Leptin Mediates a Glucose-Fatty Acid Cycle to Maintain Glucose Homeostasis in Starvation

Abstract

The transition from the fed to the fasted state necessitates a shift from carbohydrate to fat metabolism that is thought to be mostly orchestrated by reductions in plasma insulin concentrations. Here, we show in awake rats that insulinopenia per se does not cause this transition but that both hypoleptinemia and insulinopenia are necessary. Furthermore, we show that hypoleptinemia mediates a glucose-fatty acid cycle through activation of the hypothalamic-pituitary-adrenal axis, resulting in increased white adipose tissue (WAT) lipolysis rates and increased hepatic acetyl-coenzyme A (CoA) content, which are essential to maintain gluconeogenesis during starvation. We also show that in prolonged starvation, substrate limitation due to reduced rates of glucose-alanine cycling lowers rates of hepatic mitochondrial anaplerosis, oxidation, and gluconeogenesis. Taken together, these data identify a leptin-mediated glucose-fatty acid cycle that integrates responses of the muscle, WAT, and liver to promote a shift from carbohydrate to fat oxidation and maintain glucose homeostasis during starvation.

Keywords: HPA axis; glucose-alanine cycling; leptin; starvation.

Figure 1. A decreased rate of hepatic glycogenolysis is the primary determinant of the switch from glucose to fat oxidation during starvation

(A) Plasma glucose. (B) Hepatic glucose production from net hepatic glycogenolysis, gluconeogenesis from oxaloacetate (i.e. V_PC), and gluconeogenesis from glycerol. *P<0.05 vs. 8 hr fasted rats and §§§§P<0.0001 vs. 16 hr fasted rats, in both cases comparing gluconeogenesis from oxaloacetate. (C) Percent glucose oxidation in the TCA cycle [pyruvate dehydrogenase flux (V_PDH)/citrate synthase flux (V_CS)]. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. fed rats; ####P<0.0001 vs. 6 hr fasted rats, §P<0.05, §§P<0.01 vs. 16 hr fasted rats. (D)–(E) Plasma leptin and corticosterone. (F) Endogenous glucose production before and 2 hrs after treatment with a glycogen phosphorylase inhibitor. (G)–(H) Plasma leptin and corticosterone in glycogen phosphorylase inhibitor-treated rats. In panels (F)–(H), data from the same rats before and after treatment with the inhibitor were compared by the paired Student’s t-test; in all other panels, ANOVA with Bonferroni’s multiple comparisons test was used. Data are the mean±S.E.M. of n=6–8 per group. See also Fig. S1 and S2.

Figure 2. Physiologic leptin replacement reduces WAT lipolysis and hepatic gluconeogenesis in 48 hr fasted rats, but supraphysiologic leptin reverses this effect by stimulation of catecholamine secretion

(A)–(B) Plasma leptin and glucose concentrations during a 6 hr infusion of stepwise increasing doses of leptin in 48 hr fasted rats. (C) Whole-body glucose turnover. (D)–(F) Plasma corticosterone, epinephrine and norepinephrine concentrations. (G)–(H) Whole-body fatty acid and glycerol turnover. (I) Liver acetyl-CoA content. (J) Whole-body β-OHB turnover. In all panels, n=8. Paired comparisons (ANOVA with Bonferroni’s multiple comparisons test) were performed. Data are the mean±S.E.M. See also Fig. S3.

Figure 3. Increased hepatic acetyl-CoA content maintains euglycemia during starvation

(A)–(C) Whole-body fatty acid, glycerol, and β-OHB turnover. (D)–(E) Hepatic acetyl- and malonyl-CoA content. In panels (A)-(E), n=7 per time point, and data were compared by ANOVA with Bonferroni’s multiple comparisons test. (F)–(G) Plasma glucose and whole-body glucose turnover 2 hr after treatment with etomoxir. (H) Liver acetyl-CoA content. (I)–(J) Plasma leptin and corticosterone concentrations. In panels (F)–(J), data are the mean±S.E.M. of n=6 per group. Data were compared by ANOVA with Bonferroni’s multiple comparisons test (control vs. etomoxir vs. atglistatin, with these groups compared separately at each time point; control data are duplicated between Fig. 3 and Fig. 4). See also Fig. S4.

Figure 4. Increased WAT lipolysis is necessary to increase hepatic acetyl-CoA content and maintain euglycemia in the starved state

(A) Plasma glucose concentrations in control and atglistatin-treated rats. (B)–(D) Whole-body fatty acid, glycerol, and β-OHB turnover. (E) Hepatic acetyl-CoA content. (F) Whole-body glucose turnover. (G)–(H) Plasma leptin and corticosterone concentrations. In all panels, data are the mean±S.E.M. of 5–6 rats per group, and control data are duplicated from Fig. 3. Data were compared by ANOVA with Bonferroni’s multiple comparisons test (control vs. etomoxir vs. atglistatin, with these groups compared separately at each time point. See also Fig. S5.

Figure 5. Glucocorticoid activity is required to maintain WAT lipolysis and euglycemia in starvation

(A)–(B) Plasma glucose and lactate concentrations before and after treatment with mifepristone. (C)–(D) Whole-body fatty acid and glycerol turnover. (E) Hepatic acetyl-CoA content. (F)–(G) Whole-body β-OHB and glucose turnover. (H) Plasma leptin concentrations. In all panels, data are the mean±S.E.M. of n=6, with data from before and after mifepristone treatment compared by the 2-tailed paired Student’s t-test. See also Fig. S6.

Figure 6. Reductions in glucose-alanine cycling with prolonged starvation lead to suppression of hepatic gluconeogenesis and hepatic mitochondrial oxidation

(A) Whole-body alanine turnover. (B) Hepatic citrate concentrations. (C) Liver V_CS flux. (D) Plasma glucose concentrations after a 2 hr infusion of alanine [45 μmol/(kg-min)] in 48 hr fasted rats. (E)–(F) Whole-body glucose turnover and liver V_PC flux. (G) Liver citrate. (H) Hepatic mitochondrial citrate synthase (V_CS) flux. In all panels, data are the mean±S.E.M. of n=6–8 per group, with data compared by ANOVA [panels (A)–(C)] or by the 2-tailed unpaired Student’s t-test [panels (D)–(H)]. See also Fig. S7.

Figure 7. Reductions in plasma glucose concentrations from ~6 mM to ~5 mM during prolonged starvation lead to reduced glucose-alanine cycling, hypoleptinemia and HPA axis activation

(A)–(B) Plasma insulin and NEFA concentrations. (C) Muscle glucose uptake in 48 hr fasted rats with and without an infusion of glucose to increase plasma glucose concentrations from 5 to 6 mM. (D) Whole-body alanine turnover. (E) White adipose tissue glucose uptake. (F)–(H) Plasma leptin, ACTH, and corticosterone concentrations. (I)–(J) Whole-body alanine and fatty acid turnover in rats treated with an inhibitor of glycogen phosphorylase. In panels (C) and (E) data were compared by the 2-tailed unpaired Student’s t-test; otherwise the paired t-test was used. Data are the mean±S.E.M. of n=6–7 per group. See also Fig. S8.