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Protein O-Glcnacylation Is Essential for the Maintenance of Renal Energy Homeostasis and Function Via Lipolysis During Fasting and Diabetes

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Protein O-Glcnacylation Is Essential for the Maintenance of Renal Energy Homeostasis and Function Via Lipolysis During Fasting and Diabetes BASIC RESEARCH www.jasn.org Protein O-GlcNAcylation Is Essential for the Maintenance of Renal Energy Homeostasis and Function via Lipolysis during Fasting and Diabetes Sho Sugahara,1 Shinji Kume,1 Masami Chin-Kanasaki,1,2 Issei Tomita,1 Mako Yasuda-Yamahara,1 Kosuke Yamahara,1 Naoko Takeda,1 Norihisa Osawa,1 Motoko Yanagita,3 Shin-ichi Araki,1,2 and Hiroshi Maegawa1 1Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan; 2Division of Blood Purification, Shiga University of Medical Science Hospital, Otsu, Shiga, Japan; and 3Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan ABSTRACT Background Energy metabolism in proximal tubular epithelial cells (PTECs) is unique, because ATP pro- duction largely depends on lipolysis in both the fed and fasting states. Furthermore, disruption of renal lipolysis is involved in the pathogenesis of diabetic tubulopathy. Emerging evidence suggests that protein O-GlcNAcylation, an intracellular nutrient-sensing system, may regulate a number of metabolic pathways according to changes in nutritional status. Although O-GlcNAcylation in PTECs has been demonstrated experimentally, its precise role in lipolysis in PTECs is unclear. Methods To investigate the mechanism of renal lipolysis in PTECs—specifically, the role played by protein O-GlcNAcylation—we generated mice with PTECs deficient in O-GlcNAc transferase (Ogt). We analyzed their renal phenotypes during ad libitum feeding, after prolonged fasting, and after mice were fed a high- fat diet for 16 weeks to induce obesity and diabetes. Results Although PTEC-specific Ogt-deficient mice lacked a marked renal phenotype during ad libitum feeding, after fasting 48 hours, they developed Fanconi syndrome–like abnormalities, PTEC apoptosis, and lower rates of renal lipolysis and ATP production. Proteomic analysis suggested that farnesoid X receptor–dependent upregulation of carboxylesterase-1 is involved in O-GlcNAcylation’s regulation of lipolysis in fasted PTECs. PTEC-specificOgt-deficient mice with diabetes induced by a high-fat diet de- veloped severe tubular cell damage and enhanced lipotoxicity. Conclusions Protein O-GlcNAcylation is essential for renal lipolysis during prolonged fasting and offers PTECs significant protection against lipotoxicity in diabetes. JASN 30: 962–978, 2019. doi: https://doi.org/10.1681/ASN.2018090950 Mammalian cells use glucose, fatty acids (FAs), and involved in the pathogenesis of tubulopathy in ketone bodies to produce ATP, which is essential for kidney diseases, including diabetic kidney disease their survival and function. The identity of the nu- (DKD).527 Therefore, renal lipolysis has become trient used for ATP production depends on feeding status and cell type. In most cells, glucose is the principal source of ATP in the fed state, but FAs Received September 25, 2018. Accepted March 9, 2019. or ketone bodies become the main source during Published online ahead of print. Publication date available at the fasting state. Energy metabolism in kidney www.jasn.org. proximal tubular epithelial cells (PTECs) is Correspondence: Dr. Shinji Kume or Dr. Hiroshi Maegawa, De- unique, because ATP production here is thought partment of Medicine, Shiga University of Medical Science, Tsukinowa- to largely depend on lipolysis and subsequent cho, Seta, Otsu, Shiga 520-2192, Japan. Email: skume@belle. b-oxidation, regardless of feeding status.12 4 shiga-med.ac.jp or [email protected] Furthermore, a disruption of renal lipolysis is Copyright © 2019 by the American Society of Nephrology 962 ISSN : 1046-6673/3006-962 JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH the focus of an emerging research field, and better under- Significance Statement standing of renal lipolysis may contribute to the develop- ment of novel therapeutic approaches for DKD. Lipolysis is of particular importance for energy homeostasis in In general, the mechanisms by which cells take up and use proximal tubular epithelial cells (PTECs), and it is dysregulated FAs differ between the fed and fasting states. In the fed state, during the pathogenesis of diabetic kidney disease. In knockout mice lacking O-GlcNAc transferase specifically in PTECs, the authors cells obtain FAs from circulating triglycerides after lipoprotein demonstrated that protein O-GlcNAcylation, an intracellular nutri- lipase (LPL)–dependent lipolysis at the endothelial surface ent sensing system, is essential for renal lipolysis andATP production and directly transfer FAs to mitochondria for ATP produc- during prolonged fasting. They also found evidence that this novel tion (Supplemental Figure 1A).8 In contrast, during the regulatory mechanism of renal lipolysis involves farnesoid X re- – fasting state, cells take up free FAs that are released from ceptor dependent upregulation of carboxylesterase-1 and that deficiency of renal protein O-GlcNAcylation exacerbates tubulop- adipocytes and circulate bound to plasma albumin. After athy in diabetic kidney disease. These findings suggest that ma- they are transported into cells, these FAs can be stored in nipulation of the renal lipolytic mechanism to overcome the effects intracellular lipid droplets and are used for ATP production of prolonged fasting might represent a novel therapeutic approach after being once again liberated by intracellular lipolysis for diabetic kidney disease. (Supplemental Figure 1B).9 GiventhatPTECsrequirecon- tinuous lipolysis for ATP production, there must be intra- (RCALS) at Shiga University of Medical Science. All experi- cellular mechanisms that sense changes in nutritional status mental protocols were approved by the Gene Recombination and regulate lipolytic processes in the fed and fasting states. Experiment Safety Committee (approval number 28–12) and However, little is known about the physiologic mechanism the RCALS at Shiga University of Medical Science (approval underpinning lipolysis-associated energy metabolism in number 2016–8-10). PTECs. Cells have evolved a nutrient-sensing system that involves Generation of Tamoxifen-Inducible PTEC-Specific Ogt O-GlcNAcylation, a post-translational modification. Knockout Mice O-GlcNAcylation involves the addition of UDP-O–linked The Ogt gene resides on the X chromosome.20 Ogtf/f mice were n-acetylglucosamine to proteins by O-GlcNAc transferase obtained from the Jackson Laboratory (Bar Harbor, ME). This (Ogt).10212 Because UDP-O–linked n-acetylglucosamine strain originated in a B6;129 background and has been is derived from metabolites involved in FA, amino backcrossed to C57BL/6 for at least ten generations. Proximal acid, glucose, and nucleotide metabolism, the extent of tubular epithelial cell–specific O-GlcNAc transferase knock- O-GlcNAcylation is indicative of overall intracellular nutri- 2 out (PTEC-Ogty/ ) mice were generated by breeding female ent status. Accumulating evidence from experimental stud- Ogtf/f mice with male N-myc downstream-regulated gene-1 ies conducted using tissue-specificOgt-deficient mouse (NDRG1) promoter–derived tamoxifen (TM)-inducible CreERT2 models indicates that O-GlcNAcylation is required for he- mice with a C57BL/6 background21 (Figure 1A). Eight-week-old patic gluconeogenesis and lipolysis-dependent thermogen- male Ogty/f and Ogty/f mice carrying Ndrg1CreERT2 were admin- esis in brown adipose tissue during fasting.13,14 Thus, in istered 150 mg/kg per day TM for 5 consecutive days.21 Twelve manycells, O-GlcNAcylation may regulate a number of met- weeks after this induction, urine samples were collected from abolic pathways according to changes in nutritional status. 2 20-week-old Ogty/f and PTEC-Ogty/ mice using a metabolic O-GlcNAcylation in PTECs has been demonstrated experi- cage under both fed and 48-hour fasting conditions. Then, mentally15219; however, the precise role of this modification mice were euthanized, and renal cortical samples were col- in the physiology of energy homeostasis, including in lipol- lected (n=5–6). ysis in PTECs, has not been fully elucidated. We hypothesized that O-GlcNAcylation is involved in the mechanism of the continuous lipolytic activ- Mouse Models of Diabetes and Atherosclerosis ityinPTECsandthatitsdysregulationisinvolvedinthe Eighteen-week-old male db/db mice purchased from CLEA pathogenesis of diabetic tubulopathy. To address these Japan Co. (Osaka, Japan) were used as a model of type 2 di- hypotheses, we evaluated renal lipolysis and the renal phe- abetes, which is characterized by overt proteinuria in the ab- 2 2 notype of PTEC-specificOgt-deficient mice exposed to a sence of severe tubulopathy. ApoE / mice were generated by 2 prolongedfastorwithhigh-fatdiet(HFD)–induced crossbreeding male and female ApoE+/ mice.22 Their ApoE diabetes. +/+ littermates were used as controls. Eight-week-old ApoE+/+ mice were fed either a normal diet or an HFD for 24 weeks to induce obesity-related microalbuminuria without severe 2 2 METHODS tubulopathy. HFD-fed ApoE / mice were used as a model of diabetes- and atherosclerosis-associated severe tubulopathy. Ethics The normal diet (10% of total calories from fat) and HFD Animal experimentationwas conducted in accordancewith the (60% of total calories from fat) were purchased from Research guidelines of the Research Center for Animal Life Science Diets (New Brunswick, NJ). JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 963 BASIC RESEARCH www.jasn.org A B Cre ( ) Ogtf/f mice ( ) NDRG1-CreERT2 mice Ogt Ogt gene on X chromosome RL2 X LoxP Exon X LoxP NDRG1 promoter Cre ERT2 X LoxP Exon X LoxP (O-GlcNAcylation) ( ) Ogty/f β ( ) Ogty/f actin NDRG1-CreERT2 mice y/f PTEC y/f PTEC y/f Ogt Ogt y/- -Ogty/- -Ogty/- Ogt PTEC -Ogt Isolated PTECs Isolated non-PTECs Renal cortex (LTL-positive) (LTL-negative) X LoxPExon X LoxP Y X LoxP Exon X LoxP + C RL2 (O-GlcNAcylation) Y NDRG1 promoter Cre ERT2 y/f Tamoxifen (TM) Tamoxifen (TM) Ogt ( ) PTEC-Ogty/- ( ) Ogty/f y/- X LoxP LoxP X LoxP Exon X LoxP PTEC-Ogt Y Y DE F 50 TM injection 200 TM injection 5 NS 40 4 P<0.05 150 30 NS NS 3 NS 100 NS NS NS 20 NS 2 y/f 50 y/f Body weight (g) 10 Ogt Ogt 1 Food intake (g/day) PTEC-Ogty/- Blood glucose (mg/dl) PTEC-Ogty/- 0 0 0 8 12 16 20 8 12 16 20 y/f y/- (Week-old) (Week-old) Ogt PTEC -Ogt Figure 1.
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