Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes

Abstract

A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD(+)/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver.

Keywords: hepatocyte; induced pluripotent stem cell (iPS cell) (iPSC); lactic acidosis; liver; liver metabolism; mitochondrial disease.

FIGURE 1.

Generation of DMRC-iPSCs. A, homoplasmic mutation in DMRC fibroblasts (FIB) and DMRC-iPSCs. B, transcriptional expression of pluripotency-associated genes in DMRC-iPSCs. C, expression of pluripotency-associated proteins in DMRC-iPSCs. Two DMRC-iPSC lines expressed pluripotency-associated markers, including OCT4, SOX2, NANOG, SSEA4, TRA-1–60, and TRA-1–81. Scale bar, 200 μm. D, reprogramming of DNA methylation in DMRC-iPSCs. The promoter regions of these genes were highly demethylated in DMRC-iPSCs compared with DMRC fibroblasts. E, normal karyotypes of DMRC-iPSCs. F, expression of marker genes of the three germ layers in DMRC-EBs. Marker genes of three germ layers were expressed in DMRC-EBs. G, teratomas derived from DMRC-iPSCs. The teratomas formed after subcutaneous injection of DMRC-iPSCs contained diverse tissues of three germ layers. Scale bar, 50 μm. FOXA2, forkhead box protein A2; GATA2, GATA-binding protein 2; PAX6, paired box 6; SOX17, sex-determining regions Y-BOX 17; T, brachyury.

FIGURE 2.

Mitochondrial physiology in DMRC fibroblasts and DMRC-iPSCs. A, mitochondrial morphology in WT and DMRC fibroblasts. Mitochondria of DMRC fibroblasts were smaller and poorly developed compared with WT fibroblasts. Scale bar, 1000 nm. B, mitochondrial morphology of WT- and DMRC-iPSCs. No differences were observed in the mitochondrial morphology between WT- and DMRC-iPSCs. Scale bar, 1000 nm. C, increment of p-DRP1 (Se-616) in DMRC fibroblasts. The ratio of p-DRP1/DRP1 was quantified (right, n = 3). D, mtDNA copy number of DMRC fibroblasts. mtDNA copy number decreased in DMRC fibroblasts. **, p < 0.01 (n = 3). E, mtDNA copy number of DMRC-iPSCs (n = 3). FIB, fibroblasts; mtDNA, mitochondrial DNA; EM, electron microscopy; DRP1, dynamin-related protein 1.

FIGURE 3.

Differentiation of DMRC-iPSCs into hepatocytes. A, overall protocol for hepatic differentiation of hiPSCs. B, transcriptional expression of hepatic genes in DMRC hepatocytes. C, efficiency of hepatic differentiation. Hepatocytes differentiated from hiPSCs were analyzed by flow cytometry for albumin, a hepatic marker. D, expression of the hepatic marker albumin in WT and DMRC hepatocytes. Scale bar, 50 μm. E, albumin secretion in WT and DMRC hepatocytes. F, visualization of glycogen storage by PAS staining co-stained with α1-antitrypsin (AAT). Scale bar, 50 μm. G, decrease of the glycogen level in DMRC hepatocytes. ***, p < 0.001 (n = 3). HEP, hepatocytes; NC, negative control.

FIGURE 4.

Mitochondrial physiology in DMRC hepatocytes. A, mitochondrial morphology in WT and DMRC hepatocytes. Mitochondria were fragmented in DMRC hepatocytes. Mitochondria were indicated by white arrowheads. Scale bar, 500 nm. B, increment of p-DRP1 in DMRC hepatocytes. C, reduction of mtDNA copy number in DMRC hepatocytes. **, p < 0.01; ***, p < 0.001 (n = 3).

FIGURE 5.

Aberrant oxidative phosphorylation in DMRC hepatocytes. A, decrease of complex I activity in DMRC fibroblasts. **, p < 0.01 (n = 3). B, reduction of complex I activity in DMRC hepatocytes. *, p < 0.05 (n = 3). C, decreased ATP production rate in DMRC fibroblasts. **, p < 0.01 (n = 3). D, reduction of ATP production rate in DMRC hepatocytes. *, p < 0.05 (n = 3). E, decrease of cellular ATP levels in DMRC fibroblasts. *, p < 0.05 (n = 3). F, reduced cellular ATP levels in DMRC hepatocytes. *, p < 0.05 (n = 3). G, enhanced p-AMPK levels in DMRC hepatocytes.

FIGURE 6.

Abnormal metabolism in DMRC hepatocytes. A, decrease of the NAD⁺/NADH ratio in DMRC fibroblasts. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). B, reduction of the NAD⁺/NADH ratio in DMRC hepatocytes. **, p < 0.01; ***, p < 0.001 (n = 3). C, lactate secretion in DMRC fibroblasts (left) and DMRC hepatocytes (right). Lactate secretion was increased in DMRC fibroblasts but not in DMRC hepatocytes. *, p < 0.05 (n = 3). D, glucose uptake in DMRC fibroblasts (left) and DMRC hepatocytes (right) using RI. Both DMRC fibroblasts and hepatocytes showed increased glucose uptake. **, p < 0.01; ***, p < 0.001 (n = 3). E, relative expression of lactate metabolism-associated genes in DMRC hepatocytes. Transcriptional expression of Glc-6-Pase, FBP1, PEPCK, and AGXT was significantly decreased in DMRC hepatocytes. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 4). F, lactate uptake in DMRC hepatocytes using RI. DMRC hepatocytes showed decreased lactate uptake. ***, p < 0.001 (n = 3). G6Pase, glucose-6-phosphatase; FBP1, fructose-1, 9-glyoxylate aminotransferase. PEPCK, phosphoenolpyruvate carboxykinase; AGXT, alanine-glyoxylate aminotransferase.

FIGURE 7.

Effects of AMPK inhibition on the metabolism of DMRC hepatocytes. A, reduction of the AMPK phosphorylation level in WT and DMRC hepatocytes by treatment of 25 μm compound C. B, inhibitory effect of compound C on AMPK activity. ***, p < 0.001 (n = 3). C, visualization of glycogen storage after compound C treatment in DMRC hepatocytes. Scale bar, 200 μm. D, recovery of the glycogen level in DMRC hepatocytes following AMPK inhibition. *, p < 0.05; **, p < 0.01 (n = 3). E, activation of lactate metabolism-associated genes in DMRC hepatocytes after compound C treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). F, enhanced lactate turnover rate in DMRC hepatocytes by compound C treatment. **, p < 0.01 (n = 3). CC, compound C.

FIGURE 8.

Effects of AMPK knockdown on the expression of gluconeogenic genes in DMRC hepatocytes. A, decreased AMPK protein by siRNA treatment in iPSC-derived hepatocytes. Quantification of band density is shown in the right histogram. AMPK protein level was decreased to ∼50% in iPSC-derived hepatocytes by transfection with AMPK siRNAs. B, reduction of AMPK mRNA level in iPSC-derived hepatocytes by siRNA treatment. *, p < 0.05; **, p < 0.01 (n = 3). C, increased expression of lactate metabolism-associated genes in siRNA-treated DMRC hepatocytes. *, p < 0.05; **, p < 0.01 (n = 3). D, model for lactic acidosis by MTND1 mutation in the DMRC patient. siRNA, small interfering RNA.