Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

Abstract

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.

Fig. 1. Effect of obesity on plasma BCAA concentrations

Plasma BCAAs were measured using the spectrophotometric assay. Panel A. Random fed (fed) and overnight food-deprived (fasted) plasma BCAA concentrations in 13-week old ob/ob male and age-matched lean control mice fed a normal chow diet. Asterisks indicate, *p < 0.05, ***p < 0.001, compared to control in the same nutritional state, n=8/group. Panel B. Random fed (fed) and overnight food-deprived (fasted) plasma BCAA concentrations in ~13 week old Zucker fatty and age-matched lean control rats. Asterisks indicate, **p < 0.01, vs. lean controls in the same nutritional state, n=10/group.

Fig. 1. Effect of obesity on plasma BCAA concentrations

Plasma BCAAs were measured using the spectrophotometric assay. Panel A. Random fed (fed) and overnight food-deprived (fasted) plasma BCAA concentrations in 13-week old ob/ob male and age-matched lean control mice fed a normal chow diet. Asterisks indicate, *p < 0.05, ***p < 0.001, compared to control in the same nutritional state, n=8/group. Panel B. Random fed (fed) and overnight food-deprived (fasted) plasma BCAA concentrations in ~13 week old Zucker fatty and age-matched lean control rats. Asterisks indicate, **p < 0.01, vs. lean controls in the same nutritional state, n=10/group.

Fig. 2. BCAA catabolic enzymes and pS293 E1α immunoreactivity in skeletal muscle of ob/ob mice and Zucker rats

Gastrocnemius muscle was obtained from random fed or overnight fasted ob/ob mice and Zucker rats. Equal amounts of muscle lysate protein from lean control (L) and obese (O) mice (Pane A) or lean (L) and obese “fatty” (F) Zucker rats (Panel B) were then separated by SDS-PAGE and transferred to PVDF. These blots were probed for antibodies against BCATm, BCKD E1α, pS293 E1-α and BCKD kinase (arrow, bottom band is non-specific). Tubulin was used as a loading control. No statistical difference was found in the concentration of any of the antigens between the lean and obese state when replicates were analyzed (p>0.05, n=8, densitometry not shown).

Fig. 2. BCAA catabolic enzymes and pS293 E1α immunoreactivity in skeletal muscle of ob/ob mice and Zucker rats

Gastrocnemius muscle was obtained from random fed or overnight fasted ob/ob mice and Zucker rats. Equal amounts of muscle lysate protein from lean control (L) and obese (O) mice (Pane A) or lean (L) and obese “fatty” (F) Zucker rats (Panel B) were then separated by SDS-PAGE and transferred to PVDF. These blots were probed for antibodies against BCATm, BCKD E1α, pS293 E1-α and BCKD kinase (arrow, bottom band is non-specific). Tubulin was used as a loading control. No statistical difference was found in the concentration of any of the antigens between the lean and obese state when replicates were analyzed (p>0.05, n=8, densitometry not shown).

Fig. 3. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from ob/ob mice

Liver protein lysates, made from frozen lean (L) and ob/ob (O) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF for immunoblotting. Panel A shows representative immunoblots of total BCKD E1α (liver does not express BCATm), pS293 E1α, BCKD kinase and β-Actin (loading control). Other panels show densitometric analysis of replicates for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). Fed and Fasted data were initially derived from two separate blots at different times. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together, however there was insufficient material to rerun the BCKD kinase. The original data are shown for that endpoint; a t-test was used to compare the lean and obese states only (*** p < 0.001, n=6/group). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA. Asterisks indicate where statistical differences between the lean and obese condition in the corresponding nutritional state was observed based upon a Student-Newman-Keuls Multiple Comparisons post-test (*P<0.05, *** p < 0.001). No differences were observed between the lean and fasted states for any antigen. Western blotting for β-Actin was used as a loading control. The densitometry values were 7539 ± 665; 7358 ± 692; 6835 ± 532 and 6833 ± 578 for fed lean, fed obese, fasted lean and fasted obese mouse liver, respectively (N.S., p>0.05).

Fig. 3. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from ob/ob mice

Liver protein lysates, made from frozen lean (L) and ob/ob (O) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF for immunoblotting. Panel A shows representative immunoblots of total BCKD E1α (liver does not express BCATm), pS293 E1α, BCKD kinase and β-Actin (loading control). Other panels show densitometric analysis of replicates for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). Fed and Fasted data were initially derived from two separate blots at different times. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together, however there was insufficient material to rerun the BCKD kinase. The original data are shown for that endpoint; a t-test was used to compare the lean and obese states only (*** p < 0.001, n=6/group). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA. Asterisks indicate where statistical differences between the lean and obese condition in the corresponding nutritional state was observed based upon a Student-Newman-Keuls Multiple Comparisons post-test (*P<0.05, *** p < 0.001). No differences were observed between the lean and fasted states for any antigen. Western blotting for β-Actin was used as a loading control. The densitometry values were 7539 ± 665; 7358 ± 692; 6835 ± 532 and 6833 ± 578 for fed lean, fed obese, fasted lean and fasted obese mouse liver, respectively (N.S., p>0.05).

Fig. 3. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from ob/ob mice

Liver protein lysates, made from frozen lean (L) and ob/ob (O) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF for immunoblotting. Panel A shows representative immunoblots of total BCKD E1α (liver does not express BCATm), pS293 E1α, BCKD kinase and β-Actin (loading control). Other panels show densitometric analysis of replicates for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). Fed and Fasted data were initially derived from two separate blots at different times. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together, however there was insufficient material to rerun the BCKD kinase. The original data are shown for that endpoint; a t-test was used to compare the lean and obese states only (*** p < 0.001, n=6/group). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA. Asterisks indicate where statistical differences between the lean and obese condition in the corresponding nutritional state was observed based upon a Student-Newman-Keuls Multiple Comparisons post-test (*P<0.05, *** p < 0.001). No differences were observed between the lean and fasted states for any antigen. Western blotting for β-Actin was used as a loading control. The densitometry values were 7539 ± 665; 7358 ± 692; 6835 ± 532 and 6833 ± 578 for fed lean, fed obese, fasted lean and fasted obese mouse liver, respectively (N.S., p>0.05).

Fig. 3. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from ob/ob mice

Liver protein lysates, made from frozen lean (L) and ob/ob (O) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF for immunoblotting. Panel A shows representative immunoblots of total BCKD E1α (liver does not express BCATm), pS293 E1α, BCKD kinase and β-Actin (loading control). Other panels show densitometric analysis of replicates for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). Fed and Fasted data were initially derived from two separate blots at different times. To compare them together, the remaining samples were electrophoresed on the same blot and reanalyzed together, however there was insufficient material to rerun the BCKD kinase. The original data are shown for that endpoint; a t-test was used to compare the lean and obese states only (*** p < 0.001, n=6/group). The other reanalyzed samples were compared between lean and obese as well as nutritional status using an ANOVA. Asterisks indicate where statistical differences between the lean and obese condition in the corresponding nutritional state was observed based upon a Student-Newman-Keuls Multiple Comparisons post-test (*P<0.05, *** p < 0.001). No differences were observed between the lean and fasted states for any antigen. Western blotting for β-Actin was used as a loading control. The densitometry values were 7539 ± 665; 7358 ± 692; 6835 ± 532 and 6833 ± 578 for fed lean, fed obese, fasted lean and fasted obese mouse liver, respectively (N.S., p>0.05).

Fig 4. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from Zucker rats

Liver lysates made from frozen lean (L) and fa/fa Fatty Zucker (F) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCKD E1α (liver does not express BCATm), pS293 E1α and BCKD kinase from random fed lean (L) and ob/ob (O) mice. Thus the other panels show the densitometric analysis of these blots for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). An ANOVA was first used to compare the lean and obese states as well as across the fed and fasted states followed by a Student-Newman-Keuls Multiple Comparisons post test. Asterisks indicate where statistical differences between the fatty and lean condition in the corresponding nutritional state was observed (** p < 0.01, *** p < 0.001). No statistical differences were detected between expression levels of any of the antigens when random fed and fasted states were compared. Western blotting for β-Actin was used as a loading control. The densitometry values were 6270 ± 703; 6198 ± 255; 6297 ± 20 and 6786 ± 599 for fed lean, fed obese, fasted lean and fasted obese rat liver, respectively (p>0.05).

Fig 4. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from Zucker rats

Liver lysates made from frozen lean (L) and fa/fa Fatty Zucker (F) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCKD E1α (liver does not express BCATm), pS293 E1α and BCKD kinase from random fed lean (L) and ob/ob (O) mice. Thus the other panels show the densitometric analysis of these blots for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). An ANOVA was first used to compare the lean and obese states as well as across the fed and fasted states followed by a Student-Newman-Keuls Multiple Comparisons post test. Asterisks indicate where statistical differences between the fatty and lean condition in the corresponding nutritional state was observed (** p < 0.01, *** p < 0.001). No statistical differences were detected between expression levels of any of the antigens when random fed and fasted states were compared. Western blotting for β-Actin was used as a loading control. The densitometry values were 6270 ± 703; 6198 ± 255; 6297 ± 20 and 6786 ± 599 for fed lean, fed obese, fasted lean and fasted obese rat liver, respectively (p>0.05).

Fig 4. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from Zucker rats

Liver lysates made from frozen lean (L) and fa/fa Fatty Zucker (F) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCKD E1α (liver does not express BCATm), pS293 E1α and BCKD kinase from random fed lean (L) and ob/ob (O) mice. Thus the other panels show the densitometric analysis of these blots for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). An ANOVA was first used to compare the lean and obese states as well as across the fed and fasted states followed by a Student-Newman-Keuls Multiple Comparisons post test. Asterisks indicate where statistical differences between the fatty and lean condition in the corresponding nutritional state was observed (** p < 0.01, *** p < 0.001). No statistical differences were detected between expression levels of any of the antigens when random fed and fasted states were compared. Western blotting for β-Actin was used as a loading control. The densitometry values were 6270 ± 703; 6198 ± 255; 6297 ± 20 and 6786 ± 599 for fed lean, fed obese, fasted lean and fasted obese rat liver, respectively (p>0.05).

Fig 4. Total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in liver from Zucker rats

Liver lysates made from frozen lean (L) and fa/fa Fatty Zucker (F) rat liver powders, were equalized for protein content, separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCKD E1α (liver does not express BCATm), pS293 E1α and BCKD kinase from random fed lean (L) and ob/ob (O) mice. Thus the other panels show the densitometric analysis of these blots for BCKD E1α (Panel B), pS293 E1α/BCKD E1α ratio (Panel C) and BCKD kinase (Panel D). An ANOVA was first used to compare the lean and obese states as well as across the fed and fasted states followed by a Student-Newman-Keuls Multiple Comparisons post test. Asterisks indicate where statistical differences between the fatty and lean condition in the corresponding nutritional state was observed (** p < 0.01, *** p < 0.001). No statistical differences were detected between expression levels of any of the antigens when random fed and fasted states were compared. Western blotting for β-Actin was used as a loading control. The densitometry values were 6270 ± 703; 6198 ± 255; 6297 ± 20 and 6786 ± 599 for fed lean, fed obese, fasted lean and fasted obese rat liver, respectively (p>0.05).

Fig 5. BCATm, total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice

Equal amounts of protein lysates, made from frozen lean (L) and obese (O) ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and ob/ob (O) mice. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and obese states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). A “c” indicates a significant difference in comparison to the fed lean control (P<0.05), “o” a significant difference when compared to the fed obese condition (P< 0.01). Western blotting for β-Actin was used as a loading control. The densitometry values were 8293 ± 493; 8318 ± 517; 8170 ± 337 and 7712 ± 202 for fed lean, fed obese, fasted lean and fasted obese mouse adipose, respectively (p>0.05).

Fig 5. BCATm, total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice

Equal amounts of protein lysates, made from frozen lean (L) and obese (O) ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and ob/ob (O) mice. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and obese states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). A “c” indicates a significant difference in comparison to the fed lean control (P<0.05), “o” a significant difference when compared to the fed obese condition (P< 0.01). Western blotting for β-Actin was used as a loading control. The densitometry values were 8293 ± 493; 8318 ± 517; 8170 ± 337 and 7712 ± 202 for fed lean, fed obese, fasted lean and fasted obese mouse adipose, respectively (p>0.05).

Fig 5. BCATm, total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice

Equal amounts of protein lysates, made from frozen lean (L) and obese (O) ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and ob/ob (O) mice. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and obese states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). A “c” indicates a significant difference in comparison to the fed lean control (P<0.05), “o” a significant difference when compared to the fed obese condition (P< 0.01). Western blotting for β-Actin was used as a loading control. The densitometry values were 8293 ± 493; 8318 ± 517; 8170 ± 337 and 7712 ± 202 for fed lean, fed obese, fasted lean and fasted obese mouse adipose, respectively (p>0.05).

Fig 5. BCATm, total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice

Equal amounts of protein lysates, made from frozen lean (L) and obese (O) ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and ob/ob (O) mice. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and obese states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). A “c” indicates a significant difference in comparison to the fed lean control (P<0.05), “o” a significant difference when compared to the fed obese condition (P< 0.01). Western blotting for β-Actin was used as a loading control. The densitometry values were 8293 ± 493; 8318 ± 517; 8170 ± 337 and 7712 ± 202 for fed lean, fed obese, fasted lean and fasted obese mouse adipose, respectively (p>0.05).

Fig 5. BCATm, total and pS293 phosphorylated BCKD E1α and BCKD kinase immunoreactivity in adipose tissue from ob/ob mice

Equal amounts of protein lysates, made from frozen lean (L) and obese (O) ob/ob mice adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and ob/ob (O) mice. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and obese states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). A “c” indicates a significant difference in comparison to the fed lean control (P<0.05), “o” a significant difference when compared to the fed obese condition (P< 0.01). Western blotting for β-Actin was used as a loading control. The densitometry values were 8293 ± 493; 8318 ± 517; 8170 ± 337 and 7712 ± 202 for fed lean, fed obese, fasted lean and fasted obese mouse adipose, respectively (p>0.05).

Fig 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1α, BCKD kinase immunoreactivity in adipose tissue of Zucker rats

Equal amounts of protein lysates, made from frozen lean (L) and obese (F) Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and fatty (F) Zucker rats. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). An “f” indicates a significant difference in comparison to the obese fat rats in the fed state (P<0.05). Western blotting for β-Actin was used as a loading control. The densitometry values were 5876 ± 232; 6122 ± 550; 8331 ± 251 and 8029 ± 347 for fed lean, fed obese, fasted lean and fasted obese rat adipose, respectively (p>0.05).

Fig 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1α, BCKD kinase immunoreactivity in adipose tissue of Zucker rats

Equal amounts of protein lysates, made from frozen lean (L) and obese (F) Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and fatty (F) Zucker rats. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). An “f” indicates a significant difference in comparison to the obese fat rats in the fed state (P<0.05). Western blotting for β-Actin was used as a loading control. The densitometry values were 5876 ± 232; 6122 ± 550; 8331 ± 251 and 8029 ± 347 for fed lean, fed obese, fasted lean and fasted obese rat adipose, respectively (p>0.05).

Fig 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1α, BCKD kinase immunoreactivity in adipose tissue of Zucker rats

Equal amounts of protein lysates, made from frozen lean (L) and obese (F) Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and fatty (F) Zucker rats. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). An “f” indicates a significant difference in comparison to the obese fat rats in the fed state (P<0.05). Western blotting for β-Actin was used as a loading control. The densitometry values were 5876 ± 232; 6122 ± 550; 8331 ± 251 and 8029 ± 347 for fed lean, fed obese, fasted lean and fasted obese rat adipose, respectively (p>0.05).

Fig 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1α, BCKD kinase immunoreactivity in adipose tissue of Zucker rats

Equal amounts of protein lysates, made from frozen lean (L) and obese (F) Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and fatty (F) Zucker rats. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). An “f” indicates a significant difference in comparison to the obese fat rats in the fed state (P<0.05). Western blotting for β-Actin was used as a loading control. The densitometry values were 5876 ± 232; 6122 ± 550; 8331 ± 251 and 8029 ± 347 for fed lean, fed obese, fasted lean and fasted obese rat adipose, respectively (p>0.05).

Fig 6. Expression of BCATm, pS293 phosphorylated and total BCKD E1α, BCKD kinase immunoreactivity in adipose tissue of Zucker rats

Equal amounts of protein lysates, made from frozen lean (L) and obese (F) Zucker rat adipose tissue powders, were separated by SDS-PAGE and transferred to PVDF. Panel A shows representative immunoblots of BCATm, total BCKD E1α, phosphorylated BCKD (pS293 E1α) and BCKD kinase from random fed and overnight fasted lean (L) and fatty (F) Zucker rats. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). An ANOVA followed by a Student-Newman-Keuls Multiple Comparisons post test was used to compare the lean and fatty states across the fed and fasted states (n=6/group). Asterisks indicate statistical differences between the obese and lean mice (* P<0.05, *** p < 0.01, *** p < 0.001). An “f” indicates a significant difference in comparison to the obese fat rats in the fed state (P<0.05). Western blotting for β-Actin was used as a loading control. The densitometry values were 5876 ± 232; 6122 ± 550; 8331 ± 251 and 8029 ± 347 for fed lean, fed obese, fasted lean and fasted obese rat adipose, respectively (p>0.05).

Fig 7. Plasma BCAA concentrations in overnight fasted morbidly obese human subjects before and after gastric bypass surgery

Plasma was collected between 9 and 12 AM after an overnight fast after induction of general anesthesia from subjects described in table 4 before Roux-en-Y gastric bypass surgery and later when subjects returned for a second surgery. Plasma was stored at −80°C until assay for total BCAA concentration was conducted as in Fig. 1 using an enzymatic assay. **p < 0.01 before versus after surgery.

Fig. 8. Subcutaneous and Visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss

Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in table 5. The freeze clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis and transferred to PVDF. Representative immunoblots are shown in Panel A. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). A bar appears between the bargraphs for the subcutaneous and visceral depots because those blots were not run on the same gels, transferred in the same box or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery. A t-test was used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Western blotting for β-Actin was used as a loading control. The densitometry values were Before: 8989 ± 512; After: 9249 ± 388 for subcutaneous adipose tissue and Before: 9683 ± 613; After: 10046 ± 752 for visceral adipose tissue (p>0.05 in both cases).

Fig. 8. Subcutaneous and Visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss

Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in table 5. The freeze clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis and transferred to PVDF. Representative immunoblots are shown in Panel A. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). A bar appears between the bargraphs for the subcutaneous and visceral depots because those blots were not run on the same gels, transferred in the same box or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery. A t-test was used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Western blotting for β-Actin was used as a loading control. The densitometry values were Before: 8989 ± 512; After: 9249 ± 388 for subcutaneous adipose tissue and Before: 9683 ± 613; After: 10046 ± 752 for visceral adipose tissue (p>0.05 in both cases).

Fig. 8. Subcutaneous and Visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss

Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in table 5. The freeze clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis and transferred to PVDF. Representative immunoblots are shown in Panel A. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). A bar appears between the bargraphs for the subcutaneous and visceral depots because those blots were not run on the same gels, transferred in the same box or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery. A t-test was used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Western blotting for β-Actin was used as a loading control. The densitometry values were Before: 8989 ± 512; After: 9249 ± 388 for subcutaneous adipose tissue and Before: 9683 ± 613; After: 10046 ± 752 for visceral adipose tissue (p>0.05 in both cases).

Fig. 8. Subcutaneous and Visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss

Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in table 5. The freeze clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis and transferred to PVDF. Representative immunoblots are shown in Panel A. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). A bar appears between the bargraphs for the subcutaneous and visceral depots because those blots were not run on the same gels, transferred in the same box or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery. A t-test was used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Western blotting for β-Actin was used as a loading control. The densitometry values were Before: 8989 ± 512; After: 9249 ± 388 for subcutaneous adipose tissue and Before: 9683 ± 613; After: 10046 ± 752 for visceral adipose tissue (p>0.05 in both cases).

Fig. 8. Subcutaneous and Visceral adipose tissue protein expression before and after bariatric surgery-induced weight loss

Abdominal subcutaneous and visceral (omental) adipose tissue was collected longitudinally from subjects described in table 5. The freeze clamped adipose tissue was powdered under liquid nitrogen. Infranatants of lysates prepared from these under phosphorylation state preserving conditions were solubilized with SDS-PAGE sample buffer, separated by SDS gel electrophoresis and transferred to PVDF. Representative immunoblots are shown in Panel A. Other panels show densitometric analysis of BCATm (Panel B), total BCKD E1α (Panel C), pS293 E1α immunoreactivity as a ratio to total BCKD (Panel D) and BCKD E1- α kinase (Panel E). A bar appears between the bargraphs for the subcutaneous and visceral depots because those blots were not run on the same gels, transferred in the same box or necessarily exposed with the same lot of ECL reagent. Thus comparisons are limited to the samples before and after gastric bypass surgery. A t-test was used to determine significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Western blotting for β-Actin was used as a loading control. The densitometry values were Before: 8989 ± 512; After: 9249 ± 388 for subcutaneous adipose tissue and Before: 9683 ± 613; After: 10046 ± 752 for visceral adipose tissue (p>0.05 in both cases).