Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin

J O U RN A L OF P ROT EO M IC S 7 7 ( 2 01 2 ) 1 6 7 –1 75

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www.elsevier.com/locate/jprot

Proteomic analysis of liver mitochondria of apolipoprotein E knockout mice treated with metformin Aneta Stachowicza , Maciej Suskia , Rafał Olszaneckia,⁎, Józef Madeja , Krzysztof Okońb , Ryszard Korbuta a

Chair of Pharmacology, Jagiellonian University Medical College, Poland Chair of Pathomorphology, Jagiellonian University Medical College, Poland

b

AR TIC LE I N FO

ABS TR ACT

Article history:

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance.

Received 29 June 2012

Metformin, a widely known anti-diabetic drug, used for patients with type 2 diabetes

Accepted 21 August 2012

mellitus, is also claimed to be useful in treatment of NAFLD. However, both the clinical

Available online 30 August 2012

efficacy and the putative mechanisms underlying the clinical effects of metformin in

Keywords:

the primary molecular target for metformin, is a known regulator of mitochondrial

Liver

function. Thus, we used a proteomic approach to investigate the effect of metformin on

Metformin

liver mitochondria of apolipoprotein E knockout (apoE−/−) mice, an animal model of NAFLD.

Mitochondria

Two-dimensional electrophoresis coupled with mass spectrometry was applied to study

Nonalcoholic fatty liver disease

the changes in liver mitochondrial protein expression in 6-month old metformin-treated

treating NAFLD are unclear. Adenosine monophosphate‐activated protein kinase (AMPK),

Proteomics

apoE−/− mice as compared to non-treated animals. Collectively, 25 differentially expressed proteins were indentified upon metformin treatment including proteins related to metabolism, oxidative stress and cellular respiration. The most up-regulated protein was glycine N-methyltransferase (GNMT) — an enzyme, whose deficiency was shown to be directly related to the development of NAFLD. Our results clearly point to the strong mitochondrial action of metformin in NAFLD. Up-regulation of GNMT may represent an important mechanism of beneficial action of metformin in NAFLD treatment. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Nonalcoholic fatty liver disease (NAFLD), which is manifested by free fatty acid accumulation in hepatocytes (hepatic steatosis), is the most common liver disorder in the world.

Its prevalence ranges from 10% to 24% in the general population. NAFLD encompasses a wide histological spectrum of liver damage; from simple hepatic steatosis it can progress to nonalcoholic steatohepatitis (NASH), advanced fibrosis and cirrhosis and at last hepatocellular carcinoma [1]. NAFLD is

Abbreviations: AMPK, adenosine monophosphate-activated protein kinase; ApoE, apolipoprotein E; ApoE−/−, apolipoprotein E-knockout mice; CAIII, carbonic anhydrase 3; COX-IV, cytochrome c oxidase; ECHD3, enoyl-CoA hydratase domain-containing protein 3; GLP-1, glucagon-like peptide 1; GNMT, glycine N-methyltransferase; HMCS2, hydroxymethylglutaryl-CoA synthase; HSP60, 60 kDa heat shock protein; IDPc, isocitrate dehydrogenase [NADP] cytoplasmic form; IPYR, inorganic pyrophosphatase; MCP-1, monocyte chemoattractant protein-1; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PRDX-6, peroxiredoxin 6; SAH, S-adenosylhomocysteine; SAMe, S-adenosylmethionine; SCAD, short-chain specific acyl-CoA dehydrogenase; SCP-2, sterol carrier protein 2; TPP, trans-proteomic pipeline; TTBS, tris-buffered saline and Tween 20. ⁎ Corresponding author at: Laboratory of Molecular Pharmacology, Chair of Pharmacology, 16 Grzegorzecka str, 31 531, Cracow, Poland. Tel.: + 48 12 421 11 68; fax: + 48 12 4217 217. E-mail address: [email protected] (R. Olszanecki). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.08.015

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strongly associated with insulin resistance, visceral obesity, hyperlipidemia, hypertension and currently is considered to be a hepatic representation of the metabolic syndrome [2,3]. The pathogenesis of NAFLD is still not fully understood. According to classical “two hit-hypothesis”, free fatty acid accumulation in liver is a consequence of insulin resistance and leptin resistance of hepatocytes [4]. In turn, cellular surplus of fatty acids induces mitochondrial dysfunction and triggers excessive formation of reactive oxygen species (ROS), responsible for tissue damage and stimulation of inflammatory processes in the liver [5]. These, together with impairment of hepatic protein metabolism and further inhibition of insulin signaling, lead to progression of injury from simple steatosis to NASH and fibrosis [6]. Recently, it has been proposed that hepatocyte mitochondrial dysfunction characterized by augmentation of ROS production, impairment of fatty acid beta-oxidation and enhancement of apoptosis is a crucial factor in the development of NAFLD [7]. Metformin is a widely known anti-diabetic drug, often used as a first line treatment for patients with type 2 diabetes mellitus. It is broadly accepted that its beneficial effect is mainly related to the activation of adenosine monophosphate‐activated protein kinase (AMPK) – the master kinase in the regulation of cellular metabolism – in insulin-sensitive cells: hepatocytes, adipocytes and skeletal muscle cells [8]. AMPK is able to stimulate glycolysis, fatty acid oxidation and mitochondrial biogenesis; it also inhibits gluconeogenesis and synthesis of fatty acids and cholesterol [9]. Metformin was shown also to act in a way similar to glucagon-like peptide 1 (GLP-1) and exert antiinflammatory actions [10,11]. Recently, it was demonstrated that metformin not only influences mitochondrial biogenesis but also the function of mitochondrial oxidative machinery — in vitro metformin was able to inhibit the respiratory‐chain complex 1 [12], however such an effect was not present in patients treated with metformin [13]. Altogether, the action of metformin leads to increase of tissue insulin sensitivity. As insulin resistance is a key player in the pathogenesis of NAFLD, it was proposed that treatment with metformin may offer promising therapeutic options in this disease [14–16]. However, both the clinical efficacy and the mechanisms underlying the clinical effects of metformin in treating nonalcoholic fatty liver disease are unclear [17,18]. Apolipoprotein E (apoE), which is synthesized and secreted mainly by hepatocytes, plays a key role in internalization and catabolism of lipoproteins in the liver. ApoE is involved not only in passive blood cholesterol transport but also in other biological processes (e.g. activation of enzymes responsible for lipoprotein metabolism). The apoE‐knockout mice (apoE−/−) represent a well-known model of atherosclerosis [19]. Importantly, it has been demonstrated that apoE−/− mice develop hepatic steatosis and fibrosis [20]. Also, in this model the changes in liver mitoproteome were shown to accompany typical organ damage [21]. Mitochondrial proteomics represents a valuable tool for the study of metabolic disorders and mechanisms of actions of metabolic drugs [22,23]. However, so far the proteomic approach was not applied to estimate the influence of metformin on mitochondria. Thus, the aim of our study was to use the methods of differential proteomics to elucidate the effect of metformin on liver mitochondria of apoE−/− mice. We applied two-dimensional electrophoresis

coupled with mass spectrometry to investigate the changes in mitochondrial protein expression in 6-month old metformintreated apoE−/− mice as compared to non-treated transgenic animals.

2.

Methods

2.1.

Animal experiments

All animal procedures were approved by the Jagiellonian University Ethical Committee on Animal Experiments. Female apoE-knockout mice on the C57BL/6J background were obtained from Taconic (Ejby, Denmark). Mice were maintained on 12-h dark/12-h light cycles in air-conditioned rooms (22.5 ± 0.5 °C, 50 ± 5% humidity) and access to diet and water ad libitum in Animal House of Chair of Immunology of JUMC. At the age of 8 weeks mice were put on chow diet made by Ssniff (Soest, Germany). Two groups of animals were studied: control group (apoE−/− mice w/o treatment, on chow diet as above, n = 6) and metformin-treated mice (n= 6). In this group, metformin was mixed without heating with the same diet and administered to mice at a dose of 10 mg per kg of body weight per day (acc. to calculation taking into account average body mass of mouse and its daily diet requirement). At the age of 6 months mice from both groups were killed under anesthesia 5 min after injection of fraxiparine (1000 UI, Sanofi-Synthelabo, France) into the peritoneum. Next, the liver was dissected and subjected to subcellular fractionation.

2.2.

Subcellular fractionation

Isolation of mitochondria was performed at 4 °C from freshly-harvested mouse liver. Two animals were pooled for sample to obtain sufficient material for proteomic analysis. Homogenization was carried out in 250 mM sucrose, 1 mM EGTA, pH 7.8 with addition of PMSF (1 mM) and a mix of protease inhibitors (approximately 100 μl for 3 g of tissue) (Sigma, USA). Nuclei and unbroken cells were pulled down by centrifugation at 1000 g for 10 min. Then, the mitochondrial fraction was obtained by centrifugation of the supernatant at 12,000 g for 10 min. The mitochondrial pellet was then purified by 3 cycles of resuspension, homogenization and centrifugation (at 12,000 g for 15, 20, 15 min). The cytosolic fraction was obtained by further centrifugation of the supernatant (90 min at 125,000 g, 4 °C). Samples were collected and stored at − 80 °C until assayed.

2.3. Two-dimensional electrophoresis (2-DE) and gel image analysis Mitochondrial pellets were resuspended in 1 ml of lysis buffer containing 9.5 M urea, 4% CHAPS, 2% DTT, 0.5% Bio-Lyte 3–10 (Bio-Rad, USA) and a mix of protease inhibitors (100 μl for 3 mg of sample). Samples were vortexed and left at 25 °C for 30 min to ensure maximal protein solubilization, then centrifuged at 12,000 g for 15 min. The supernatant was harvested and the protein concentration was determined with the Bradford method [24]. The supernatant was then divided into aliquots containing appropriate amount of protein for single IPG strips

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(150 μg for analytical gels) and stored at −80 °C. Before loading, samples were purified by precipitation with 2-DE Clean-up kit (GE Healthcare, USA) and resuspension in 300 μl of rehydration buffer (8 M urea, 0.5% CHAPS, 0.2% DTT and 0.2% Bio-Lyte 3–10). Then, samples were loaded on linear 3–10 immobilized pH gradient 17-cm strips (Bio-Rad, USA) using an in-gel rehydration method and were rehydrated overnight in a reswelling tray. The strips were focused with a multistep voltage gradient from 400 to 3500 V (max 50 mA/IPG strip, 20 °C) for a total of 66 kVh. Once IEF was completed the strips were equilibrated in buffer (6 M urea, 30% glycerol, 2% SDS and 0.01% bromophenol blue) with addition of 1% w/v DTT (20 min) and 4.8% w/v iodoacetamide (20 min), to ensure sufficient reduction and alkylation of proteins. Second dimension (SDS-PAGE) was performed using 12% T (total acrylamide concentration), 2.6% C (degree of cross-linking) polyacrylamide gels without a stacking gel, using the Protean II xi system (Bio-Rad, USA). After electrophoresis the gels were fixed overnight in ethanol:acetic acid:water solution (4:1:5 v/v/v). Finally, protein profiles were visualized by silver staining, using the Plus One silver staining kit (GE Healthcare, USA) with modifications to provide compatibility with subsequent mass spectrometry analysis [25]. For analysis, silver-stained gel images were taken using a Gel Doc XR scanner (Bio-Rad, USA). PDQuest™ 8.0.1 (Bio-Rad, USA) was used for gel image analysis, quantification and statistical validation. In total, n = 3 gel images representing each group from the total number of n = 2 replicates of two individual samples (n= 6 gel images per group) were analyzed. All results were carefully verified manually, including verification of housekeeping proteins chosen for normalization (local regression model) to ensure accurate quantification. Student's T-test with the level of significance set at p < 0.05 was used to reveal statistically significant differences in the protein expression. Significant differences were further analyzed with LC MS/MS system to identify proteins of interest.

2.4.

LC MS/MS

Gel pieces containing protein spots of interest were destained, reduced, alkylated and digested with modified trypsin (Sigma, USA) according to the protocol described by Shevchenko et al. [26]. Peptide mixtures were lyophilized and stored at −80 °C for further LC/MS analysis. Each sample was resuspended in 0.1% TFA and injected in an Acclaim PepMap100 RP C18 75 μm i.d.× 25 cm column (LC Packings/Dionex) via trap column (PepMap100 RP C18 300 μm i.d.× 5 mm column, LC Packings/ Dionex). The peptides were separated in an acetonitrile gradient (buffer A — 5% acetonitrile and 0.1% formic acid; buffer B — 95% acetonitrile and 0.1% formic acid) with at a flow rate of 300 nl/ min with a Switchos/UltiMate 3000 RSLC nano HPLC system (LC Packings/Dionex, USA) and applied on-line to a LCQ (Thermo Finnigan, USA) ion-trap mass spectrometer. The mobile phase gradient was started with increase from 0 to 25% B within 35 min, then increasing to 40% B in 40 min, next rapidly increasing to 80% B within 12 s, followed by 80% B isocratic for 5 min. Finally B phase was rapidly decreased to 0% and left isocratic 100% A for column equilibration for 30 min. The main working liquid-junction ESI ion source parameters were as follows: ion spray voltage 1.5 kV, capillary voltage 10 V, and capillary temperature 200 °C. Spectra were collected in full scan

169

mode (270–1600 Da), followed by three MS/MS scans of three most intense ions from full scan using dynamic exclusion criteria. Collected data were analyzed by X!Tandem search algorithm (The GPM Organization) and statistically validated with Trans-Proteomic Pipeline (TPP) software (Institute for Systems Biology). Search parameters were set as follows: taxonomy: mouse (SwissProt), enzyme: trypsin, missed cleavage sites allowed: 3, fixed modification: carbamidomethyl, variable modifications: oxidation of methionine, peptide fragment mass tolerance: 0.4 Da and selected device and parent δm: ion trap (4 Da).

2.5.

Immunoblotting

The purity of the fractions was assessed by immunoblotting of cytochrome c oxidase (COX-IV) and α-tubulin. Isolated mitochondria were lysed in PBS containing 1% Triton X-100, 0.1% SDS, 1 mM PMSF, 100 μM leupeptin, and 50 μM pepstatin A. Samples, containing equal amounts of total protein (as estimated with the Bradford method) were mixed with gel loading buffer (50 mM Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/ml bromophenol blue) in a ratio 4:1 (v/v) and incubated at 95 °C for 5 min. Samples (50 μg of protein) were separated on SDS-polyacrylamide gels (7.5–15%) (Mini Protean II, Bio Rad, USA) using the Laemmli buffer system and proteins were semidry transferred to nitrocellulose membranes (Amersham Biosciences, USA). Membranes were blocked overnight at 4 °C with 5% (w/v) non-fat dried milk in TTBS and incubated 3 h at room temperature with specific primary antibodies: 1:5000 ANTI-COX-IV (Abcam, USA), 1:250 ANTI-alpha-tubulin (Sigma, USA), 1:1000 ANTIPRDX-6 (Sigma, USA) and 1:500 ANTI-GNMT (Santa Cruz, USA), then for 1 h with HRP-conjugated secondary antibodies (Amersham Biosciences, USA). Bands were developed with the use of ECL-system reagents (Amersham Biosciences, USA). Rainbow markers (Amersham Biosciences, USA) were used for molecular weight determinations. Protein pattern images were taken using a Gel Doc XR scanner (Bio-Rad, USA).

2.6.

Histology

Samples of liver tissue were formalin fixed, routinely processed and embedded in paraffin. From paraffin blocks 4 μm sections were prepared and stained with the hematoxylin–eosin method.

3.

Results

Accuracy of the isolation protocol and purity of the mitochondrial fractions were assessed by immunoblotting for α-tubulin and COX-IV (Fig. 1C). The representative 2-DE gel image of liver mitochondrial proteins of apoE −/−, as well as selected pairs of spots showing differences between apoE −/− and metformin-treated apoE−/− mice are presented in Fig. 1A and B, respectively. Protein identifications obtained by LC– MS/MS analysis from numbered spots showing significant differences are listed in Table 1. For detailed information about MS/MS identification of peptide sequences see electronic Supplementary material Table 1. Collectively, 25

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a)

b) pH 3

pH 10 GNMT

GNMT

14

14 17

18

12

5

16

apoE -/-

metformin

PRDX-6

PRDX-6

15 8

22 11

14

10 4

metformin

ATPA

ATPA

23 apoE -/-

20

metformin

c)

3 21 9

23

23

6 13

1

apoE -/-

24

7 2

9

9

25

α -tubulin

52 kDa

19 17 kDa COX-IV

52 kDa β -actin

38 kDa cytosol

mitochondria

Fig. 1 – Representative 2-D map of mitochondrial proteins expressed in 6-month-old apolipoprotein E deficient mice (a), with magnifications of spot pairs corresponding to glycine N-methyltransferase, peroxiredoxin-6 and ATP synthase subunit alpha (b). Purity of mitochondrial fraction was assessed by Western blot method, showing the absence of cytosolic α-tubulin l in mitochondrial fraction (c).

differentially expressed spots were detected and identified by PDQuest™ and analyzed by mass spectrometry. Quantitative differences in expression of proteins are listed in Fig. 2. Furthermore, the differences in glycine N-methyltransferase (GNMT) and peroxiredoxin-6 (PRDX-6) expression in mitochondria were verified by Western blot (Fig. 3). In addition to proteomic approach we performed standard hematoxylin/eosin (HE) staining of the livers of C57BL/6J mice, apoE−/− mice and metformin-treated apoE−/− mice. In HE staining C57BL/6J mice showed normal liver structure (Fig. 4). In apoE−/− animals, the cytoplasm of the liver cells was finely vacuolated, testifying diffuse steatosis. Moreover, several hepatocytes, especially those with centrolobular location, were binucleated. Focally, the nuclei were hyperchromatic and enlarged, up to twice the normal size. In the group of metformin-treated mice the morphological changes in liver were similar, but less pronounced (Fig. 4).

4.

Discussion

Here, for the first time the differential proteomic approach was applied to study the influence of metformin on liver mitoproteome in an animal model of NAFLD–apoE−/− mice. In our previous study we have shown that changes in liver mitoproteome of apoE−/− mice compared to C57BL/6J mice accompany typical organ damage [21]. Our current results clearly point to the strong mitochondrial action of metformin. We identified 25 differentially expressed proteins, which can

be divided into three main groups: proteins related to metabolism (esp. beta-oxidation of fatty acids), oxidative stress and cellular respiration (esp. citric acid cycle and electron transport chain). The protein related to metabolism to be up-regulated by metformin to the largest extent was glycine N-methyltransferase (GNMT) — its expression increased 3.2‐fold. GNMT plays a crucial role in methionine metabolism. It catalyzes methylation of glycine by using S-adenosylmethionine (SAMe) to form N-methylglycine (sarcosine) with the concomitant production of S-adenosylhomocysteine (SAH). The role of GNMT is to remove excess SAMe and maintain a constant SAMe/SAH ratio in order to avoid aberrant methylation [27]. Interestingly, it has been discovered that GNMT-knockout mice develop liver steatosis, fibrosis and hepatocellular carcinoma [28]. Patients with liver cirrhosis show attenuated GNMT expression. Importantly, genetic deficiency of GNMT sensitizes the liver to the injury caused by inflammatory stimuli (bacterial lipopolysaccharide, TNF-α) [29]. As deficiency of GNMT plays a crucial role in the development of fatty liver disease, its up-regulation may represent an important mechanism of the beneficial action of metformin in NAFLD. Interestingly, so far GNMT was considered as a cytosolic protein; our report also points to a mitochondrial localization of this protein. Clearly, further investigations are required to study the molecular background of metformin influence on GNMT expression and the biological meaning of its mitochondrial localization. Several proteins related to beta-oxidation of fatty acids including short-chain specific acyl-CoA dehydrogenase (SCAD),

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Table 1 – Differentially expressed proteins in liver mitochondria of metformin-treated apoE−/− mice. No.

Protein

UniProtKB accession number

Molecular mass (kDa)

pI a

Unique peptides

Total peptides

Protein coverage (%)

Tandem score b

1

Phosphatidylethanolamine-binding protein 1 60 kDa heat shock protein, mitochondrial ATP synthase subunit beta, mitochondrial Inorganic pyrophosphatase Component of pyruvate dehydrogenase complex (E2), mitochondrial 3-hydroxyisobutyrate dehydrogenase, mitochondrial Ester hydrolase C11orf54 homolog Selenium-binding protein 1 Peroxiredoxin-6 Probable 4-hydroxy-2-oxoglutarate aldolase, mitochondrial Short-chain specific acyl-CoA dehydrogenase, mitochondrial Carbamoyl-phosphate synthase, mitochondrial Enoyl-CoA hydratase domain-containing protein 3, mitochondrial Glycine N-methyltransferase Hydroxymethylglutaryl-CoA synthase, mitochondrial Non-specific lipid-transfer protein Carbamoyl-phosphate synthase, mitochondrial Aconitate hydratase, mitochondrial Cytochrome b-c1 complex subunit 2, mitochondrial Carbonic anhydrase 3 ES1 protein homolog, mitochondrial Alcohol dehydrogenase [NADP +] ATP synthase subunit alpha, mitochondrial Carbamoyl-phosphate synthase, mitochondrial Isocitrate dehydrogenase [NADP], cytoplasmic

P70296

20.8

5.19

2

4

13.0

−13.6

P63038 P56480 Q9D819 Q8BMF4

60.9 56.3 32.6 67.9

5.91 5.19 5.37 8.81

4 8 4 5

5 14 8 9

6.3 18.0 18.0 7.6

−28.7 −68.8 −29.6 −37.6

Q99L13

35.4

6.01

4

8

9.6

−38.0

Q91V76 P17563 O08709 Q9DCU9

35.0 52.5 24.9 34.6

5.89 5.87 5.71 7.61

3 9 7 6

7 18 10 9

9.8 20.0 41.0 15.0

−24.6 −91.4 −57.7 −46.5

Q07417

44.9

7.12

8

12

19.0

−75.8

Q8C196

164.5

6.48

7

10

5.1

−54.6

Q9D7J9

32.4

8.60

3

6

10.0

−23.9

Q9QXF8 P54869

32.7 56.8

7.09 7.47

6 17

9 45

19.0 28.0

−49.3 −159.4

P32020 Q8C196

59.1 164.5

7.16 6.48

8 18

13 26

12.0 9.5

−71.7 −173.1

Q99KI0 Q9DB77

85.4 48.2

8.08 9.26

7 5

10 7

9.7 12.0

−52.0 −44.0

P16015 Q9D172 Q9JII6 Q03265 Q8C196

29.3 28.1 36.6 59.7 164.5

6.89 9.00 6.90 9.22 6.48

12 3 13 8 6

21 4 43 14 13

44.0 8.6 38.0 14.0 4.5

−97.5 −25.9 −133.4 −76.7 −55.6

O88844

50.9

8.88

12

16

24.0

−106.5

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

a b

pI — isoelectric point. Tandem score — log10 of expectation value, which reflects the number of matches expected to be found by chance in a database.

enoyl-CoA hydratase domain-containing protein 3 (ECHD3) and inorganic pyrophosphatase (IPYR) were also up-regulated after treatment with metformin of apoE−/− mice. SCAD and ECHD3 take part in the first and second step of mitochondrial beta-oxidation, respectively, while IPYR, by promoting the rapid hydrolysis of pyrophosphate, provides the driving force for the activation of fatty acids destined for oxidation [30]. The impairment of liver fatty acid oxidation is a hallmark of NAFLD. Noteworthy, Zhang et al. have shown that down-regulation of enoyl-CoA hydratase is associated with hepatic steatosis in rats after exposure to a high fat diet [31]. Clearly, up-regulation by metformin of proteins involved in mitochondrial beta oxidation could be of potential benefit in hepatic steatosis. Another protein related to metabolism, which was down-regulated in metformin-treated apoE−/− mice, was hydroxymethylglutaryl-CoA synthase (HMCS2, mitochondrial form). This enzyme participates in ketogenesis — the process of formation of ketone bodies out of fatty acids. The excessive generation of ketone bodies causes ketoacidosis —

the most severe complication in patients with type 1 diabetes mellitus [32]. Interestingly, it has been demonstrated that HMCS2 was increased fourfold in the kidneys of type 2 diabetic db/db mice [33]. Whether by metformin might down-regulation of HMCS2 decrease the risk of severe ketoacidosis in diabetic patients or patients with excessive formation of ketone bodies (e.g. after bariatric surgery) remains to be tested. Mitochondrial oxidative stress plays a pivotal role in the development of NAFLD [34]. Thus, there are some interesting changes in expression of proteins related to oxidative stress in liver mitochondria of metformin-treated apoE −/− mice. Peroxiredoxin 6 (PRDX-6), an important antioxidant protein, was up-regulated 1.7-fold. This cytosolic protein is involved in detoxification of peroxides, especially hydrogen peroxide and phospholipid hydroperoxides [35]. It was shown that Prdx-6-knockout mice undergone liver ischemia-reperfusion had significantly more hepatocellular injury compared with wild-type mice [36]. Interestingly, as it was shown in the

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ATP synthase subunit alpha, mitochondrial [23] Non-specific lipid-transfer protein [16] Carbamoyl-phosphate synthase, mitochondrial [12] Carbamoyl-phosphate synthase, mitochondrial [24] Hydroxymethylglutaryl-CoA synthase, mitochondrial [15] Carbamoyl-phosphate synthase, mitochondrial [17] Cytochrome b-c1 complex subunit 2, mitochondrial [19] Aconitate hydratase, mitochondrial [18] ATP synthase subunit beta, mitochondrial [3] Component of pyruvate dehydrogenase complex (E2), mitochondrial [5] 60 kDa heat shock protein, mitochondrial [2] Short-chain specific acyl-CoA dehydrogenase, mitochondrial [11] Selenium-binding protein 1 [8] 3-hydroxyisobutyrate dehydrogenase, mitochondrial [6] Enoyl-CoA hydratase domain-containing protein 3, mitochondrial [13] Isocitrate dehydrogenase (NADP), cytoplasmic [25] ES1 protein homolog, mitochondrial [21] Inorganic pyrophosphatase [4] Probable 4-hydroxy-2-oxoglutarate aldolase, mitochondrial [10] Peroxiredoxin-6 [9] Phosphatidylethanolamine-binding protein 1 [1] Alcohol dehydrogenase (NADP+) [22] Carbonic anhydrase 3 [20] Ester hydrolase C11orf54 homolog [7] Glycine N-methyltransferase [14]

-4

-3

-2

-1

0

1

2

3

4

5

factor of change in expression of mitochondrial proteins

Fig. 2 – Relative changes in expression of mitochondrial proteins in 6-montH-old metformin-treated apoE−/− group compared to apoE−/− mitochondria. Corresponding spot numbers are shown in brackets.

same study, PRDX-6 can shuttle from the cytoplasm to the mitochondria and protect hepatocytes against mitochondrial dysfunction. Carbonic anhydrase 3 (CAIII) was another protein up-regulated by metformin in liver mitochondria of apoE −/− mice. CAIII catalyzes reversible hydration of carbon dioxide and is responsible for maintaining acid–base balance in the tissue [37]. In addition, CAIII might function as oxyradical scavenger and protect cells from hydrogen

peroxide-induced apoptosis [38]. We found isocitrate dehydrogenase [NADP] cytoplasmic form (IDPc) slightly upregulated in mitochondria of apoE−/− mice. Clearly, the presence of IDPc – a cytoplasmic protein - in mitochondria is somewhat surprising and requires further investigation. Noteworthy, the activity of IDPc is related to protection against oxidative stress: IDPc produces α-ketoglutarate, CO2 and NADPH from isocitrate and plays a role in providing

MITOCHONDRIA apoE-/-

apoE-/-

+ metformin

GNMT 31 kDa 24 kDa

PRDX-6

COX-IV 12 kDa

GNMT

3

0.4

Intensity of GNMT/COX-IV

Intensity of GNMT/COX-IV

0.5

0.3 0.2 0.1

PRDX-6

2.4 1.8 1.2 0.6 0.0

0.0 control

metformin

control

metformin

Fig. 3 – Western blot of GNMT and PRDX-6 in liver's mitochondria of 6-montH-old apoE−/− mice and metformin-treated apoE−/− mice. COX-IV was used as a reference. *p < 0.05 vs. apoE−/−.

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C57BL/J6

apoE -/-

apoE -/- + metformin

Fig. 4 – Representative images of livers of 6-month old C57BL/6J mice, apoE−/− mice and metformin-treated apoE−/− mice. Hematoxylin and eosin, magnification 400 ×.

NADPH for the regeneration of reduced glutathione [39]. Indeed, IDPc was shown to be up-regulated by hyperglycemia in renal cells and protect them from oxidative stress [40]. Next, non-specific lipid-transfer protein [sterol carrier protein 2 (SCP-2)] was down-regulated about 2.5-fold in liver mitochondria of metformin-treated apoE−/− mice. SCP-2 is responsible for cholesterol transferring between membranes. Kriska et al. have shown that SCP-2 can traffic cholesterol hydroperoxides and thus mediate oxidative injury in cells [41]. Consistently, our results indicate that metformin administration leads to substantial improvement of anti-

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oxidant defenses, which seems to be very favorable in NAFLD treatment. In our hands, treatment with metformin resulted in a significant decrease in mitochondrial expression of 60 kDa heat shock protein (HSP60), which is a chaperone protein responsible for transport and refolding of proteins from cytoplasm into the mitochondrial matrix [42]. HSP60 could be also considered as a pro-inflammatory protein. It has been lately discovered that HSP60 (present in blood) induces release of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) from adipocytes [43]. Moreover, HSP60 was shown to aggravate insulin resistance of adipose tissue. Thus, HSP60 might be a mediator of adipose tissue inflammation and obesity-associated metabolic disorders [44]. In our study metformin treatment leads to a slight decrease of HSP60 expression in liver. It is tempting to speculate that downregulation of HSP60 may play a local anti-inflammatory role in liver and/or remotely in adipose tissue. This, however remains an attractive hypothesis to be tested. Proteins related to cellular respiration were down-regulated in liver mitochondria of metformin-treated apoE−/− mice. The levels of the proteins belonging to the electron transport chain like ATP synthase subunit α, ATP synthase subunit β and cytochrome b-c1 complex subunit 2 as well as proteins belonging to the citric acid cycle: aconitate hydratase and component of pyruvate dehydrogenase complex (E2) were decreased about 1.2– 2.87-fold. It is known that metformin inhibits respiratory-chain complex 1 [12]. Our results may point to overall decrease of oxidative respiration in mitochondria induced by metformin treatment. This is in keeping with previous results obtained in vitro [45,46]. Interestingly, no inhibitory effect of metformin was detectable in patients [13], thus further studies are required to clarify the mechanism of metformin action on mitochondrial respiration. In summary, the differential proteomic approach allowed the identification of important changes in mitochondrial protein expression upon metformin treatment in the liver of apoE−/− mice. The proteins related to beta-oxidation of fatty acids and protection against oxidative stress were up-regulated, while proteins related to cellular respiration were downregulated. Importantly, GNMT – an enzyme, whose deficiency was shown to be directly related to the development of NAFLD – was the protein up-regulated by metformin to the largest extent. Collectively, the pattern of changes suggests beneficial effects on mitochondria of metformin administration in NAFLD, however the exact functional consequences of the revealed alterations as well as molecular mechanisms of metformin influence on mitochondrial proteins require further investigation. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2012.08.015.

Acknowledgment This study was supported by the grants from National Science Centre (NCN): 2011/01/N/NZ2/00089 and N N401 124339. Aneta Stachowicz acknowledges the financial support from the project Interdisciplinary PhD Studies “Molecular sciences for

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medicine” (co-financed by the European Social Fund within the Human Capital Operational Programme). Duality of interest The authors declare that there is no duality of interest associated with this manuscript. Contribution statement RO and RK were responsible for the conception and design of the study. AS, MS, JM and KO were responsible for analysis and interpretation of the data. AS drafted the article. All authors revised the paper critically for important intellectual content and gave final approval of the version to be published.

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