Fig 2: Activation of mTOR requires both growth stimulus and metabolic checkpoint. a NRCMs with preincubation of wortmannin (2 µM, 1 h) or vehicle were treated with phenylephrine (PE, 100 µM) or vehicle for 6 h. Immunoblots of total cell lysates (left) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA, and PP2Cm (right) are shown (*p < 0.05 vs. Vehicle, n = 5). b, c NRCMs transduced with indicated adenovirus were preincubated with 2 µM wortmannin for 1 h and subsequently treated with phenylephrine (PE, 100 µM) or vehicle. b Immunoblots of total cell lysates (left) and statistical analyses of densitomeric measurements of p-p70 S6K (Thr 389) and p-mTOR (Ser 2448) (right) are shown (*p < 0.05 vs. sh-con/Vehicle, #p < 0.05 vs. sh-con/PE, n = 4). c 48 h after PE treatment, myocytes were fixed and stained with anti-Troponin T. Cell surface area in each group was quantified and expressed relative to the control (*p < 0.05 vs. sh-con/Vehicle, #p < 0.05 vs. sh-con/PE, n = 4). Scale bar, 25 µm. d Schematic illustration of the working hypothesis on how glucose regulates cell growth through modulating KLF15 mediated transcriptional control of BCAA degradation. Data shown as mean ± s.e.m. P values were determined using one-way ANOVA followed by Newman-Keuls comparison test (a, b) or Kruskal–Wallis test followed by Dunn’s comparison test (c)

Fig 3: Glucose negatively regulates BCAA degradation. a GO enrichment analysis of differentially regulated genes in Glut1-TG hearts using DAVID. Top 5 clusters of either downregulated or upregulated genes are shown. b qRT-PCR analyses of selected BCAA degradation genes in Glut1-TG and WT mouse hearts. The expression was normalized to 18S rRNA and reported as fold change over WT (*p < 0.05 vs. WT, n = 6). Bckdhb branched-chain keto acid dehydrogenase E1 subunit beta, Dbt dihydrolipoamide branched-chain transacylase E2, Ivd isovaleryl-CoA dehydrogenase, Mccc2 Methylcrotonoyl-CoA Carboxylase 2, Mccc1 Methylcrotonoyl-CoA Carboxylase 1, Pccb Propionyl Coenzyme A Carboxylase, Beta Polypeptide; Pcca Propionyl Coenzyme A Carboxylase, Alpha Polypeptide; Mut Methylmalonyl Coenzyme A Mutase. c Schematic illustration of the BCAA degradation pathway. Enzymes examined in Fig. 1b are shown in blue. Degradation of Leu, Ile, and Val share the same initial steps catalyzed by Bcat2, Bckdha, Bckdhb, PP2Cm, and Dbt. Leu leucine, Ile isoleucine, Val valine, KIV a-ketoisovalerate, 3-MB-CoA 3-Methylbutanoyl-CoA, 2-MB-CoA 2-Methylbutanoyl-CoA, IB-CoA Isobutyryl-CoA, MC-CoA 2-Methylcrotonyl-CoA, MG-CoA 2-Methylglutaconyl-CoA, MA-CoA 2-Methylbutanoyl-CoA, MM-CoA Methylmalonyl-CoA. d Representative immunoblots of BCAT2, BCKDHA, PP2Cm, and a-tubulin in heart tissue homogenates (left) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA, and PP2Cm (right) are shown (*p < 0.05 vs. WT, n = 4). e NRCMs were incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. Representative immunoblots of cell lysates (left) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA, and PP2Cm (right) are shown (*p < 0.05 vs. 5.5 mM Glc, n = 4). Glc glucose. f The relative intensity of indicated BCAAs and their metabolites measured by targeted metabolomics of Glut1-TG and WT mouse hearts (*p < 0.05 vs. WT, n = 4). g The intracellular BCAA concentration was quantified in Glut1-TG and WT hearts (*p < 0.05 vs. WT, n = 6). h Cellular BCAA levels after 24 h incubation with DMEM containing 5.5 mM or 25 mM glucose (*p < 0.05 vs. 5.5 mM Glc, n = 6). Data shown as mean ± s.e.m. P values were determined using unpaired Student’s t-test (b, d, e, f, g, h) or Mann–Whitney test (b, d, e, f)

Fig 4: Glucose negatively regulates KLF15 through a CREB dependent mechanism. a NRCMs transfected with 1 µg indicated KLF15 promoter luciferase vectors or empty vector were incubated with DMEM containing 5.5 mM or 25 mM glucose for 36 h. The luciferase activity was measured (*p < 0.05 vs. -1068-luc/5.5 mM Glc, n = 6). b Upper panel: a schematic illustration of two potential CREB DNA-binding elements in the KLF15 promoter region, referred to as CRE I and CRE II. Lower panel: ChIP analysis of CREB binding of the KLF15 promoter in vivo. Results are representative of three independent experiments. c NRCMs were incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. Protein-bound chromatin was prepared and immunoprecipitated with IgG and CREB antibodies. The relative occupancy on the promoter was compared with the input signal (*p < 0.05 vs. 5.5 mM Glc, n = 4). d NRCMs transfected with 1 µg CRE-luc vector were incubated with DMEM containing 5.5 mM or 25 mM glucose or transfected with Glut1 or control (GFP) plasmid for 36 h. The luciferase activity was measured (*p < 0.05 vs. 5.5 mM Glc or GFP, n = 6). e NRCMs were transduced with indicated adenovirus or incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. Representative immunoblots of cell lysates for p-CREB (Ser 133), CREB and ß-actin (left) and statistical analysis of densitomeric measurement of p-CREB (Ser 133) (right) are shown (*p < 0.05 vs. 5.5 mM Glc or LacZ, n = 4). f, g NRCMs transduced with indicated adenovirus were incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. f Representative immunoblots of cytosolic and nuclear fractions (left) and statistical analysis of densitomeric measurement of nuclear KLF15 (right) are shown (*p < 0.05 vs. LacZ/5.5 mM Glc, #p < 0.05 vs. LacZ/25 mM Glc, n = 4). g Immunoblots of cell lysates (left) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA and PP2Cm (right) are shown (*p < 0.05 vs. LacZ/5.5 mM Glc, #p < 0.05 vs. LacZ/25 mM Glc, n = 4). Data shown as mean ± s.e.m. P values were determined using unpaired Student’s t-test (a, d, e), Mann–Whitney test (c, d), or one-way ANOVA followed by Newman-Keuls comparison test (f, g)

Fig 5: KLF15 is essential for glucose-mediated downregulation of BCAA degradation. a qRT-PCR analysis of KLF15 mRNA in Glut1-TG and WT hearts. The expression was normalized to 18S rRNA and reported as fold change over WT (*p < 0.05 vs. WT, n = 6). b Representative immunoblots of KLF15 and a-tubulin in heart tissue homogenates (left) and statistical analysis of densitomeric measurement of KLF15 (right) are shown (*p < 0.05 vs. WT, n = 4). c The mRNA level of KLF15 in NRCMs incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h (*p < 0.05 vs. 5.5 mM Glc, n = 4). d NRCMs were incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. Representative immunoblots from cytosolic and nuclear fractions (left) and statistical analysis of densitomeric measurement of nuclear KLF15 (right) are shown (*p < 0.05 vs. 5.5 mM Glc, n = 4). e NRCMs transduced with indicated adenovirus were incubated with DMEM containing 5.5 mM or 25 mM glucose for 24 h. Immunoblots of cell lysates (left) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA, and PP2Cm (right) are shown (*p < 0.05 vs. LacZ/5.5 mM Glc, #p < 0.05 vs. LacZ/25 mM Glc, n = 4). Data shown as mean ± s.e.m. P values were determined using unpaired Student’s t-test (a, b, c, d) or one-way ANOVA followed by Newman–Keuls comparison test (e)