Glucose negatively regulates BCAA degradation
Previously we have demonstrated that overexpression of an insulin independent glucose transporter Glut1 in cardiomyocytes (Glut1-TG) increased glucose uptake and utilization but did not alter cardiac function or survival of the mice under unstressed condition . RNA microarray and gene ontology analysis of the Glut1-TG hearts revealed that downregulation of the “branched-chain amino acids (BCAAs) degradation” pathway was among the most enriched terms (Fig. 1a ), which was confirmed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis (Supplementary Table 1 ). A total of 26 out of 46 genes in the BCAA degradation pathway was downregulated in the Glut1-TG heart (Supplementary Fig. 1a, b ), and the changes of the key enzymes were confirmed by real-time PCR (Fig. 1b, c ) . The mRNA levels of branched-chain amino acid transaminase 2 (BCAT2) and branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, two enzymes that catalyzed the initial and committed steps of BCAA degradation to branched-chain acyl-coA, were significantly downregulated, so were multiple enzymes involved in downstream reactions for converting the CoA products into the TCA cycle (Fig. 1b, c ). The mRNA level of mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm), that dephosphorylates and activates BCKDH complex, was also decreased (Fig. 1b, c). The protein levels of the BCAT2, branched-chain keto acid dehydrogenase E1 Alpha Polypeptide (BCKDHA), and PP2Cm were also significantly decreased in Glut1-TG hearts (Fig. 1d). No reduction was seen in the mRNA and protein expression of BCKD kinase (BCKDK) in Glut1-TG hearts (Supplementary Fig. 1c, d).
Fig. 1
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 α-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 α-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 )
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Consistent with the in vivo observations in mice, increasing glucose uptake by either overexpression of Glut1 or high glucose medium (HG, 25 mM) reduced the expressions of BCAT2, BCKDHA, and PP2Cm, the three key proteins in BCAA degradation pathway, in both primary cells and proliferating cell lines (Fig. 1e , Supplementary Fig. 1e-h). Taken together, these data indicate that high glucose suppresses the expression of BCAA degradation enzymes.
We subsequently performed targeted metabolomics analysis to determine whether the downregulation of gene expression in Glut1-TG hearts affected the BCAA degradation in vivo. Increased glucose uptake in Glut1-TG hearts led to a higher level of glycolytic metabolites (Supplementary Fig. 1i, j ), as expected. Importantly, the level of BCAAs was also increased while the downstream BCAA metabolites, e.g., α-keto-β-methylvalerate (KMV) and α-ketoisocaproate (KIC) were decreased in Glut1-TG hearts (Fig. 1f). Notably, levels of other amino acids exhibited the opposing trend compared to that of the BCAAs (Supplementary Fig. 1i ), suggesting that high glucose selectively inhibited BCAA degradation. Independent biochemical assays showed a 2.5-fold increase of BCAA level in Glut1-TG hearts (Fig. 1g ). In addition, high glucose medium or Glut1 overexpression also increased the BCAA content in both myocyte and non-myocyte cell types (Fig. 1h, Supplementary Fig. 1k ). Taken together, these observations strongly suggest that glucose negatively regulates intracellular BCAA degradation.
KLF15 is the target of high intracellular glucose
We next searched for the upstream regulatory mechanisms mediating the transcriptional suppression of BCAA degradation pathway. The microarray data showed ~2-fold downregulation of KLF15 in Glut1-TG hearts (Supplementary Fig. 2a ), which was confirmed by RT-PCR and immunoblot (Fig. 2a, b ). Furthermore, both high glucose medium and Glut1 overexpression reduced the KLF15 mRNA and protein levels in neonatal rat cardiomyocytes (NRCMs) (Fig. 2c, d, Supplementary Fig. 2b ). Similar observations were also made in other cell lines including H9C2 and HEK-293 (Supplementary Fig. 2c, d). Previously, KLF15 has been shown to mediate the transcription of genes for BCAA degradation in multiple organs, including skeletal muscle, liver, and heart . We found that overexpression of KLF15 in the presence of high glucose is sufficient to normalize both mRNA and protein expression of BCAA degradation enzymes (Fig. 2e , Supplementary Fig. 2e, f ), suggesting that glucose negatively regulates BCAAs degradation through a KLF15 dependent mechanism.
Fig. 2
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 α-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)
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To investigate the mechanisms by which high glucose suppressed KLF15 expression, the KLF15 promoter region containing ~1 kb upstream sequence from the transcription start site or its serially truncated segments was cloned into a luciferase vector and the luciferase activity was examined (Fig. 3a ). High glucose medium or Glut1 overexpression significantly reduced the luciferase activity containing 1 kb promoter sequence and the combination of the two showed synergetic effects (Supplementary Fig. 2 g). However, the suppression effect on luciferase activity by high glucose was no longer observed in smaller promoter constructs (Fig. 3a ), indicating that the glucose response element(s) is located between the −1068 to −541 bp regions. We found two potential binding elements (CREI and CREII) for the cAMP response element binding protein (CREB) in this region ( http://jaspar.genereg.net/ ); both exhibited high similarity with CRE consensus sequence and were conserved across species (Fig. 3b). The chromatin immunoprecipitation (ChIP) revealed that endogenous CREB bound to both CREI and CREII sites (Fig. 3b). Importantly, high glucose significantly reduced CREB binding occupancy of CREI and CREII on the KLF15 promoter (Fig. 3c). To further test if high glucose suppressed the CREB transcriptional activity, cells expressing a luciferase vector containing three CREB consensus elements (CRE-luc) were subjected to high glucose medium or Glut1 overexpression. Both measures significantly decreased CRE luciferase activity (Fig. 3d). We also observed that the phosphorylation of CREB at Ser 133, critical for CREB transcriptional activation , was significantly decreased in cells cultured in high glucose medium or overexpressing Glut1 (Fig. 3e ). Moreover, overexpression of CREB was sufficient to attenuate high glucose induced downregulation of KLF15, as well as BCAA degradation enzymes (Fig. 3f, g ). These data indicated that high glucose inhibited KLF15 transcription by negatively regulating the binding of CREB to its promoter region.
Fig. 3
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 β-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)
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Glucose promotes cell growth through the KLF15-BCAA pathway
Previous studies have identified KLF15 as a negative regulator of cardiac hypertrophy . In pathological cardiac hypertrophy caused by pressure overload in vivo or by phenylephrine (PE) treatment of NRCMs in vitro, the expressions of KLF15 and BCAA degradation enzymes were decreased (Supplementary Fig. 3a–d), whereas glucose utilization increased . Physiological stimuli for growth that caused increased glucose uptake e.g., insulin or insulin like growth factor 1 (IGF-1) also induced similar changes of KLF15 and BCAA degradation enzymes (Supplementary Fig. 3e–g) . While overexpression of Glut1 did not further increase myocyte cell size in response to PE stimulation (Supplementary Fig. 3h), reducing glucose uptake by knocking down Glut1 (sh-Glut1, Supplementary Fig. 3i, j ) was sufficient to attenuate the downregulation of KLF15 and BCAA degradation enzymes in PE stimulated cells (Fig. 4a, b ). Furthermore, the increases in cell size and atrial natriuretic factor (ANF) promoter activity was significantly reduced by knocking down Glut1 (sh-Glut1) in the presence of PE (Fig. 4c, d). Knockdown of KLF15 reversed the anti-hypertrophy effect by sh-Glut1 in PE treated groups (Fig. 4c, d, Supplementary Fig. 3k ), suggesting that KLF15 was an important mediator of glucose-dependent cell growth. We also found that overexpression of KLF15 prevented the downregulation of BCAA degradation enzymes in PE treated cells, in parallel with the reductions in cell size and ANF promoter luciferase activity (Fig. 4e–g , Supplementary Fig. 3l ). The anti-hypertrophy effect of KLF15 overexpression was partially abolished when BCAA degradation pathway was inhibited by knocking down PP2Cm or BCAT2 (Fig. 4f, g , Supplementary Fig. 3m, n ). Taken together, these data reveal a mechanism that links glucose reliance in cell growth to BCAA degradation pathway through KLF15.
Fig. 4
Suppression of BCAA degradation by high glucose is required for cardiomyocyte growth in response to PE. a, b NRCMs transduced with indicated adenovirus for 72 h were treated with phenylephrine (PE, 100 μM) or vehicle for 6 h. a Representative immunoblots of cytosolic and nuclear fractions (left) and statistical analysis of densitomeric measurement of KLF15 (right) are shown (*p < 0.05 vs. sh-con/Vehicle, p < 0.05 vs. sh-con/PE, n = 4). b Representative immunoblots of cell lysates (left) and statistical analyses of densitomeric measurements of BCAT2 and BCKDHA (right) are shown (*p < 0.05 vs. sh-con/Vehicle, p < 0.05 vs. sh-con/PE, n = 5). c NRCMs transduced with indicated adenovirus were treated with phenylephrine (PE, 100 μM) or vehicle for 48 h. Cellular 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, p < 0.05 vs. sh-Glut1/PE, n = 4). Scale bar, 25 μm. d NRCMs transfected with 1 μg ANF promoter luciferase reporter (ANF-luc) were transduced with indicated adenovirus for 36 h and further incubated with phenylephrine (PE, 100 μM) or vehicle. The luciferase activity was measured after 36 h of PE (*p < 0.05 vs. sh-con/Vehicle, p < 0.05 vs. sh-con/PE, p < 0.05 vs. sh-Glut1/PE, n = 4). e NRCMs transduced with indicated adenovirus were treated with phenylephrine (PE, 100 μM) or vehicle for 6 h. Representative immunoblots of cell lysates (upper) and statistical analyses of densitomeric measurements of BCAT2, BCKDHA and PP2Cm (lower) are shown (*p < 0.05 vs. LacZ/Vehicle, p < 0.05 vs. LacZ/PE, n = 4). f NRCMs transduced with indicated adenovirus were treated with phenylephrine (PE, 100 μM) or vehicle for 48 h. Cellular 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, p < 0.05 vs. KLF15/PE, n = 4). Scale bar, 25 μm. g NRCMs transfected with 1 μg ANF-luc were transduced with indicated adenovirus followed by incubation with phenylephrine (PE, 100 μM) or vehicle for 36 h. The luciferase activity was measured (*p < 0.05 vs. sh-con/Vehicle, p < 0.05 vs. sh-con/PE,
p < 0.05 vs. KLF15/PE, n = 4). Data shown as mean ± s.e.m. P values were determined using one-way ANOVA followed by Newman–Keuls comparison test (a, b , g ) or Kruskal–Wallis test followed by Dunn’s comparison test (c , d, e, f )
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Glucose modulates mTOR in an energy independent manner
In searching for the end effector of glucose mediated cell growth, we found that knockdown of Glut1 attenuated PE induced mTOR activation in NRCMs (Supplementary Fig. 4a ). Consistent with previous observations, inhibition of mTOR activity by rapamycin treatment is sufficient to prevent PE induced cardiomyocyte growth (Supplementary Fig. 4b, c). As glucose is an important energy substrate and the mTOR activity is highly sensitive to cellular energy status, we investigated whether glucose is required to maintain cellular energy balance during the growth. Replacing glucose with either pyruvate (12 mM) or lactate (12 mM) in the culture medium attenuated NRCMs’ growth in response to hypertrophic stimulus (Fig. 5a ) with no change in viability or intracellular ATP level compared with cells cultured with glucose (Supplementary Fig. 4d, e ). Moreover, only NRCMs cultured with glucose showed mTOR activation in response to PE treatment (Fig. 5b , Supplementary Fig. 4f). However, we observed that NRCMs cultured with pyruvate or lactate exhibited increased phosphorylation of AMP-activated protein kinase (AMPK) indicating that AMPK was activated (Supplementary Fig. 4f ). AMPK is an energy sensor and a negative regulator of mTOR activity and cell growth . To determine whether restricting glucose inhibited mTOR through activation of AMPK, we prevented AMPK activation by including both lactate (6 mM) and acetate (6 mM) as the replacement of glucose in the culture (Supplementary Fig. 4d, e , Fig. 5b). NRCMs cultured with lactate and acetate, nevertheless, remained resistant to mTOR activation and hypertrophic growth by PE (Fig. 5a, b ), suggesting that AMPK activation is not required for mTOR inhibition under these conditions. Similarly, rat adult cardiomyocytes cultured with pyruvate or lactate showed no activation of AMPK but showed attenuated mTOR activation and reduced hypertrophic growth during PE stimulation (Supplementary Fig. 4g, h ). Taken together, these observations show that independent of its function in energy provision, glucose plays an essential role in mediating mTOR activation in response to growth stimulation.
Fig. 5
Glucose removal inhibits mTOR activation and cell growth. a NRCMs were pretreated with DMEM containing either glucose or non-glucose substrates for one hour and then incubated with phenylephrine (PE, 100 μM) or vehicle for 48 h. Myocytes were fixed and stained with anti-Troponin T to visualize CMs. Cellular surface area in each group was quantified and expressed relative to the control (*p < 0.05 vs. Glucose/Vehicle, p < 0.05 vs. Glucose/PE, n = 4). Scale bar, 25 μm. b NRCMs were pretreated with DMEM containing either glucose or non-glucose substrates for one hour and then incubated with phenylephrine (PE, 100 μM) or vehicle for 6 h. Immunoblots of cell lysates (left) and statistical analyses of densitomeric measurements of p-p70 S6K (Thr 389), p-mTOR (Ser 2448), p-mTOR (Ser 2481), and p-AMPK α (Thr 172) (right) are shown (*p < 0.05 vs. Glucose/PE(-), p < 0.05 vs. Glucose/PE(+), n = 4). Data shown as mean ± s.e.m. P values were determined using one-way ANOVA followed by Newman–Keuls comparison test (b ) or Kruskal-Wallis test followed by Dunn’s comparison test (a)
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mTOR activation requires the inhibition of BCAA degradation
To test if the suppression of BCAA degradation by glucose was required for mTOR activation during cell growth, we first examined the intracellular BCAAs amount in response to PE treatment at various time points. PE induced a transient accumulation of intracellular BCAA that coincided with activation of mTOR, which was prevented by overexpression of KLF15 (Fig. 6a, b , Supplementary Fig. 4i ). This transient accumulation of BCAA in cultured cardiomyocytes is consistent with the observation in the hearts in response to pressure overload or myocardial infarction, in which the increase of myocardial BCAA level peaks at one week after the stimulus . Knockdown of BCAT2 or PP2Cm, the key enzymes for BCAA degradation, induced the intracellular BCAA accumulation (Supplementary Fig. 4j ) and restored mTOR activation by PE in KLF15 overexpressing cells (Fig. 6c, Supplementary Fig. 4k ). Similarly, the downregulation of BCAA degradation enzymes by PE was abolished in cardiomyocytes cultured with non-glucose medium (Supplementary Fig. 4l ). Knocking down PP2Cm or BCAT2 was sufficient to reactivate mTOR during PE treatment in both neonatal and adult rat cardiomyocytes cultured even in non-glucose medium and restored the growth response to the same extent as in cells cultured with glucose (Fig. 6d, e , Supplementary Fig. 4m-p). Taking together, these data suggest that suppression of BCAA degradation pathway by glucose is required for mTOR activation during cell growth.
Fig. 6
BCAA accumulation is required for mTOR activation during cardiomyocyte growth. a Cellular BCAA level was measured in NRCMs treated with phenylephrine (PE, 100 μM) or vehicle at indicated time points (*p < 0.05 vs. Vehicle, n = 5). b Cellular BCAA level was examined in NRCMs transduced with indicated adenovirus and treated with phenylephrine (PE, 100 μM) or vehicle for 3 h (* p < 0.05 vs. LacZ/Vehicle, p < 0.05 vs. LacZ/PE, n = 5).
c NRCMs transduced with indicated adenovirus were treated with phenylephrine (PE, 100 μM) or vehicle for 6 h. Immunoblots of 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/LacZ/Vehicle, p < 0.05 vs. sh-con/LacZ/PE, p < 0.05 vs. sh-con/KLF15/PE, n = 4). SE for short exposure. d, e NRCMs transduced with indicated adenovirus were incubated with DMEM containing either glucose or non-glucose substrates for one hour and then treated with phenylephrine (PE, 100 μM) or vehicle. d Immunoblots of 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/Glucose/Vehicle, p < 0.05 vs. sh-con/Glucose/PE, p < 0.05 vs. sh-con/Lactate/PE, n= 4). e 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/Glucose/Vehicle, p < 0.05 vs. sh-con/Glucose/PE, p < 0.05 vs. sh-con/Lactate/PE, n = 4). Scale bar, 25 μm. Data shown as mean ± s.e.m. P values were determined using one-way ANOVA followed by Newman-Keuls comparison test (a , b , c , d) or Kruskal–Wallis test followed by Dunn’s comparison test (e)
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Crosstalk between growth stimulus and metabolic response
Although glucose is required for cell growth, high glucose alone did not stimulate mTOR activation in cardiomyocytes in the absence of hypertrophic stimuli (Supplementary Fig. 5a ). The Glut1-TG hearts did not develop hypertrophy without stress , and high glucose medium did not increase cell size in NRCMs (Supplementary Fig. 5b). These findings suggest that BCAA accumulation induced by increased glucose reliance is required but not sufficient to stimulate cell growth, i.e., the activation of mTOR requires additional input even in the presence of metabolic reprogramming. We thus investigated the cross talk between glucose and the two upstream regulators of mTOR signaling: PI3K and MAPK pathways. As expected, PE treatment stimulated both PI3K and MAPK pathways (Supplementary Fig. 5c), while high glucose had no effect on either (Supplementary Fig. 5d). Similarly, removing glucose or overexpression of KLF15 had no effect on activation of either PI3K or MAPK in response to PE treatment (Fig. 7a, b‚ Supplementary Fig. 5e ), but strongly suppressed mTOR activation and cardiomyocyte growth in response to PE (Figs. 4f,
5a, b , 6c , Supplementary Fig. 4i ). In addition, inhibition of PI3K pathway by wortmannin or inhibition of MAPK by U0126 was sufficient to inhibit mTOR activation during PE stimulation but had no effect on BCAA degradation pathway (Fig. 8a , Supplementary Fig. 5f). Knockdown of KLF15 or PP2Cm in cells subjected to PE stimulation, which effectively suppressed BCAA degradation pathway, failed to restore mTOR activation or hypertrophic growth when PI3K or MAPK was inhibited (Fig. 8b, c, Supplementary Fig. 5g-l), indicating that PE induced mTOR activation also required intact PI3K or MAPK signaling. Taken together, these data suggest that the activation of mTOR during a growth response requires input signal via the PI3K and/or MAPK pathway, as well as the metabolic response via increased intracellular glucose (Fig. 8d ). Either component is indispensable, and they act cooperatively to sustain a growth response.
Fig. 7
Glucose does not affect signaling upstream of mTOR. a NRCMs incubated with DMEM medium containing glucose or non-glucose substrates were treated with phenylephrine (PE, 100 μM) or vehicle for 6 h. Immunoblots of whole cell lysates (left) and statistical analyses of densitomeric measurements of p-Akt (Thr 308), p-TSC2 (Ser 939), p-TSC2 (Thr 1462), and p-p44–42 MAPK (Thr 202/Tyr 204) (right) are shown (*p < 0.05 vs. Glucose/PE(−),
n = 4). b NRCMs transduced with indicated adenovirus were treated with phenylephrine (PE, 100 μM) or vehicle for 6 h. Immunoblots of whole cell lysates (left) and statistical analyses of densitomeric measurements of p-Akt (Thr 308), p-TSC2 (Thr 1462), and p-p44–42 MAPK (Thr 202/Tyr 204) (right) are shown (*p < 0.05 vs. LacZ/Vehicle, n = 4). 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 (a)
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Fig. 8
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 )
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Glucose-BCAA circuit regulates cardiac hypertrophy in mice
Increased glucose reliance, downregulation of KLF15 and impaired BCAA degradation have been demonstrated in hearts with pathological hypertrophy , but their role as a regulatory circuit for hypertrophic response has never been tested. If our hypothesis is correct, the anti-hypertrophy effect of KLF15 will not be observed in mice deficient of BCAA degradation such as PP2Cm KO. We thus sought to increase KLF15 expression in the heart of WT and PP2Cm KO mice, via retro-orbital injection of an adeno-associated virus serotype 9 vector carrying KLF15 and directed by the cardiac-specific cTNT promoter (AAV9-KLF15) . This approach showed high efficiency and specificity of gene expression in the heart (Supplementary Fig. 6a, b ). As high level overexpression of KLF15 in transgenic mice causes arrhythmia due to a regulatory role of KLF15 on potassium channel , we titrated the dosage of AAV9-KLF15 to limit the KLF15 overexpression to ~2-fold (Fig. 9a , Supplementary Fig. 6c ). The expression level of KLF15 was stable for up to 5 weeks and did not cause any overt phenotype in WT mice. Importantly, AAV9-KLF15 administration was sufficient to increase multiple enzymes involved in BCAA degradation pathway (Fig. 9a ).
Fig. 9
Glucose and KLF15 mediated BCAA degradation is essential for cardiomyocyte growth in vivo. a Immunoblots of cardiac tissue homogenates from WT mice injected with AAV9-KLF15 or PBS for indicated time period are shown. b –f PP2Cm KO and WT mice were subjected to TAC surgery one week after retro-orbital injection of AAV9-KLF15 or control virus (AAV9-GFP). b The heart weight/body weight (HW/BW) ratio of PP2Cm KO and WT hearts with indicated AAV injection 4 weeks after TAC or sham operation (*p< 0.05 vs. WT/AA9-GFP/sham, p < 0.05 vs. WT/AAV9-GFP/TAC,
p < 0.05 vs. WT/AAV9-KLF15/TAC, n = 5–11). c Representative wheat germ agglutinin staining and quantification of cardiomyocyte cross-sectional area in indicated hearts 4 weeks after TAC or sham operation (*p < 0.05 vs. WT/AA9-GFP/sham,
p < 0.05 vs. WT/AAV9-GFP/TAC, &p < 0.05 vs. WT/AAV9-KLF15/TAC, n = 3–4). Scale bar, 50 μm. d Left ventricles from indicated hearts were collected 3 days post surgery. Immunoblots of tissue homogenates (left) and statistical analyses of densitomeric measurements of p-p70 S6K (Thr 389), p-mTOR (Ser 2448) and p-mTOR (Ser 2481) (right) are shown (*p < 0.05 vs. WT/AA9-GFP/sham, p < 0.05 vs. WT/AAV9-GFP/TAC, p < 0.05 vs. WT/AAV9-KLF15/TAC, n = 3). e qRT-PCR measurements of ANP and BNP levels in PP2Cm KO and WT hearts with indicated AAV injection 4 weeks after surgery (*p < 0.05 vs. WT/AA9-GFP/sham, p < 0.05 vs. WT/AAV9-GFP/TAC, p < 0.05 vs. WT/AAV9-KLF15/TAC, n = 3–4). f Left ventricular ejection fraction (LVEF%) assessed by echocardiography at 2 weeks and 4 weeks post TAC surgery (*p < 0.05 vs. WT/AA9-GFP/sham, p < 0.05 vs. WT/AAV9-GFP/TAC,
p < 0.05 vs. WT/AAV9-KLF15/TAC, n = 5–12). Data shown as mean ± s.e.m. P values were determined using one-way ANOVA followed by Newman–Keuls comparison test (b , c , d, e, f )
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We found that the increases in heart weight and myocyte cross-sectional area, induced by TAC, were significantly reduced by KLF15 overexpression in WT but not in PP2Cm KO mice (Fig. 9b , c). Immunoblot analysis demonstrated that AAV9-KLF15 treatment significantly reduced the phosphorylation of p70 S6K and mTOR after TAC in WT mice but not in PP2Cm KO mice (Fig. 9d). Importantly, PI3K and MAPK signaling was activated to a similar extent in all the groups subjected to TAC compared with sham operated mice (Supplementary Fig. 6c). In addition, molecular markers for pathological cardiac hypertrophy ANP and BNP, were markedly elevated in WT-TAC and a greater increase observed in PP2Cm KO-TAC hearts (Fig. 9e ). AAV9-KLF15 treatment significantly reduced the expression of ANP and BNP in WT but not in PP2Cm KO hearts after TAC (Fig. 9e ). Echocardiographic measurements demonstrated that TAC induced cardiac dysfunction in both WT and PP2Cm KO mice compared with sham operated mice, which was rescued by AAV9-KLF15 in WT only (Fig. 9f, Supplementary Table 2). Collectively, these data demonstrate that suppression of KLF15-BCAA degradation pathway is required for activation of mTOR and development of pathological hypertrophy of adult hearts in vivo.
MY CONCLUSION::::::
TAKING MODERATE GLUCOSE - I TEND TO TAKE 3 TO 4 CARBOHYDRATES TO 1 PROTEIN RATIO, ALONG WITH BCAA, HELPS LOSE FAT. WHICH MEANS I SHOULD GET 35 g OF CARBOHYDRATES ON EACH PROTEIN SHAKE I TAKE. I NOW TAKE TWO TO THREE NEYMAR PROTEIN SHAKES ON WORKOUT DAYS(3 DAYS A WEEK). BUT EACH SHAKE HAS CARBPHYDRATES TO PROTEIN RATIO OF 4:1. SIMPLE, BUT EFFECTIVE. PLEASE NOTE I GET 8 g OF PROTEIN ON EACH SHAKE USING ONE EGG ONLY PER SHAKE. 3 SHAKES EQUATE TO 3 EGGS, WHICH HAVE A LOT OF BCAA IN THEM. AND ONE MORE THING: PLEASE ENSURE YOU GET A MINIMUM OF 1.5 g/kg Protein per bodyweight. Also, do not go below 4 g/kg per bodyweight in Carbohydrates. I already lost more than 12% body fat using Branch Chain Amino Acids alone, taken with low glucose. But beware of this approach, I also started having insomnia, muscle tissue loss and constipation, fatigue and yes... Lose motion, because I was not taking enough Glycogen on Cheat days. GLYCOGEN REPLENISHMENT is number one key to success in fat-loss, as above detailed study can suggest.
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