The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice

2620 Diabetes Volume 63, August 2014 Jelske N. van der Veen,1,2 Susanne Lingrell,1,2 Robin P. da Silva,1,3 René L. Jacobs,1,3 and Dennis E. Vance1,2 The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice Diabetes 2014;63:2620–2630 | DOI: 10.2337/db13-0993 Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) of type 2 diabetes is increasing, and therefore under- catalyzes the synthesis of phosphatidylcholine (PC) standing the underlying physiology is of major impor- in the liver. Mice lacking PEMT are protected against tance. Up to 75% of patients with obesity and diabetes diet-induced obesity and insulin resistance. We in- also suffer from nonalcoholic fatty liver disease (NAFLD) vestigated the role of PEMT in hepatic carbohydrate me- (1,2), indicating that the liver plays an important role in tabolism in chow-fed mice. A pyruvate tolerance test the etiology of obesity-associated diabetes. Steatosis in fi revealed that PEMT de ciency greatly attenuated gluco- the liver is often associated with hepatic IR; the exact neogenesis. The reduction in glucose production was mechanisms by which these conditions are related remain specific for pyruvate; glucose production from glycerol METABOLISM unclear. IR may cause accumulation of triacylglycerol in was unaffected. Mitochondrial PC levels were lower and the liver (3,4). In an IR state, elevated plasma concentra- PE levels were higher in livers from Pemt2/2 compared +/+ tions of both glucose and fatty acids can lead to increased with Pemt mice, resulting in a 33% reduction of the 2/2 hepatic de novo lipogenesis and steatosis (3,4). Intracel- PC-to-PE ratio. Mitochondria from Pemt mice were also smaller and more elongated. Activities of cytochrome c lular accumulation of lipids, such as diacylgycerol or oxidase and succinate reductase were increased in mito- ceramide, can activate protein kinase C and subsequently chondria of Pemt2/2 mice. Accordingly, ATP levels in hep- impair insulin signaling (5,6). Hence, metabolism of fat atocytes from Pemt2/2 mice were double that in Pemt+/+ and glucose in the liver is connected, particularly in the hepatocytes. We observed a strong correlation between mitochondria, where fatty acid oxidation supplies acetyl- mitochondrial PC-to-PE ratio and cellular ATP levels in hep- CoA to the tricarboxylic acid (TCA) cycle for ATP produc- atoma cells that expressed various amounts of PEMT. tion. The TCA cycle also provides a source of carbons for Moreover, mitochondrial respiration was increased in cells gluconeogenesis. Therefore, abnormal lipid and glucose lacking PEMT. In the absence of PEMT, changes in mito- metabolism associated with IR and type 2 diabetes may chondrial phospholipids caused a shift of pyruvate toward be related to abnormalities in mitochondria. decarboxylation and energy production away from the car- Hepatic phosphatidylcholine (PC) synthesis also has boxylation pathway that leads to glucose production. a role in the development of IR (7). PC is synthesized via the cytidine diphosphate–choline pathway (8). Alterna- tively, hepatocytes can methylate phosphatidylethanol- Obesity is often accompanied by hepatic steatosis and amine (PE) via PE N-methyltransferase (PEMT) (9). The insulin resistance (IR) or type 2 diabetes. The prevalence PEMT pathway is responsible for ;30% of hepatic PC 1Group on Molecular and Cell Biology of Lipids, the Alberta Diabetes Institute, the Received 25 June 2013 and accepted 18 March 20014. Mazankowski Alberta Heart Institute, Edmonton, Alberta, Canada © 2014 by the American Diabetes Association. Readers may use this article as 2 Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada long as the work is properly cited, the use is educational and not for profit, and 3 Department of Agricultural, Food and Nutritional Science, University of Alberta, the work is not altered. Edmonton, Alberta, Canada Corresponding author: Dennis E. Vance, [email protected]. diabetes.diabetesjournals.org van der Veen and Associates 2621 synthesis; the remaining 70% is generated via the cytidine 5 mmol/L KCl, and 7.5 mmol/L MgCl2 (pH 7.4). The diphosphate–choline pathway (10). Unexpectedly, we re- homogenate was centrifuged at 1,000g for 10 min at ported that mice lacking PEMT are protected against 4°C. The pellet was resuspended in 10 mmol/L Tris-HCl, high-fat diet (HFD)-induced obesity and IR (7). This pro- 10 mmol/L KCl, and 0.15 mmol/L MgCL2 (pH 6.7) and tection was completely blunted when the diet was supple- frozen at 280°C. Thawed cells were disrupted using mented with choline, leading to the conclusion that PEMT a Dounce homogenizer, mixed with one-seventh the vol- provides an endogenous source of choline that supports ume of 2 mol/L sucrose and centrifuged at 1,000g for 10 2 2 normal weight gain (7). Although Pemt / mice were com- min. The supernatant was centrifuged at 12,000g for 15 pletely protected against IR, hepatomegaly and steatosis min at 4°C. The pellet was resuspended in 10 mmol/L Tris- developed when the mice were fed an HFD. This protection HCl, 0.15 mmol/L MgCl2, and 0.25 mol/L sucrose (pH 6.7). 2 2 against IR in the presence of hepatic steatosis led us to Mitochondria from livers of Pemt+/+ and Pemt / mice investigate the role of PEMT in hepatic carbohydrate me- were isolated as previously described (11). 3 tabolism, independent of body weight or fatty liver. In Vivo 2-[ H]deoxy-D-Glucose Uptake 2/2 We report that in livers of Pemt mice, the concen- Nonfasted animals were injected with 0.1 units/kg insulin tration of PE in mitochondria is increased, leading to in- 3 i.v., 2 g/kg glucose i.v., and 300 mCi/kg 2-[ H]deoxy-D- fl creased ux of pyruvate into the TCA cycle for subsequent glucose i.v. (12). Fifteen minutes after injection, blood energy production. Consequently, hepatic glucose produc- was collected by cardiac puncture into EDTA-containing tion from pyruvate was strongly diminished, suggesting tubes, plasma was prepared by centrifugation, and organs that these changes may contribute to the protection 2 2 were harvested and snap-frozen in liquid nitrogen. Plasma against the development of IR in Pemt / mice. proteins were precipitated with 0.3 N Ba(OH)2 and 0.3 N RESEARCH DESIGN AND METHODS ZnSO4. Radioactivity in the supernatant was divided by the plasma glucose concentration to calculate the specific Animals 2 2 radioactivity of glucose. White adipose, skeletal muscle, Female C57Bl/6 Pemt+/+ and Pemt / mice (backcrossed and liver were homogenized in 0.5% perchloric acid, and .7 generations) (7) were fed standard rodent chow (cat. the homogenate was centrifuged for 20 min at 2,000g. no. 5001; LabDiet) ad libitum with free access to water. Half of the supernatant was treated with Ba(OH) and Experimental procedures were approved by the University 2 ZnSO4. Radioactivity was measured in both aliquots, of Alberta’s Institutional Animal Care Committee in ac- 3 and radioactivity in 2-[ H]deoxy-D-glucose was calculated cordance with the Canadian Council on Animal Care. as the difference between the supernatants divided by In Vivo Tolerance Tests sample weight. This value was divided by the specific ra- Mice (4–6 months old) were nonfasted before the gluca- dioactivity of glucose to calculate glucose uptake per gram gon tolerance tests, fasted for 4 h before insulin tolerance of tissue (micromoles per gram of tissue). tests and for 12 h before glucose, pyruvate, and glycerol Phospholipid Labeling in Primary Hepatocytes tolerance tests. Subsequently, the mice were injected in- 2 2 Hepatocytes were isolated from Pemt+/+ and Pemt / mice traperitoneally with bovine insulin (1 units/kg body wt after perfusion of the liver with collagenase (13) and cul- i.p.), glucagon (140 mg/kg body wt i.p.), sodium pyruvate tured in 60-mm collagen-coated dishes in Dulbecco’smod- (2 g/kg body wt i.p.), or glycerol (2 g/kg body wt i.p.) or ified Eagle’s medium (DMEM) containing 10% FBS for 18 h. alternatively, glucose (2 g/kg body wt i.p.) was adminis- tered by oral gavage. Blood glucose levels were measured Cells were washed twice with serum-free DMEM and pulsed m 3 using a glucometer (Accu-Chek). with 10 Ci [ H]serine/dish for 1 h, followed by a chase of 0, 1, 2, or 4 h in the presence of 1 mmol/L hydroxylamine Real-Time Quantitative PCR and Analysis of to inhibit phosphatidylserine decarboxylase (14). Mitochon- Mitochondrial DNA Content dria were isolated and lipids extracted with chloroform/ RNA isolation, cDNA synthesis, and real-time quantitative methanol (2:1 v/v). Phospholipids were separated by thin- PCR were performed as described (7). mRNA levels were layer chromatography (15), followed by methanolysis with normalized to cyclophilin mRNA. For determination of mi- 3 CH3OH/sulphuric acid at 80°C for 2 h. Since [ H]serine is tochondrial DNA copy number, total DNA was extracted also incorporated into fatty acids (16), radioactivity in only from livers using a DNEasy kit (Qiagen). The mitochon- the phospholipid head groups was measured. drial DNA content was calculated using real-time quan- Analytical Procedures titative PCR by measuring a mitochondrial gene (Nd1, Mitochondrial PC, PE, and phosphatidylserine were forward: ACA CTT ATT ACA ACC CAA GAA CAC AT, re- quantified by phosphorous assay (15) after separation verse: TCA TAT TAT GGC TAT GGG TCA GG) versus by thin-layer chromatography. ATP in McArdle-RH7777 a nuclear gene (Lpl, forward: GAA AGG TGT GGG GAG cells and primary hepatocytes was measured using a lucif- ACA AG, reverse: TCT GTC AAA GGC ACT GAA CG). erase assay kit (Sigma-Aldrich) according to the manufac- Isolation of Mitochondria turer’s instructions.

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