LEPROT and LEPROTL1 cooperatively decrease hepatic growth hormone action in mice

Abstract

Growth hormone (GH) is a major metabolic regulator that functions by stimulating lipolysis, preventing protein catabolism, and decreasing insulin-dependent glucose disposal. Modulation of hepatic sensitivity to GH and the downstream effects on the GH/IGF1 axis are important events in the regulation of metabolism in response to variations in food availability. For example, during periods of reduced nutrient availability, the liver becomes resistant to GH actions. However, the mechanisms controlling hepatic GH resistance are currently unknown. Here, we investigated the role of 2 tetraspanning membrane proteins, leptin receptor overlapping transcript (LEPROT; also known as OB-RGRP) and LEPROT-like 1 (LEPROTL1), in controlling GH sensitivity. Transgenic mice expressing either human LEPROT or human LEPROTL1 displayed growth retardation, reduced plasma IGF1 levels, and impaired hepatic sensitivity to GH, as measured by STAT5 phosphorylation and Socs2 mRNA expression. These phenotypes were accentuated in transgenic mice expressing both proteins. Moreover, gene silencing of either endogenous Leprot or Leprotl1 in H4IIE hepatocytes increased GH signaling and enhanced cell-surface GH receptor. Importantly, we found that both LEPROT and LEPROTL1 expression were regulated in the mouse liver by physiologic and pathologic changes in glucose homeostasis. Together, these data provide evidence that LEPROT and LEPROTL1 influence liver GH signaling and that regulation of the genes encoding these proteins may constitute a molecular link between nutritional signals and GH actions on body growth and metabolism.

Figure 1. Transgenic expression of LEPROT and LEPROTL1 induces growth retardation.

(A) Top: human transgenic and mouse endogenous LEPROT and LEPROTL1 mRNA were detected by RT-PCR in the indicated tissues of WT and transgenic (left, LEPROT-Tg; right, LEPROTL1-Tg) mice. The mouse Leprot mRNA samples were run on the same gel but were noncontiguous. Bottom: LEPROT and LEPROTL1 proteins were analyzed by Western blot in muscle and liver of WT and transgenic (Tg: left, LEPROT; right, LEPROTL1) mice. (B) Growth curves of WT (circles), LEPROT-Tg (squares), and LEPROTL1-Tg (triangles) male and female mice. Data shown are mean ± SEM.

Figure 2. LEPROT and LEPROTL1 decrease GH responsiveness and cell-surface GH binding in liver.

(A) Liver phospho-STAT5/STAT5 ratio after intravenous GH or saline (PBS) injection (n = 4 mice per group), (B) specific hGH binding in primary hepatocytes at 4°C (n = 3 mice per group), and (C) liver endogenous mouse Ghr mRNA expression (n = 3 mice per group) in WT and LEPROT-Tg and LEPROTL1-Tg mice. *P < 0.05 versus WT; Wilcoxon’s t test. Data shown are mean ± SD.

Figure 3. LEPROT and LEPROTL1 decrease GH signaling.

Mouse primary hepatocytes (A and B) and mouse C2C12 myoblast cells (C) were infected with GFP-, LEPROT-, or LEPROTL1-expressing adenoviruses and subsequently incubated with (+) or without (–) GH. Basal and GH-induced p-STAT5/STAT5 ratio (A) and Socs2 mRNA (B and C) were measured by Western blot and Q-PCR, respectively. The blot is representative of 3 independent experiments; lanes were run on the same gel but were noncontiguous. Quantifications were performed from 3 independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05 versus respective control, Wilcoxon’s t test. Data shown are mean ± SD.

Figure 4. Leprot and Leprotl1 silencing increase GH signaling.

H4IIE hepatic cells were transfected with control, Leprot, or Leprotl1 siRNAs. (A) Specific hGH binding at room temperature (n = 3 per group). (B) p-JAK2, JAK2 (C), p-STAT5, STAT5, and (D) Socs2 mRNA expression in control, LEPROT, or LEPROTL1 siRNA–transfected cells after incubation with (+) or without (–) GH were measured by Western blot and Q-PCR, respectively. The lanes on panel C were run on different gels. ***P < 0.001; *P < 0.05 versus respective treated control, Wilcoxon’s t test (n = 3). Data shown are mean ± SD.

Figure 5. LEPROT and LEPROTL1 decrease cell-surface GH binding but not hEGF binding without increasing soluble GH binding formation.

COS-7 cells were cotransfected with GHR (A, C, and D) or EGFR (B). Specific hGH binding (A and D) and hEGF binding (B) at 4°C confined to the cell surface were determined in the presence of LEPROT or LEPROTL1 expression vectors (1.25 μg) compared with control vectors (A and B) or both LEPROT and LEPROTL1 expression vectors (0.5 μg each) compared with single transfection of each LEPROT or LEPROTL1 (0.5 μg; D). Specific hGH binding corresponding to soluble GHR in medium from A was determined (C). ***P < 0.001; **P < 0.01 versus control transfected cells; ^††P < 0.01 versus single transfected cells, Wilcoxon’s t test (n = 3). Data shown are mean ± SD.

Figure 6. Regulation of Leprot and Leprotl1 mRNA expression by nutritional status, diabetes status, and insulin treatment.

(A) Endogenous Leprot and Leprotl1 mRNA levels in livers of WT female mice fed ad libitum, subjected to 24 hours fasting, and refed for 12 or 24 hours with a high-carbohydrate diet (n = 8 mice per group) were measured by Q-PCR. (B) Endogenous Leprot and Leprotl1 protein levels in livers of WT female mice fed ad libitum or subjected to a 24 hour fasting were measured by Western blot, using antibodies against the C-ter of both Leprot and Leprotl1 (n = 3). (C) Endogenous Leprot and Leprotl1 mRNA levels were measured in livers from control or STZ-induced T1DM mice (STZ; glycemia > 400 mg/dl) (n = 8 mice per group). (D) Endogenous Leprot and Leprotl1 mRNA levels were measured in H4IIE hepatocytes treated for 24 hours with insulin (10 nM) by Q-PCR. **P < 0.01; *P < 0.05 versus control, Wilcoxon’s t test (n = 5). Data shown are mean ± SEM.

Figure 7. LEPROT/LEPROTL1 double-transgenic mice display an exacerbated growth defect phenotype and liver GH resistance.

(A) Picture of 10-week-old male WT and LEPROT/LEPROTL1 Tg mice. (B) Liver Igf1 and Als mRNA expression levels after 6 hours of fasting in WT (n = 6) and LEPROT/LEPROTL1-Tg mice (n = 4). (C) Specific cell-surface hGH binding (n = 3 mice per group) and (D) Socs2 mRNA levels (with or without GH) in primary hepatocytes isolated from LEPROT-Tg and LEPROT/LEPROTL1-Tg mice. *P < 0.05, LEPROT/LEPROTL1-Tg versus WT (B) or versus LEPROT-Tg (C and D), Wilcoxon’s t test. Data shown are mean ± SD.