Rap1 in the VMH regulates glucose homeostasis

Abstract

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

Keywords: G proteins; Glucose metabolism; Metabolism; Neuroscience; Signal transduction.

Figure 1. Rap1 mediates hyperglycemia in the hypothalamus.

(A and B) Increased hypothalamic Rap1 activity aggravates hyperglycemia under HFD conditions. Body weight (A) and blood glucose (B) of Rap1^V12 mice are shown. Six weeks after AAV-Rap1^V12 injection, mice were subjected to HFD feeding (n = 12/group). (C and D) Hypothalamic deletion of Rap1a and Rap1b prevents HFD-induced hyperglycemia. Shown are the body weight (C) and blood glucose (D) of Rap1^ΔHYP mice. One week after AAV2-Cre injection into the medial hypothalamus of the Rap1a and Rap1b double-floxed mice, HFD feeding started (n = 12–13/group). Values are presented as the mean ± SEM. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 compared with each group on day 0, by 1-way ANOVA followed by Dunnett’s multiple comparisons test, and *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group, by repeated measures 2-way ANOVA with Bonferroni’s multiple comparisons test.

Figure 2. Improved glucose homeostasis in Rap1^ΔSF1 mice.

(A) Representative images (original magnification, ×100; inset, original magnification, ×180) and quantification of Rap1 immunoreactivity in the hypothalamus of Rap1^ΔSF1 mice and control mice. The number of Rap1-positive cells in the ARC and the VMH was counted and is represented as a fold change relative to control (n = 3). (B) Representative Western blots and densitometric quantification of VMH Rap1 expression normalized to β-actin. (n = 7–8/group). See complete unedited blots in the supplemental material. (C–E) Glucose profiles of Rap1^ΔSF1 or control mice under normal chow conditions. Blood glucose (C, n = 6–15), glucose tolerance testing (D, n = 8), and insulin tolerance testing (E, n = 8) were measured. (F–H) Glucose profiles of HFD-fed Rap1^ΔSF1 or control mice (14 weeks of HFD feeding, n = 6–7). Glucose (F), glucose tolerance testing (G), and insulin tolerance testing (H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for 2-tailed t tests (A–C and F) or 2-way ANOVA followed by Bonferroni’s multiple comparisons tests (D, E, G, and H). All error bars are mean ± SEM.

Figure 3. Improved insulin sensitivity in Rap1^ΔSF1 mice.

(A–C) Hyperinsulinemic-euglycemic clamp studies in Rap1^ΔSF1 and littermate control mice fed an HFD for 18 weeks (n = 4–5). Shown are the GIR (A), peripheral glucose disposal rate (B), and 2-deoxy-d-glucose (2DG) uptake (C). (D) Western blot (left) and quantification (right) of AKT (Thr308) and glycogen synthase kinase-3β (Ser9) phosphorylation in liver, fat, and muscle at 10 minutes after a bolus injection of insulin (1 U/kg, i.p.) or saline into Rap1^ΔSF1 or control mice fed an HFD for 35 weeks. See complete unedited blots in the supplemental material. *P < 0.05 for 2-tailed t tests (A–C) or 2-way ANOVA followed by Bonferroni’s multiple comparisons tests (D). All error bars are mean ± SEM.

Figure 4. Body weight and adiposity of Rap1^ΔSF1 mice.

Shown are the weekly body weight (A) and body composition (B) in normal chow–fed male Rap1^ΔSF1 mice or control mice (n = 9–14). Additionally, shown are the weekly body weight (n = 10–15) (C) and body composition (n = 6–7) (D) under HFD conditions. The HFD was initiated at 7 weeks of age, and body composition was measured after 17 weeks of HFD feeding. All error bars are mean ± SEM.

Figure 5. Food intake and energy expenditure of Rap1^ΔSF1 mice.

Metabolic profiles of male Rap1^ΔSF1 mice or control mice (n = 7 per group) with respect to body weight (A), lean mass (B), fat mass (C), food intake (D), O₂ consumption (E), CO₂ production (F), heat production (G), ambulatory activity (H), rearing (I), and respiratory exchange ratio (J). *P < 0.05 for 2-tailed t tests (A–C and E–J) or 2-way ANOVA followed by Bonferroni’s multiple comparisons tests (D). All error bars are mean ± SEM.

Figure 6. Leptin responsiveness is increased in Rap1^ΔSF1 mice.

(A and B) Leptin (3 mg/kg, twice a day, i.p.) or vehicle was administered to normal chow–fed lean Rap1^ΔSF1 or control mice (n = 5–6). Shown are the body weight (A) and cumulative food intake (B). (C and D) HFD-fed Rap1^ΔSF1 or control mice (15 weeks of HFD) were injected with leptin (3 mg/kg, twice per day, i.p.) or vehicle (n = 6–7). Body weight (C) and food intake (D) were measured every day. (E and F) Leptin (3 mg/kg, i.p.) was administered to the indicated mice (n = 3 per group). (E) Representative immunohistochemistry images for phosphorylated STAT3, original magnification, ×100. (F) Quantification of immunohistochemistry. Age- and body weight–matched cohorts were used (A–F). *P < 0.05, **P < 0.01, and ***P < 0.001 for 2-tailed t tests (F), 1-way ANOVA followed by Tukey’s multiple comparison test (B) or 2-way ANOVA followed by Bonferroni’s multiple comparisons test (A, C, and D). All error bars are mean ± SEM.

Figure 7. ESI-05 improves glucose homeostasis independent of leptin action.

(A and B) Centrally administered ESI-05 (0.2 nmol/mouse, twice a day) lowered blood glucose (A) and improved glucose tolerance (B) in HFD-fed C57BL/6J mice (21 weeks of HFD). Age- and body weight–matched cohorts were used (n = 7–10). (C and D) The glucose-lowering effect of ESI-05 (0.2 nmol, twice a day) was not observed in brain-specific Rap1-deficient mice (Rap1^ΔCNS) fed an HFD for 5 weeks (n = 5–7) or in 6-week HFD-fed Rap1^ΔSF1 mice (n = 4–5). (E and F) Effect of ESI-05 (i.c.v., 0.2 nmol/mouse, twice a day) on blood glucose and glucose tolerance in ob/ob mice. Shown are the fed blood glucose (E) and glucose tolerance (F). *P < 0.05, **P < 0.01, and ***P < 0.001 for 2-tailed t tests (A–E) or 2-way ANOVA followed by Bonferroni’s multiple comparisons test (B and F). All error bars are mean ± SEM.