Limits...
Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

Munusamy S, Saba H, Mitchell T, Megyesi JK, Brock RW, Macmillan-Crow LA - BMC Endocr Disord (2009)

Bottom Line: Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury.Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria.The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology & Toxicology, Division of Nephrology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. munusamyshankar@uams.edu

ABSTRACT

Background: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).

Methods: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.

Results: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.

Conclusion: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

No MeSH data available.


Related in: MedlinePlus

Diabetes-induced alterations in the activity of mitochondrial complexes and ATP levels. A) Blue Native Gel Electrophoresis (BN-PAGE) showing alterations in mitochondrial complex proteins during diabetes. The box represents Complexes-III and V that differ in control and diabetic (5-week post-STZ) mitochondria. B) Densitometric analysis of BN-PAGE showing a decrease in Complex III levels during diabetes. (C-H) Bar graphs indicating inactivation of mitochondrial Complex-III (C), and induction of F0, F1-ATPase (Complex-V) activity (D), and ATP levels (E) in diabetic rat kidney as compared to controls. Activities of renal mitochondrial complexes: F) Complex-I, G) Complex-II, and H) Complex-IV, were not altered by diabetes. All values are expressed as percentage Mean ± SEM (n = 5) of controls (set to 100). * P < 0.05 compared with age-matched controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2636815&req=5

Figure 2: Diabetes-induced alterations in the activity of mitochondrial complexes and ATP levels. A) Blue Native Gel Electrophoresis (BN-PAGE) showing alterations in mitochondrial complex proteins during diabetes. The box represents Complexes-III and V that differ in control and diabetic (5-week post-STZ) mitochondria. B) Densitometric analysis of BN-PAGE showing a decrease in Complex III levels during diabetes. (C-H) Bar graphs indicating inactivation of mitochondrial Complex-III (C), and induction of F0, F1-ATPase (Complex-V) activity (D), and ATP levels (E) in diabetic rat kidney as compared to controls. Activities of renal mitochondrial complexes: F) Complex-I, G) Complex-II, and H) Complex-IV, were not altered by diabetes. All values are expressed as percentage Mean ± SEM (n = 5) of controls (set to 100). * P < 0.05 compared with age-matched controls.

Mentions: The BN-PAGE data indicated that Complex-III was decreased (77.8% of control) and Complex-V was increased in the diabetic kidneys compared to the control group (Figure 2A and 2B). In addition to BN-PAGE, respiratory complex activity assays were performed which further confirmed that Complex-III activity was significantly reduced in diabetic renal mitochondria (66.32 ± 1.93%) as compared to the age-matched controls (100 ± 3.58%, Figure 2C); while F0F1-ATPase (Complex-V) activity was increased (148.71 ± 6.45% in diabetic Vs 100 ± 3.37% in control; Figure 2D). Consistent with increased F0F1-ATPase activity, ATP levels were also significantly increased in diabetic rat kidneys (143.39 ± 11.56%) compared to the age-matched controls (100 ± 2.01%; Figure 2E). Interestingly, no significant changes were observed in the activities of Complexes I, II and IV between the diabetic and control groups (Figure 2F–H). It is important to realize that Complex III contains 11 subunits. Hence, further studies were performed to identify the specific subunits within Complex-III that might be altered during hyperglycemia using Complex III immunoprecipitation.


Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

Munusamy S, Saba H, Mitchell T, Megyesi JK, Brock RW, Macmillan-Crow LA - BMC Endocr Disord (2009)

Diabetes-induced alterations in the activity of mitochondrial complexes and ATP levels. A) Blue Native Gel Electrophoresis (BN-PAGE) showing alterations in mitochondrial complex proteins during diabetes. The box represents Complexes-III and V that differ in control and diabetic (5-week post-STZ) mitochondria. B) Densitometric analysis of BN-PAGE showing a decrease in Complex III levels during diabetes. (C-H) Bar graphs indicating inactivation of mitochondrial Complex-III (C), and induction of F0, F1-ATPase (Complex-V) activity (D), and ATP levels (E) in diabetic rat kidney as compared to controls. Activities of renal mitochondrial complexes: F) Complex-I, G) Complex-II, and H) Complex-IV, were not altered by diabetes. All values are expressed as percentage Mean ± SEM (n = 5) of controls (set to 100). * P < 0.05 compared with age-matched controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636815&req=5

Figure 2: Diabetes-induced alterations in the activity of mitochondrial complexes and ATP levels. A) Blue Native Gel Electrophoresis (BN-PAGE) showing alterations in mitochondrial complex proteins during diabetes. The box represents Complexes-III and V that differ in control and diabetic (5-week post-STZ) mitochondria. B) Densitometric analysis of BN-PAGE showing a decrease in Complex III levels during diabetes. (C-H) Bar graphs indicating inactivation of mitochondrial Complex-III (C), and induction of F0, F1-ATPase (Complex-V) activity (D), and ATP levels (E) in diabetic rat kidney as compared to controls. Activities of renal mitochondrial complexes: F) Complex-I, G) Complex-II, and H) Complex-IV, were not altered by diabetes. All values are expressed as percentage Mean ± SEM (n = 5) of controls (set to 100). * P < 0.05 compared with age-matched controls.
Mentions: The BN-PAGE data indicated that Complex-III was decreased (77.8% of control) and Complex-V was increased in the diabetic kidneys compared to the control group (Figure 2A and 2B). In addition to BN-PAGE, respiratory complex activity assays were performed which further confirmed that Complex-III activity was significantly reduced in diabetic renal mitochondria (66.32 ± 1.93%) as compared to the age-matched controls (100 ± 3.58%, Figure 2C); while F0F1-ATPase (Complex-V) activity was increased (148.71 ± 6.45% in diabetic Vs 100 ± 3.37% in control; Figure 2D). Consistent with increased F0F1-ATPase activity, ATP levels were also significantly increased in diabetic rat kidneys (143.39 ± 11.56%) compared to the age-matched controls (100 ± 2.01%; Figure 2E). Interestingly, no significant changes were observed in the activities of Complexes I, II and IV between the diabetic and control groups (Figure 2F–H). It is important to realize that Complex III contains 11 subunits. Hence, further studies were performed to identify the specific subunits within Complex-III that might be altered during hyperglycemia using Complex III immunoprecipitation.

Bottom Line: Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury.Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria.The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology & Toxicology, Division of Nephrology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. munusamyshankar@uams.edu

ABSTRACT

Background: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).

Methods: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.

Results: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.

Conclusion: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

No MeSH data available.


Related in: MedlinePlus