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. 2019 Sep 6;294(36):13366-13377.
doi: 10.1074/jbc.RA119.008967. Epub 2019 Jul 19.

The peptide hormone adropin regulates signal transduction pathways controlling hepatic glucose metabolism in a mouse model of diet-induced obesity

Su Gao  1 , Sarbani Ghoshal  2 , Liyan Zhang  3 , Joseph R Stevens  2 , Kyle S McCommis  4 , Brian N Finck  4 , Gary D Lopaschuk  3 , Andrew A Butler  5
Affiliations

The peptide hormone adropin regulates signal transduction pathways controlling hepatic glucose metabolism in a mouse model of diet-induced obesity

Su Gao et al. J Biol Chem. .

Abstract

The peptide hormone adropin regulates energy metabolism in skeletal muscle and plays important roles in the regulation of metabolic homeostasis. Besides muscle, the liver has an essential role in regulating glucose homeostasis. Previous studies have reported that treatment of diet-induced obese (DIO) male mice with adropin34-76 (the putative secreted domain) reduces fasting blood glucose independently of body weight changes, suggesting that adropin suppresses glucose production in the liver. Here, we explored the molecular mechanisms underlying adropin's effects on hepatic glucose metabolism in DIO mice. Male DIO B6 mice maintained on a high-fat diet received five intraperitoneal injections of adropin34-76 (450 nmol/kg/injection) over a 48-h period. We found that adropin34-76 enhances major intracellular signaling activities in the liver that are involved in insulin-mediated regulation of glucose homeostasis. Moreover, treatment with adropin34-76 alleviated endoplasmic reticulum stress responses and reduced activity of c-Jun N-terminal kinase in the liver, explaining the enhanced activities of hepatic insulin signaling pathways observed with adropin34-76 treatment. Furthermore, adropin34-76 suppressed cAMP activated protein kinase A (PKA) activities, resulting in reduced phosphorylation of inositol trisphosphate receptor, which mediates endoplasmic reticulum calcium efflux, and of cAMP-responsive element-binding protein, a key transcription factor in hepatic regulation of glucose metabolism. Adropin34-76 directly affected liver metabolism, decreasing glucose production and reducing PKA-mediated phosphorylation in primary mouse hepatocytes in vitro Our findings indicate that major hepatic signaling pathways contribute to the improved glycemic control achieved with adropin34-76 treatment in situations of obesity.

Keywords: adropin; c-Jun N-terminal kinase (JNK); endoplasmic reticulum stress (ER stress); energy metabolism; gluconeogenesis; insulin receptor substrate 1 (IRS1); insulin resistance; lipogenesis; protein kinase A (PKA); type 2 diabetes.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Adropin34–76 treatment enhanced IRS signaling in the liver. A, the phosphorylation levels of Ser307 in IRS1 (n = 3–5) and total IRS1 protein levels (n = 4–5) as well as the phosphorylation levels of Tyr608 in IRS1 immunoprecipitates (IP) (n = 4) were measured by Western blotting (IB). The Western blotting of the phosphorylation levels of Ser307 in IRS1 were repeated (n = 4–5), and similar changes were detected. α-Tubulin was used as the loading control for pIRS1 (Ser307) and total IRS1. The same α-tubulin band serving as the loading control for total IRS1 was used as the loading control for the blots of pAKT (Ser473) and total AKT (Fig. 2A) and the blots of pIKK (α/β) (Ser176/180) and total IKK (α/β) (Fig. S6). B, IRS2 protein levels (n = 4–5) and message levels (Irs2) (n = 5–6) were determined by Western blotting and RT-PCR, respectively. In Western blotting, GAPDH was used as the loading control for IRS2. The same GAPDH band was used as the loading control for the blots of p-c-Jun (Ser63) and total c-Jun (Fig. 4E) and the blots of pCREB (Ser133) and total CREB (Fig. 8B). *, p ≤ 0.05; ***, p < 0.0005, adropin versus vehicle. Error bars, S.E.
Figure 2.
Figure 2.
Adropin34–76 treatment enhanced AKT signaling in the liver. A and B, the phosphorylation levels of Ser473 in AKT and total AKT protein levels (n = 4) (A) and the phosphorylation levels of Ser9 in GSK 3β and total GSK 3β protein levels (n = 4–5) (B) were determined by Western blotting. In A, α-tubulin was used as the loading control. The same α-tubulin band was used as the loading control for the blot of total IRS1 (Fig. 1A) and the blots of pIKK (α/β) (Ser176/180) and total IKK (α/β) (Fig. S6). In B, GAPDH was used as the loading control. C, glycogen levels were determined and were normalized to tissue masses (n = 8). D, nuclear levels (n = 4–5) and whole-tissue level (n = 4) of FoxO1 were measured by Western blotting. Histone H3 was used as the loading control in the blot of nuclear lysates. The same histone H3 band was used as the loading control for the blots of (n)SREBP1c (Fig. 6A), (n)CRTC2 (Fig. 8B), and (n)NF-κB p65 (Fig. S6). GAPDH was used as the loading control in the blot of whole-tissue lysates. *, p ≤ 0.05, adropin versus vehicle. Error bars, S.E.
Figure 3.
Figure 3.
Adropin34–76 treatment altered the expression of glucose metabolic genes in the liver. The message levels of genes in glycolysis (A), including glucokinase (Gck) (n = 6) and liver pyruvate kinase (Pklr) (n = 6), and genes in glucose production (B), including G6Pase (G6pc) (n = 6) and PEPCK-1 (Pck1) (n = 6–7) were determined by real-time RT-PCR. *, p ≤ 0.05, adropin versus vehicle. Error bars, S.E.
Figure 4.
Figure 4.
Adropin34–76 treatment alleviated ER stress responses and diminished JNK signaling in the liver. A and B, the phosphorylation levels of Ser51 in eIF2α and total eIF2α levels in whole-tissue lysates (A) and the levels of XBP-1s in nuclear lysates (n = 4–5) and whole-tissue lysates (n = 4) (B) were determined by Western blotting (n = 4–5). In A, α-tubulin was used as the loading control. In B, histone H3 was used as the loading control for nuclear XBP1s, and GAPDH was used as the loading control for whole-tissue XBP1s. C, BiP message (Hspa5) levels (n = 6) and protein levels in whole-tissue lysates (n = 4–5) were determined by real-time RT-PCR and Western blotting, respectively. In Western blotting, GAPDH was used as the loading control for BiP. D and E, the phosphorylation levels of Thr183/Tyr185 in JNK and total JNK levels (n = 4–5) (arrows indicating JNK splice isoforms) (D) and the phosphorylation levels of Ser63 in c-Jun and total c-Jun levels (n = 4–5) in whole-tissue lysates (E) were determined by Western blotting (n = 4–5). In D, α-tubulin was used as the loading control. The same α-tubulin band was used as the loading control for the blot of whole-tissue IP3R1 (Fig. 7). In E, GAPDH was used as the loading control. The same GAPDH band was used as the loading control for the blot of total IRS2 (Fig. 1B) and the blots of pCREB (Ser133) and total CREB (Fig. 8B). *, p ≤ 0.05; ****, p < 0.0001, adropin versus vehicle. Error bars, S.E.
Figure 5.
Figure 5.
Adropin34–76 treatment reduced the expressions of lipogenic genes in the liver. A, triacylglycerol contents were measured and were normalized to tissue masses (n = 8). Real-time RT-PCR was performed to determine the message levels of genes in de novo fatty acid synthesis, including acetyl-CoA carboxylase-α (Acaca) (n = 6), fatty acid synthase (Fasn) (n = 5–6), stearoyl-CoA desaturase (Scd1) (n = 6), and Elovl6 (elongase) (n = 6) (B); de novo TAG synthesis, including mitochondrial glycerol-3-phosphate acyltransferase (Gpam) (n = 6) and diacylglycerol acyltransferase-2 (Dgat2) (n = 6) (C); and acetyl-CoA carboxylase-β (Acacb) (n = 5) (D). *, p ≤ 0.05, adropin versus vehicle Error bars, S.E.
Figure 6.
Figure 6.
Adropin34–76 treatment reduced the nuclear level of SREBP1c in the liver. A, the nuclear levels of SREBP1c (n = 4–5) and the levels of precursor SREBP1c in whole-tissue lysates (n = 4–5) were measured by Western blotting. GAPDH and histone H3 were used as the loading control in the blot of whole-tissue lysates and nuclear lysates, respectively. The same histone H3 band was used as the loading control for the blots of (n)FoxO1 (Fig. 2D), (n)CRTC2 (Fig. 8B), and (n)NF-κB p65 (Fig. S6). B, BiP protein levels in the immunoprecipitates (IP) of precursor SREBP1c from microsomal fractions were determined by Western blotting (IB) (n = 4–5). The blotting was repeated twice, and the blot with 3 samples/treatment was presented. *, p ≤ 0.05; **, p < 0.01, adropin versus vehicle. Error bars, S.E.
Figure 7.
Figure 7.
Adropin34–76 treatment decreased PKA phosphorylation and increased AKT phosphorylation of IP3R in the liver. A, the phosphorylation levels of PKA substrate sites (n = 4) and the phosphorylation levels of AKT substrate sites in IP3R1 following immunoprecipitation (IP) of IP3R1 as well as total IP3R1 levels in whole-tissue lysates (n = 4–5) were determined by Western blotting (IB). α-Tubulin was used as the loading control for whole-tissue IP3R1. The same α-tubulin band was used as the loading control for the blots of pJNK (Thr183/Tyr185) and total JNK (Fig. 4D). **, p < 0.01, adropin versus vehicle. Error bars, S.E.
Figure 8.
Figure 8.
Adropin34–76 treatment decreased cAMP level and the phosphorylation level of CREB in the liver. A, cAMP contents were measured and were normalized to tissue masses (n = 8). B, the phosphorylation levels of Ser133 in CREB and total CREB levels in whole-tissue lysates (n = 4–5) as well as the nuclear levels of CRTC2 (n = 4–5) were measured by Western blotting. GAPDH and histone H3 were used as the loading control in whole-tissue lysates and nuclear lysates, respectively. The same GAPDH band was used as the loading control for the blot of total IRS2 (Fig. 1B) and the blots of p-c-Jun (Ser63) and total c-Jun (Fig. 4E). The same histone H3 band was used as the loading control for the blots of (n)FoxO1 (Fig. 2D), (n)SREBP1c (Fig. 6A), and (n)NF-κB p65 (Fig. S6). *, p ≤ 0.05, adropin versus vehicle. Error bars, S.E.
Figure 9.
Figure 9.
Adropin34–76 treatment suppresses glucose production in primary mouse hepatocyte. A, glucose production from the hepatocytes was determined by quantifying glucose levels in culture media. The assay was performed from three hepatocyte preparations, and the data were pooled and presented as a percentage of the vehicle-treated values (n = 10). The levels of glucose production in the vehicle-treated group were around 0.1 mg/mg of protein/h. B, cAMP levels in HEPG2 liver cells were measured in the presence of increasing levels of forskolin (an activator of adenylate cyclase) in the culture media. The experiments were repeated three times. C, the phosphorylation levels of Ser133 in CREB and total CREB levels, and the phosphorylation levels of PKA substrates in the hepatocytes were determined by Western blotting (n = 2). Heat shock protein 90 (Hsp90) was used as the loading control. D, the message levels of glucose production genes, including G6Pase (G6pc) (n = 5) and PEPCK (Pck1) (n = 2–3), in the hepatocytes were determined by real-time PCR. The quantitation of Pck1 was repeated in another experiment (n = 3), and the levels of Pck1 in the adropin-treated group were below the detection limit. Hypoxanthine guanine phosphoribosyltransferase was used as the reference gene. *, p ≤ 0.05, adropin versus vehicle. Error bars, S.E.
Figure 10.
Figure 10.
A model of adropin actions in regulating hepatic glucose metabolism. AC, adenylate cyclase.
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