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, 63 (10), 3242-52

Regulation of Substrate Oxidation Preferences in Muscle by the Peptide Hormone Adropin

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Regulation of Substrate Oxidation Preferences in Muscle by the Peptide Hormone Adropin

Su Gao et al. Diabetes.

Abstract

Rigorous control of substrate oxidation by humoral factors is essential for maintaining metabolic homeostasis. During feeding and fasting cycles, carbohydrates and fatty acids are the two primary substrates in oxidative metabolism. Here, we report a novel role for the peptide hormone adropin in regulating substrate oxidation preferences. Plasma levels of adropin increase with feeding and decrease upon fasting. A comparison of whole-body substrate preference and skeletal muscle substrate oxidation in adropin knockout and transgenic mice suggests adropin promotes carbohydrate oxidation over fat oxidation. In muscle, adropin activates pyruvate dehydrogenase (PDH), which is rate limiting for glucose oxidation and suppresses carnitine palmitoyltransferase-1B (CPT-1B), a key enzyme in fatty acid oxidation. Adropin downregulates PDH kinase-4 (PDK4) that inhibits PDH, thereby increasing PDH activity. The molecular mechanisms of adropin's effects involve acetylation (suggesting inhibition) of the transcriptional coactivator PGC-1α, downregulating expression of Cpt1b and Pdk4. Increased PGC-1α acetylation by adropin may be mediated by inhibiting Sirtuin-1 (SIRT1), a PGC-1α deacetylase. Altered SIRT1 and PGC-1α activity appear to mediate aspects of adropin's metabolic actions in muscle. Similar outcomes were observed in fasted mice treated with synthetic adropin. Together, these results suggest a role for adropin in regulating muscle substrate preference under various nutritional states.

Figures

Figure 1
Figure 1
A: The AdrKO mice and WT littermates were maintained in a fed condition. RER was monitored in one dark-light cycle (n = 4–9). B: Food was removed from the AdrTG mice and the WT littermates before the dark onset, and RER was monitored in the following dark-light cycle (n = 6–10). The six-period moving average trend line was calculated to represent the data points. *P < 0.05.
Figure 2
Figure 2
A: In the AdrKO group, complete FAO, indicated by CO2 production, and incomplete FAO, indicated by ASM production, in muscle lysate were measured (n = 7–8). B: Cpt1b message levels were measured (n = 6–7). C: Muscle mitochondria were isolated and CPT-1 activity measured. The activity assay was performed in three separate groups. The results in individual groups were converted to % WT and pooled (n = 11–13). D: LC-ACs, including 16:0 (palmitoylcarnitine), 18:0 (stearoylcarnitine), and 18:1 (oleoylcarnitine), in muscle were quantified (n = 3–5). E: In the AdrTG group, complete FAO and incomplete FAO in muscle lysate were measured (n = 4–11). F: Cpt1b message levels were measured (n = 5–8). G: Muscle CPT-1 activity assay was performed in two separate groups. The results in individual groups were converted to % WT and pooled (n = 6–10). H: LC-ACs in muscle were quantified (n = 4–7). *,**, and ***P < 0.05.
Figure 3
Figure 3
The AdrKO group was maintained in the fed state, whereas the AdrTG group was fasted overnight (16 h) before tissue samples were collected. A: Whole-muscle lysates containing intact mitochondria were prepared. Pyruvate oxidation or PDH activity was measured (AdrKO group, n = 8; AdrTG group, n = 6–10). B: Pdk4 message levels were measured (AdrKO, n = 6; AdrTG, n = 5–7). C: PDK4 protein levels were measured (AdrKO, n = 6; AdrTG, n = 6). D: The ratios of the free CoA/acetyl-CoA (CoA/Ac-CoA) were calculated (AdrKO, n = 7–8; AdrTG, n = 6–10). *,**, and ***P < 0.05.
Figure 4
Figure 4
The AdrKO group was maintained in the fed state, and the AdrTG group was fasted overnight (16 h). A: PGC-1α protein was immunoprecipitated from whole-tissue lysate, and the immunoprecipitated products were immunoblotted for acetylated lysine (Ac-K) (AdrKO group, n = 5–6; AdrTG, n = 6–8). The light chain of IgG (IgG-LC) was used as the loading control. (Note: the blotting of the two groups was performed in separate runs.) B: PGC-1α protein was immunoprecipitated from whole-tissue lysate, and the immunoprecipitated products were immunoblotted for PGC-1α protein (AdrKO group, n = 4; AdrTG, n = 4). The IgG-LC was used as the loading control. C: The PGC-1α protein levels in the same lysate were directly measured by immunoblotting (AdrKO group, n = 4–6; AdrTG group, n = 6). IB, immunoblotting; IP, immunoprecipitation. *P < 0.05.
Figure 5
Figure 5
The AdrKO group was maintained in the fed state, and the adropin TG group was fasted overnight (16 h). A: SIRT1 protein levels in whole-muscle lysates were measured (AdrKO group, n = 6–7; AdrTG, n = 6–7). (Note: the blotting of the two groups was performed in separate runs.) B: SIRT deacetylase activities in muscle nuclear extracts were measured (AdrKO group, n = 6–7; AdrTG, n = 5–6). The activity of the fasted WT mice in the AdrTG group is significantly higher than that of the fed WT mice in the AdrKO group. C: Acetylated p53 (Ac-p53) levels and p53 levels in muscle nuclear extracts were measured (Ac-p53, n = 4; p53, n = 3–5). D: The NAD+ levels in whole-muscle extracts were measured (AdrKO group, n = 6; AdrTG, n = 6). *,**, and ***P < 0.05.
Figure 6
Figure 6
Food was removed in the early light cycle, and the WT lean mice were fasted for the next 24 h. Two intraperitoneal injections of adropin (or vehicle) separated by 8 h were administered during the light cycle. At the end of fasting on the next day, the third injection of adropin (or vehicle) was given. The mice were euthanized 2 h following the third injection and muscle tissues isolated. A: SIRT1 protein levels in whole-tissue lysates were measured (n = 3–5). PGC-1α protein was immunoprecipitated from whole lysate, and the immunoprecipitated products were immunoblotted for acetylated lysine (Ac-K) (n = 4–5). The light chain of IgG (IgG-LC) was used as the loading control. B and C: The message level of Cpt1b (n = 5) and Pdk4 (n = 5) were measured. D: Mitochondria were isolated and CPT-1 activity measured. The activity assay was performed in two separate groups. The results in individual groups were converted to % vehicle-treated and pooled (n = 8). E: LC-ACs, including 16:0 (palmitoylcarnitine) and 18:1 (oleoylcarnitine), were quantified (n = 4–5). F: FAO levels in the lysate containing intact mitochondria were measured, and the production of ASM is shown (n = 7–8). G: Pyruvate oxidation or PDH activity was measured (n = 6–8). IB, immunoblotting; IP, immunoprecipitation. *P < 0.05.

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