of nutritional (P < 0.01) but not of physiological (P = 0.856) status was observed. However, there was no interaction between nutritional and physiological status (P = 0.377) (Fig. 4A). The PKC{alpha} content of islets did not differ in any of the groups of rats (Fig. 4B); hence, there was no effect of nutritional (P = 0.067) or physiological (P = 0.269) status, or any interaction between them (P = 0.070).



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  		FIGURE 4 Western blot analysis of PKA{alpha} subunit (A) and PKC{alpha} content (B) in islets from CN, CP, LPN, and LPP rats. The columns are the means ± SEM of 4 independent experiments. Means without a common letter differ, P < 0.05.

 
Immunohistochemistry revealed a greater expression of PKA{alpha} in islet cells than in the exocrine parenchyma; this enzyme was also detected in ductal cells and nerves (data not shown). Dual immunostaining for PKA{alpha} and insulin in serial pancreatic sections showed that this kinase was expressed by ß-cells and non-ß-cells. The cytoplasmic distribution of PKA{alpha} in islet cells was not modified by pregnancy and/or protein deprivation (Fig. 5).



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  		FIGURE 5 PKA{alpha} distribution in endocrine pancreas of CN (a), CP (b), LPN (c), and LPP (d) rats. Immunolocalization of PKA{alpha} (a–d) and insulin (e–h) was done in pairs of serial sections of pancreata of all experimental groups using a standard biotin-avidin-peroxidase method. Note that the PKA{alpha} isoform was immunodetected within all endocrine cells of the islets, including the insulin-secreting ß-cells. These cells presented a cytoplasmic distribution of this enzyme that was not modified by pregnancy and/or protein deprivation. Scale bar = µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Weight loss and hypoalbuminemia are frequently observed in rats fed a low-protein diet for a long time (31). After the short period of low-protein diet consumption (15 d) used here, these classic alterations were not yet present; however, there was a significant alteration in the capacity of the islets to secrete insulin in response to glucose. When islets from LPN rats were challenged with a physiological concentration of glucose (8.3 mmol/L), insulin secretion was lower than that for islets from CN rats.

During pregnancy, insulin production and the sensitivity of the endocrine pancreas to glucose increase to allow an appropriate response to increased peripheral resistance to insulin (32,33). These observations were confirmed here by comparing the insulin secretion of islets from the CP and CN groups at both glucose concentrations. In rats fed the low-protein diet, pregnancy improved the responsiveness of ß-cells to a physiological concentration of glucose (Fig. 1A and B). Similarly, in a previous study (1) we verified that the dose-response curve to glucose in islets from LPN rats was shifted to the right compared with islets from CN rats. Pregnancy increased glucose sensitivity in both nutritional states with the insulin secretion curves to glucose displaced to the left compared with those observed in islets from nonpregnant rats. The insulin content of islets from LPP rats did not differ from that of islets from CP rats (unpublished data). Changes in the glucokinase, hexokinase, and GLUT2 levels, as well as in glucose metabolism, are central to the adaptative process in pregnancy (16), and it is well accepted that placental lactogens play a crucial role in this process.

Because the generation of cAMP is important for reducing the glucose stimulatory threshold of ß-cells (11), we examined the insulin secretory response to 2.8 mmol glucose/L in the presence of IBMX, a phosphodiesterase inhibitor, and in the presence of increasing concentrations of forskolin, an adenylyl cyclase activator that increases the intracellular cAMP levels in ß-cells. IBMX induced a less potent secretory response in islets from LPP rats than in those from CP rats. In addition, the EC50 showed that the potency of forskolin was significantly reduced in islets from rats fed a low-protein diet compared with those fed a normal diet. Pregnancy decreased the EC50 in both nutritional states, but to a lesser degree in protein-deprived rats than in control rats.

In the absence of glucose or at low concentrations of this sugar, an increase in cAMP content has little if any effect on insulin secretion (34). In normal rats, cAMP increases the insulin secretion just above the threshold for glucose (~5.6 mmol/L), which is also the threshold for the stimulation of cAMP metabolism in ß-cell islets (35). In islets from pregnant rats, as well as in islets from prolactin-treated rats, the threshold for glucose-stimulated insulin secretion is reduced to ~3 mmol/L (11). This phenomenon could explain the higher sensitivity of islets from CP rats to lower concentrations of forskolin compared with islets from CN rats (0.01 vs. 1.0 µmol/L). Forskolin did not stimulate secretion in islets from protein-restricted rats, regardless of the physiological status, indicating that the cAMP/PKA system in islets from these rats is inoperative or is severely attenuated. A reduced generation of cAMP by islets from rats fed low protein may be a consequence of a lower rate of glucose metabolism and the generation of coupling factors, with a subsequent reduction in the intracellular Ca2+ concentration and in the levels of the Ca2+-calmodulin complex that ultimately stimulates adenylyl cyclase (36,37).

The reduced response to IBMX and forskolin by islets from protein-deprived rats could indicate a decrease in the amount of the {alpha}-catalytic subunit of PKA, the major mediator of the cAMP signal transduction pathway. Because a low-protein diet reduces PKA{alpha} expression in rat islets (3), we examined the participation of this kinase in the control of insulin secretion by islets from protein-deprived rats that were or were not pregnant. The PKA{alpha} isoform was detected in all endocrine cells of the islets, including the ß-cells. The cytoplasmic distribution of this enzyme in ß-cells was not modified by pregnancy and/or protein deprivation. However, immunoblotting showed that PKA{alpha} expression was significantly lower in islets from rats fed low protein than in islets from control rats, regardless of the physiological status.

There are 2 possible explanations for the decrease in PKA{alpha} levels in islets from rats fed a low-protein diet. First, diet could directly affect the expression of several genes and their encoded proteins, including key enzymes involved in the secretory process. Second, a low-protein diet could influence the neuronal-endocrine axis (10) and alter the regulation of the expression of PKA{alpha} by different hormones (8,9). Thus, the reduced insulin secretion in response to IBMX and forskolin that occurred in islets from protein-restricted rats may result from a reduced production of cAMP, secondary to a diminished rate of glucose metabolism and a decrease in PKA{alpha} levels.

A low-protein diet also reduces the expression of PKC in pancreatic islets (2) and its activity in several tissues (38). However, as presented here, protein deprivation for a short period of time did not alter PKC{alpha} expression. Indeed, islets from pregnant protein-deprived rats secreted proportionally more insulin in response to PMA than did islets from control rats. Because the activation of PKC is involved in insulin secretion by rat islets in response to stimulatory concentrations of glucose (39), and because pregnancy activates the PLC/PKC pathway in the pancreas (40,41), the increased insulin secretion by islets from pregnant rats fed low protein compared with pregnant control rats, in the presence of PMA, may have occurred at the expense of increased PKC activity.

In conclusion, our results indicate that the response of islets from rats fed low protein during pregnancy is similar to that of islets from control rats, at least for the physiologic glucose concentration. However, the decreased response to IBMX and forskolin indicates diminished production and/or sensitivity of cAMP; this is associated with a decrease in PKA expression, which may result in lower PKA activity.


   ACKNOWLEDGMENTS
 
The authors thank L. D. Teixeira, C. R. Afonso, and M. B. Leonardo for technical assistance, and S. Hyslop and N. Conran for English editing.


   FOOTNOTES
 
1 Part of a dissertation presented by Marciane Milanski as partial fulfillment of the requirements for a master’s degree in health science at the Faculdade de Ciências Médicas, Universidade Federal de Mato Grosso. Back

2 Supported in part by the Brazilian foundations FAPESP (grant no. 02/13218-0), CNPq (grant no. 479138/2003-6), and FINEP/PRONEX (grant no. 134/97). Back

4 Abbreviations used: CN, control nonpregnant; CP, control pregnant; DAG, diacylglycerol; EC50, 50% effective concentration; GLUT, glucose transporter; IBMX, isobutylmethylxanthine; LPN, low-protein nonpregnant; LPP, low-protein pregnant; PK, protein kinase; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; TBS, Trizma-buffered saline; TTBS, Tween-Tris-buffered saline. Back

Manuscript received 11 January 2005. Initial review completed 11 February 2005. Revision accepted 13 May 2005.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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A. Chamson-Reig, S. M Thyssen, E. Arany, and D. J Hill
Altered pancreatic morphology in the offspring of pregnant rats given reduced dietary protein is time and gender specific.
J. Endocrinol., October 1, 2006; 191(1): 83 - 92.
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