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. 2017 Oct 25:8:853.
doi: 10.3389/fphys.2017.00853. eCollection 2017.

Branched-Chain Amino Acid Negatively Regulates KLF15 Expression via PI3K-AKT Pathway

Yunxia Liu  1 , Weibing Dong  1 , Jing Shao  1 , Yibin Wang  1 , Meiyi Zhou  1 , Haipeng Sun  1
Affiliations

Branched-Chain Amino Acid Negatively Regulates KLF15 Expression via PI3K-AKT Pathway

Yunxia Liu et al. Front Physiol. .

Abstract

Recent studies have linked branched-chain amino acid (BCAA) with numerous metabolic diseases. However, the molecular basis of BCAA's roles in metabolic regulation remains to be established. KLF15 (Krüppel-like factor 15) is a transcription factor and master regulator of glycemic, lipid, and amino acids metabolism. In the present study, we found high concentrations of BCAA suppressed KLF15 expression while BCAA starvation induced KLF15 expression, suggesting KLF15 expression is negatively controlled by BCAA.Interestingly, BCAA starvation induced PI3K-AKT signaling. KLF15 induction by BCAA starvation was blocked by PI3K and AKT inhibitors, indicating the activation of PI3K-AKT signaling pathway mediated the KLF15 induction. BCAA regulated KLF15 expression at transcriptional level but not post-transcriptional level. However, BCAA starvation failed to increase the KLF15-promoter-driven luciferase expression, suggesting KLF15 promoter activity was not directly controlled by BCAA. Finally, fasting reduced BCAA abundance in mice and KLF15 expression was dramatically induced in muscle and white adipose tissue, but not in liver. Together, these data demonstrated BCAA negatively regulated KLF15 expression, suggesting a novel molecular mechanism underlying BCAA's multiple functions in metabolic regulation.

Keywords: Akt; BCAA; Klf15; PI3K; regulation.

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Figures

Figure 1
Figure 1
BCAA negatively controls KLF15 expression. Quantitative PCR results of KLF15 in MEFs. (A) MEFs were cultured in BCAA-free DMEM for indicated time before harvesting. (B) MEFs were cultured with different concentrations of BCAAs for 6 h before harvesting. (C) MEFs were cultured without BCAA (−) for 4 h followed with or without (−) BCAA replenishment for extra 2 h before harvesting. (D,E), AML12 liver cell line (D) and C2C12 muscle cell line (E) were cultured with (+) or without (−) BCAA for 4 h before harvesting. Data are means ± SE for three individual samples. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
BCAA but not their catabolites regulate KLF15 expression. (A,B) Quantitative PCR results of BCAT2 (A) or KLF15 (B) in MEFs with Bcat2 silencing using shRNA. (C) Quantitative PCR results of KLF15 in MEFs with Bcat2 silencing with or without BCKA (500 μM) or 800 μM BCAAs replenishment for 2 h after 4-h BCAA starvation (−). *p < 0.05, **p < 0.01.
Figure 3
Figure 3
BCAA starvation induces AKT phosphorylation. Measurements of Ser473 or Thr308 phosphorylation of AKT (pAKT) or total AKT (60KD band) using immunoblots. (A) MEF cells were treated with BCAA deprivation (−) for indicated time. (B) MEF cells were treated with different concentrations of BCAAs for 2 h. Data showed 1 experiment representative of 3.
Figure 4
Figure 4
Inhibitors of PI3K and AKT attenuate BCAA-starvation-induced KLF15 expression. (A) Measurements of Ser473 or Thr308 phosphorylation of AKT (pAKT) or total AKT (60KD band) using immunoblots. Cells were pretreated 60 min with 10 μM LY294002 or 1 μM Wortmannin before BCAA deprivation (−) for 2 h. Data showed 1 experiment representative of 3. (B) Cells were pretreated 60 min with 10 μM LY294002 or 1 μM Wortmannin before BCAA deprivation (−) for 4 h and then harvested for measurement of KLF15 mRNA by quantitative PCR. (C) Measurements of Ser473 or Thr308 phosphorylation of AKT (pAKT) or total AKT (60KD band) using immunoblots. Cells were pretreated 60 min with 10 μM AKT inhibitor MK-2206 before BCAA deprivation (−) for 2 h. Data showed 1 experiment representative of 3. (D) Cells were pretreated 60 min with 10 μM AKT inhibitor MK-220 before BCAA deprivation (−) for 4 h and then harvested for measurement of KLF15 mRNA by quantitative PCR. Data are means ± SE for three individual samples. *p < 0.05, **p < 0.01.
Figure 5
Figure 5
BCAA regulates KLF15 expression at transcriptional level. (A) Quantitative PCR was performed to measure the mRNA level of KLF15 in cells pretreated with actinomycin D (5 μg/ml) or DMSO for 30 min before BCAA starvation (−) for 6 h. (B) Luciferase assay results from MEF cells transfected with luciferase reporter vectors with (+) or without (−) KLF15 3'UTR with (+) or without BCAA (−). (C) Quantitative PCR was performed to measure the relative mRNA level of KLF15 in cells treated with actinomycin D (5 μg/ml) for indicated time in the presence (con) or absence of BCAA (BCAA starvation). Data are means ± SE for three individual samples. **p < 0.01.
Figure 6
Figure 6
BCAA does not directly affect KLF15 promoter activity. Quantitative PCR was performed to measure the mRNA level of luciferase (A) or KLF15 (B) in cells transfected with empty PGL3-basic or KLF15-promoter-driving luciferase constructs and treated with BCAA starvation (−) or dexamethasone for 24 h. Data are means ± SE for three individual samples. *p < 0.05.
Figure 7
Figure 7
Fasting leads to lower BCAA abundance and higher KLF15 expression in vivo. Mice at 10-week of age were fasted for 12 h. Plasma levels of BCAAs were measured (A, n = 7 in each group) and mRNA level of KLF15 in different tissues were analyzed with quantitative PCR (B, n = 4 in each group). *p < 0.05, **p < 0.01.
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