Pituitary Cell Types Affected by Food Deprivation
The pilot studies done on the first 3 groups of rats (5 rats/group) showed a weight loss in the fasted rats of 30 gm over the 24 h period. Serum leptin in the fed group averaged 1.866 ± 0.467 ng/ml and that in the fasted group averaged 2.3 ± 0.7 ng/ml. There were no significant differences in serum leptin between the two groups.
The first objective of this study was to determine the effect of short term fasting on the pituitary cells themselves. illustrates the cell counts, showing that fasting caused declines in percentages of pituitary cells with GH (by 31%, p<0.01), prolactin (by 53% p<.001) and LH (by 50%, p<0.001). In contrast however, fasting increased percentages of corticotropes over 2X (p<0.001) from 10 to 21% of AP cells. The percentages of thyrotropes appeared unaffected by fasting. illustrate the changes in immunolabeled corticotropes, somatotropes and gonadotropes.
Effect of Food Deprivation on Each Pituitary Cell type
Focused Studies of Changes in Gonadotropes and Somatotropes
The studies then focused on other changes in gonadotropes and somatotropes, because they were affected most severely by the food deprivation. Detection of GH mRNA showed that somatotropes had not disappeared from the population. The counts showed a slight increase from 33 ± 2% to 40 ± 2% (SD, p<.001) of anterior pituitary cells. In contrast, cells with LH mRNA detected by in situ hybridization showed a 50% decline in expression from 14 ± 2% to 7 ± 3% (±SD, p<.001).
Tests of gonadotrope and somatotrope function also included the detection of changes in binding sites for GnRH or GHRH with protocols that detect biotinylated analogs of GnRH or GHRH (Childs et al. 1983a
; Childs et al. 1994a
; Childs et al. 1999
). shows that fasting caused an 80% reduction in expression of GHRH receptive cells (p<0.001) and a 73% reduction in GnRH receptive cells (p<0.001). illustrate these cell populations in fed and fasted rat pituitary populations.
Effect of Food Deprivation on the Expression of Pituitary GHRH or GnRH receptors
Fasting Effects on Leptin Proteins and mRNA
Food deprivation brought about a significant 64% reduction in the overall percentages of pituitary cells with leptin mRNA from 33 ± 2 to 12 ± 1% of anterior pituitary cells (p<0.001). Image analyses also showed a 40% reduction in the integrated optical density of label (p=0.03) (data not shown). In these first groups of fasted animals, there was also a 22% reduction in the percentages of cells with leptin proteins, from 36 ± 2% to 29 ± 2 % of AP cells (±SE; p<0.029) (). illustrate the in situ hybridization and the reduction in cells with leptin mRNA.
Effect of Food Deprivation on Pituitary Cells with Leptin mRNA or Proteins
Effect of Fasting on Differential Expression of Leptin by AP Cells
The next objective of these studies was to learn which pituitary cell types were most affected by the food deprivation, specifically in their production of leptin. illustrates the changes in the percentages of cells with leptin and each pituitary hormone in the anterior pituitary. In normal, fed populations, cells that co-express leptin and GH are 22% of the anterior pituitary population and this subset represents 84% of GH cells; cells that co-express leptin with LH, or TSH, or ACTH are 2-3% of the pituitary population (20-30% of each of these cell types); those with leptin and prolactin constitute only 1% of pituitary cells or 0.6% of prolactin cells..
Figure 4 Pituitary cells that express leptin: Effects of food deprivation Dual labeling for leptin proteins and each of the pituitary hormones was done on freshly dispersed pituitary cells. shows a significant decrease in percentages of AP cells with (more ...)
When the analysis focused on the population of leptin-bearing cells, the dual labeling accounted for over 90% of cells with leptin proteins, with 61% of leptin-bearing cells co-expressing GH, 8-10% co-expressing LH, TSH, or ACTH and only 3% co-expressing prolactin. The remaining 10% could be folliculostellate cells, or monohormonal FSHβ cells, which were not detected in these dual labeling studies.
also shows that food deprivation brought about major losses in leptin protein expression by somatotropes or gonadotropes, reducing the percentages of AP cells with leptin and GH from 22% to 13% and those with leptin and LH from 3% to 1% (p<0.001). In contrast, corticotropes maintained their expression of leptin at 30% of ACTH cells. The overall increase in corticotropes resulted in a 1.94 increase in AP cells with leptin and ACTH, from 3.6% to 7% of AP cells (p=0.03). illustrates this increased expression of leptin proteins in corticotropes. The analysis of leptin in fasted rat pituitary populations accounted for 86% of the leptin cell population, with 45% of leptin cells storing GH (reduced from 61%) and 24% storing ACTH (increased from 8%).
Because gonadotropes and somatotropes were most severely affected by the food deprivation, the studies then determined if the reduced leptin mRNA was seen in these populations. shows an 80% decline in cells that co-expressed leptin mRNA and GH proteins from 25 ± 3% to 5 ± 2% of AP cells (p=<0.014). Similarly, fasting caused a significant decline in the co-expression of leptin mRNA in gonadotropes from 3 ± 0.5% of AP cells to 0.7 ± 0.02% of AP cells (p<0.029). The sum of the losses in gonadotropes and somatotropes accounted for the overall loss in leptin mRNA. illustrate the dual labeling for leptin mRNA and GH in fed and fasted rat populations.
Effect of fasting on % of AP cells with leptin mRNA and GH or LH
Effects of Glucose on Pituitary Leptin-bearing Cells, in vivo
After the parallel fasting-induced reductions in leptin, LH and GH were detected, the experiments were expanded to test the hypothesis that nutrients, such as glucose might regulate this system. This involved the addition of a third set of fasted animals provided with 10% glucose water during the period of food deprivation. shows data from 3 groups of rats (3 rats/group). Both groups of fasted rats lost weight, but serum glucose was restored in the animals given 10% glucose water.
Whole pituitaries from 3 separate sets of animals were committed to assays for mRNA by QRT-PCR, in order to obtain the entire pituitary for the mRNA extracts. This precluded any sampling errors that might be caused by regional differences in the pituitary. shows that leptin mRNA detected by QRT-PCR was reduced in the fasted animals (p<0.026) and restored in the fasted animals given 10% glucose water (p<0.03). The data are the ratio of number of leptin transcripts to the number of transcripts of the Hprt housekeeping gene. [A similar result was seen if Rps9 was used as the housekeeping gene; data not shown]. Glucose also restored leptin mRNA, detected by in situ hybridization (p<.001 fasted vs glucose-fasted) ().. also shows a parallel restoration of LH mRNA, as detected by in situ hybridization and counts of mRNA-bearing cells (p=0.02 fed vs fasted and p=0.004 fasted vs glucose-fasted). QRT-PCR assays of GH mRNA showed that there were no significant changes with fasting or with glucose water (data not shown).
Effect of Food Deprivation With or Without Glucose Water on Pituitary Leptin mRNA and Cells with Leptin mRNA
Finally, when additional sets of fresh cultures of pituitary cells were committed to immunolabeling for leptin, LH and GH, glucose also restored expression of all three protein hormones, as detected by cell counts. illustrates this restoration graphically. Note that the fasting-mediated reduction in leptin bearing cells in these groups of rats is more dramatic than in the first group (). depict immunolabeled cultures from fasted rats with and without glucose water, showing the restoration of somatotropes () and gonadotropes () in the population from fasted rats given 10% glucose.
Restorative effects of glucose, in vivo, or leptin, in vitro, on GH, LH and leptin expression
Effects of Leptin on Pituitary Cells, in vitro
The final set of experiments tested the efficacy of leptin, in vitro
on the possible restoration of GH and LH hormone expression in 3 groups of freshly dispersed cells from additional fasted animals (6 rats total). Leptin was added for 1 h, with a concentration range (10-100 pg/ml) that matched those assayed in pituitary cell culture media (Akhter et al. 2007
). shows that, as little as 10 pg/ml leptin for 1 h restored the percentages of LH and GH hormone-bearing cells to values not different from those in cultures from fed or fasted rats treated with glucose, in vivo
. Exogenous leptin for 1 h partially restored the percentages of leptin-bearing cells, with values midway between those from fed rats and fasted rats. illustrate the restoration of GH or LH cells in the fasted groups treated with leptin, in vitro