Reduced renal sympathetic nerve activity contributes to elevated glycosuria and improved glucose tolerance in hypothalamus-specific Pomc knockout mice

Abstract

Objective: Hypothalamic arcuate nucleus-specific pro-opiomelanocortin deficient (ArcPomc^-/-) mice exhibit improved glucose tolerance despite massive obesity and insulin resistance. We demonstrated previously that their improved glucose tolerance is due to elevated glycosuria. However, the underlying mechanisms that link glucose reabsorption in the kidney with ArcPomc remain unclear. Given the function of the hypothalamic melanocortin system in controlling sympathetic outflow, we hypothesized that reduced renal sympathetic nerve activity (RSNA) in ArcPomc^-/- mice could explain their elevated glycosuria and consequent enhanced glucose tolerance.

Methods: We measured RSNA by multifiber recording directly from the nerves innervating the kidneys in ArcPomc^-/- mice. To further validate the function of RSNA in glucose reabsorption, we denervated the kidneys of WT and diabetic db/db mice before measuring their glucose tolerance and urine glucose levels. Moreover, we performed western blot and immunohistochemistry to determine kidney GLUT2 and SGLT2 levels in either ArcPomc^-/- mice or the renal-denervated mice.

Results: Consistent with our hypothesis, we found that basal RSNA was decreased in ArcPomc^-/- mice relative to their wild type (WT) littermates. Remarkably, both WT and db/db mice exhibited elevated glycosuria and improved glucose tolerance after renal denervation. The elevated glycosuria in obese ArcPomc^-/-, WT and db/db mice was due to reduced renal GLUT2 levels in the proximal tubules. Overall, we show that renal-denervated WT and diabetic mice recapitulate the phenotype of improved glucose tolerance and elevated glycosuria associated with reduced renal GLUT2 levels observed in obese ArcPomc^-/- mice.

Conclusion: Hence, we conclude that ArcPomc is essential in maintaining basal RSNA and that elevated glycosuria is a possible mechanism to explain improved glucose tolerance after renal denervation in drug resistant hypertensive patients.

Keywords: GLUT2; Glucose tolerance; Glycosuria; Hypothalamic POMC; Renal denervation; Sympathetic nervous system.

Graphical abstract

Figure 1

Improved glucose tolerance and elevated glycosuria associated with reduced renal sympathetic nerve activity in obese 22–24-wk old female ArcPomc^−/− mice. A) Improved glucose tolerance in ArcPomc^−/− mice; B) Elevated glycosuria in ArcPomc^−/− mice; C) Normal blood pressure in ArcPomc^−/− mice; D) Reduced renal sympathetic nerve activity (RSNA) in ArcPomc^−/− mice. *P < 0.05, ***P < 0.001, ****P < 0.0001, 2-tailed Student's t-test or 2-way RMANOVA followed by Tukey's multiple comparison test was used for comparisons as appropriate; n = 6. Error bars reflect mean ± SEM.

Figure 2

Immunofluorescence detection of GLUT2 and SGLT2 levels in 24-wk ArcPomc^−/− mice. A) Reduced GLUT2 levels in proximal tubular cells, but no change in its distribution or location, in ArcPomc^−/− mice; B) No change in proximal tubular SGLT2 levels in ArcPomc^−/− mice. *P < 0.05, 2-tailed Student's t-test was used for comparisons; n = 6. Error bars reflect mean ± SEM. Scale bar = 33 μm; images were taken under 40× objective lens field.

Figure 3

Glomerular morphology and urine electrolytes in 24-wk old ArcPomc^−/− mice. A) Glomerular hypertrophy in obese ArcPomc^−/− mice (H&E staining); Elevated B) Natriuresis; C) Kaliuresis; D) Chloriuresis in obese and weight-matched female ArcPomc^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student's t-test was used for comparisons; n = 6. Error bars reflect mean ± SEM. Scale bar = 33 μm; images were taken under 40× objective lens field. The red arrows point out the glomeruli.

Figure 4

Impact of high-fat diet (HFD) on glycemia in female ArcPomc^−/− mice. A) Reduced mass of daily food intake in HFD fed WT and ArcPomc^−/− mice relative to their regular chow (RC) fed controls, *P < 0.05 for RC vs. HFD; ^##P < 0.01 for RC ArcPomc^−/− vs. WT (n = 6), 2-way ANOVA followed by Tukey's multiple comparisons test (F_{1, 17} = 3.3, P < 0.05, interaction of genotype and diet); B) No change in daily calorie intake between HFD and RC fed WT or ArcPomc^−/− mice. Increased calorie intake in ArcPomc^−/− mice relative to WT mice, ^∗∗P < 0.01 for ArcPomc^−/− vs. WT (n = 6), 2-way ANOVA followed by Tukey's multiple comparisons test; C) HFD increases body weight in WT and ArcPomc^−/− mice, *P < 0.05 for ArcPomc^−/− RC vs. HFD, ^#P < 0.01 for WT RC vs HFD, 2-way RMANOVA followed by Tukey's multiple comparisons test (F_{24, 112} = 91.6, P < 0.05, interaction of time and genotype); D) No change in fasting blood glucose levels in HFD fed ArcPomc^−/− mice relative to their RC controls, but an increase in fasting blood glucose levels in HFD fed WT mice compared to their RC controls, *P < 0.05 for WT RC vs. HFD, 2-way ANOVA followed by Tukey's multiple comparison test; E) No impairment in glucose tolerance in HFD fed ArcPomc^−/− mice; bar-graph shows AUC for the glucose tolerance test, *P < 0.05 for WT RC vs. HFD, ^##P < 0.05 for ArcPomc^−/− RC or HFD vs WT, 2-way ANOVA followed by Tukey's multiple comparison test; F) Elevated glycosuria in HFD fed ArcPomc^−/− mice. ^∗∗P < 0.01 for ArcPomc^−/− vs. WT, 2-way ANOVA followed by Tukey's multiple comparisons test. KO, ArcPomc^−/− mice; RC, regular chow; HFD, high-fat diet; AUC, area under the curve. Error bars reflect mean ± SEM.

Figure 5

Effects of renal denervation on glycemia in male WT and db/db mice. A) Renal denervation improves glucose tolerance in 8–10-wk old WT mice; B) Elevated glycosuria in renal-denervated WT mice; C) No change in insulin sensitivity after renal denervation in WT mice; D) Renal denervation improves glucose tolerance in db/db mice; E) Elevated glycosuria in renal-denervated 9-wk old db/db mice; F) Reduced glycosuria in renal-denervated 12-wk old db/db mice. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student's t-test or 2-way RMANOVA followed by Tukey's multiple comparison test was used for comparisons as appropriate; n = 6. RD, renal denervation; AUC, area under the curve. Error bars reflect mean ± SEM.

Figure 6

Representative western blots showing renal cortical GLUT2 and SGLT2 levels in renal-denervated WT and db/db mice. A) Decreased renal cortical GLUT2 levels in renal-denervated WT and db/db mice; B) No change in renal cortical SGLT2 levels in renal-denervated WT and db/db mice. *P < 0.05, 2-tailed Student's t-test was used for comparisons; n = 8 (4 samples per group on each blot). Error bars reflect mean ± SEM.

Figure 7

Effects of renal denervation on urine electrolytes in male WT and db/db mice. Elevated A) Natriuresis; B) Kaliuresis; C) Chloriuresis in renal-denervated WT and db/db mice. *P < 0.05, **P < 0.01, 2-tailed Student's t-test was used for comparisons; n = 6. Error bars reflect mean ± SEM.