Figure 1. Hepatocyte MCU Activity Regulates FAO-Coupled Mitochondrial Respiration.

(A) Generation and confirmation of MCU^Δhep mice by PCR genotyping (left) and western blotting (right).

(B) Schematic representation of the patch-clamp technique for measuring MCU channel activity.

(C) Representative I_MCU traces derived from MCU^fl/fl and MCU^Δhep mitoplasts.

(D) I_MCU densities (picoamperes/picofarads) in mitoplasts. n = 6.

(E) Representative traces of extramitochondrial Ca²⁺ ([Ca²⁺]_out) clearance and DJm in permeabilized hepatocytes from MCU^fl/fl and MCU^Δhep. n = 3.

(F) _mCa²⁺ uptake rate calculated from (E). n = 3.

(G) Representative traces of [Ca²⁺]_out rise from MCU^fl/fl and MCU^Δhep. n = 3.

(H) Quantification of _mCa²⁺ after addition of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) from (G). n = 3.

(I) Immunoblot for the reconstitution of MCU in MCU^Δhep mice using adenovirus-mediated delivery. n = 2.

(J) Reconstitution of MCU restores [Ca²⁺]_out clearance ability. n = 4.

(K) _mCa²⁺ uptake rate calculated from (J). n = 4.

(L) MCU reconstitution restores the matrix Ca²⁺ in MCU^Δhep. Shown are representative traces of [Ca²⁺]_out rise in response to FCCP. n = 4.

(M) Quantification of _mCa²⁺ calculated after addition of FCCP from (L). n = 4.

(N) MCU reconstitution restores the OCR in MCU KO hepatocytes. n = 3.

(O) Quantification of basal, ATP-coupled, and maximal OCRs in hepatocytes from (N). n = 3.

(P) The FAO OCR was measured in MCU^fl/fl and MCU^Δhep hepatocytes using palmitate as a substrate with or without etomoxir (40 mM). n = 3.

(Q) Quantification of basal, ATP-coupled, and maximal FAO-coupled OCRs from (P). n = 3.

(R) Measurement of hepatocyte ATP. n = 3.

(S) Measurement of AMP:ATP ratio in hepatocytes. n = 3. Statistical analysis: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant.

(T) Schematic of hepatocyte deletion of MCU, showing reduction in _mCa²⁺ and bioenergetic parameters.