Leptin-mediated changes in hepatic mitochondrial metabolism, structure, and protein levels

Abstract

Leptin reduces body weight in ob/ob mice by decreasing food intake and increasing energy expenditure; however, the mechanisms by which it does the latter are not known. Here we report that 30% of the weight loss induced by leptin treatment of ob/ob mice is due to changes in energy expenditure. In assessing leptin's effects on specific tissues, we found that hepatic basal metabolic rate was paradoxically decreased 1.7-fold with leptin treatment, which was the result of a 1.6-fold reduction in mitochondrial volume density and altered substrate oxidation kinetics. The altered kinetics were associated with a decrease in protein levels of 2 mitochondrial respiratory chain components--cytochrome c oxidase subunit VIa and cytochrome c oxidase subunit IV. In addition to reduced hepatic metabolism, there was reduced long chain fatty acid production and a 2.5-fold increase in hepatic lipid export, both of which explain the reduced steatosis in leptin-treated animals. These data help clarify the role of the liver in leptin-mediated weight loss and define the mechanisms by which leptin alters hepatic metabolism and corrects steatosis.

Fig. 1.

Components of leptin-mediated weight loss and leptin-mediated correction of hepatic steatosis. (A) ob/ob mice have a lower core body temperature than wild-type mice (measured by rectal thermometer), which is corrected by leptin infusion. Housing the mice at thermoneutral temperatures increases the core body temperature of ob/ob mice and diminishes the significance of body temperature correction by leptin. Ω, P < 0.001; *, P < 0.0001. (B) By leptin treating and pair feeding mice at room and thermoneutral temperatures, we were able to distinguish different components of leptin-mediated weight loss. The difference between each pair-fed and leptin-treated group at a given temperature represents weight loss due to leptin-mediated increases in energy expenditure and basal metabolic rate (P < 0.0001). The difference between the same treatment administered at different temperatures represents weight loss due to ambient temperature dependent effects on satiety (P < 0.0001). (C) Leptin-mediated correction of hepatic steatosis has 1 component that is correlated with ambient temperature dependent induction of satiety by leptin, which is represented by the difference between each pair-fed and leptin-treated group at a given temperature (P < 0.01). It also has another component that is correlated with leptin induced increases in energy expenditure, which is represented by the difference between the same treatment administered at different temperatures (P < 0.01) (D) Plasma levels of triglyceride are increased much faster in 12-day leptin-treated ob/ob mice (■) than in saline-treated littermates (♦) after the injection of 0.5 mg/kg tyloxapol, an inhibitor of VLDL hydrolysis, via the tail vein. The slope of the line indicates the rate of VLDL production, which is increased by 2.48-fold with leptin treatment. *, P < 0.05. Error bars show SEM in all figures.

Fig. 2.

Leptin decreases hepatocyte basal metabolic rate, nonmitochondrial respiration, and alters hepatic mitochondrial respiration rates. (A) Twelve day leptin treatment of ob/ob mice reduces primary hepatocyte basal metabolic rate (respiration in an isotonic medium containing 10 mM glucose) as compared to saline treatment. (B) Nonmitochondrial respiration (respiration in the presence of myxothiazol) is also decreased with 12 days of leptin treatment. (C) Hepatic mitochondrial respiratory capacity (respiration in the presence of FCCP) is increased in ob/ob mice in relation to their wild-type littermates, and 12 days of leptin treatment brings it back toward wild-type levels. This suggests an increase in the substrate oxidation system during leptin deficiency, and its normalization with leptin treatment. (D and E) State 2 and state 4 rates are both increased in ob/ob mice in relation to wild-type littermates as well, indicating that mitochondrial proton leak rate is increased with leptin deficiency. Twelve days of leptin treatment decreases state 2 and 4 rates in ob/ob mice back to wild-type levels. n = 5 for all points, and each was done in duplicate. α,▵, P < 0.01; β, P < 0.05.

Fig. 3.

Leptin directly modifies hepatic mitochondrial substrate oxidation kinetics. Hepatic mitochondrial leak kinetics (A) and phosphorylation system kinetics (B) do not differ between ob/ob and wild-type mice, and are not affected by 12 days of leptin treatment. (C) The substrate oxidation system, however, is significantly increased in ob/ob mice in comparison to their wild-type littermates (P < 0.0001). Twelve days of leptin treatment of these ob/ob mice significantly reduces the substrate oxidation system back toward wild-type levels (P < 0.0001). (E) Mitochondrial respiratory capacity is increased in hypoleptinemic mice (NC) with respect to saline-treated controls (PBS). Restoration of leptin levels in these mice (FF) brings respiratory capacity back to control levels. (D) Hepatic mitochondrial state 4 respiration, however, is unchanged in both wild-type hypoleptinemic mice (NC) and in those animals with restored levels of leptin (FF) with respect to saline-treated controls (PBS). α,β, P < 0.01. n = 5 for all points, and each was done in duplicate.

Fig. 4.

Electron microscopic analysis of hepatic mitochondrial structure and size. (A) Mitochondria of 12 day leptin treated ob/ob mice look lighter and ballooned compared to those of 12 day pair-fed (B) and 12 day PBS-treated (C) ob/ob mice. Cristae structure also looks quite different in (A) compared with (B and C). (D) Mitochondrial volume density is significantly lower in leptin-treated ob/ob mice versus pair-fed and PBS-treated ob/ob mice. (E) Leptin treatment of ob/ob mice does not change the area of individual mitochondria, however pair feeding significantly increases it. A total of 168 sections from n = 5 mice in each group were taken and yielded similar results. α,▵, P < 0.01; β,γ = P < 0.05