Glucagon regulates hepatic mitochondrial function and biogenesis through FOXO1

in Journal of Endocrinology
Correspondence should be addressed to S Guo: shaodong.guo@tamu.edu
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Glucagon promotes hepatic glucose production maintaining glucose homeostasis in the fasting state. Glucagon maintains at high level in both diabetic animals and human, contributing to hyperglycemia. Mitochondria, a major place for glucose oxidation, are dysfunctional in diabetic condition. However, whether hepatic mitochondrial function can be affected by glucagon remains unknown. Recently, we reported that FOXO1 is an important mediator in glucagon signaling in control of glucose homeostasis. In this study, we further assessed the role of FOXO1 in the action of glucagon in the regulation of hepatic mitochondrial function. We found that glucagon decreased the heme production in a FOXO1-dependent manner, suppressed heme-dependent complex III (UQCRC1) and complex IV (MT-CO1) and inhibited hepatic mitochondrial function. However, the suppression of mitochondrial function by glucagon was largely rescued by deleting the Foxo1 gene in hepatocytes. Glucagon tends to reduce hepatic mitochondrial biogenesis by attenuating the expression of NRF1, TFAM and MFN2, which is mediated by FOXO1. In db/db mice, we found that hepatic mitochondrial function was suppressed and expression levels of UQCRC1, MT-CO1, NRF1 and TFAM were downregulated in the liver. These findings suggest that hepatic mitochondrial function can be impaired when hyperglucagonemia occurs in the patients with diabetes mellitus, resulting in organ failure.

 

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    Glucagon decreases mitochondrial function and oxygen consumption via FOXO1. (A) Oxygen consumption rate (OCR) was measured in HepG2 cells treated with 100 nM glucagon. HepG2 cells were cultured in DMEM medium supplied with 10% FBS and 1% P/S. Cells were treated with 100 nM glucagon for 2, 6, and 10 h. OCR was then measured. **P < 0.01 vs vehicle; #P < 0.05, ##P < 0.01 vs vehicle; $P < 0.05, $$P < 0.01 vs vehicle, n = 4. (B) ATP production was measured in control (CNTR) and L-F1KO primary mouse hepatocytes cultured in DMEM medium with 10% FBS and 1% P/S. Cells were treated with 100 nM glucagon for 10 h and ATP production measured by the ATP lite Luminescence, *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon, n = 5. (C) OCR was measured in the control primary mouse hepatocytes treated with or without 100 nM glucagon. Hepatocytes were cultured in DMEM medium supplied with 10% FBS and 1% P/S. The control hepatocytes were treated with or without 100 nM Glucagon for 10 h and OCR measured by seahorse analyzer. *P < 0.05, **P < 0.01 vs CNTR-Glucagon. (D) Basal respiration, maximal respiration, ATP production, and respiration capacity were calculated by OCR in control hepatocytes. *P < 0.05, **P < 0.01 vs CNTR-Vehicle, n = 3–5. (E) OCR was measured in L-F1KO hepatocytes with or without glucagon. L-F1KO hepatocytes were cultured in DMEM medium supplied with 10% FBS and 1% P/S, then treated with or without 100 nM glucagon for 10 h and OCR measured. (F) Basal respiration, maximal respiration, ATP production, and respiration capacity were calculated by OCR in L-F1KO hepatocytes. (G) OCR was measured in the control and db/db primary mouse hepatocytes. Hepatocytes were cultured in DMEM medium supplied with 10% FBS and 1% P/S, then OCR measured by seahorse analyzer. *P < 0.05, **P < 0.01 vs CNTR, n = 4–7. (H) Basal respiration, maximal respiration, ATP production, and respiration capacity were calculated by OCR in control and db/db hepatocyte. *P < 0.05, **P < 0.01 vs CNTR, n = 4–7. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0081.

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    Heme biogenesis genes, Fxn and Urod, are regulated by FOXO1 in the liver. (A) Relative mRNA level of Foxo1 was determined by qPCR in control primary mouse hepatocytes treated with or without 100 nM glucagon. *P < 0.05, **P < 0.01 vs 0 min treatment, n = 3. (B) FOXO1 protein level was measured by Western blot in primary mouse primary hepatocytes. *P < 0.05, **P < 0.01 vs 0 min treatment, n = 3. (C) Scatter plot of differentially expressed genes between livers from 18 h-fasted control and L-F1KO male mice. (D) Functional analysis of differentially expressed genes in the liver of 18 h-fasted control and L-F1KO male mice. (E) Relative mRNA levels of Foxo1, Fxn, and Urod between livers of 18 h-fasted control and L-F1KO male mice. **P < 0.01 vs CNTR, n = 3. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0081.

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    Glucagon reduces hepatic heme pool by decreasing gene expression of Fxn and Urod. (A) Relative mRNA levels of Fxn and Urod in control and L-F1KO primary mouse hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon, n = 3. (B) Relative mRNA expression of FXN and UROD was detected in HepG2 cells transfected with 4 µg plasmid DNA expressing control green fluorescence protein (GFP) or FOXO1 and treated with 100 nM glucagon for 10 h. FXN and UROD mRNA levels were measured by qPCR. *P < 0.05 vs group without Glucagon treatment and FOXO1 overexpression. (C) Principal component analysis of metabolites in the liver from random-fed control and L-F1KO male mice i.p. injected with 16 µg/kg glucagon for 1 h. (D) The relative value of Heme in the livers of random-fed control and L-F1KO male mice i.p. injected with 16 µg/kg glucagon for 1 h. *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon, n = 5. (E) Relative mRNA levels of Fxn and Urod in the fasted control and db/db mice livers. **P < 0.01 vs CNTR, n = 3–4. (F) The relative value of Heme in the fasted livers of control and db/db mice livers. *P < 0.05 vs CNTR, n = 3–4. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0081.

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    Glucagon decreases hepatic mitochondrial ETC integrity. (A) The relative mRNA expression of Mt-nd6 (complex I), Sdha (complex II), Uqcrc1 (complex III), Mt-co1 (complex IV), Atp5a (complex V) in control and L-F1KO primary hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon. (B and C) The protein levels of FOXO1, UQCRC1 and MT-CO1 in control and L-F1KO primary hepatocytes with or without 100 nM glucagon for 10 h were determined by Western blotting and (C) quantification of FOXO1, UQCRC1 and MT-CO1 protein levels. *P < 0.05, **P < 0.01 vs CNTR-Vehicle; #P < 0.05, ##P < 0.01 vs CNTR-Glucagon. (D and E) The protein levels of UQCRC1 and MT-CO1 in the fasted control and db/db mice livers were determined by Western blotting and (E) quantification of UQCRC1 and MT-CO1 protein levels. *P < 0.05, **P < 0.01 vs CNTR.

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    The expression of FXN, UROD, UQCRC1, and MT-CO1 in the mice livers under fasting and feeding state. (A and B) Relative mRNA levels of Fxn and Urod in liver from 18 h-fasted and random-fed control and L-F1KO male mice. *P < 0.05 vs CNTR-Vehicle. (C and D) The protein levels of MT-CO1 and UQCRC1 in the livers of random-fed and 18 h-fasted control and L-F1KO male mice were detected via Western blotting and (D) quantification of UQCRC1 and MT-CO1 protein levels. *P < 0.05 vs Fasted-CNTR.

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    Glucagon promotes fatty acid oxidation to compensate energy deficit. (A, B, C and D) Fatty acid oxidation (FAO) was measured in primary hepatocytes. The hepatocytes were cultured in substrate-limited medium 24 h prior to the assay and treated with 100 nM glucagon for 10 h. FAO assay medium was used to culture hepatocytes 45 min before the assay and then palmitate was added into wells just before assay, followed by OCR measurement. Fatty acid oxidation in control primary hepatocytes (A). *P < 0.05; **P < 0.01 vs CNTR-Vehicle; ##P < 0.01 vs CNTR-Glucagon. Fatty acid oxidation in L-F1KO primary hepatocytes (C). **P < 0.01 vs L-F1KO-Glucagon; ##P < 0.01 vs L-F1KO-Vehicle; $P < 0.05 vs L-F1KO-Vehilce-Palmitate. Basal respiration and ATP production were calculated by OCR in control (B) and L-F1KO (D) hepatocytes. *P < 0.05, **P < 0.01 vs indicated; #P < 0.05 vs L-F1KO-Vehicle-Palmitate. (E) The mRNA expressions of Hadha, Cpt1a, Vlcad, and Acadm were measured in control and L-F1KO hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05, **P < 0.01 vs CNTR-Vehicle; ##P < 0.01 vs L-F1KO Vehicle. (F) The mRNA expressions of Hadha, Cpt1a, Vlcad, and Acadm in livers of random-fed and 18 h-fasted control and L-F1KO male mice were measured by qPCR. *P < 0.05, **P < 0.01 vs indicated. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0081.

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    Glucagon attenuates mitochondrial biosynthesis in the liver. (A) mtDNA copy numbers were measured relative to the nuclear DNA in control and L-F1KO hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon, n = 3. (B) Electron microscopy study of mitochondrial biogenesis and morphology in the livers from random-fed control and L-F1KO male mice injected with or without 16 μg/kg glucagon for 1h. Scale bar, 500 nm. (C) The mRNA expressions of TFAM and NRF1 were measured in control and L-F1KO hepatocytes treated with or without 100 nM glucagon for 10 h by qPCR. *P < 0.05 vs CNTR; #P < 0.05 vs CNTR-Glucagon. (D) The mRNA expressions of TFAM and NRF1 in livers of the fasted control and db/db mice by qPCR. *P < 0.05 vs CNTR. (E and F) Western blots (F) and corresponding quantification (G) of TFAM and NRF1 in control and L-F1KO hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05 vs CNTR-Vehicle; #P < 0.05 vs CNTR-Glucagon. (G and H) Western blots (G) and corresponding quantification (H) of TFAM and NRF1 in livers of fasted control and db/db mice. **P < 0.01 vs CNTR. (I) The mRNA expressions of Ppargc1a and Sirt1 were measured in control and L-F1KO primary hepatocytes treated with or without 100 nM glucagon for 10 h. *P < 0.05, **P < 0.01 vs CNTR-Vehicle; #P < 0.05, ##P < 0.01 vs CNTR-Glucagon. (J) The mRNA expression of Fis1, Drp1, Mfn1, and Mfn2 were detected in control and L-F1KO hepatocytes treated with or without 100 nM glucagon for 10 h by qPCR. *P < 0.05 vs CNTR-Glucagon.

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    A schematic diagram representing the function of glucagon in diabetic state. In diabetic condition, glucagon maintains at high level to significantly increase hepatic glucose production by glycogenolysis and gluconeogenesis. Meanwhile, high level of glucagon decreases the expression of FXN and UROD in a FOXO1-dependent manner to reduce the heme pool, impairing heme-dependent UQCRC1 (complex III) and MT-CO1 (complex IV). Moreover, glucagon also decreases NRF1 and TFAM to attenuate the hepatic mitochondrial biogenesis in a FOXO1-dependent manner. Therefore, hyperglucagonemia suppresses the hepatic mitochondrial function and biogenesis. The hyperglycemia and dysfunctional hepatic mitochondria rendered by hyperglucagonemia will further lead to other liver diseases. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0081.

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