Studies of nuclear encoded mitochondrial proteins in metabolism

Mitochondrial function is vital for human health. Inherited genetic disease can cause mitochondrial DNA (mtDNA) deficiency, disorders with generally poor prognosis. The majority of the genesinvolved in keeping a normal mitochondrial function are nuclear encoded. Deficiency in the nuclear encoded enzymes that provide building blocks for mtDNA synthesis, thymidine kinase 2 (TK2) and deoxyguanosine kinase (DGUOK), cause myopathy, encephalomyopathy and hepatocerebral disorders in humans. The nuclear encoded SLC25A10 is located in the mitochondrial inner membrane and is involved in regulation of cell metabolism.

In the first study we hypothesized that SLC25A10 had a regulatory role in cancer metabolism. Since the antidiabetic drug metformin was known to reduce the risk for cancer and to alter cell energy production, we used the siSLC25A10 model to investigate effects of metformin. In the siSLC25A10 cell line, metformin significantly downregulated the SLC25A10 carrier, especially at low glucose conditions, at both mRNA and protein levels. Since SLC25A10 is a mitochondrial transporter, this lower expression affects the exchange of nutrients with the potential to alter metabolic pathways of cancer cells.

In addition to cell culture studies, animal models are important tools to study mitochondrial functions. We constructed a DGUOK complete knockout mouse model to investigate the phenotype with the aim to find a model for mechanistic studies and treatment strategies. Interestingly, the Dguok−/− mice survived for more than 20 weeks despite very low mtDNA levels in liver tissue. Lipid metabolism as well as the de novo serine synthesis and the folate cycle were altered in the long surviving Dguok−/− mice. Two pyruvate kinase genes, PKLR and PKM, were active to supply pyruvate for the mitochondrial citric acid cycle (TCA cycle), which may be an explanation for the long-term survival although severely affected mitochondrial function.

We also constructed a skeletal and cardiac muscle specific TK2 knockout mouse (mTk2 KO) and a liver specific TK2 knockout mouse (livTK2 KO). The mTk2 KO mice showed dilated hearts and markedly reduced adipose tissue, but livTK2 KO mice were not different compared to the control group. A severe decrease of mtDNA was found only in skeletal muscle and heart tissue in the mTk2 KO mice. The mTk2 KO mice survived for maximum 16 weeks, but livTK2 KO mice survived for more than one and a half years. The data suggested that TK2 was vital for mtDNA maintenance in cardiac and skeletal muscle, while Tk2 deficiency in liver could be compensated for. Despite low mtDNA levels in the liver of the livTK2 KO mice we did not observe any difference compared to the control mice. The receptor for angiotensin-converting enzyme 2 (ACE2), was also affected by mtDNA deficiency in mTk2 KO mice. Since ACE2 is a receptor for the SARS-CoV-2 virus, its regulation in relation to mitochondrial function may have important clinical implications.

List of scientific papers

I. Zhao, Q; Zhou, X; Curbo, S; Karlsson, A. Metformin downregulates the mitochondrial carrier SLC25A10 in a glucose dependent manner. Biochemical Pharmacology. 444-450, volume 156, (2018).
https://doi.org/10.1016/j.bcp.2018.09.015

II. Zhou, X; Curbo, S; Zhao, Q; Krishnan, S; Kuiper, R; Karlsson, A. Severe mtDNA depletion and dependency on catabolic lipid metabolism in DGUOK knockout mice. Human Molecular Genetics. 2874-2884, volume 28, issue 17, (2019).
https://doi.org/10.1093/hmg/ddz103

III. Zhou, X, Mikaeloff, F; Curbo S, Zhao, Q, Kuiper R, Végvári A, Neogi U and Karlsson A. Coordinated pyruvate kinase activity is crucial for metabolic adaptation and cell survival during mitochondrial dysfunction. Human Molecular Genetics. 2012-2026, volume 30, issue 21, (2021).
https://doi.org/10.1093/hmg/ddab168

IV. Zhao, Q; Zhou, X; Kuiper, R; Curbo, S; Karlsson, A. Mitochondrial dysfunction is associated with lipid metabolism disorder and upregulation of angiotensin-converting enzyme 2. [Submitted]

V. Zhao, Q; Zhou, X; Kuiper, R; Curbo, S; and Karlsson, A. Specific depletion of thymidine kinase 2 in liver tissue does not affect mice survival. [Manuscript]