Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Nov;155(11):4316-28.
doi: 10.1210/en.2014-1172. Epub 2014 Aug 13.

Adipocyte versus pituitary leptin in the regulation of pituitary hormones: somatotropes develop normally in the absence of circulating leptin

Angela K Odle  1 , Anessa Haney, Melody Allensworth-James, Noor Akhter, Gwen V Childs
Affiliations

Adipocyte versus pituitary leptin in the regulation of pituitary hormones: somatotropes develop normally in the absence of circulating leptin

Angela K Odle et al. Endocrinology. 2014 Nov.

Abstract

Leptin is a cytokine produced by white fat cells, skeletal muscle, the placenta, and the pituitary gland among other tissues. Best known for its role in regulating appetite and energy expenditure, leptin is produced largely by and in proportion to white fat cells. Leptin is also important to the maintenance and function of the GH cells of the pituitary. This was shown when the deletion of leptin receptors on somatotropes caused decreased numbers of GH cells, decreased circulating GH, and adult-onset obesity. To determine the source of leptin most vital to GH cells and other pituitary cell types, we compared two different leptin knockout models with Cre-lox technology. The global Lep-null model is like the ob/ob mouse, whereby only the entire exon 3 is deleted. The selective adipocyte-Lep-null model lacks adipocyte leptin but retains pituitary leptin, allowing us to investigate the pituitary as a potential source of circulating leptin. Male and female mice lacking adipocyte leptin (Adipocyte-lep-null) did not produce any detectable circulating leptin and were infertile, suggesting that the pituitary does not contribute to serum levels. In the presence of only pituitary leptin, however, these same mutants were able to maintain somatotrope numbers and GH mRNA levels. Serum GH trended low, but values were not significant. However, hypothalamic GHRH mRNA was significantly reduced in these animals. Other serum hormone and pituitary mRNA differences were observed, some of which varied from previous results reported in ob/ob animals. Whereas pituitary leptin is capable of maintaining somatotrope numbers and GH mRNA production, the decreased hypothalamic GHRH mRNA and low (but not significant) serum GH levels indicate an important role for adipocyte leptin in the regulation of GH secretion in the mouse. Thus, normal GH secretion may require the coordinated actions of both adipocyte and pituitary leptin.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
A diagram of the floxed leptin gene. A, Representation of the floxed leptin exon 3 (Taconic) used to create the tissue-specific leptin knockout mice. Leptin exon 3 is flanked by two loxP sites (represented by black arrowheads). Also contained within these loxP sites is the selection neocassette, flanked by two Flippase recognition target sites (diagonal striped boxes). B, Floxed leptin gene after the neocassette has been deleted, by crossing with an animal bearing the Flippase recombination enzyme. One Flippase recognition target site remains, along with lep exon 3 and both loxP sites. C, Final product of recombination after the floxed leptin gene has been exposed to Cre-recombinase. Exon 3 has been deleted, and only a single loxP site remains.
Figure 2.
Figure 2.
Sample genotyping of Global-lep-null and Adipocyte-lep-null mutant and control organs. Figure 2A contains a sample of the extensive organ genotyping done on the Global-lep-null animals and littermate Leptinfl/fl controls. The first panel demonstrates the genotyping for detection of Cre-recombinase (166 bp), which is not present in either the three control samples or the three Global-lep-null samples of adipose tissue. Because the EIIa-cre always sorted with the wild-type lep gene, we used Cre-negative Leptinfl/Δ males and females to create Cre-negative LeptinΔ/Δ animals. The second panel shows genotyping for the floxed lep gene (327 bp), which is present in the control pituitaries but absent in the Global-lep-null pituitaries. This is because both alleles of lep have been excised. The final three panels indicate the excision of lep (535 bp band) in deletion pituitaries, fat samples, and stomachs but not in any of the control samples. B, Sample organ genotyping in the Adipocyte-lep-null line. All Adipocyte-lep-null samples (but none of the control samples) are Cre positive, as indicated by the adipose samples in the first panel. Floxed lep alleles are present in both control and deletion pituitaries because the Adipoq-cre is specific only to adipose tissue. The final three panels show no lep excision in control or deletion pituitaries or stomach samples but strong excision in Adipocyte-lep-null adipose tissue (but not control tissue).
Figure 3.
Figure 3.
Analysis of serum and fat leptin protein levels. Panel A contains serum leptin levels for control and Global-lep-null males (left) and females (right). A one-tailed Student's t test was used to compare values between groups within sexes. Values are expressed as average leptin levels (picograms per milliliter) ± SEM. Significance is set at P < .05. B, Serum leptin levels (picograms per milliliter ± SEM) for control and Adipocyte-lep-null males (left) and females (right). A two-tailed Student's t test was used to determine differences. C, Control and deletion Adipocyte-lep-null male (left) and female (right) adipose leptin protein levels. Values are displayed as picograms per milliliter per microgram of protein ± SEM, and differences were determined with one-tailed Student's t tests. Significance (for this figure and all others) is indicated as follows: *, P = .01 to P = .05; **, P = .01 to P = .001; ***, P = .001 to P = .0001; ****, P < .0001.
Figure 4.
Figure 4.
Characterization of weight over time in the Global- and Adipocyte-lep-null mutants and controls. A, Weekly average weights from weaning to 5 months of age in male (top) and female (bottom) Global-lep-null mutants (squares) and controls (circles). Values are expressed as averages of at least five animals per group per sex at each time point ± SEM. Student's t tests were used to determine the differences, with significance set as P < .05. Weights for Adipocyte-lep-null mutants are shown in a similar manner in panel B.
Figure 5.
Figure 5.
Pituitary and hypothalamic mRNA analysis. A, Male (left) and female (right) control and deletion Adipocyte-lep-null pituitary mRNA levels. For all mRNA results, levels were determined using qRT-PCR and Δ-Δ-cycle threshold analysis, with cyclophilin A as an internal control. Relative quantification values are expressed with ± SEM, with control mRNA levels set at 1. A Student's t test (two tailed) was used to determine differences within sexes, with significance set at P < .05. Target pituitary mRNAs included FSH, GH, ghrelin, GHRH, GHS-R, LH, POMC, prolactin, and TSH. At least five pituitaries were used per experimental group per sex. B, mRNA analysis for male (left) and female (right) control and Adipocyte-lep-null mutant hypothalami, and at least five whole hypothalamus samples were analyzed per group per sex. Target hypothalamus mRNAs were GHRH, ghrelin, leptin receptor (all isoforms), and somatostatin. C, mRNA analysis for male (left) and female (right) control and Global-lep-null hypothalami, and n is at least five per group per sex.
Figure 6.
Figure 6.
Immunolabeling of GH and leptin in primary pituitary cells. A, Results from immunolabeling of male Adipocyte-lep-null and Global-lep-null control and mutant primary pituitary cell cultures. At least two coverslips for each of three animals per group were immunolabeled for either GH or leptin (amino acids 22–40). Ten fields per coverslip were analyzed for labeled vs unlabeled pituitary cells. Values are expressed as the average percentage of cells immunolabeled ± SEM. Student's t tests were used to determine differences in GH or leptin labeling. Representative fields of GH-labeled pituitary cells from a control and an Adipocyte-lep-null male are shown in panel B. Fields showing leptin-labeled pituitary cells from a control and a Global-lep-null animal are shown in panel C.
Figure 7.
Figure 7.
Serum levels of pituitary hormones. Male and female pituitary hormone levels from Adipocyte-lep-null and Global-lep-null males and females are shown in a series of graphs. Values for FSH, GH, and LH (top row) and prolactin and TSH (bottom row) are displayed on individual charts. Values are expressed as nanograms per milliliter ± SEM, with significance set at P < .05. Samples from at least five animals per group per sex are represented for each hormone. Student's t tests were used to determine differences.
See this image and copyright information in PMC

References

    1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994;372:425–432. - PubMed
    1. Ingalls AM, Dickie MM, Snell GD. Obese, a new mutation in the house mouse. J Heredity. 1950;41:317–318. - PubMed
    1. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 1996;98:1101–1106. - PMC - PubMed
    1. Tartaglia LA, Dembski M, Weng X, et al. Identification and expression cloning of a leptin receptor, OB-R. Cell. 1995;83:1263–1271. - PubMed
    1. Schwartz MW, Baskin DG, Bukowski TR, et al. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes. 1996;45:531–535. - PubMed

Publication types