Gipr is essential for adrenocortical steroidogenesis; however, corticosterone deficiency does not mediate the favorable metabolic phenotype of Gipr(-/-) mice - PubMed (original) (raw)
Gipr is essential for adrenocortical steroidogenesis; however, corticosterone deficiency does not mediate the favorable metabolic phenotype of Gipr(-/-) mice
Holly E Bates et al. Diabetes. 2012 Jan.
Abstract
Glucose-dependent insulinotropic polypeptide (GIP) promotes glucose-dependent insulin secretion. However, GIP also enhances glucocorticoid secretion and promotes adiposity. Because obesity and diabetes are glucocorticoid dependent, we examined whether the effects of GIP on energy balance and glycemia are regulated by glucocorticoids using pharmacological activation of GIP receptor (GIPR) signaling with [d-Ala(2)]GIP in mice and in Y1 adrenocortical cells. Genetic elimination of GIPR activity was also studied in normal- and high-fat (HF)-fed Gipr-deficient (Gipr(-/-)) mice. [d-Ala(2)]GIP increased murine corticosterone levels in a GIPR-dependent manner. Conversely, basal corticosterone levels were reduced, whereas food deprivation resulted in significantly enhanced plasma corticosterone levels in Gipr(-/-) mice. [d-Ala(2)]GIP increased cAMP levels, activated extracellular signal\x{2013}related kinase (ERK)1/2, increased expression of steroidogenic genes, and increased neutral lipid storage in Y1GIPR cells. Gipr(-/-) adrenal glands demonstrated a twofold upregulation of the ACTH receptor mRNA and increased sensitivity to ACTH ex vivo. Although HF-fed Gipr(-/-) mice exhibited significantly lower plasma corticosterone, glucocorticoid-treated HF-fed Gipr(-/-) mice had similar energy balance and glycemia compared with Gipr(+)(/+) controls. Hence, although the Gipr is essential for adrenal steroidogenesis and links HF feeding to increased levels of corticosterone, reduced glucocorticoid levels do not significantly contribute to the enhanced metabolic phenotypes in HF-fed Gipr(-/-) mice.
Figures
FIG. 1.
GIPR signaling alters plasma corticosterone levels in mice. A: [
d
-Ala2]GIP induces a rise in corticosterone levels. [
d
-Ala2]GIP (24 nmol/kg) was injected intraperitoneally into overnight-fasted C57BL/6 mice and corticosterone sampled from the tail vein at 15, 30, 45, or 60 min after injection (n = 4–6 per group). B: Intraperitoneal injection of [
d
-Ala2]GIP (24 nmol/kg) increases corticosterone in Gipr+/+ but not _Gipr_−/− mice 30 min after injection (n = 10 per group). C: Twice-daily injection of [
d
-Ala2]GIP (24 nmol/kg) for 60 days in streptozotocin-injected mice (26) increases corticosterone levels more than threefold (n = 8–12). D: Basal, unstressed corticosterone levels are reduced in _Gipr_−/− mice compared with wild-type littermates (n = 7–10). *P < 0.05, **P < 0.01 [
d
-Ala2]GIP vs. saline-injected control or _Gipr_−/− vs. Gipr+/+.
FIG. 2.
Food deprivation stress selectively enhances corticosterone responses in _Gipr_−/− mice. Gipr+/+ and _Gipr_−/− mice were exposed to various stress paradigms with at least 2 weeks between experiments, and corticosterone was measured in blood samples obtained from the tail vein. For food deprivation stress, blood samples were obtained in the morning between 0900 and 1100 h, after a 24-h fast. Mice were subjected to restraint stress by placement in a perforated 50-mL plastic conical tube for 30 min. For ACTH stimulation, ACTH (Sigma-Aldrich) was injected at a dose of 5 units/kg i.p. at 0900 h. For insulin-induced hypoglycemia, mice were fasted for 5 h and given 1.0 unit/kg insulin (Humulin R, 100 units/mL; Lilly) by intraperitoneal injection, and corticosterone samples were obtained between 1300 and 1600 h. **P < 0.01 vs. Gipr+/+ mice (n = 6–14 per group).
FIG. 3.
Corticosterone levels are reduced in HF-fed _Gipr_−/− mice. Gipr+/+ and _Gipr_−/− mice were fed a 45% HF diet for 2–3 weeks, and basal, unstressed HPA parameters were measured by real-time qRT-PCR. A: Plasma corticosterone levels were reduced in _Gipr_−/− mice without changes in plasma ACTH levels. Gene expression of pomc in the pituitary gland (B) and crh in the hypothalamus (C) were not altered in _Gipr_−/− mice. In a similar manner, GR expression was not changed in the pituitary (B), hypothalamus (C), or epididymal white adipose tissue (D) of _Gipr_−/− mice. Gene expression of the glucocorticoid synthesizing enzyme 11β-HSD1 (D) was not changed in Gipr−/− mice. *P < 0.05 vs. Gipr+/+ mice (n = 4–11 per group).
FIG. 4.
Altered expression of steroidogenic genes and increased sensitivity to ACTH in adrenal glands of _Gipr_−/− mice. A: Expression of genes involved in adrenal steroidogenesis were measured by real-time qRT-PCR in adrenal glands from Gipr+/+ and _Gipr_−/− mice after 2 weeks of HF feeding (n = 8–9 per group). B: Ex vivo adrenal sensitivity to ACTH (1 nmol/L) was measured in adrenal glands from Gipr+/+ and _Gipr_−/− mice after 5 weeks HF feeding (n = 5 per group). *P < 0.05, ** P < 0.01 vs. Gipr+/+ mice.
FIG. 5.
GIP regulates lipid accumulation in adrenocortical cells. A: Expression of genes involved in adrenal cholesterol homeostasis was measured in adrenal glands from Gipr+/+ and _Gipr_−/− mice by real-time qRT-PCR after 2 weeks HF feeding (n = 8–9 per group). B: The intensity of Oil Red O staining for neutral lipids is reduced in adrenal glands from _Gipr_−/− mice compared with Gipr+/+ littermates after 9 weeks of HF feeding (n = 3 per group; original magnification ×10). C, cortex; M, medulla. C: Elution of Oil Red O from Y1GIPR adrenocortical cells is increased after 7 days treatment with [
d
-Ala2]GIP (n = 3 per group). *P < 0.05 vs. Gipr+/+ mice, **P < 0.01 vs. saline control. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 6.
[
d
-Ala2]GIP stimulates steroidogenesis in Y1 cells. A: GIP stimulates an increase in cAMP production by Y1 mouse adrenocortical cells stably transfected with the GIPR in a dose-dependent manner. B: Incubation of 72-h serum-starved Y1GIPR cells with 100 nmol/L [
d
-Ala2]GIP increases ERK1/2 phosphorylation (P-ERK1/2; n = 6 per group). S, saline; G, [
d
-Ala2]GIP; A, ACTH. C: Treatment (24 h) of Y1 cells with [
d
-Ala2]GIP stimulates progesterone production. D: Incubation (6 h) of Y1 adrenocortical cells with [
d
-Ala2]GIP increases StAR, cyp11a1, and sr-b1 mRNA transcripts measured by real-time qRT-PCR (n = 5–7 per group). **P < 0.01 vs. saline, ***P < 0.001 vs. empty vector/saline.
FIG. 7.
Corticosterone supplementation does not alter energy balance in _Gipr_−/− mice. HF-fed Gipr+/+ and _Gipr_−/− mice were supplemented with 5 μg/mL corticosterone in the drinking water for 22 weeks. A: Adrenal gland weights were reduced in mice supplemented with corticosterone in their drinking water (main effect corticosterone, P < 0.05). B: _Gipr_−/− mice gained weight more slowly than wild-type littermates, but this weight gain was not affected by corticosterone supplementation. V
o
2 (C) was not altered, whereas activity (D) was increased in _Gipr_−/− mice compared with wild-type littermates during the beginning of the dark phase (main effect genotype, P < 0.05). E: Assessment of fat mass by magnetic resonance imaging after 16 weeks of corticosterone supplementation and HF feeding demonstrated that _Gipr_−/− mice had reduced fat mass (main effect genotype, P = 0.03), which was not altered by corticosterone supplementation (n = 7–8 per group). *P < 0.05 vs. Gipr+/+. Cort, corticosterone supplementation.
FIG. 8.
Corticosterone supplementation does not worsen glucose homeostasis in _Gipr_−/− mice. Glucose excursion and 20–30 min plasma insulin responses after oral gavage (n = 7–8) (A) and intraperitoneal injection (n = 4–7) (B) of 1.5 mg/g glucose. Corticosterone supplementation reduced glucose excursion after oral glucose in wild-type mice (main effect corticosterone, P = 0.03) but not in Gipr−/− mice. Plasma insulin was reduced at 20–30 min in _Gipr_−/− mice after oral but not intraperitoneal glucose (n = 7–8). Insulin sensitivity (n = 6–7) (C) assessed by ITT was not affected by corticosterone supplementation in Gipr+/+ vs. _Gipr_−/− mice. **P < 0.01 vs. Gipr+/+. Cort, corticosterone supplementation.
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