Wildtype C57BL/6 mice were purchased from SLAC Laboratory Animals Company Limited, Shanghai, China. All animals were housed at with a 12-h light, 12-h dark cycle with free access to water and standard chow. This study was carried out in accordance with the guidelines of the Committee for Humane Treatment of Animals at Shanghai Jiao Tong University School of Medicine. The protocol was approved by the Committee for Humane Treatment of Animals at Shanghai Jiao Tong University School of Medicine.

HepG2, NIH 3T3, and MEF cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, Beijing) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, MD), penicillin (100 IU/mL) and streptomycin (100 μg/mL) in a humidified 5% CO2-95% air incubator at 37°C. Custom BCAA-free DMEM was provided by Invitrogen. BCAA and BCKA chemicals were purchased from Sigma.

The 3′UTR of mouse KLF15 was identified using the UCSC Genome Browser (http://genome.ucsc.edu/) and cloned into the psiCHECK_2 plasmid (Promega) with XhoI and NotI sites, respectively. The 3'UTR fragment (921 bp) was located in the 3′ flanking region of the synthetic Renilla luciferase gene. Thus, Renilla luciferase expression can be regulated by the downstream 3′UTR activity. The promoter region of Klf15 (−5 kb before the translation start site in the second exon) was cloned into PGL3 reporter vector (Promega) (Jeyaraj et al., 2012). Plasmids were transfected using lipofectimine2000 (Invitrogen) according to the manufacturer's protocol. The transfected cells were rinsed once with cold PBS and lysed with passive lysis buffer (Promega) after transfection for 24 h. Luciferase activity was measured using the GloMax-Multi Detection System (Promega) and the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase expression was normalized to the expression of firefly luciferase. Data are represented as means ± SE from three biological replicates representing three independent experiments.

Total RNA was extracted using the Trizol (Invitrogen) according to the manufacturer's instructions. Total RNA (2 μg) was reverse transcribed using random primers and MMLV (Promega). Each cDNA sample was analyzed in triplicate with the Applied Biosystems Prism7900HT Real-Time PCR System using Absolute SYBR Green (ABI) with the specific primers. The relative amount of specific mRNA was normalized by 18sRNA. Mouse KLF15 primer sequences are: forward, 5′-TCTCGTCACCGAAATGCTCA-3′ and reverse, 5′-GAGTCAGGGCTGGCACAAGA-3′.

Proteins from tissue or cells were harvested in buffer (50 mM HEPES [pH7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM glycerophosphate, 2.5 mM sodium pyrophosphate 1 mM Na3VO4, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL of aprotinin, leupeptin, and pepstatin). Samples were separated on 4–12% Bis-Tris gels (Invitrogen), and transferred onto a nitrocellulose blot (Amersham). The blot was probed with the indicated primary antibodies. Protein signals were detected using conjugated secondary antibodies and enhanced chemiluminescence (ECL) western blotting detection regents (Pierce). The BCKDK antibody (AV52131) was purchased from sigma. Phospho-Akt (#4051 for pAKT Ser473 and #2965 for pAKT Thr308) and total AKT (#9272 for AKT1,2,3) antibodies were purchased from Cell Signaling Technology. β-actin antibody was purchased from Sigma Chemical Co.

Fifty microliter of plasma was precleared of protein by addition of an equal volume of methanol, followed by two rounds of centrifugation to precipitate protein. The supernatant (90% recovery) was lyophilized and resuspended in 45 μl dH₂O. Standards (L-valine, L-leucine, L-isoleucine, L-allo-isoleucine) were prepared from powder. 10 mM stocks were made in dH₂O and serial pooled dilutions prepared. A 250 μM stock of D3-Leucine was also prepared in dH₂O. 2 μl of standard or 2 μl of sample was diluted in 50 μl of butanolic HCL in 1.5 ml tube, heated to 60°C for 20 min and then speed vacuumed to dryness. The samples were resuspended in 200 ul of 50:50 dH₂O:acetonitrile containing 0.1% formic acid. The samples were analyzed by LC-MS/MS.

Unless otherwise specified, statistical analyses were performed with Student's t-test (two groups) or one-way ANOVA (>2 groups) where appropriate. Data are calculated as the mean ± SE (standard error) unless otherwise indicated. A p-value of less than 0.05 was considered statistically significant.

To verify the KLF15 regulation by BCAAs identified in gene expression profiles of MEF (data not shown), we examined the time course of KLF15 expression following BCAA deprivation. In commercial Dulbecco's Modified Eagle's Medium (DMEM), the BCAAs concentration is 800 μM. A customized DMEM containing all components except for BCAAs was used to deprive BCAA. In MEFs, KLF15 mRNA demonstrated a clear increase at 2 h after BCAA deprivation, and remained elevated for at least 10 h (Figure 1A). Further study showed that BCAA suppressed KLF15 expression in a dose-dependent way (Figure 1B). To further verify the suppression of BCAA on KLF15 expression, we starved the cells with BCAA-free DMEM and then replenished with BCAA. BCAA replenishment clearly inhibited the BCAA-deprivation-induced KLF15 expression (Figure 1C). KLF15 induction by BCAA starvation was also observed in other cell types, such as liver (AML12) and muscle (C2C12) cell lines (Figures 1D,E).

BCAA can be catabolized in cells. BCAA and their catabolites such as BCKA and 3-hydroxyisobutyrate exert various physiological and pathological functions (Lynch and Adams, 2014; Jang et al., 2016). We then explored whether BCAA or their catabolic intermediates affected the KLF15 expression. Branched-chain amino acid transaminase 2 (BCAT2) catalyzes the first reaction of BCAA catabolism. If the BCAT2 expression is abolished, BCAA can't be catalyzed into BCKA and other intermediates. When BCAT2 expression was silenced by siRNA in NIH-3T3 cell (Figure 2A), KLF15 expression was not affected (Figure 2B). Meanwhile, In the BCAT2 knockdown cell, KLF15 expression was induced by BCAA deprivation, which was inhibited by replenishing BCAA (Figure 2C). Thus, BCAA, but not BCKA or downstream catabolites, suppressed the expression of KLF15.

We then explored the signaling pathway(s) involved in KLF15 regulation by BCAA. A recent study showed that amino acid starvation activated PI3K/AKT signaling (Tato et al., 2011). PI3K is a key signaling molecule to transduce extracellular signals to regulate gene expression in a variety of cell types. AKT is a well-known downstream target of PI3K and its activation can be measured by phosphorylation at signature sites Ser473 or Thr308. To address whether BCAA deprivation activated PI3K pathway, we determined the AKT phosphorylation at Ser473 and Thr308 in response to BCAA starvation. BCAA deprivation caused significant AKT activation at 1 and 2 h (Figure 3A; Figure S1). BCAAs deprivation induced AKT activation in a dose-dependent way (Figure 3B; Figure S2).

Pharmacological inhibitors of PI3K were then used to determine whether PI3K signaling pathway participated in BCAA-deprivation-induced KLF15 expression. The basal level and BCAA-starvation-induced Akt phosphorylation at Thr308 and Ser473 were blocked by LY294002 and Wortmannin, demonstrating a clear inhibition of PI3K signaling (Figure 4A; Figure S3). Both LY294002 and Wortmannin abolished BCAA-starvation-induced KLF15 expression (Figure 4B). Similarly, AKT inhibitor MK-2206 inhibited AKT activation (Figure 4C; Figure S4) and the KLF15 mRNA expression induced by BCAA starvation (Figure 4D). Together, these data support a critical role of PI3K-AKT signaling pathway in BCAA-starvation-induced KLF15 expression.

We then investigated whether BCAA starvation induced KLF15 expression at transcriptional or post-transcriptional level. Actinomycin D, an inhibitor of RNA synthesis, prevented KLF15 induction by BCAA starvation (Figure 5A), suggesting a transcriptional regulation. To investigate whether BCAAs affected KLF15 expression at post-transcriptional level, we cloned mouse KLF15 3'UTR fragments into the reporter vector p-siCHECK_2, where Renilla luciferase expression is regulated by downstream 3′UTR, and generated Luc-3′UTR reporters. After transfecting this reporter vector into cells, the luciferase activity was measured. The results showed that BCAA starvation did not affect the expression of the luciferase fused with mouse KLF15 3'UTR (Figure 5B). Meanwhile, BCAA starvation failed to affect the half-life of KLF15 mRNA (Figure 5C). These data suggest that BCAA starvation affect the KLF15 expression at transcriptional but not post-transcriptional level.

A common mechanism of transcriptional regulation is promoter activation. A chimeric construct of luciferase gene under the control of KLF15 promoter was introduced into cells by transient transfection (Jeyaraj et al., 2012). KLF15-promoter contains a well-defined GRE for the transactivity by glucocorticoid receptor (Shimizu et al., 2011). Unexpectedly, BCAA starvation failed to increase the KLF15-promoter-driven luciferase activity (data not shown). Meanwhile, the KLF15-promoter-driven luciferase mRNA expression was also not induced by BCAA starvation (Figure 6A) even BCAA starvation induced endogens KLF15 mRNA expression (Figure 6B). On the other hand, activation of KLF15 promoter and mRNA were observed following dexamethasone treatment (Figure 6). Thus, BCAA starvation induced the KLF15 transcription without directly affecting KLF15 promoter activation.

We further investigated the relationship between BCAA and KLF15 expression in vivo. Plasma BCAA levels significantly decreased after 12-h fasting (Figure 7A). We measured the KLF15 expression in different tissues in fasted mice. KLF15 expression was significantly induced in muscle and white adipose tissue, but not in liver (Figure 7B). These data suggest that, in vivo, BCAA abundance was negatively associated with KLF15 expression.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.