Journal: bioRxiv

Article Title: Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands

doi: 10.1101/2023.11.27.568879

Figure Lengend Snippet: ECAR, OCR, and ATP production rate increase acutely at 24-hours following radiation in salivary acinar cells. ( a ) Primary acinar cells were isolated from parotid salivary glands of untreated female FVB mice (n=3/group), cultured for 5 days, and then irradiated at 24 and 48 hours prior to the Seahorse XF Real-Time ATP Rate Assay. ( b ) Individual ECAR readings for untreated (UT), 24-hour IR (24hr IR), and 48-hour IR (48hr IR) groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( c ) Average basal, oligo, and rot/AA ECAR readings for UT, 24hr IR, and 48hr IR. ( d ) Individual OCR readings for UT, 24hr IR, and 48hr IR groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( e ) Average basal, oligo, and rot/AA OCR readings for UT, 24hr IR, and 48hr IR. ( f ) Basal ATP production rates of UT, 24hr IR, and 48hr IR groups. ( g ) The proportion of mitochondrial (mito) ATP production rate and glycolytic (glyco) ATP production rate for UT, 24hr IR, and 48hr IR groups. Each panel is representative of 3 independent assays (n=3/group). Significant differences were determined using one-way ANOVA and Bonferroni post-hoc test, p<0.05. Treatment groups with the same letter are not significantly different from each other.

Article Snippet: Female FVB/NJ mice (wild type; stock no. 001800) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Isolation, Cell Culture, Irradiation, Injection

Journal: bioRxiv

Article Title: Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands

doi: 10.1101/2023.11.27.568879

Figure Lengend Snippet: ECAR, OCR, and ATP production increase at 5 days and decrease chronically following radiation in salivary acinar cells. ( a ) Female FVB mice received 5 Gy IR targeted to the head and neck region (n=3/group) and primary acinar cells were isolated from parotid salivary glands of untreated (UT) and IR mice at day 5, 30, and 60 post-IR. Cells were cultured for 2 days prior to running the Seahorse XF Real-Time ATP Rate Assay. ( b ) Individual ECAR readings for untreated UT and day 5 IR (D5 IR) groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( c ) Average basal, oligo, and rot/AA ECAR readings for UT and D5 IR. ( d ) Individual OCR readings for UT and D5 IR groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( e ) Average basal, oligo, and rot/AA OCR readings for UT and D5 IR. ( f ) Basal ATP production rates of UT and D5 IR groups. ( g ) The proportion of mitochondrial (mito) ATP production rate and glycolytic (glyco) ATP production rate for UT and D5 IR groups. ( h ) Individual ECAR readings for UT, day 30 IR (D30 IR), and day 60 IR (D60 IR) groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( i ) Average basal, oligo, and rot/AA ECAR readings for UT, D30 IR, and D60 IR. ( j ) Individual OCR readings for UT, D30 IR, and D60 IR groups with the responses to 2.0uM oligomycin (oligo) injection and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( k ) Average basal, oligo, and rot/AA OCR readings for UT, D30 IR, and D60 IR. ( l ) Basal ATP production rates of UT, D30 IR, and D60 IR groups. ( m ) The proportion of mitochondrial (mito) ATP production rate and glycolytic (glyco) ATP production rate for UT and D5 IR groups. Each panel is representative of 3 independent assays (n=3/group). Significant differences were determined using one-way ANOVA and Bonferroni post-hoc test, p<0.05. Treatment groups with the same letter are not significantly different from each other.

Article Snippet: Female FVB/NJ mice (wild type; stock no. 001800) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Isolation, Cell Culture, Injection

Journal: bioRxiv

Article Title: Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands

doi: 10.1101/2023.11.27.568879

Figure Lengend Snippet: Glycolytic hexokinase protein levels and enzymatic activity increase at day 3 following radiation and lactate concentration increases at day 5 and day 14 following radiation in the salivary gland. Female FVB mice were untreated (UT) or exposed to IR and parotid salivary glands were removed at 3 (D3 IR), 5 (D5 IR), 14 (D14 IR), and 30 (D30 IR) days after radiation treatment (see ). ( a ) Cropped representative western blots probing for hexokinase 1 (HK1), phosphofructokinase-muscle (PFKM), and pyruvate kinase muscle isozyme-1 (PKM1) using untreated, D3 IR, D5 IR, and D30 tissue samples (n=3/group). Extracellular signal-regulated kinase 1/2 (ERK 1/2) was used as a loading control. Original blots shown in Supplementary Figure S5. ( b-d ) Quantification of (a). ( e ) A hexokinase enzyme activity assay was performed on tissue samples (n=4/group) and activity was measured over 60 minutes. ( f ) A pyruvate kinase enzyme activity assay was performed on tissue samples (n=4/group) and activity was measured over 60 minutes. ( g ) A lactate assay kit was used to measure lactate concentration in tissue samples (n=5/group). Data are presented as mean ± standard error of the mean (SEM). Significant differences were determined using one-way ANOVA and Bonferroni post-hoc test, p<0.05. Treatment groups with the same letter are not significantly different from each other.

Article Snippet: Female FVB/NJ mice (wild type; stock no. 001800) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Activity Assay, Concentration Assay, Western Blot, Enzyme Activity Assay, Lactate Assay

Journal: bioRxiv

Article Title: Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands

doi: 10.1101/2023.11.27.568879

Figure Lengend Snippet: Mitochondrial complex I and III protein subunit levels and spare respiratory capacity decrease at day 5 following radiation and mitochondrial DNA copy number increases chronically following radiation in the salivary gland. Female FVB mice were untreated or exposed to IR and parotid salivary glands were removed at D3, D5, and D30 after radiation (see ). ( a ) Cropped representative western blot probing for complex I-75 kDa subunit using tissue samples (n=3/group). Original blots shown in Supplementary Figure S6. ( b ) Quantification of (a). Data presented as mean ± standard error of the mean (SEM). ( c ) Cropped representative western blot probing for ubiquinol-cytochrome-C reductase complex core protein 2 (UQCRC2) using tissue samples (n=3/group). ( d ) Quantification of (c). Data presented as mean ± standard error of the mean (SEM). ( e-g ) Female FVB mice received 5 Gy IR targeted to the head/neck region (n=3/group) and primary acinar cells were isolated from parotid salivary glands of UT and IR mice at day 5 and cultured for 2 days prior to the Seahorse XF Cell Mito Stress Test. ( e ) Individual OCR readings for UT and D5 IR groups with responses to 2.0uM oligomycin (oligo) injection, 2.0uM FCCP injection, and 0.5uM rotenone/antimycin A (rot/AA) injection shown. ( f ) Average basal, oligo, FCCP, and rot/AA OCR readings for UT and D5 IR. ( g ) Spare respiratory capacity of UT and D5 IR groups. Each panel is representative of 2 independent assays (n=3/group). Significant differences were determined using the two-tailed unpaired T-test, p<0.05 in panel (g). ( h-i ) DNA was isolated from tissue samples (n=5/group) and RT-qPCR was performed with primers specific for the mitochondrial gene cyclooxygenase-2 ( COX2 ) and the ribosomal gene ribosomal protein S18 ( Rsp18 ) (h), and primers specific for the mitochondrial gene NADH:ubiquinone oxidoreductase core subunit 1 ( ND1 ) and the nuclear gene hexokinase 2 ( HK2 ) in (i). Data were normalized to 15S ribosomal protein as an internal control and fold change was calculated relative to DNA content in UT mice. Mitochondrial DNA (mtDNA) copy number was estimated using the ratio of COX2/Rsp18 DNA content (h) and the ratio of ND1/HK2 DNA content (i). Data presented as interquartile range of the data with the median indicated by the line ± standard error of the mean (SEM). Significant differences were determined using one-way ANOVA and Bonferroni post- hoc test, p<0.05. Treatment groups with the same letter are not significantly different from each other.

Article Snippet: Female FVB/NJ mice (wild type; stock no. 001800) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Western Blot, Isolation, Cell Culture, Injection, Two Tailed Test, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands

doi: 10.1101/2023.11.27.568879

Figure Lengend Snippet: Dependency on long-chain fatty acids as a fuel source increases at day 5 following radiation treatment compared to untreated in salivary acinar cells. Female FVB mice received 5 Gy IR targeted to the head and neck region (n=3/group) and primary acinar cells were isolated from parotid salivary glands of untreated (UT) and IR mice at day 5, cultured for 2 days, and then the Seahorse XF Mito Fuel Flex Test Kit was performed (see ). ( a ) Schematic diagram of glucose, glutamine, and long-chain fatty acid metabolism and where the assay inhibitors block fuel oxidation. Image created in Biorender.com. ( b ) Glucose dependency and flexibility in untreated and day 5 IR (D5 IR) groups. ( c ) Glutamine dependency and flexibility in untreated and D5 IR groups. ( d ) Long-chain fatty acid (LCFA) dependency and flexibility in untreated and D5 IR groups. Data is expressed as % of total oxidation of glucose, glutamine, and long-chain fatty acids ± standard error of the mean (SEM). Significant differences were determined using two-way ANOVA and Bonferroni post-hoc test, p<0.05. Treatment groups with the same letter are not significantly different from each other. Abbreviations: MPC = mitochondrial pyruvate carrier complex, CPT1A = carnitine palmitoyltransferase 1A, TCA cycle = tricarboxylic acid cycle, α-KG = alpha-ketoglutarate, GLS1 = glutaminase-1, BPTES = (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide).

Article Snippet: Female FVB/NJ mice (wild type; stock no. 001800) were purchased from Jackson Laboratories (Bar Harbor, ME).

Techniques: Isolation, Cell Culture, Blocking Assay