Hypothalamic MC4R regulates glucose homeostasis through adrenaline-mediated control of glucose reabsorption via renal GLUT2 in mice

Abstract

Aims/hypothesis: Melanocortin 4 receptor (MC4R) mutation is the most common cause of known monogenic obesity in humans. Unexpectedly, humans and rodents with MC4R deficiency do not develop hyperglycaemia despite chronic obesity and insulin resistance. To explain the underlying mechanisms for this phenotype, we determined the role of MC4R in glucose homeostasis in the presence and absence of obesity in mice.

Methods: We used global and hypothalamus-specific MC4R-deficient mice to investigate the brain regions that contribute to glucose homeostasis via MC4R. We performed oral, intraperitoneal and intravenous glucose tolerance tests in MC4R-deficient mice that were either obese or weight-matched to their littermate controls to define the role of MC4R in glucose regulation independently of changes in body weight. To identify the integrative pathways through which MC4R regulates glucose homeostasis, we measured renal and adrenal sympathetic nerve activity. We also evaluated glucose homeostasis in adrenaline (epinephrine)-deficient mice to investigate the role of adrenaline in mediating the effects of MC4R in glucose homeostasis. We employed a graded [¹³C₆]glucose infusion procedure to quantify renal glucose reabsorption in MC4R-deficient mice. Finally, we measured the levels of renal glucose transporters in hypothalamus-specific MC4R-deficient mice and adrenaline-deficient mice using western blotting to ascertain the molecular mechanisms underlying MC4R control of glucose homeostasis.

Results: We found that obese and weight-matched MC4R-deficient mice exhibited improved glucose tolerance due to elevated glucosuria, not enhanced beta cell function. Moreover, MC4R deficiency selectively in the paraventricular nucleus of the hypothalamus (PVH) is responsible for reducing the renal threshold for glucose as measured by graded [¹³C₆]glucose infusion technique. The MC4R deficiency suppressed renal sympathetic nerve activity by 50% in addition to decreasing circulating adrenaline and renal GLUT2 levels in mice, which contributed to the elevated glucosuria. We further report that adrenaline-deficient mice recapitulated the increased excretion of glucose in urine observed in the MC4R-deficient mice. Restoration of circulating adrenaline in both the MC4R- and adrenaline-deficient mice reversed their phenotype of improved glucose tolerance and elevated glucosuria, demonstrating the role of adrenaline in mediating the effects of MC4R on glucose reabsorption.

Conclusions/interpretation: These findings define a previously unrecognised function of hypothalamic MC4R in glucose reabsorption mediated by adrenaline and renal GLUT2. Taken together, our findings indicate that elevated glucosuria due to low sympathetic tone explains why MC4R deficiency does not cause hyperglycaemia despite inducing obesity and insulin resistance. Graphical abstract.

Keywords: Diabetes; Endocrinology; Hypothalamus; Melanocortin 4 receptor; Mouse model; Obesity.

Fig. 1

Improved glucose tolerance despite insulin resistance in 6h fasted obese male Mc4r null 24-week-old C57Bl/6 mice. (a) Body weight, (b) OGTT, (c) fasting plasma insulin levels, (d) ITT in obese Mc4r null 24-week-old C57Bl/6 mice. Bar graphs in (b) and (d) represent the corresponding AUC. Two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. **p<0.01, ***p<0.001;. Error bars are mean ± SEM. Mc4r^−/−, Mc4rloxTB/loxTB

Fig. 2

Elevated glucosuria but no enhancement in beta cell function post glucose administration in obese male Mc4r null 24-week-old C57Bl/6 mice. (a) Blood glucose levels during the IVGTT (mice were fasted for 6 h before the test); n=7 WT and n=6 Mc4r^−/−, (b) plasma insulin levels during IVGTT; n=7 (c) 24 h urine glucose concentration at baseline, (d) 24 h urine glucose concentration after administering 250 mg glucose by oral gavage, (e) 24 h urine glucose concentration after administering 200 mg glucose by i.p. injection. Two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. **p<0.01, ***p<0.001 vs WT; ^†p<0.01 vs 0 min, WT. Error bars are mean ± SEM. Mc4r^−/−, Mc4r^loxTB/loxTB

Fig. 3

Improved glucose tolerance and elevated glucosuria in male 18- to 24-week-old hypothalamus-specific Mc4r knockout mice. (a) Body weight, (b) OGTT (mice were fasted for 6 h before the test), (c) 24 h urine glucose concentration in Mc4r^loxP/loxP;Sim1^Cre mice. (d) Representative images of fluorescence in situ hybridisation showing reduced Mc4r in the PVH; n=5, four sections per mouse and four areas of interest per section were analysed. Scale bar, 100 μm. (e) Body weight, (f) OGTT, (g) urine [¹³C₆]glucose levels in Mc4r^loxP/loxP+AAV-Cre mice during the graded glucose infusion procedure; n=5. Bar graphs in (b) and (f) represent the corresponding AUC. Two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. *p<0.05, **p<0.01, ***p<0.001. Error bars are mean ± SEM. 3V, third ventricle

Fig. 4

Reduced renal sympathetic nerve activity and adrenal Pnmt expression in male 8- to 16-week-old Mc4r^loxP/loxP;Sim1^Cre mice vs Mc4r^loxP/loxP mice. (a) Plasma adrenaline, (b) plasma noradrenaline. (c) Representative traces of blood pressure, renal sympathetic nerve activity (RSNA) and adrenal sympathetic nerve activity (ADSNA). (d) RSNA, (e) ADSNA and (f) adrenal Pnmt expression in male 8- to 16-week-old Mc4r^loxP/loxP;Sim1^Cre mice vs Mc4r^loxP/loxP mice. Two-tailed unpaired Student’s t test was used for comparisons. *p<0.05, **p<0.01, ***p<0.001;. Error bars are mean ± SEM

Fig. 5

Adrenaline increases glucose transport in the mouse primary renal tubular epithelial (RPTE) cells and reverses improved glucose tolerance and elevated glucosuria in hypothalamus-specific MC4R-deficient mice. (a) Schematic of the experiment setup and cell culture inserts. (b) [¹³C₆]Glucose levels after treatment with adrenaline or noradrenaline in the RPTE cells; n=5 (c) OGTT, (d) 24 h urine glucose concentration at baseline, (e) 24 h urine glucose concentration after administering 250 mg glucose by oral gavage in 18- to 24-week-old Mc4r^loxP/loxP+AAV-Cre mice whose plasma adrenaline levels are restored. Two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. *p<0.05, **p<0.01, ***p<0.001 for adrenaline vs noradrenaline and HBSS groups in part (b) and for adrenaline group vs saline group in (c–e). Adr, adrenaline; Nor-Adr, noradrenaline; Sal, saline

Fig. 6

Blood and urine glucose levels in male 6- to 9-week-old-mice adrenaline-deficient (Pnmt-KO) mice before (a–g) and after (h–j) restoration of their plasma adrenaline levels. (a) Plasma adrenaline, (b) plasma noradrenaline, (c) OGTT, (d) 24 h urine glucose concentration at baseline, (e) 24 h urine glucose concentration after administering 250 mg glucose by oral gavage in adrenaline-deficient mice, (f) ITT, (g) plasma insulin levels in adrenaline-deficient mice, (h) plasma adrenaline (to validate its restoration), (i) OGTT, (j) 24 h urine glucose concentration at baseline and (k) 24 h urine glucose concentration after administering 100 mg glucose by i.p. injection, following restoration of plasma adrenaline in otherwise adrenaline-deficient mice. Bar graphs in (c), (f) and (i) represent the corresponding AUC. Two-tailed unpaired Student’s t test or repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test were used for comparisons. *p<0.05, **p<0.01, ***p<0.001.. Error bars are mean ± SEM. Adr, adrenaline; Sal, saline

Fig. 7

Decreased renal GLUT2 and Na⁺/K⁺-ATPase levels/activity in hypothalamus-specific MC4R- and adrenaline-deficient mice. (a, b) Decreased renal cortical GLUT2 levels in hypothalamus-specific MC4R-deficient (Mc4r^PVH) (a) and adrenaline-deficient (Pnmt-KO) (b) mice. (c, d) Increased renal cortical GLUT2 levels after treatment with adrenaline in hypothalamus-specific MC4R-deficient (c) and Pnmt-KO (d) mice. (e, f) Decreased renal Na⁺/K⁺-ATPase levels in hypothalamus-specific MC4R-deficient (e) and Pnmt-KO (f) mice. (g, h) Decreased renal Na⁺/K⁺-ATPase activity in hypothalamus-specific MC4R-deficient (g) and Pnmt-KO (h) mice. Proteins of interest and internal control were probed on the same membrane as described in the Methods. Two-tailed unpaired Student’s t test was used for comparisons. *p<0.05, **p<0.01, ***p<0.001. Error bars are mean ± SEM. Adr, adrenaline; Sal, saline; Mc4r^PVH, Mc4r^loxP/loxP with AAV-Cre; Ctrl, Mc4r^loxP/loxP with AAV-GFP