Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1

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Abstract

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1.

Introduction

SIR2 protein is an NAD-dependent deacetylase [1]. In yeast, increasing the dosage of the SIR2 gene extends life span, whereas disruption of the SIR2 gene shortens it [2]. SIR2 determines life span not only in yeast but also in Caenorhabditis elegans[3] and Drosophila melanogaster[4]. Mammals have seven SIR2 homologues, SIRT1-7 [5]. SIRT1 deacetylates and regulates the activities of many proteins in the nucleus. SIRT1 upregulates expression of gluconeogenic genes and downregulates glycolytic genes through deacetylation of PPARγ coactivator-1α and FOXO1 [6], [7].

SIRT3, SIRT4, and SIRT5 proteins are known to be localized to mitochondria [8]. In vitro, SIRT3 protein deacetylates and activates mitochondrial enzymes such as glutamate dehydrogenase, isocitrate dehydrogenase 2, and acetyl CoA synthetase 2 (AceCS2) [9], [10], [11]. While SIRT4 does not have NAD-dependent deacetylase activity, it does have ADP-ribosyl transferase activity. SIRT4 inhibits insulin secretion by repression of glutamate dehydrogenase activity through ADP-ribosylation [12], [13]. SIRT5 protein also exhibits NAD-dependent deacetylase activity on histone H4 peptide in vitro[13]. Recently, Nakagawa et al. reported that SIRT5 interacts with carbamoyl phosphate synthetase 1 (CPS1), and that deacetylated CPS1 is decreased and CPS1 activity is downregulated in livers of SIRT5 knockout (KO) mice [14].

During fasting or starvation, circulating amino acids are derived mainly from catabolism of skeletal muscle, and are used in gluconeogenesis in liver to maintain blood glucose levels. The ammonia co-generated in liver from these amino acids is toxic; the urea cycle detoxifies this ammonia by converting it to non-toxic, water-soluble urea, which is readily excreted from kidney [15], [16].

CPS1 is the mitochondrial protein that catalyzes the first step of the urea cycle, the condensation of ammonia with bicarbonate to form carbamoyl phosphate [15], [16]. Patients with CPS1 deficiency exhibit lethally severe hyperammonemia in the neonatal period [16], which suggests a critical role for CPS1 in the urea cycle.

In the present study, to investigate the physiological role of SIRT5, we generated SIRT5-overexpressing transgenic (SIRT5 Tg) mice and attempted to identify the target protein of SIRT5 regulation in liver. We show here that SIRT5 protein might regulate urea production by deacetylation and activation of mitochondrial CPS1, complementing the previous study of SIRT5 KO mice [14].

Section snippets

Materials and methods

Animal Experiments: The mice were housed in an air-controlled (temperature 25 °C) room with dark–light cycle (10 h; 14 h). Animal care and procedures were approved by the Animal Care Committee of Kyoto University.

Isolation of total RNA and quantitative RT-PCR: Total RNA was isolated from livers, kidneys and hearts of 11 week-old C57BL/6 mice using Trizol (Invitrogen), and cDNA was prepared by reverse transcriptase (Superscript II; Invitrogen) with an oligo (dT) primer. SIRT5 mRNA levels were

Upregulation of SIRT5 mRNA levels by fasting

Expression levels of SIRT1 mRNA are known to be increased in liver and heart by fasting [25]. However, it is unclear whether the expression levels of SIRT5 are regulated by nutrient conditions. To evaluate alteration of SIRT5 mRNA expression levels in different nutrient conditions, total RNA was extracted from organs including liver, kidney, and heart in C57BL/6 mice fed ad libitum, fasted for 24 h, or refed for 24 h after 24-h fasting, and quantitative RT-PCR was carried out. SIRT5 mRNA levels

Discussion

In the present study, SIRT5 mRNA levels in liver were found to be increased by fasting. To investigate the function of SIRT5 in liver, we established SIRT5 Tg mice and identified CPS1 as the target protein of SIRT5 by analyses of liver mitochondrial proteins using two-dimensional electrophoresis and MALDI-TOF-MS. We found that CPS1 is deacetylated and that CPS1 activity is significantly increased in the liver of SIRT5 Tg mice. CPS1 is the first and key enzyme of the urea cycle, condensing

Acknowledgments

This study was supported by Scientific Research Grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, from the Ministry of Health, Labor, Welfare, Japan, and by the Kyoto University Global COE Program “Center for Frontier Medicine”.

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