Obesity dysregulates fasting-induced changes in glucagon secretion

Abstract

Hyperglucagonemia, a hallmark in obesity and insulin resistance promotes hepatic glucose output, exacerbating hyperglycemia and thus predisposing to the development type 2 diabetes. As such, glucagon signaling is a key target for new therapeutics to manage insulin resistance. We evaluated glucagon homeostasis in lean and obese mice and people. In lean mice, fasting for 24 h caused a rise in glucagon. In contrast, a decrease in serum glucagon compared to baseline was observed in diet-induced obese mice between 8 and 24 h of fasting. Fasting decreased serum insulin in both lean and obese mice. Accordingly, the glucagon:insulin ratio was unaffected by fasting in obese mice but increased in lean mice. Re-feeding (2 h) restored hyperglucagonemia in obese mice. Pancreatic perfusion studies confirm that fasting (16 h) decreases pancreatic glucagon secretion in obese mice. Consistent with our findings in the mouse, a mixed meal increased serum glucagon and insulin concentrations in obese humans, both of which decreased with time after a meal. Consequently, fasting and re-feeding less robustly affected glucagon:insulin ratios in obese compared to lean participants. The glucoregulatory disturbance in obesity may be driven by inappropriate regulation of glucagon by fasting and a static glucagon:insulin ratio.

Keywords: fasting; glucagon; insulin resistance; obesity.

Conflict of interest statement

DECLARATION OF INTEREST

All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Figures

Figure 1.

Serum A) Glucagon, B) Insulin, C) Glucagon:Insulin ratio, D) Glucose, and E) Glucose:Insulin ratio in lean (fed: n=33 for Glucagon and Insulin, n= 14 for glucose), 4h: n=8, 8h: n=9, 16h: n=27, and 24h fasted: n=8 ) and diet-induced obese (fed: n=29, 4h: n=12, 8h: n=11, 16h: n=24, and 24h fasted: n=9) mice fed ad libitum, and fasted for 4, 8, 16, and 24 hours. Letters that differ indicate differences within group (a,b,c Lean, x,y,z Obese), P < 0.05; ****, ***,**,* indicates when obese differs from lean within timepoint, ****P≤0.0001, ***P<0.001, **P <0.01, *P<0.05. Bars indicate means ± SEM.

Figure 2.

Serum A) Glucagon, B) Insulin, C) Glucagon:Insulin Ratio, and D) Glucose in lean (n=17 for insulin and glucagon, n=6 for glucose) and diet-induced obese (n=23 for all) mice fed ad-libitum, fasted for 16h, and re-fed for 2h. Letters that differ indicate differences within group (a,b,c Lean, x,y,z Obese), P<0.05; ****, ***,**,* indicates when obese differs from lean within timepoint, ****P≤0.0001, ***P<0.001, **P <0.01, *P<0.05. Bars indicate means ± SEM.

Figure 3.

Liver A) cAMP in lean (fed: n=12, 4h: n=8, 8h: n=8, 16h: n=6, 24h fasted: n=8) and diet-induced obese (fed: n=13, 4h: n=11, 8h: n=12, 16h: n=7, 24h fasted: n=9) mice and B) glycogen in lean (fed: n=7, 4h: n=7, 8h: n=8, 16h: n=6, 24h fasted: n=6) and diet-induced obese (fed: n=7, 4h: n=11, 8h: n=10, 16h: n=6, 24h fasted: n=7 mice) mice fed ad libitum, or fasted for 4, 8, 16, or 24 hours. Letters that differ indicate differences within group (a,b,c Lean, x,y,z Obese), P < 0.05; **,* indicates when obese differs from lean within timepoint, **P <0.01, *P<0.05. Bars indicate means ± SEM.

Figure 4.

Pearson’s correlation (95% confidence interval) between A&C) serum glucagon and hepatic cAMP and B&D) serum insulin and hepatic cAMP in lean (n=41) and diet-induced obese mice (n=50).

Figure 5.

Phosphorylation levels of A,B,C) Ser133 in CREB, Ser63 in ATF-1, and total CREB protein, as well as D&E) phosphorylated Ser473 in AKT and total AKT protein in livers of lean (fasted: n=6, re-fed: n=8) and diet-induced obese (fasted: n=6, re-fed: n=6) mice fasted for 16h or re-fed for 2h after a 16h fast. A&D) Representative Western blots and B,C&E) densitometric quantification normalized to Histone H3. F (fasted), RF (re-fed); *P

Figure 6.

Relative mRNA expression of glucagon-responsive genes, A) Phosphoenolpyruvate Carboxykinase (PEPCK), B) Glucose 6-Phosphatase, C) Tyrosine Aminotransferase, and D) Carnitine Palmitoyl Transferase-1a (CPT-1a) in lean (fed: n=8, 4h: n=8, 8h: n=5, 16h: n=6, 24h fasted: n=4) and diet-induced obese (fed: n=12, 4h: n=12, 8h: n=10, 16h: n=6, 24h fasted: n=8) mice fed ad libitum, or fasted for 4, 8, 16, or 24 hours. Gene expression was normalized to the housekeeping gene ACTβ and presented as relative expression compare to the lean- fed group. Letters that differ indicate differences within group (a,b,c Lean, x,y,z Obese), P < 0.01; ****, ***,**,* indicates when obese differs from lean within timepoint, ****P≤0.0001, ***P<0.001, **P <0.01, *P<0.05. Bars indicate means ± SEM.

Figure 7.

Pancreatic secretion of A) Glucagon (pmol/L), B) Glucagon (% change from baseline), C) Somatostatin (ng/mL), and D) Somatostatin (% change from baseline) in response to insulin perfusion in lean fed (n=5), obese fed (n=7), and obese 16h fasted (n=8) mice. *Differs from fed obese, within timepoint P

Figure 8.

Plasma concentrations of A) Glucagon, B) Insulin, C) Glucagon:Insulin ratio, D) Glucose, and E) Glucose:Insulin ratio before (time 0) and for 6 h after ingesting a mixed meal consumed over 30 min in lean (n=9) and obese (n=17) participants. Letters that differ indicate differences within group (a,b Lean, x,y Obese) in response to the meal, P < 0.05; **,* indicates when obese differs from lean within timepoint, **P <0.01, *P<0.05. Values are means ± SEM.