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. 2010 Jun;298(6):E1226-35.
doi: 10.1152/ajpendo.00033.2010. Epub 2010 Mar 16.

Progressive Adaptation of Hepatic Ketogenesis in Mice Fed a High-Fat Diet

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Progressive Adaptation of Hepatic Ketogenesis in Mice Fed a High-Fat Diet

Nishanth E Sunny et al. Am J Physiol Endocrinol Metab. .
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Abstract

Hepatic ketogenesis provides a vital systemic fuel during fasting because ketone bodies are oxidized by most peripheral tissues and, unlike glucose, can be synthesized from fatty acids via mitochondrial beta-oxidation. Since dysfunctional mitochondrial fat oxidation may be a cofactor in insulin-resistant tissue, the objective of this study was to determine whether diet-induced insulin resistance in mice results in impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis. Ketone turnover (micromol/min) in the conscious and unrestrained mouse was responsive to induction and diminution of hepatic fat oxidation, as indicated by an eightfold rise during the fed (0.50+/-0.1)-to-fasted (3.8+/-0.2) transition and a dramatic blunting of fasting ketone turnover in PPARalpha(-/-) mice (1.0+/-0.1). C57BL/6 mice made obese and insulin resistant by high-fat feeding for 8 wk had normal expression of genes that regulate hepatic fat oxidation, whereas 16 wk on the diet induced expression of these genes and stimulated the function of hepatic mitochondrial fat oxidation, as indicated by a 40% induction of fasting ketogenesis and a twofold rise in short-chain acylcarnitines. Together, these findings indicate a progressive adaptation of hepatic ketogenesis during high-fat feeding, resulting in increased hepatic fat oxidation after 16 wk of a high-fat diet. We conclude that mitochondrial fat oxidation is stimulated rather than impaired during the initiation of hepatic insulin resistance in mice.

Figures

Fig. 1.
Fig. 1.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of plasma acetoacetate and β-hydroxybutyrate isotopomers. A: LC-MS/MS spectrum of β-hydroxybutyrate isotopomers isolated from mouse blood. B: relationship between infused ketone tracers and mass isotopomers detected in processed blood. M + 2 acetoacetate and M + 4 β-hydroxybutyrate were infused but are interconverted to M + 4 acetoacetate and M + 2 β-hydroxybutyrate by biological exchange through β-hydroxybutyrate dehydrogenase. Upon processing of the blood with sodium borodeuteride, plasma acetoacetate is chemically reduced to β-hydroxybutyrate but with a single mass unit increase in its molecular weight due to the addition of a deuterium. Thus, detection of M + 0, M + 2, and M + 4 mass isotopomers of β-hydroxybutyrate quantifies blood enrichment of β-hydroxybutyrate, whereas detection of M + 1, M + 3, and M + 5 mass isotopomers of β-hydroxybutyrate quantifies blood enrichment of acetoacetate. C: enrichment curves for acetoacetate (M + 3) and β-hydroxybutyrate (M + 4) tracers constructed with increasing tracer-to-tracee ratios for correction of measured enrichments by LC-MS/MS. D: a spillover curve was constructed with varying proportions of acetoacetate and β-hydroxybutyrate to calculate the contribution of M + 1 acetoacetate to the M + 2 of β-hydroxybutyrate after processing with sodium borodeuteride. The M + 1/M on the x-axis is a function of acetoacetate/β-hydroxybutyrate, whereas the M + 2/M on the y-axis is the resulting spillover correction for M + 2. This correction was applied as a function of M + 1/M in blood samples.
Fig. 2.
Fig. 2.
Ketone tracer enrichment measured by LC-MS/MS is corroborated by NMR measurements. A: comparison of a previously validated NMR method for determining ketone enrichment with the LC-MS/MS method demonstrates that the 2 methods provide identical measurements of β-hydroxybutyrate and acetoacetate tracer enrichments in rat plasma. B: ketone turnover in 24-h-fasted Long-Evans rats was not different when determined by NMR or LC-MS/MS.
Fig. 3.
Fig. 3.
Steady-state infusion of ketone tracers demonstrates induction of ketogenesis during fasting in mice. A: enrichment curves of β-hydroxybutyrate isotopomers in mouse blood during a 105-min infusion of ketone tracers demonstrates that steady state is reached by 75 min of infusion. B: 3 separate infusion experiments were repeated in the same C57BL/6 mice during the fed-to-24-h-fasted transition. Ketone turnover was stimulated substantially during the transition from feeding (n = 6) to 16 h of fasting (n = 5) but did not increase further by 24 h of fasting (n = 3) (attrition over the experiment was due to loss of catheter patency). C: total ketone concentration continued to rise throughout the fed-to-fasted transition despite a leveling of ketone turnover. The data are represented by means ± SE. *P ≤ 0.05.
Fig. 4.
Fig. 4.
In vivo ketone turnover in mice reflects alterations of hepatic fat oxidation associated with PPARα loss of function. A: fasting for 16 h induced an 8-fold rise in total plasma ketone bodies in wild-type mice, which were severely blunted in PPARα−/− mice. B: ketone turnover measured by ketone tracer dilution was likewise impaired in PPARα−/− mice after a 16-h fast, consistent with the known effects of PPARα loss of function and indicative that the method accurately reports alterations of hepatic ketogenesis. The data are represented by means ± SE. *P ≤ 0.05 between fed and fasted groups; **P ≤ 0.05 between PPARα+/+ and PPARα−/−.
Fig. 5.
Fig. 5.
High-fat feeding induces insulin resistance and increased hepatic fat oxidation in mice. A: mice fed a high-fat diet for either 8 or 16 wk had a dramatically impaired hepatic insulin sensitivity index. B: ketone turnover was unchanged after 8 wk of a 60% high-fat diet but was significantly elevated after 16 wk of the high-fat diet. C: liver acetylcarnitine was assayed by LC-MS/MS in mice fed a synthetic control diet (10% fat calories) or a high-fat diet (60% fat calories) for either 8 or 16 wk. D: high-fat feeding for 8 wk resulted in normal expression of genes that regulate hepatic fat oxidation relative to cyclophilin, whereas 16 wk on the diet induced expression of these genes. Data are presented as means ± SE. *P ≤ 0.05 between control and high-fat diet. **P ≤ 0.05 between 8 and 16 wk of diet intervention; #P ≤ 0.1 between control and high-fat diet; ##P ≤ 0.1 between 8 and 16 wk of diet intervention. HMGCS, hydroxylmethylglutaryl-CoA synthase; CPT Ia, carnitine palmitoyltransferase Ia.
Fig. 6.
Fig. 6.
Correlations of ketone turnover with liver and plasma metabolite concentrations. A: ketone turnover in all mice reported in this study (57 mice) correlates with plasma ketone concentration. The best correlation was obtained by a polynomial trend line, indicating that the highest ketone concentrations may be influenced by suppressed ketone utilization rather than increased hepatic ketogenesis. B: liver acetylcarnitine levels determined by LC-MS/MS correlated positively with ketone turnover in mice fed control or high-fat diets for 8 and 16 wk. C: ketone turnover correlated positively with nonesterified fatty acids (NEFA) in plasma of mice fed a control or high-fat diet for 8 and 16 wk. D: ketone turnover and hepatic triglyceride content trended toward a positive correlation but did not reach significance from mice fed a control or high-fat diet for 8 and 16 wk. Correlations were determined by Pearson's correlation and regression analysis. P < 0.05 was considered significant.

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