Figures and data
Pan-tissue mitochondrial respiration atlas overview, work-flow, and analysis pipeline.
A. Study overview highlighting the 33 different tissues collected from four different groups of mice (n = 10 / group) for respirometry analysis and mitochondrial content quantification. The four groups of mice are young male, young female, old male, and old female. Young = 10-week-old; old = 80-week-old. B. General schematic showing the preparation of samples for respirometry analysis. C. General representation of the electron transport chain to illustrate the key components of the respirometry assay used to assess mitochondrial function, the associated data, and examples of subsequent data analysis. AA, Antimycin A; Rot, Rotenone; TMPD, N, N, N’, N’-tetramethyl-p-phenylenediamine; Asc, Ascorbate; NADH, nicotinamide adenine dinucleotide.
Tissue-by-tissue analysis of mitochondrial function in male and female mice.
A. Young male mitochondrial respiration through complex I (CI, NADH-stimulated), complex II (CII, Succinate stimulated in the presence of Rotenone, a CI inhibitor), and at complex IV (CIV, via TMPD + Ascorbate). B. Young (10 weeks) female mitochondrial respiration through CI, CII, and at CIV. C. Old (80 weeks) male mitochondrial respiration through CI, CII, and at CIV. D. Old (80 weeks) female respiration through CI, CII, and at CIV, respectively. n = 10 mice per tissue. All data is represented as the mean alone and organized highest (left) to lowest (right). BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.
Tissue-by-tissue analysis of young male and female mitochondrial function.
A. (left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all young tissues. B. (left) Systems-level view of mitochondrial respiration through complex II (CII, Succinate-stimulated in the presence of rotenone to inhibit CI). (right) Mitochondrial respiration through CII across all tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all tissues. All data is presented as the mean with standard error, and organized highest to lowest for young male values. D. Heat map view of mitochondrial function across all tissues assayed (omitting reproductive organs). Data is represented as young male/female. Tissues with elevated respiration in males appear red while the same for females appear blue. Data is organized highest to lowest by summation of young male CI, CII, and CIV respiration values. n = 10 young male (YM, 10 weeks) and 10 young female (YF, 10 weeks) per tissue assayed. Statistical significance is represented as: a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.
Tissue-by-tissue analysis of young and old male mitochondrial function.
A. (left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all male tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all male tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all male tissues. All data is presented as the mean with standard error and organized highest to lowest for old male values. D. Heat map view of mitochondrial function across all male tissues assayed. Data is presented as old/young male. Tissues with elevated respiration in old males appear red while the same for young males appear blue. Data is organized highest to lowest by summation of old male CI, CII, and CIV respiration values. n = 10 old male (OM, 80 weeks) and 10 young male (YM, 10 weeks) per tissue assayed. Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.
Tissue-by-tissue analysis of young and old female mitochondrial function.
A. (left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all female tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all female tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all female tissues. All data is organized highest to lowest for old female values. D. Heat map view of mitochondrial function across all female tissues assayed. Data is presented as old/young female, so tissues with elevated mitochondrial respiration in old females appear red while the same for young females appear blue. Data is organized highest to lowest by summation of old female CI, CII, and CIV respiration values. n = 10 old female (OF, 80 weeks) and 10 young female (YF, 10 weeks) per tissue assayed. Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.
Tissue-by-tissue analysis of old male and female mitochondrial function.
A. (left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all old tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all tissues. C. (left) Systems level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all tissues. All data is presented as the mean with standard error and organized highest to lowest for male values. D. Heat map view of mitochondrial function across all tissues assayed. Data is represented as old male/female, so tissues with elevated mitochondrial respiration in males appear red while the same for females appear blue. Data is organized highest to lowest by summation of male CI, CII, and CIV respiration values. n = 10 old male (OM, 80 weeks) and 10 old female (OF, 80 weeks) per tissue assayed. Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles
Mitochondrial respiration is affected by sex but dominated by age.
A. Total number of statistically significant differences across all tissue-by-tissue comparisons, grouped by the component measured (CI = respiration through complex I, CII = respiration through complex II, or CIV = respiration through complex IV). B. Total number of significant differences across tissue-by-tissue comparisons grouped by the specific comparison, colored to represent the mitochondrial component affected (CI, CII, or CIV), and summed to the right of each histogram to highlight the number of significant findings per comparison. To underscore the number of statistically significant sex- or age-associated differences, the total number of significant findings from YM-by-YF and OM-by-OF (sex effect), and OM-by-YM and OF-by-YF (age effect) were summed. C. Quantification of the total number of tissues affected in each tissue-by-tissue comparison. Data are colored based on the specific comparison and grouped as sex- or age-associated to illustrate the effect-type. D. Cumulative absolute difference of means for CI, CII, and CIV (from left to right) grouped by effect-type, sex (originating from YM-by-YF or OM-by-OF) or age (originating from OM-by-YM or OF-by-YF). These graphs do not indicate directionality of the change, only the absolute cumulative magnitude. Each box within the histogram represents a unique tissue. All histograms are organized lowest (left) to highest (right) in degree of tissue contribution to total change given the comparison type. The top contributors to sex- or age-associated changes are highlighted with non-greyscale colors and their relative percentage of contribution to the total cumulative difference is provided. E. Principal Component Analysis (PCA) of all tissues and groups for CI, CII, and CIV (from left to right). F. Pearson correlation heat maps of all tissues combined from young and old, male and female mice. From top to bottom and left to right the samples are organized by group as follows: YF, YM, OF, and OM (n = 10 mice per group). YM = young male, YF = young female, OM = old male, OF = old female. Young = 10 weeks, Old = 80 weeks. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; At, atria; Vent, ventricles; Diaphr, diaphragm muscle; Med, kidney medulla.
The effects of age on mitochondrial respiration occur in sex-specific ways.
A-B. Systems-level view of the total relative change (sum of all relative changes) present at each mitochondrial parameter (respiration via CI, CII, or CIV) for males (M) and females (F), respectively. All change is old (O) compared to young (Y). C. Heat maps of O / Y data for each mitochondrial parameter per tissue across lifespan. Male and female values are grouped and organized by greatest (top) to least (bottom) sum of relative change per tissue. Tissues with the largest positive relative change as a result of aging are located on the top-most region of the heat maps, while those with the largest negative relative change are at the bottom. The middle of each heat map represents tissues with little relative response to aging or with opposite effect directions across sex. D. Graphs showing magnitude of sex-effect with age across all tissues and mitochondrial parameters (CI, CII, or CIV). The blue line and circles represent log2(old male/young male) data and the red line and boxes represent log2(old female/young female) data; both linked to the left y-axis. The black line and triangles represent the absolute difference of male and female relative change data (linked to the right y-axis). All data is organized from left to right by highest to lowest absolute difference of relative change across sex. Tissues with the greatest relative difference across sex are located to the left-most region of the graph, while those with the most similar response to aging are located to the right. A hollow blue circle, red box, and black triangle represent a tissue with a divergent sex-specific aging response. E. Summary diagrams classifying the relative trends of each mitochondrial parameter assayed per tissue as age- or age-and-sex-specific. Age-specific effects have a shared relative change direction across sex. Tissues in the red “Shared Increase” box have a positive relative change for both males and females. Tissues in the blue “Shared Decrease” box have a negative relative change for both males and females. Tissues in the Purple box labeled “Divergent” have opposing log2(O/Y) directions (signs, + or -) for male and female values; these tissues display relative mitochondrial changes that are age-and-sex-specific. F. Summary diagram showing tissues with a shared increase (red box) or decrease (blue box) consistent across all complexes (CI, CII, and CIV). Tissues within the shared increase or decrease classifications are grouped by their tissue-type, for example. PL, DI, QD, SL, and HV are all muscle, CO, HI, HY, and CE are all brain and so on. CI = respiration measured through complex I stimulated by NADH; CII = respiration through complex II stimulated by succinate in the presence of rotenone; CIV = respiration at complex IV stimulated by TMPD and ascorbate in the presence of rotenone and antimycin A. Tissues: BT = brown adipose tissue, CC = cecum, CE = cerebellum, CO =brain cortex, DI = diaphragm, DC = distal colon, DU = duodenum, EY = eyes, GS = gastrocnemius, GW = gonadal white adipose tissue, HS = hamstring, HA = heart atria, HV = heart ventricles, HI = hippocampus, HY = hypothalamus, IL = ileum, IW = inguinal white adipose tissue, JE = jejunum, KC = kidney cortex, KM = kidney medulla, LV = liver, LN = lung, MW = mesenteric white adipose tissue, PN = pancreas, PL = plantaris, PC = proximal colon, QD = quadriceps, SK = skin, SL = soleus, SP = spleen, ST = stomach, TN = tongue. Reproductive organs are omitted from cross-sex analysis.
Assay parameters for Seahorse-based respirometry analysis across all tissues.
Table shows tissues used, approximate size per tissue homogenized for analysis, amount of 1X MAS buffer used for homogenization, and the amount of protein used for respiration analysis across all tissues.
Mitochondrial respiration of brown adipose tissue (BAT) across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of cecum across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of brain cerebellum across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of brain cortex across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of diaphragm muscle across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of distal colon across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of duodenum across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of eyes across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of fallopian tubes and testicles across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the two primary comparisons are 1) young (10 weeks) female vs. old (80 weeks) female, 2) young male (10 weeks) vs. old (80 weeks) male. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of gastrocnemius muscle across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of gonadal white adipose tissue (gWAT) across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of hamstring (biceps femoris) muscles across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of heart atria across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of heart ventricles across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of brain hippocampus across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of brain hypothalamus across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of ileum across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, *** p < 0.001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of inguinal white adipose tissue (iWAT) across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of jejunum across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. ** p < 0.01. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of kidney cortex across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. ** p < 0.01. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of kidney medulla across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of liver across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of lung across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of mesenteric white adipose tissue (MesWAT) across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of pancreas across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of plantaris muscles across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of proximal colon across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of Quadricep muscle complex across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of skin across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of soleus muscles across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of spleen across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of stomach across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Mitochondrial respiration of tongue across sex and age.
A. Average oxygen consumption rate (OCR) traces per group, normalized to the amount of tissue homogenate protein input. From left to right the four primary comparisons are 1) young (10 weeks) male vs. young female, 2) old (80 weeks) male vs. old female, 3) young male vs. old male, and 4) young female vs. old female. Each group tracing represents the average trace of n = 10 samples. Each tracing shows the entire process of the Seahorse-based respirometry assay with injection compounds listed at the time of introduction to the sample. The sequence is as follows: i) basal reads, ii) addition of NADH (activation of respiration through complex I), iii) addition of antimycin A (AA, inhibitor of complex III) and rotenone (Rot, inhibitor of complex I), iv) addition of TMPD and ascorbate (to activate complex IV via electron donation to cytochrome c), and finally v) addition of azide (inhibitor of complex IV). B. The same information as presented in (A) conducted on the same samples, but the NADH injection step is replaced with the injection of succinate (to activate respiration through complex II) and rotenone (to inhibit complex I). C. Average values of all data presented in (A) and (B). Each dot represents the average of three technical replicates measured at three separate times. Both independent measurements of complex IV (CIV) were used to determine average CIV respiration. D. Average respiration values normalized to mitochondrial content as determined by MitoTracker Deep Red (MTDR) staining. E. Mitochondrial content quantification per group. n = 10 mice per group. * p < 0.05, ** p < 0.01, **** p < 0.0001. CI = complex I, CII = complex II, CIV = complex IV. YM = young male, YF = young female, OM = old male, OF = old female.
Tissue-centric view of mitochondrial changes in the brain across sex and age.
A. Depiction of whole mouse brain denoting the specific regions assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all brain regions assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 40 samples. Data point color indicates the specific brain region, and bar color indicates the group. C. Individual brain region average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are four background colors indicating brain region (from left to right: cerebellum (CE), cortex (CO), hippocampus (HI), and hypothalamus (HY)). D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old- dominant effect (respectively) within a specific brain region. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values.
Tissue-centric view of mitochondrial changes in the kidneys across sex and age.
A. Depiction of the kidney regions assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all kidney regions assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 20 samples. Data point color indicates the specific kidney region, and bar color indicates the group. C. Individual kidney region average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are two background colors indicating the specific region (from left to right: cortex then medulla). D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old-dominant effect (respectively) of a kidney region. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values. KC = kidney cortex, KM = kidney medulla; both regions collected and assayed bilaterally.
Tissue-centric view of mitochondrial changes in the heart across sex and age.
A. Depiction of the heart regions assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all heart regions assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 20 samples. Data point color indicates the specific heart region, and bar color indicates the group. C. Individual heart region average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are two background colors indicating the specific region (from left to right: atria then ventricles). D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old-dominant effect (respectively) of a heart region. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values. HA = heart atria (both left and right), HV = heart ventricles (both left and right).
Tissue-centric view of mitochondrial changes in skeletal muscle across sex and age.
A. Depiction of the skeletal muscles assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all skeletal muscles assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 70 samples. Data point color indicates the specific skeletal muscle, and bar color indicates the group. C. Individual skeletal muscle average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are seven background colors indicating the specific skeletal muscle [from left to right: diaphragm (DI), soleus (SL), tongue (TN), plantaris (PL), quadriceps (QD), hamstring (HS), and gastrocnemius (GS)]. D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old-dominant effect (respectively) of a skeletal muscle. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values. All muscles collected and assayed bilaterally.
Tissue-centric view of mitochondrial changes in the white adipose tissue across sex and age.
A. Depiction of white adipose tissue (WAT) regions assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all WAT regions assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 30 samples. Data point color indicates the specific WAT region, and bar color indicates the group. C. Individual WAT region average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are three background colors indicating the specific region (from left to right: gonadal WAT, inguinal WAT, mesenteric WAT). D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old-dominant effect (respectively) of a WAT region. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values. gW = gonadal WAT, iW = inguinal WAT, mW = mesenteric WAT.
Tissue-centric view of mitochondrial changes in the gastrointestinal tract across sex and age.
A. Depiction of the gastrointestinal tract (GI) denoting the specific regions assayed in this study. B. Average mitochondrial respiration through complex I (CI), respiration through complex II (CII), and respiration at complex IV (CIV) for all GI regions assayed. Data are grouped by sex and age (young (10 weeks) male, young female, old (80 weeks) male, old female which are represented as YM, YF, OM, and OF, respectively). Each data point represents the average of n = 10 mice, each bar represents n = 70 samples. Data point color indicates the specific GI region, and bar color indicates the group. C. Individual GI region average values of each group. Each bar represents the average of n = 10 mice. Under each mitochondrial parameter assayed (CI, CII, and CIV), there are seven background colors indicating the specific GI region (from left to right: stomach, duodenum, jejunum, ileum, cecum, proximal colon, and distal colon). D. Graphs depicting the total change in CI, CII, and CIV (from left to right) as a result of sex or age. Light blue or light grey bars to the left of the butterfly plot indicate a male- or young-dominant effect (respectively), while the light red and dark grey bars to the right indicate a female- or old-dominant effect (respectively) within a specific GI region. Male values were calculated by averaging young and old male. Female values were calculated by averaging young and old female. Young values were calculated by averaging young male and female. Old values were calculated by averaging old male and female. The change in a particular mitochondrial parameter was calculated by subtracting either the male and female or young and old values. ST = stomach, DU = duodenum, IL = ileum, JE = jejunum, CE = cecum, PC = proximal colon, DC = distal colon.
