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-Ala2]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-Ala2]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-Ala2]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.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42–amino acid peptide hormone released from intestinal K cells upon nutrient ingestion (1). The original description of GIP bioactivity encompassing inhibition of gastric emptying was subsequently expanded to include potentiation of glucose-dependent insulin secretion. GIP also can stimulate lipogenesis (2,3) and increase the activity of lipoprotein lipase in adipocytes in part through stimulatory effects on resistin secretion (4,5), thus enhancing fatty acid incorporation into adipose tissue.

Complementary insights into the physiological importance of endogenous GIP action have been obtained from studies using antagonists, elimination of GIP-producing K cells, or receptor knockout mice. Reduction or elimination of GIP action promotes resistance to genetic and high-fat (HF) diet–induced obesity, increased energy expenditure and activity, reduced fat deposition in nonadipose and adipose depots, improved insulin sensitivity, and lower plasma resistin levels (59). Therefore, it remains unclear whether enhancement or reduction of GIP action is the preferred therapeutic strategy for the treatment of obesity-associated type 2 diabetes.

An additional extrapancreatic locus of GIP action is the adrenal cortex. Expression of the GIP receptor (GIPR) has been demonstrated in the adrenal cortex by radioligand binding (10) and RT-PCR (11) in rodents; however, the GIPR is not expressed in normal human adrenal glands (12). Ectopic expression of the GIPR in the human adrenal gland causes significant hypercortisolemia after meal ingestion and leads to Cushings syndrome (13), demonstrating that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. Indeed, GIP administration increases corticosterone levels in rats (10), and isolated rat adrenocortical zona fasciculata/reticularis cells respond to GIP in a cAMP-dependent protein kinase manner (10). However, the mechanisms through which GIP stimulates glucocorticoid secretion and the physiological importance of adrenal GIP action remains poorly understood.

Chronic elevations of glucocorticoid levels promote obesity and/or metabolic syndrome, and many rodent models of obesity or insulin resistance, including ob/ob and db/db mice, Zucker and ZDF rats, HF-fed rodents, and streptozotocin-induced diabetic rodents, exhibit elevations in glucocorticoids (1417). Furthermore, adrenalectomy or glucocorticoid receptor (GR) antagonists ameliorate or in some cases prevent diabetes and obesity phenotypes (18,19). Accordingly, we hypothesized that antagonism or removal of the GIPR-dependent stimulation of corticosterone secretion after nutrient ingestion may contribute to one or more of the phenotypes exhibited by Gipr−/− mice. We now show that activation or disruption of the GIPR modulates glucocorticoid levels in mice, and GIP directly enhances glucocorticoid synthesis and secretion in adrenocortical cells. However, the reduction in glucocorticoid levels observed in Gipr−/− mice does not substantially contribute to the phenotypes of improved glucose tolerance and resistance to diet-induced obesity in HF-fed Gipr−/− mice.

All experiments were done using male mice acclimatized to handling before commencement of experiments. Wild-type C57BL/6 mice were obtained from the Toronto Centre for Phenogenomics (TCP; Toronto, ON, Canada). The generation and characterization of Gipr−/− mice has been described previously (20). Mice were housed under a 12-h light/dark cycle in the TCP animal facility with free access to standard rodent diet (2018, 18% kcal from fat; Harlan Teklad, Mississauga, ON, Canada) and water, except where otherwise noted. All procedures were conducted according to protocols and guidelines approved by the TCP Animal Care Committee.

Acute stimulation of corticosterone by [d-Ala2]GIP.

Overnight fasted C57BL/6 mice or ad libitum fed Gipr+/+ and Gipr−/− mice were injected intraperitoneally with 24 nmol/kg [d-Ala2]GIP (California Peptide Research Inc., Napa, CA) or saline. Blood for corticosterone was obtained from the tail vein.

Ex vivo ACTH sensitivity.

Adrenal glands from Gipr+/+ and Gipr−/− mice were obtained after 5 weeks of HF diet, and responsiveness to ACTH was measured as described (21). Briefly, adrenals were excised, cleaned, halved, and incubated in vitro in Dulbecco’s modified Eagle’s medium with 25 mmol/L HEPES and 0.1% w/v BSA. After preincubation for 120 min, the medium was changed and adrenals were incubated for 60 min to determine basal corticosterone production. Medium was changed again and 1 nmol/L ACTH (Sigma-Aldrich, St. Louis, MO) was applied for 60 min. Medium was frozen for analysis of corticosterone levels.

Basal corticosterone measurements.

Basal blood samples were obtained at the diurnal peak (1800 h) and trough (0900 h) of the corticosterone rhythm in Gipr+/+ and Gipr−/− mice. Mice were in a quiet room for at least 1 h prior to sampling, and blood for corticosterone was obtained from the tail vein within 1 min of opening the cage.

Stress experiments.

Gipr+/+ and Gipr−/− mice were exposed to various stress regimens, including 24-h food deprivation, 30-min restraint stress, ACTH stimulation test (5 units/kg), and insulin-induced hypoglycemia (1.0 unit/kg Humulin; Lilly, Toronto, ON, Canada), with at least 2 weeks between experiments for recovery. For dietary stress, mice were fed a HF diet with 45% fat (Research Diets, New Brunswick, NJ) for 2 weeks after which blood was obtained by cardiac puncture after cervical dislocation. The adrenal glands, pituitary, and hypothalamic wedge were immediately dissected and stored in RNAlater (Qiagen, St. Louis, MO) until frozen at −80°C.

Y1 adrenocortical cells.

Mouse Y1 adrenocortical cells were a gift from B. Schimmer (University of Toronto). Cells were cultured in Ham’s F10 Media (Invitrogen, Burlington, ON, Canada) supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated FBS, and penicillin/streptomycin. The rat GIPR cDNA (22) was transfected into Y1 cells using lipofectamine 2,000 transfection reagent (Invitrogen, Burlington, ON, Canada). After selecting for cells stably expressing the vector through incubation in 100 μg/mL G418 (Invitrogen, Burlington, ON, Canada), cells were maintained as a pooled population of mixed G418-resistant clones (Y1GIPR cells). Cells for experiments were plated in Primaria-coated six-well culture plates (BD Falcon, Mississauga, ON, Canada) and treated with human GIP (California Peptide Research Inc.), [d-Ala2]GIP (California Peptide Research Inc.), 10 μmol/L Forskolin (Sigma-Aldrich), or 1 nmol/L ACTH. cAMP production was measured as described (22) after serum starvation for 24 h and assayed by radioimmunoassay (Biomedical Technologies, Stoughton, MA). Progesterone secretion was measured in cells incubated in complete media for 24 h and assayed by enzyme immunosorbent assay (Cayman Chemical Company, Ann Arbor, MI). Neutral lipid content of Y1 cells was quantified by elution of Oil Red O in isopropanol and measurement of the absorbance of the eluant at 492 nm (23).

Immunoblotting of Y1 cells.

Cells were grown to ∼70% confluence in six-well culture plates. Prior to the experiment, cells were serum starved in α–minimum essential medium (Invitrogen, Burlington, ON, Canada) for 72 h. Cells were lysed and immunoblotted as previously described (24). Membranes were immunoblotted for phosphorylated extracellular signal–related kinase (ERK; 1:500) and total ERK (1:500, Cell Signaling Technologies, Danvers, MA) and for Hsp90 (1:3,500; BD Transduction Laboratories, Mississauga, ON, Canada), followed by the appropriate secondary antibodies.

Adrenal gland histology.

Adrenal glands were isolated from Gipr+/+ and Gipr−/− mice and cleaned of extraneous fat. Adrenals were fixed in 10% neutral buffered formalin for hematoxylin-eosin staining or frozen in Tissue-Tek (Optimal Cutting Temperature Compound; Sakura Finetek, Torrance, CA) for staining of neutral lipids with Oil Red O.

Corticosterone supplementation experiments.

At aged 10–13 weeks, Gipr+/+ and Gipr−/− littermates were fed ad libitum a HF diet, with 45% kcal from fat (Research Diets) for 22 weeks. Concurrently, mice were randomized to receive water supplemented with 5 μg/mL corticosterone (Sigma-Aldrich) dissolved in ethanol or vehicle (0.04% ethanol) for 22 weeks. This dose was chosen on the basis of preliminary experiments demonstrating that this dose increased plasma corticosterone levels but did not cause dark-phase hypercorticosteronemia or significantly suppress plasma ACTH levels. Assessment of fat and lean mass was done using a whole-body magnetic resonance analyzer (Echo Medical Systems, Houston, TX) after 16 weeks of treatment. For assessment of food and water intake after 14 weeks of treatment, mice were individually housed for the preceding week. Mice were fasted during the day (0800 to 1600 h), weighed, and then given preweighed rodent food. Food was reweighed at 0900 h (nocturnal food intake) and then at 0900 h 3 days later for ad libitum food intake. After 14 and 20 weeks of corticosterone supplementation, water intake was measured in individually housed mice over the course of 3 or 7 days, respectively. Vo2 and respiratory exchange ratio were determined after 15 weeks of treatment by indirect calorimetry using an Oxymax System (Columbus Instruments, Columbus, OH). Vo2 was measured at 15-min intervals between 1200 and 0700 h for 19 h after at least 2-h acclimatization to the metabolic chamber where mice had free access to food and the appropriately supplemented water. Locomotor activity was simultaneously measured as the number of independent beam breaks (ambulatory activity) in Opto M3 activity monitors (Columbus Instruments). After 16 and 17 weeks of treatment, an oral glucose tolerance test and intraperitoneal glucose tolerance test were performed (1.5 mg/g glucose), respectively, after an overnight 16-h fast. After 18 weeks of treatment, an insulin tolerance test (ITT) was performed (1.2 units/kg insulin, Humulin R, 100 units/mL; Lilly) after a 5-h fast. Blood was sampled from the tail vein, and blood glucose levels were measured using a Glucometer Elite blood glucose meter (Bayer, Toronto, ON, Canada). Basal plasma corticosterone samples were obtained from the tail vein of individually housed mice after 10 and 20 weeks of treatment at the diurnal peak (1900 h) and trough (0800 h) of the corticosterone rhythm. Mice were transported to the sampling room 3 h prior to the 1900-h sample to acclimatize and left in the room overnight for the 0800-h sample.

RNA isolation.

Total RNA was extracted from adrenal glands, hypothalamus, adipose tissue, or pituitary using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada). RNA from Y1 cells was extracted using TriReagent (Molecular Research Center Inc., Cincinnati, OH).

Real-time quantitative RT-PCR.

First-strand cDNA was synthesized from total RNA using the SuperScript II or III synthesis system (Invitrogen, Carlsbad, CA) and random hexamers. Real-time quantitative (q)RT-PCR was carried out with the ABI Prism 7900 Sequence Detection System using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The relative mRNA transcript levels were quantified with the 2-ΔΔCt method (25), using cyclophillin or gapdh as internal control genes.

Hormone assays.

Plasma was separated by centrifugation and stored at −80°C until assayed. Corticosterone and ACTH (ICN Biomedicals, Montreal, QC, Canada) and plasma insulin (ALPCO, Salem, NH) were assayed by radioimmunoassay.

Statistical analysis.

All results are expressed as means ± SE. Statistical significance was assessed by ANOVA and where appropriate, a Student t-test using GraphPad Prism 4 (GraphPad Software, San Diego, CA). P < 0.05 was considered to be statistically significant.

The GIPR agonist [d-Ala2]GIP increases plasma corticosterone in mice via the GIPR.

[d-Ala2]GIP administration to overnight fasted C57BL/6 mice induced a brisk rise in corticosterone levels (Fig. 1A); in contrast, no increase in plasma corticosterone levels occurred after [d-Ala2]GIP administration in Gipr−/− mice (Fig. 1B). To determine if chronic GIPR activation alters corticosterone levels, we assessed diabetic mice treated with saline or [d-Ala2]GIP twice daily for 60 days (26). Corticosterone levels were more than threefold higher in mice chronically injected with [d-Ala2]GIP (Fig. 1C).

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. 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+/+.

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Basal corticosterone levels are reduced in Gipr−/− mice.

We next assessed plasma corticosterone in Gipr−/− mice. Corticosterone levels were reduced in Gipr−/− mice (Fig. 1D) during the peak of the circadian corticosterone rhythm at 1800 h, 1 h prior to their dark cycle. The lower corticosterone levels in Gipr−/− mice were not associated with simultaneous differences in glycemia (data not shown). We next examined the corticosterone response to stress in Gipr−/− mice. Plasma corticosterone responses to restraint stress or insulin-induced hypoglycemia were comparable across genotypes (Fig. 2). In a similar manner, exogenous administration of ACTH produced robust yet comparable increases in plasma corticosterone in Gipr+/+ versus Gipr−/− mice (Fig. 2). In contrast, corticosterone levels were more than threefold higher in Gipr−/− mice after 24 h of food deprivation (Fig. 2). Glucose levels were not different between Gipr+/+ versus Gipr−/− mice during these experiments (data not shown).

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. 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).

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Corticosterone levels are reduced in HF-fed Gipr−/− mice in the absence of reduced hypothalamic–pituitary axis activation.

HF feeding increases basal corticosterone levels (17). Accordingly, we examined whether the Gipr links excess nutrients to increased plasma corticosterone. Remarkably, basal corticosterone levels were reduced by >50% in Gipr−/− mice after 2 weeks of HF feeding (Fig. 3A). However, levels of plasma ACTH (Fig. 3A), pituitary proopiomelanocortin (pomc) (Fig. 3B), and hypothalamic corticotrophin-releasing hormone (crh) mRNA transcripts (Fig. 3C) were comparable in Gipr+/+ versus Gipr−/− mice. GR expression also was similar in the pituitary, hypothalamus, and epididymal white adipose tissue of Gipr+/+ versus Gipr−/− mice (Fig. 3B–D), and expression of the glucocorticoid synthesizing enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) was similar between genotypes (Fig. 3D). Hence, elimination of the Gipr is not associated with generalized perturbations in key molecular components of the hypothalamic–pituitary axis (HPA) despite expression of Gipr mRNA at all levels of the HPA (Supplementary Fig. 1), nor is it associated with alterations in several genes regulating glucocorticoid synthesis and action in key target tissues.

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. 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).

Close modal

Gipr−/− mice exhibit altered adrenal expression of steroidogenic genes and increased sensitivity to ACTH.

To delineate mechanism(s) through which GIPR signaling controls corticosterone in mice, we assessed the expression of genes regulating adrenal steroidogenesis in Gipr+/+ versus Gipr−/− mice after 2 weeks of HF feeding. Levels of StAR, cyp11A1, and dax1 mRNA transcripts were comparable in Gipr−/− mice, sf1 mRNA transcripts were significantly reduced (P < 0.05), creb1 mRNA transcripts were increased (P = 0.07), and mc2r mRNA transcripts were significantly higher (P < 0.01) in Gipr−/− adrenals (Fig. 4A). To assess whether increased levels of mRNA transcripts for the ACTH receptor (mcr2) reflected functionally increased sensitivity to ACTH, we administered a low dose of ACTH (1 nmol/L) to Gipr+/+ and Gipr−/− mouse adrenals ex vivo. A significantly greater corticosterone response to ACTH was detected from Gipr−/− mouse adrenals, consistent with enhanced ACTH sensitivity (Fig. 4B).

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. 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.

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GIP regulates lipid accumulation in adrenocortical cells.

Because GIP enhances adrenal corticosterone secretion, we hypothesized that the Gipr regulates expression of genes controlling adrenal cholesterol homeostasis. Although the majority of mRNA transcripts encoding genes within the cholesterol biosynthetic and transport pathways were not differentially expressed in Gipr−/− versus Gipr+/+ adrenal glands (Fig. 5A), expression of sr-b1 was significantly reduced in Gipr−/− adrenal glands (Fig. 5A). Since SR-B1 is responsible for selective uptake of cholesterol esters from HDL, the primary source of adrenal cholesterol stores in mice (27,28), we examined levels of neutral lipids in adrenal glands of Gipr−/− mice using Oil Red O histochemistry. The intensity of Oil Red O staining in the adrenal cortex was clearly reduced after 9 weeks of a HF diet in Gipr−/− mice (Fig. 5B). Conversely, activation of the GIPR with [d-Ala2]GIP significantly increased incorporation of Oil Red O in Y1GIPR cells (Fig. 5C).

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. 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.)

Close modal

[d-Ala2]GIP directly stimulates steroidogenesis and increases expression of steroidogenic genes in Y1 cells.

As native Y1 adrenocortical cells do not express an endogenous GIPR (Supplementary Fig. 1), we used Y1 mouse adrenocortical cells stably transfected with the rat GIPR cDNA. Y1GIPR cells exhibited a robust dose-dependent increase in cAMP levels in response to GIP (Fig. 6A). In a similar manner, ERK1/2 phosphorylation was rapidly increased after exposure to [d-Ala2] GIP (P < 0.05) (Fig. 6B). Because Y1 cells do not produce corticosterone (29), we assessed progesterone secretion in response to [d-Ala2]GIP. A fivefold increase in progesterone secretion was observed after treatment with [d-Ala2]GIP (P < 0.001) (Fig. 6C). Steroidogenesis is under tight control by StAR, a protein that transports cholesterol from the outer to inner mitochondrial membrane to Cyp11A1, which cleaves the cholesterol side chain to initiate the rate-limiting step of corticosterone synthesis. Therefore, we assessed whether [d-Ala2]GIP regulates this pathway in Y1GIPR cells. [d-Ala2]GIP significantly increased StAR, cyp11a1, and sr-b1 mRNA transcript levels and increased sf-1 transcript levels (Fig. 6D). This stimulatory effect of [d-Ala2]GIP on gene expression was not significant for ppia (cyclophillin), mc2r, ldlr, or acat.

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. 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.

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Corticosterone supplementation does not exacerbate body weight gain in Gipr−/− mice.

To determine if lower corticosterone levels in Gipr−/− mice contribute to their metabolic phenotype, Gipr+/+ and Gipr−/− mice were supplemented with 5 μg/mL corticosterone in their drinking water and fed a HF diet for 22 weeks. Water and corticosterone intake were not affected by genotype (data not shown). Adrenal weights after 22 weeks were not different between vehicle-treated Gipr+/+ and Gipr−/− mice. However, adrenal weights (Fig. 7A) were significantly reduced in both genotypes as a result of corticosterone supplementation (P < 0.05), consistent with feedback inhibition of the HPA from exogenous corticosterone administration. Body weights increased over time with HF feeding (Fig. 7B), and Gipr−/− mice gained less weight than wild-type littermates. However, weight gain was not affected by corticosterone supplementation in Gipr+/+ versus Gipr−/− mice. Nocturnal food intake was slightly reduced in Gipr−/− mice (P < 0.02), as was ad libitum food intake (P = 0.02) (Supplementary Fig. 2), although this was no longer significant when normalized to body weight. Food intake was not altered by corticosterone supplementation. Locomotor activity was increased in Gipr−/− mice during the dark phase compared with Gipr+/+ mice; however, neither Vo2 nor locomotor activity was affected by corticosterone supplementation (Fig. 7C and D). Gipr−/− mice had reduced fat mass; however, corticosterone supplementation had no effect on fat (Fig. 7E) or lean mass (data not shown) in Gipr−/− mice.

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. Vo2 (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. 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. Vo2 (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.

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Corticosterone supplementation does not impair glucose homeostasis in Gipr−/− mice.

Overnight fasting glucose levels (∼16-h fast) were not different between genotypes or affected by corticosterone supplementation (Fig. 8A and B). Corticosterone supplementation had no effect on oral or intraperitoneal glucose tolerance in Gipr+/+ or Gipr−/− mice (Fig. 8A and B). Despite comparable glucose levels, insulin levels (Fig. 8A and B) and insulin-to-glucose ratios (P < 0.05) (data not shown) to oral but not intraperitoneal glucose were reduced in Gipr−/− mice. Insulin sensitivity assessed by ITT was neither different between genotypes nor affected by corticosterone supplementation (Fig. 8C).

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.

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.

Close modal

We have shown that activation of the GIPR increases plasma glucocorticoid levels in mice and that GIP directly activates steroidogenic gene expression in mouse adrenocortical cells. It seems likely that GIP also promotes steroidogenesis via increasing uptake of cholesterol since Gipr−/− mouse adrenal glands expressed lower levels of sr-b1 mRNA transcripts and had reduced neutral lipid staining, whereas Y1GIPR cells stimulated with [d-Ala2]GIP expressed higher transcript levels of sr-b1 and stored more neutral lipid. Since glucocorticoids are diabetogenic and obesigenic in rodents (16,18), we hypothesized that reduced glucocorticoid levels in Gipr−/− mice might contribute to their resistance to diet-induced obesity and preservation of insulin sensitivity and glucose tolerance observed after HF feeding. However, supplementation of drinking water with low-dose corticosterone did not alter energy balance, insulin sensitivity, or glucose tolerance in HF-fed Gipr−/− mice.

Pharmacological levels of GIP stimulate ACTH secretion from AtT20 mouse corticotrope cells (30), and the Gipr is expressed in the hypothalamus and pituitary (11). Although HF-fed Gipr−/− mice exhibited a twofold reduction in basal corticosterone levels, plasma ACTH, pituitary pomc, and hypothalamic crh mRNA levels were not different. Nevertheless, the increased adrenal expression of the ACTH receptor and enhanced adrenal sensitivity to ACTH in adrenal glands from Gipr−/− mice suggest the evolution of adaptive compensatory mechanisms that mask the impact of loss of adrenal GIP action. Enhanced ACTH sensitivity in Gipr−/− mice likely diminishes the extent of corticosterone deficiency and metabolic effects that might otherwise arise pursuant to elimination of GIPR action in the adrenal gland.

Further evidence for a direct effect of GIP on the adrenal gland was obtained in experiments using Y1GIPR cells. [d-Ala2]GIP directly stimulated steroidogenesis, increased cAMP, activated the ERK pathway, and increased StAR, cyp11a1, sf-1, and sr-b1 gene expression. Conversely, mRNA levels of sf-1, a transcription factor critical for regulation of adrenal steroidogenesis (31), and levels of sr-b1, a downstream target of SF-1 (32), were reduced in adrenal glands from Gipr−/− mice. GIP likely mediates some of its effects on the adrenal gland via modifying cholesterol uptake and/or storage. Gipr−/− mice had less neutral lipid staining in the adrenal cortex after 9 weeks of HF feeding, whereas [d-Ala2]GIP increased neutral lipid accumulation in Y1GIPR cells. Since sr-b1 mediates selective uptake of cholesterol from HDL, the most important pathway for adrenal cholesterol uptake in mice, and depletion of sr-b1 leads to deficits in corticosterone (28,33), it is likely that GIP mediates adrenal steroidogenesis in part via this cholesterol uptake pathway.

Although the corticosterone responses to restraint stress, insulin-induced hypoglycemia, and high-dose ACTH were normal in Gipr−/− mice, the more chronic stressor of 24-h food deprivation elicited a threefold greater corticosterone response in Gipr−/− mice. In contrast to potent stressors such as restraint and insulin, food deprivation does not significantly increase ACTH levels, but it does increase basal corticosterone levels by reducing hepatic glucocorticoid clearance (34,35). Hence, at the low ACTH levels associated with food deprivation, the greater adrenal sensitivity to ACTH in Gipr−/− mice may be unmasked, leading to differentially greater increases in corticosterone responses in Gipr−/− mice. In contrast, no differences in corticosterone levels are apparent under situations where ACTH levels would already be expected to be high, as exemplified by restraint and insulin. Thus, it is likely that food deprivation is associated with reduced hepatic clearance of corticosterone and enhanced sensitivity to lower levels of ACTH, leading to hypercorticosteronemia in Gipr−/− mice.

Glucocorticoids regulate metabolism through stimulation of hepatic glucose production (36), impairment of insulin sensitivity and glucose tolerance (37), inhibition of insulin secretion (38), and facilitation of lipolysis (23). Chronic elevations of glucocorticoids promote fat deposition (23,39), insulin resistance (39), hepatosteatosis, hyperphagia, and decreased locomotion (39). Many commonly used rodent models of obesity or insulin resistance, including ob/ob and db/db mice, Zucker and ZDF rats, HF-fed rodents, and streptozotocin-induced diabetic rodents, exhibit elevations in glucocorticoids (1417). Adrenalectomy or administration of a glucocorticoid antagonist can ameliorate or in some cases reverse the abnormal metabolic phenotype (18,19), illustrating the importance of glucocorticoids in the development of rodent obesity and diabetes. Administration of a peptide GIPR antagonist in HF-fed mice lowers corticosterone levels in association with reduced body weight and fat deposition, increased locomotion, and improved glucose homeostasis (7,8,40). However, our current data in HF-fed Gipr−/− mice suggest that modest reductions in plasma corticosterone do not appear to substantially modify the dominant metabolic phenotypes arising after HF feeding in Gipr−/− mice.

Corticosterone supplementation did not significantly modify food intake, energy expenditure, weight gain, or fat mass in Gipr+/+ and Gipr−/− mice. However, corticosterone supplementation slightly improved oral but not intraperitoneal glucose tolerance in wild-type mice. This modest improvement was likely related to the small reductions in body weight and fat mass induced by corticosterone supplementation that would in turn improve glucose disposal. This reduction in fat mass may be related to the lipolytic effects of corticosterone at low concentrations (23). It is likely that the upregulation of ACTH sensitivity in Gipr−/− mice resulted in only a subtle reduction in plasma corticosterone that was not sufficient to lower weight gain or modify energy balance and glucose homeostasis. Consistent with our current data, Irwin et al. (7) demonstrated that administration of the GIP antagonist Pro(3)GIP to ob/ob mice for 60 days significantly improved glucose control and insulin sensitivity, without concomitant changes in circulating corticosterone.

In summary, we show that GIP stimulates plasma glucocorticoid levels in mice and demonstrate that GIP directly activates steroidogenesis through stimulation of steroidogenic genes as well as sr-b1 expression and consequent adrenocortical lipid deposition. Gipr−/− mice compensate for disruption of GIP action in the adrenal gland via upregulation of mcr2 expression and enhanced ACTH sensitivity. Corticosterone supplementation did not reverse the beneficial metabolic phenotype of HF-fed Gipr−/− mice, including resistance to diet-induced obesity and maintenance of oral glucose tolerance with lower levels of plasma insulin, implicating organs such as the brain and adipose tissue as potential mediators of the favorable Gipr−/− phenotype. Our findings may have relevance for envisioned therapeutic strategies using GIPR antagonists for the treatment of obesity and diabetes in human subjects. Because the GIP–adrenal axis does not appear to be functional in normal human subjects, the observation that manipulation of corticosterone levels does not substantially abrogate the beneficial metabolic phenotype of Gipr−/− mice has positive implications for targeting the GIPR in human subjects.

H.E.B. has received fellowships from the Banting and Best Diabetes Centre and the Canadian Diabetes Association. J.E.C. has received fellowships from the Banting and Best Diabetes Centre and the Canadian Institutes of Health Research. J.R.U. has received fellowships from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research. A.M. has received a fellowship from the Canadian Institutes of Health Research. D.J.D. has received funding from the Canadian Institutes of Health Research (MOP 82700) and is supported in part by a Canada Research Chair in Regulatory Peptides and a Banting and Best Diabetes Centre Novo Nordisk Chair in Incretin Biology.

No potential conflicts of interest relevant to this article were reported.

H.E.B. was primarily responsible for the experimental design, experiments, interpretation, and writing of the manuscript. J.E.C., J.R.U., L.L.B., and A.M. contributed significantly to the experiments as well as data interpretation. Y.S. provided Gipr−/− mice and reviewed data. D.J.D. contributed to the experimental design, interpretation of data, and writing of the manuscript and is the guarantor of the manuscript and takes responsibility for all aspects of the work.

The authors would like to thank Bernard Schimmer (University of Toronto) for kindly donating the Y1 mouse adrenocortical cell line and Carolyn Cummins (University of Toronto) for her helpful discussions.

1.
Vilsbøll
T
,
Krarup
T
,
Sonne
J
, et al
.
Incretin secretion in relation to meal size and body weight in healthy subjects and people with type 1 and type 2 diabetes mellitus
.
J Clin Endocrinol Metab
2003
;
88
:
2706
2713
[PubMed]
2.
Hauner
H
,
Glatting
G
,
Kaminska
D
,
Pfeiffer
EF
.
Effects of gastric inhibitory polypeptide on glucose and lipid metabolism of isolated rat adipocytes
.
Ann Nutr Metab
1988
;
32
:
282
288
[PubMed]
3.
Oben
J
,
Morgan
L
,
Fletcher
J
,
Marks
V
.
Effect of the entero-pancreatic hormones, gastric inhibitory polypeptide and glucagon-like polypeptide-1(7-36) amide, on fatty acid synthesis in explants of rat adipose tissue
.
J Endocrinol
1991
;
130
:
267
272
[PubMed]
4.
Kim
SJ
,
Nian
C
,
McIntosh
CH
.
Resistin is a key mediator of glucose-dependent insulinotropic polypeptide (GIP) stimulation of lipoprotein lipase (LPL) activity in adipocytes
.
J Biol Chem
2007
;
282
:
34139
34147
[PubMed]
5.
Hansotia
T
,
Maida
A
,
Flock
G
, et al
.
Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure
.
J Clin Invest
2007
;
117
:
143
152
[PubMed]
6.
Miyawaki
K
,
Yamada
Y
,
Ban
N
, et al
.
Inhibition of gastric inhibitory polypeptide signaling prevents obesity
.
Nat Med
2002
;
8
:
738
742
[PubMed]
7.
Irwin
N
,
McClean
PL
,
O’Harte
FP
,
Gault
VA
,
Harriott
P
,
Flatt
PR
.
Early administration of the glucose-dependent insulinotropic polypeptide receptor antagonist (Pro3)GIP prevents the development of diabetes and related metabolic abnormalities associated with genetically inherited obesity in ob/ob mice
.
Diabetologia
2007
;
50
:
1532
1540
[PubMed]
8.
McClean
PL
,
Irwin
N
,
Cassidy
RS
,
Holst
JJ
,
Gault
VA
,
Flatt
PR
.
GIP receptor antagonism reverses obesity, insulin resistance, and associated metabolic disturbances induced in mice by prolonged consumption of high-fat diet
.
Am J Physiol Endocrinol Metab
2007
;
293
:
E1746
E1755
[PubMed]
9.
Althage
MC
,
Ford
EL
,
Wang
S
,
Tso
P
,
Polonsky
KS
,
Wice
BM
.
Targeted ablation of glucose-dependent insulinotropic polypeptide-producing cells in transgenic mice reduces obesity and insulin resistance induced by a high fat diet
.
J Biol Chem
2008
;
283
:
18365
18376
[PubMed]
10.
Mazzocchi
G
,
Rebuffat
P
,
Meneghelli
V
, et al
.
Gastric inhibitory polypeptide stimulates glucocorticoid secretion in rats, acting through specific receptors coupled with the adenylate cyclase-dependent signaling pathway
.
Peptides
1999
;
20
:
589
594
[PubMed]
11.
Usdin
TB
,
Mezey
E
,
Button
DC
,
Brownstein
MJ
,
Bonner
TI
.
Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain
.
Endocrinology
1993
;
133
:
2861
2870
[PubMed]
12.
Chabre
O
,
Liakos
P
,
Vivier
J
, et al
.
Gastric inhibitory polypeptide (GIP) stimulates cortisol secretion, cAMP production and DNA synthesis in an adrenal adenoma responsible for food-dependent Cushing’s syndrome
.
Endocr Res
1998
;
24
:
851
856
[PubMed]
13.
Lacroix
A
,
Ndiaye
N
,
Tremblay
J
,
Hamet
P
.
Ectopic and abnormal hormone receptors in adrenal Cushing’s syndrome
.
Endocr Rev
2001
;
22
:
75
110
[PubMed]
14.
Guillaume-Gentil
C
,
Rohner-Jeanrenaud
F
,
Abramo
F
,
Bestetti
GE
,
Rossi
GL
,
Jeanrenaud
B
.
Abnormal regulation of the hypothalamo-pituitary-adrenal axis in the genetically obese fa/fa rat
.
Endocrinology
1990
;
126
:
1873
1879
[PubMed]
15.
Chan
O
,
Inouye
K
,
Vranic
M
,
Matthews
SG
.
Hyperactivation of the hypothalamo-pituitary-adrenocortical axis in streptozotocin-diabetes is associated with reduced stress responsiveness and decreased pituitary and adrenal sensitivity
.
Endocrinology
2002
;
143
:
1761
1768
[PubMed]
16.
Saito
M
,
Bray
GA
.
Diurnal rhythm for corticosterone in obese (ob/ob) diabetes (db/db) and gold-thioglucose-induced obesity in mice
.
Endocrinology
1983
;
113
:
2181
2185
[PubMed]
17.
Tannenbaum
BM
,
Brindley
DN
,
Tannenbaum
GS
,
Dallman
MF
,
McArthur
MD
,
Meaney
MJ
.
High-fat feeding alters both basal and stress-induced hypothalamic-pituitary-adrenal activity in the rat
.
Am J Physiol
1997
;
273
:
E1168
E1177
[PubMed]
18.
Langley
SC
,
York
DA
.
Effects of antiglucocorticoid RU 486 on development of obesity in obese fa/fa Zucker rats
.
Am J Physiol
1990
;
259
:
R539
R544
[PubMed]
19.
Saito
M
,
Bray
GA
.
Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse
.
Am J Physiol
1984
;
246
:
R20
R25
[PubMed]
20.
Miyawaki
K
,
Yamada
Y
,
Yano
H
, et al
.
Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice
.
Proc Natl Acad Sci U S A
1999
;
96
:
14843
14847
[PubMed]
21.
Kotelevtsev
Y
,
Holmes
MC
,
Burchell
A
, et al
.
11beta-hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress
.
Proc Natl Acad Sci U S A
1997
;
94
:
14924
14929
[PubMed]
22.
Baggio
LL
,
Huang
Q
,
Brown
TJ
,
Drucker
DJ
.
Oxyntomodulin and glucagon-like peptide-1 differentially regulate murine food intake and energy expenditure
.
Gastroenterology
2004
;
127
:
546
558
[PubMed]
23.
Campbell
JE
,
Peckett
AJ
,
D’souza
AM
,
Hawke
TJ
,
Riddell
MC
.
Adipogenic and lipolytic effects of chronic glucocorticoid exposure
.
Am J Physiol Cell Physiol
2011
;
300
:
C198
C209
[PubMed]
24.
Koehler
JA
,
Drucker
DJ
.
Activation of glucagon-like peptide-1 receptor signaling does not modify the growth or apoptosis of human pancreatic cancer cells
.
Diabetes
2006
;
55
:
1369
1379
[PubMed]
25.
Livak
KJ
,
Schmittgen
TD
.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
.
Methods
2001
;
25
:
402
408
[PubMed]
26.
Maida
A
,
Hansotia
T
,
Longuet
C
,
Seino
Y
,
Drucker
DJ
.
Differential importance of glucose-dependent insulinotropic polypeptide vs glucagon-like peptide 1 receptor signaling for beta cell survival in mice
.
Gastroenterology
2009
;
137
:
2146
2157
[PubMed]
27.
Temel
RE
,
Trigatti
B
,
DeMattos
RB
,
Azhar
S
,
Krieger
M
,
Williams
DL
.
Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells
.
Proc Natl Acad Sci U S A
1997
;
94
:
13600
13605
[PubMed]
28.
Hoekstra
M
,
Ye
D
,
Hildebrand
RB
, et al
.
Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production
.
J Lipid Res
2009
;
50
:
1039
1046
[PubMed]
29.
Rainey
WE
,
Saner
K
,
Schimmer
BP
.
Adrenocortical cell lines
.
Mol Cell Endocrinol
2004
;
228
:
23
38
[PubMed]
30.
Westendorf
JM
,
Schonbrunn
A
.
Peptide specificity for stimulation of corticotropin secretion: activation of overlapping pathways by the vasoactive intestinal peptide family and corticotropin-releasing factor
.
Endocrinology
1985
;
116
:
2528
2535
[PubMed]
31.
Parker
KL
,
Rice
DA
,
Lala
DS
, et al
.
Steroidogenic factor 1: an essential mediator of endocrine development
.
Recent Prog Horm Res
2002
;
57
:
19
36
[PubMed]
32.
Cao
G
,
Garcia
CK
,
Wyne
KL
,
Schultz
RA
,
Parker
KL
,
Hobbs
HH
.
Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1
.
J Biol Chem
1997
;
272
:
33068
33076
[PubMed]
33.
Cai
L
,
Ji
A
,
de Beer
FC
,
Tannock
LR
,
van der Westhuyzen
DR
.
SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance
.
J Clin Invest
2008
;
118
:
364
375
[PubMed]
34.
Kiss
A
,
Jezova
D
,
Aguilera
G
.
Activity of the hypothalamic pituitary adrenal axis and sympathoadrenal system during food and water deprivation in the rat
.
Brain Res
1994
;
663
:
84
92
[PubMed]
35.
Woodward
CJ
,
Hervey
GR
,
Oakey
RE
,
Whitaker
EM
.
The effects of fasting on plasma corticosterone kinetics in rats
.
Br J Nutr
1991
;
66
:
117
127
[PubMed]
36.
Friedman
JE
,
Yun
JS
,
Patel
YM
,
McGrane
MM
,
Hanson
RW
.
Glucocorticoids regulate the induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes
.
J Biol Chem
1993
;
268
:
12952
12957
[PubMed]
37.
Wajngot
A
,
Giacca
A
,
Grill
V
,
Vranic
M
,
Efendic
S
.
The diabetogenic effects of glucocorticoids are more pronounced in low- than in high-insulin responders
.
Proc Natl Acad Sci U S A
1992
;
89
:
6035
6039
[PubMed]
38.
Lambillotte
C
,
Gilon
P
,
Henquin
JC
.
Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexamethasone effects in mouse islets
.
J Clin Invest
1997
;
99
:
414
423
[PubMed]
39.
Karatsoreos
IN
,
Bhagat
SM
,
Bowles
NP
,
Weil
ZM
,
Pfaff
DW
,
McEwen
BS
.
Endocrine and physiological changes in response to chronic corticosterone: a potential model of the metabolic syndrome in mouse
.
Endocrinology
2010
;
151
:
2117
2127
[PubMed]
40.
Gault
VA
,
McClean
PL
,
Cassidy
RS
,
Irwin
N
,
Flatt
PR
.
Chemical gastric inhibitory polypeptide receptor antagonism protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat and cafeteria diets
.
Diabetologia
2007
;
50
:
1752
1762
[PubMed]

Supplementary data