Steatotic livers are susceptible to normothermic ischemia-reperfusion injury from mitochondrial Complex-I dysfunction

Table 1 Content of high-fat/high-sucrose diet (D03021303; Research Diets, Inc., NJ, United States)

	Gram%	Kcal%
Protein	23.7	20
Carbohydrate	41.4	35
Fat	23.6	45
Total		100
Kcal/g	4.73
Ingredient
Casein, 80 Mesh	200	800
L-Cystine	3	12
Corn Starch	50	200
Maltodextrin 10	45.6	182
Sucrose	250	1000
Cellulose, BW200	50	0
Soybean Oil	25	225
Lard	177.5	1598
Mineral Mix S10026	10	0
DiCalcium Phosphate	13	0
Calcium Carbonate	5.5	0
Potassium Citrate. 1 H20	16.5	0
Vitamin Mix V10001	10	40
Choline Bitartrate	2	0
FD and C Blue Dye #1	0.05	0
Total	858.15	4057

Table 2 Mitochondrial respiration media

Chemical	Final concentration (mmol/L)
EGTA	0.5
MgCl2	3
K-Lactobionate	60
Taurine	20
KH2PO4	10
Sucrose	110
Bovine serum albumin	1 mg/mL
HEPES	20 (pH 7.0 at 37  °C)

Table 3 Mitochondrial respiration assay protocol

Reagents added	Final concentration in oxygraph chamber (mmol/L)	Action of reagent	Measurement output
Step 1
Glutamate	10	CI substrates	CI leak respiration
Malate	5		(CILeak)
Pyruvate	10
Step 2
ADP	1.25	Substrate for ATP generation	CI oxidative phosphorylation
Step 3
Succinate	10	CII substrate	CI+CII oxidative phosphorylation1
Step 4
Rotenone	0.001	CI inhibitor	Isolate flux to CII [CII(rot)]
Step 5
Oligomycin	0.0025	ATP-Synthase inhibitor	CI+CII leak respiration (CI,IILeak)
Step 6
FCCP	0.0015	Mitochondrial uncoupler	ETS capacity
Step 7
Antimycin A	0.0050	CIII inhibitor	Residual oxidase consumption

¹The individual contribution of CII to oxidative phosphorylation (CII-OXPHOS) can also be derived (CI+II-OXPHOS minus CI-OXPHOS). ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; CI: Complex I; CI-OXPHOS: Complex I oxidative phosphorylation; CI+II-OXPHOS: Complex I + Complex II oxidative phosphorylation; CI,II_Leak: Complex I + Complex II leak respiration; CI_Leak: Complex I leak respiration; CII: Complex II; CIII: Complex III; ETS: Electron transfer system; FCCP: Carbonylcyanide p-trifluoromethoxy-phenylhydrazone; Rot: Rotenone.