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Molecular Endocrinology, doi:10.1210/me.2006-0249
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Molecular Endocrinology 21 (4): 972-986
Copyright © 2007 by The Endocrine Society

Overexpression of Angiopoietin-Like Protein 4 Alters Mitochondria Activities and Modulates Methionine Metabolic Cycle in the Liver Tissues of db/db Diabetic Mice

Yu Wang, Karen S. L. Lam, Janice B. B. Lam, Michael C. Lam, Priscilla T. Y. Leung, Mingyan Zhou and Aimin Xu

Genome Research Center (Y.W., P.T.Y.L., M.Z.) and Departments of Biochemistry (Y.W., M.Z.) and Medicine (K.S.L.L., J.B.B.L., M.C.L., A.X.), Research Center of Heart, Brain, Hormone, and Healthy Aging (K.S.L.L., M.C.L., A.X.), University of Hong Kong, Hong Kong, China; and School of Biological Sciences (J.B.B.L.), University of Auckland, Auckland 1003, New Zealand

Address all correspondence and requests for reprints to: Yu Wang, Genome Research Center, Faculty of Medicine Building, the University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong Special Administrative Region, China. E-mail: yuwanghk{at}hku.hk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly produced from fat tissue and liver. Recent data from others and our laboratory have demonstrated this protein to be an important player in energy metabolism and insulin sensitivity. However, the molecular mechanisms underlying its metabolic actions remain elusive. In this study, we have employed a two-dimensional fluorescence difference gel electrophoresis technique to study the protein profiles in the livers of db/db mice treated with or without ANGPTL4. When compared with those of lean mice, 118 proteins were found to be up- or down-regulated in db/db mice. Adenovirus-mediated overexpression of ANGPTL4 could reverse a large portion of the up- or down-regulated proteins to control levels. Especially, a number of mitochondria proteins were down-regulated by ANGPTL4 to a great extent. Chronic treatment with ANGPTL4 resulted in an elevated activity of mitochondria respiratory chain complexes II–III and IV in db/db mice. Additionally, several key enzymes in the methionine/homocysteine metabolic cycle were found to be increased in db/db diabetic mice but decreased by ANGPTL4 treatment. HPLC analysis consistently revealed that ANGPTL4 could significantly restore the augmented S-adenosylmethionine levels and S-adenosylmethionine/S-adenosylhomocysteine ratios in livers of db/db mice. In summary, our results suggest that ANGPTL4 might elicit its metabolic effects through modulating the mitochondria functions and methionine metabolic cycles in the liver tissue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TYPE 2 DIABETES MELLITUS (T2DM) is one of the most common endocrine diseases in developed countries (1). Excessive hepatic glucose production is thought to be a major contributor to the T2DM status (2, 3, 4). Prolonged elevation of blood glucose levels can lead to many diabetic complications, such as cardiovascular disease, stroke, and diabetic neuropathy and retinopathy, etc. (1). Thus the discovery of antidiabetic agents that can inhibit hepatic glucose production is a popular research area for the pharmaceutical research community. Although some hypoglycemic drugs have been developed for the treatment of this disease (5, 6), more efficacious agents need to be sought. Moreover, despite decades of efforts, the detailed mechanisms underlying this disease remain mysterious.

Proteins secreted from adipose tissue (adipokines) are increasingly recognized to play an important role in the regulation of glucose and lipid metabolism in other metabolic organs. Abnormal expression and secretion of many adipokines have been implicated in the pathogenesis of T2DM (7, 8). Angiopoietin-like protein 4 (ANGPTL4), a recently discovered adipokine (9, 10), was originally identified as the target gene of peroxisome proliferator-activated receptors (PPARs), including both the insulin-sensitizing PPAR{gamma} agonists, thiazolidinediones, and the lipid-lowering PPAR{alpha} agonists, fenofibrates (11, 12). The expression of ANGPTL4 is under nutritional control, with its plasma abundance increased by fasting and decreased by high-fat feeding (9). Transgenic mice with ANGPTL4 overexpression have approximately 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat (13). ANGPTL4 is also an inhibitor of lipoprotein lipase and can physically associate with plasma lipoproteins (13, 14). This evidence suggests that ANGPTL4, like most of the other adipokines such as leptin and adiponectin, could serve as a blood-borne hormone involved in regulating energy metabolism and insulin sensitivity.

Recently, we have reported that adenovirus-mediated expression of ANGPTL4 potently reduced hyperglycemia and markedly alleviated glucose intolerance and hyperinsulinemia in db/db diabetic mice (15). Ex vivo studies revealed that ANGPTL4 could act directly on primary rat hepatocytes to decrease hepatic glucose production and enhance insulin-mediated inhibition of gluconeogenesis (15). Furthermore, our clinical evidence revealed that serum levels of ANGPTL4 inversely correlated with plasma glucose concentrations and HOMA IR, the homeostasis model assessment of insulin resistance (15). Serum levels of ANGPTL4 were significantly lower in patients with T2DM than those in healthy subjects, suggesting that the decreased ANGPTL4 could be a causative factor of this disease. Nevertheless, the cellular mechanisms that underlie the metabolic actions of adiponectin remain largely uncharacterized.

In this study, we have employed two-dimensional differential gel electrophoresis (2D-DIGE) to investigate the global protein changes in the livers of obese diabetic db/db mice treated with or without ANGPTL4. Compared with those of the lean mice, 76 protein spots have been identified to be significantly up- or down-regulated in the livers of db/db diabetic mice. These proteins are involved in amino acid/fatty acid/glucose metabolism, oxidative stress, and several other signaling pathways. Notably, ANGPTL4 treatment alters a number of mitochondria proteins and caused elevated mitochondria complex activities. In particular, we have found that the dysregulated methionine/homocysteine metabolic cycle in the liver of db/db mice could be effectively reversed by ANGPTL4 treatment, which might represent one of the potential mechanisms that underlie ANGPTL4-mediated effects in regulating the mitochondria functions in liver.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
2D-DIGE Profiles of Mouse Liver Proteins
To investigate the metabolic functions of ANGPTL4 in vivo, we have previously generated the recombinant adenovirus that encodes the mouse full-length ANGPTL4 (15). Recombinant adenovirus (5 x 109 plaque-forming units) that expresses ANGPTL4 (Adv-ANGPTL4) or luciferase (Adv-Luc) was introduced into C57BLKS lean or db/db mice through tail vein injection. Adenovirus-mediated ANGPTL4 overexpression did not significantly affect food intake or body weight of these animals but caused a marked transient elevation of triglycerides and total cholesterol in the circulation. In sharp contrast to its hyperlipidemia effect, ANGPTL4 overexpression significantly decreased blood glucose levels and alleviated hyperinsulinemia and glucose intolerance associated with db/db diabetic mice (15). Moreover, our ex vivo study demonstrated that ANGPTL4 could directly inhibit gluconeogenesis and increase hepatic glycogen contents in rat primary hepatocytes, suggesting the liver to be the target tissue of this protein.

In this study, we further analyzed the protein expression profiles of the liver tissues derived from the lean mice and db/db mice treated with or without ANGPTL4 using 2D-DIGE approaches. Total soluble liver proteins were labeled with Cy3 or Cy5 dyes and mixed to form random pairs on each 2D DIGE gel as described in Materials and Methods. As shown in the representative images (Fig. 1Go), protein spots were evenly distributed across the 4–7 pI range in the first dimension and between 15 and 160 kDa on the 12.5% SDS-PAGE in the second dimension. An average of about 2000 spots was detected in each image by DeCyder DIA image analysis, with pixel intensities ranging from 100 to 50000 (data not shown). For DeCyder biological variation analysis, a gel with the most detected protein spots was chosen as the master image, and the spots of all the other gel images were matched to the master gel. The volume of each matched spot was calculated against the internal control and then used for obtaining the average ratios for each group. The standardized volume ratio between two groups was used for statistical analysis using Student’s t test. After analyzing the 16 gel images (four images from each group), we have found 196 protein spots that are significantly (>1.5-fold) changed in at least one group of animal compared with another group (Fig. 2Go). With the use of peptide mass finger printing and tandem mass spectrometry analysis, 130 proteins have been identified. Further investigation using PathwayStudio software (Ariadne Genomics, Rockville, MD) revealed that these protein species belong to several functional groups, including cytoskeletons, extracellular matrix proteins, GTPase regulators, metabolic enzymes, methyltransferases, phosphatases, transcription factors, and transporters, etc. (data not shown).


Figure 1
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  		Fig. 1. Representative 2D-DIGE Images of Liver Lysates from Lean and db/db Mice Treated with or without ANGPTL4

Protein lysates (50 µg) labeled with Cy3, Cy5, or Cy2 (for internal control) were mixed and separated as described in Materials and Methods using pH 4–7 IEF strips in the first dimension and 12.5% SDS-PAGE in the second dimension. The fluorescence images were obtained using a Typhoon 9410 scanner.

 

Figure 2
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  		Fig. 2. Master Gel Generated after Analyzing with DeCyder Software

The annotated spots are those found to be significantly up- or down-regulated at equal or more than 1.5-fold in at least one experimental group compared with another group. Statistical analysis was performed using the Student’s t test.

 
Identification of the Differentially Expressed Proteins in the Livers of db/db Mice
Comprehensive analysis using the DeCyder software revealed that there were 40 up-regulated and 78 down-regulated protein spots (1.5-fold or more) in the liver samples of db/db mice compared with those of lean mice. Of these protein spots, 76 were identified by MALDI-TOF and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis (Table 1GoGo). Notably, a large portion of down-regulated proteins belong to cytoskeleton and molecular chaperones, whereas most of the up-regulated proteins were enzymes involved in the glucose, lipid, and amino acid metabolism. Some of these protein spots were different isoforms derived from one protein species. For examples, five isoforms of formyltetrahydrofolate dehydrogenase (FTHFD, spot 497, 519, 520, 532, 504) were found to be up-regulated with the average fold ratio of 2.6 (Fig. 3Go), suggesting its total protein levels were increased in db/db mice. On the other hand, the expression patterns of five isoforms of selenium binding protein (spot 1240, 1232, 1217, 1222, and 1218 with the pI values of 5.5, 5.55, 5.8, 5.85, and 6.0, respectively) were different, with the ratios (db/db vs. lean mice) gradually decreased from basic to acidic end (Fig. 4Go). The expression levels of spot 1218 and 1222 were not significantly different from those of lean mice. On the other hand, the spots 1240, 1232, and 1217 were significantly decreased by –2.3-, –1.68-, and –1.57-fold, respectively. These results suggest that the selenium-binding protein might be posttranslationally modified and the modification might be altered in diabetic states.


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  		Table 1. Up- or Down-Regulated Proteins in the Livers of db/db Mice Compared with Those in the Livers of Lean Mice

 

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  		Table 1A. Continued

 

Figure 3
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  		Fig. 3. Quantitative Analysis by DeCyder Software

Five different isoforms of FTHFD were up-regulated in the livers of db/db diabetic mice (right panel) compared with those in lean mice (left panel).

 

Figure 4
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  		Fig. 4. The Pattern Changes of Different Isoforms of Selenium-Binding Protein in the Livers of db/db Mice

Five isoforms of selenium-binding proteins with different isoelectric points were identified (A), and their abundances were quantified by DeCyder software and compared with those of the lean mice (B).

 
Interestingly, among the list of up- or down-regulated proteins (Table 1GoGo), there were 14 spots corresponding to key enzymes involved in methionine and homocysteine metabolic cycles (Fig. 5Go), including methionine adenosyltransferase I {alpha} (MAT1A; spot 1373, 1419, and 1471 with 3.31-, 3.2-, and 2.44-fold respectively), S-adenosylhomocysteine hydrolase (SAHH; spot 1458, 2.41-fold), FTHFD, betaine-homocysteine methyltransferase (spot 1594, –1.5-fold) and two folate-binding proteins, dimethylglycine dehydrogenase (DMDH; spot 631 and 588 with 1.5- and 2.11-fold, respectively) and sarcosine dehydrogenase (SDH; spot 553 and 552 with 2.89- and 3.08-fold, respectively). MAT1A is responsible for the adenylation of L-methionine to form S-adenosylmethionine (SAM), the methyl donor in virtually all known biological methylation reactions, and the transmethylation reactions to produce a methylated substrate and S-adenosylhomocysteine (SAH) (16, 17, 18). SAH is subsequently cleaved to homocysteine and adenosine by SAHH. Homocysteine has several possible fates: remethylation to methionine, export to the extracellular space, or transsulfuration into cysteine, the rate-limiting precursor of glutathione synthesis (19, 20). The increased plasma level of homocysteine is recognized as an independent risk factor for the development of vascular disease. Notably, betaine-homocysteine methyltransferase, an enzyme involved in the remethylation of homocysteine to methionine, is moderately down-regulated (~1.5-fold). FTHFD, DMDH, and SDH are mitochondria folate-binding proteins critically involved in folate metabolism. FTHFD catalyzes the rate-limiting step in hepatic folate-dependent formate oxidation. DMDH and SDH are flavoproteins catalyzing the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these processes, the bound tetrahydrofolate interacts directly with the dehydrogenases and serves as an acceptor for the one-carbon unit to form 5,10-methylenetetrahydrofolate. Taken together, alterations of these enzymes might cause a dysregulated folate transportation and methionine metabolic cycle in the livers of db/db diabetic mice (Fig. 5Go).


Figure 5
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  		Fig. 5. Several Dysregulated Enzymes in db/db Mice Are Involved in the Metabolic Pathways of Methionine

MAT, Methionine adenosyltransferase; MTs, methyltransferase; MS, methionine synthase; BHMT, betaine methyltransferase.

 
Analysis using the KEGG pathway database (21) revealed that several enzymes involved in amino acid metabolisms were also significantly changed in db/db mice. Two up-regulated enzymes, 3-hydroxyanthranilate 3,4-dioxygenase and phenyalanine hydroxylase, are involved in the biosynthesis of tryptophan, phenylalanine, and tyrosine, the glucogenic and ketogenic amino acids. On the other hand, 2-oxoisovalerate dehydrogenase, an enzyme responsible for the degradation of the three branched amino acids (valine, leucine, and isoleucine), was down-regulated by 2.32-fold. In addition to proteins involved in amino acid metabolism, we have also identified differentially expressed enzymes in lipid and glucose metabolism. For instance, the cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc), an enzyme that plays a critical role in fat and cholesterol biosynthesis (22), was markedly increased by 7.7-fold in db/db mice. 2-Hydroxyphytanoyl-coenzyme A lyase (2HPCL), a key enzyme in the phytanic acid {alpha}-oxidation pathway localized in peroxisomes (23, 24), was also up-regulated by 1.77-fold, whereas glycerol kinase, an enzyme involved in fatty acid degradation, was down-regulated by 1.63-fold. In addition, phosphoglucomutase, fructose-1,6-bisphosphatase, ornithine aminotransferase, arginase, {alpha}-enolase, and ketohexokinase, several enzymes involved in glycogen metabolism, glycolysis, and urea cycle were found to be up-regulated (Table 1GoGo).

Notably, a large portion of the proteins that are down-regulated in the liver of db/db mice are cytoskeletal proteins and chaperones. Keratins are the intermediate filament proteins that can provide mechanical integrity and cytoprotection to the liver epithelial cells (25, 26). Here, our results showed that the expression of both type I and type II keratins (K8/18) were significantly decreased. In addition, the binding protein of keratin, different isoforms of 14-3-3 protein (27, 28), were also down-regulated. Glucose-regulated protein (78 kDa) (GRP78) is an abundant member of molecular chaperone family in the lumen of the endoplasmic reticulum participating in the quality control of secretory proteins and endoplasmic reticulum stress. Consistent with a previous report that the level of GRP78 protein was reduced in Zucker obese/diabetic rats (29), we also found a decreased expression of different isoforms of GRP78 in the livers of db/db mice (Table 1GoGo).

Altered Protein Expression Profiles in the Livers of db/db Mice Treated with ANGPTL4
Our previous report showed that ANGPTL4 treatment significantly alleviated hyperglycemia in db/db diabetic mice (15). In this study, we further evaluated the effects of ANGPTL4 on protein expression profiles in the liver tissues of db/db mice. Among the 76 up- and down-regulated proteins in this diabetic model, 47 were significantly restored by ANGPTL4 treatment to a level that was not significantly (P ≥ 0.05) different from lean mice (Table 1GoGo). For some proteins, such as ornithine aminotransferase, senescence marker protein-30, phenylalanine hydroxylase, 3-hydroxyanthranilate 3,4-dioxygenase, major urinary protein, transferrin, and DMDH etc., ANGPTL4 treatment could down-regulate them to a level (>1.5-fold) that was significantly different from lean mice samples (P < 0.05). On the other hand, ANGPTL4 treatment had not much effect on the increased expression levels of IDPc in db/db diabetic liver. In lean mice treated with ANGPTL4, 24 of the 76 identified spots showed significant changes compared with those of the lean mice treated with luciferase (1.5-fold or more with P < 0.05) (Table 1GoGo). Interestingly, about 50% of these proteins corresponded to enzymes involved in the methionine and homocysteine metabolic cycle.

We next used Western blotting analysis to validate the data of our proteomic study. Our results showed that the expression of several cytoskeletal proteins and chaperones, including GRP78, tissue transglutaminase (TTG), keratin 18, 14-3-3, and actin, were decreased in db/db mice, and the expression of glutathione-S-transferase (GST) was significantly increased in db/db mice. These changes were reversed after ANGPTL4 treatment (Fig. 6Go), a pattern similar to that observed by 2D-DIGE analysis. To further confirm whether these changes at the protein levels were also correlated to their gene expressions, we performed quantitative real-time PCR analysis for a number of genes selected from Table 1GoGo, using the commercial Tagman expression kits. Notably, this analysis confirmed that several genes involved in the methionine metabolic cycle and glucose and lipid metabolism were up-regulated in db/db mice, and these changes were reversed after ANGPTL4 treatment (Fig. 7Go). Similar effects of ANGPTL4 on the expressions of these enzymes, cytoskeletal proteins, and chaperones were also observed in primary mouse hepatocytes infected with adenovirus-encoding luciferase or ANGPTL4 (data not shown). On the other hand, we also found that the expression levels of some genes, such as keratin 18 and tissue transglutaminase, were not significantly different in mice and primary hepatocytes treated with ANGPTL4 (data not shown), suggesting that changes of their protein abundance might be attributed to the modifications at the posttranscriptional level.


Figure 6
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  		Fig. 6. Western Blotting Analysis of the Expression of Several Cytoskeletal Proteins in the Liver Tissues of Lean and db/db Mice Treated with Luciferase or ANGPTL4

Equal amounts (50 µg) of liver protein lysates were separated by 12.5% SDS-PAGE. After transferring to nylon membranes and incubating with the first and secondary antibodies, the immune complexes were detected by enhanced chemiluminescence as described in Materials and Methods. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

 

Figure 7
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  		Fig. 7. Quantitative PCR Analysis of the Gene Expression Levels of Cytoskeletal Proteins, Enzymes, and Chaperones

RNA was extracted from liver tissues collected from lean mice and db/db diabetic mice treated with or without ANGPTL4. Real-time PCR analysis was performed according to the procedures described in Materials and Methods. #, P < 0.01 vs. lean mice treated with luciferase; *, P < 0.01 vs. db/db mice treated with luciferase. OAT, Ornithine aminotransferase.

 
Restoration of SAM/SAH Metabolic Profiles in Livers of db/db mice by ANGPTL4 Treatment
As we described above, several up-regulated enzymes in the livers of db/db mice were involved in the methionine/homocysteine metabolic cycles, and ANGPTL4 treatment resulted in a significant decrease of their expression levels (Table 1GoGo). For example, MAT1A, the only known enzyme catalyzing SAM synthesis, and SAHH, which is responsible for the cleavage of SAH to generate homocysteine, were up-regulated by an average of 2.983- and 2.41-fold, respectively, in db/db mice, and the augmented expression of these two enzymes was largely diminished by ANGPTL4 treatment. Our real-time PCR analysis also demonstrated that the gene expression levels of both enzymes were significantly increased in the livers of db/db diabetic mice and decreased after ANGPTL4 treatment (Fig. 7Go). It is known that SAM serves as the methyl donor for many biological methylation reactions of molecules such as hormones, neurotransmitters, DNA, RNA, and phospholipids. The SAM/SAH ratio plays an important role in determining the metabolic status in liver (17, 18). We subsequently measured the levels of SAM and SAH in these liver samples using HPLC analysis. In agreement with the proteomic profiling data, HPLC analysis (Table 2Go) revealed that SAM and the ratio of SAM/SAH were significantly increased in db/db diabetic mice, whereas ANGPTL4 reversed these changes to a level close to that of the controls. Taken together, these results suggest that ANGPTL4 might play an important role in regulating the metabolism of methionine/homocysteine cycles in the livers of db/db diabetic mice.


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  		Table 2. SAM and SAH Concentrations in the Livers of Lean, db/db Mice Treated with or without ANGPTL4

 
Modulation of Mitochondrial Respiratory Chain Activities in the Livers of db/db Mice by ANGPTL4
Because a large number of the dysregulated proteins in db/db mice were localized in mitochondria, we subsequently purified the liver mitochondria organelles from lean mice, as well as from db/db mice treated with luciferase or ANGPTL4, and tested the enzyme activities of the mitochondria respiratory chain (MRC). As shown in Fig. 8AGo, the enzyme activities of complexes I–V were significantly decreased in the livers of db/db mice. ANGPTL4 treatment resulted in an increased activity of complexes II–III and IV in both db/db mice and the lean control mice. These results suggest that the improved mitochondria functions might partly contribute to the beneficial metabolic effects of ANGPTL4. Moreover, our results also revealed that primary hepatocytes treated with adenovirus encoding ANGPTL4 showed significant increased MRC enzyme activities of complexes II–III and IV compared with those treated with adenovirus encoding luciferase (Fig. 8BGo). On the other hand, ANGPTL4 treatment had no significant effects on mitochondrial DNA copy numbers in both mice livers and primary hepatocyte (data not shown), suggesting that the improved MRC activities might be due to mechanisms other than the mitochondrial genome-mediated gene expression.


Figure 8
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  		Fig. 8. ANGPTL4 Modulates Hepatic Mitochondrial Enzyme Activities in Vivo and in Vitro

The mitochondria organelle was purified from liver tissues (A) or from mouse primary hepatocytes (B) under different treatments as indicated. The MRC enzyme activities were measured as described in Materials and Methods. *, P < 0.05 vs. lean mice or hepatocytes treated with luciferase; #, P < 0.05 vs. db/db mice treated with luciferase.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Compared with the traditional two-dimensional gel electrophoresis analysis, 2D-DIGE technology offers an increased capacity of spot matching and accurate quantitative analysis for multiple groups of samples (30, 31). Here, we have used the 2D-DIGE technology to investigate the protein expression profiles in the liver tissues of lean mice and db/db obese/diabetic mice treated with or without ANGPTL4 (Fig. 1Go) (15). Comprehensive analysis using DeCyder software revealed that 118 protein spots were significantly up- or down-regulated in samples from db/db mice relative to those in the lean mice (Fig. 2Go). Among them, 76 were identified by both peptide mass fingerprinting and peptide fragment sequencing (Table 1GoGo). Pathway analysis of the protein changes indicated an increased gluconeogenesis, decreased glycolysis, and a dysregulated methionine metabolism in the db/db mouse liver (data not shown). Importantly, we found that chronic treatment with ANGPTL4 reversed most of the up- or down-regulated proteins to a level that was not significantly different from that of lean mice. Moreover, ANGPTL4 treatment significantly affected the protein expressions and respiratory chain activities of liver mitochondria (Table 1GoGo and Fig. 8Go). A number of mitochondria enzymes involved in glucose and amino acid metabolism were dramatically down-regulated by ANGPTL4 in both db/db and lean mice (Table 1GoGo). On the other hand, ANGPTL4 treatment significantly increased the activities of the MRC complexes II–III and IV (Fig. 8Go). In addition, both protein profiling and metabolite HPLC analysis pointed to a dysregulated methionine metabolic cycle in the livers of db/db mice, which could also be restored by ANGPTL4 treatment (Tables 1GoGo and 2Go). In contrast to several previous proteomic studies using ob/ob leptin deficiency, obese mice treated with glucose- and lipid-lowering drugs, including PPAR{alpha} or PPAR{gamma} agonists (32, 33, 34, 35), our results revealed that the hypoglycemic activities of ANGPTL4 might not be related to its effects on fatty acid combustion, especially peroxisomal fatty acid ß-oxidation.

Consistent with our proteomic data, several recent metabolic profiling studies in obese Zucker fa/fa rats suggest that the altered methionine metabolism and mitochondrial functions are associated with insulin resistance and early T2DM (36, 37). nuclear magnetic resonance analysis of the liver extracts from obese Zucker rats suggested a significant alteration in the ratio of SAM/SAH, which may be in part responsible for the development of steatosis and induction of mitochondria dysfunction. The levels of MAT1A enzyme and SAM were also increased in the ZDF fa/fa rats. Impaired mitochondrial activity and methionine/homocysteine metabolism have been found in human subjects with T2DM and in the insulin-resistant offspring of patients with T2DM (38, 39). In fact, a growing body of evidence has demonstrated a link between various disturbances in mitochondrial functioning and T2DM (40). Interestingly, Santamaria et al. (41) have reported that cellular SAM levels could directly regulate mitochondrial protein expressions and its functionalities, suggesting that altered methionine metabolic cycle might contribute to the development of T2DM through modulating mitochondria oxidative phosphorylation and metabolism. Liver is a key organ for the maintenance of systemic glucose homeostasis in mammals through regulating blood glucose levels within a very narrow range under various nutritional conditions. Dysfunctions of the liver mitochondria biogenesis may play critical roles in the progression to T2DM. In this study, we have confirmed that ANGPTL4 could significantly decrease the elevated SAM and SAM/SAH ratios associated with db/db diabetic livers (Table 2Go), as well as the augmented enzymes responsible for generating these metabolites. Moreover, our results indicated that ANGPTL4 treatment could improve the MRC activities (Fig. 8Go) and modulate the expression of a number of mitochondria proteins (Table 1GoGo). In addition, our evidence also suggested that the improved mitochondrial functions in the livers of mice treated with ANGPTL4 could be a consequence of the improved energy homeostasis or methionine metabolism. Nevertheless, it is necessary to perform more prospective and intervention studies to clarify the independent risk of methionine metabolites on mitochondria functions and thus apply alternative treatments in patients with T2DM.

Although we have not identified any enzymes involved in ß-oxidation, a peroxisomal enzyme involved in fatty acid {alpha}-oxidation, 2HPCL, was found to be increased in db/db mice. {alpha}-Oxidation provides an alternative pathway for ß-oxidation of the fatty acids that contain a 3-methyl group (23). In db/db mice, the increased SAM levels could facilitate the methylation reactions on protein, lipids, DNA, and other potential targets. However, it is not known whether increased SAM levels could directly affect fatty acid oxidation by methylation. If this is the case, an increased 2HPCL level could be the result of an adaptive response to elevated methylated fatty acids. Notably, ANGPTL4 treatment significantly decreased both SAM and 2HPCL levels (Table 1GoGo), suggesting a possible link between these two changes. Oxidative stress is a common link for hyperglycemic damage and plays a key mediatory role in the development and progression of diabetes and its complications. Some of the proteins listed in Table 1GoGo are involved in controlling of cytosolic and mitochondrial redox balance, including GST, glutathione peroxidase, selenium-binding protein, IDPc, etc. It is known that apart from fat and cholesterol biosynthesis, IDPc also plays important regulatory roles in cellular defense against oxidative stress (42). The increased IDPc and GST might reflect a cellular defense mechanism against the altered oxidative stress. Here, we found that the protein levels in ANGPTL4-treated db/db mice were significantly different from those in control mice, suggesting that whereas this hormone showed dramatic effects on methionine metabolism and mitochondria activities, it had little effect on the antioxidative system in the liver. These phenomena might partly explain the hepatomegaly associated with ANGPTL4-treated livers. Similarly, the hyperlipidemia and fatty liver observed in ANGPTL4-treated mice were also observed in the IDPc transgenic mice (43).

Most of the down-regulated proteins in db/db mice are found to be the components of cytoskeletons involved in maintaining normal structures/functions of the hepatocytes, such as keratin 8/18, tubulin, actin, 14-3-3 protein, etc. It is known that keratins 8 and 18 can protect the liver from stress, and the decreased keratin expression is associated with increased oxidative stress (25, 26, 44). 14-3-3 Proteins are able to bind to the phosphorylated keratin and regulate its filament solubility and reorganization, which, in turn, play important roles in a variety of physiological conditions such as cell cycle regulation, cell stress, cell signaling, apoptosis, cell integrity, and cell compartment-specific roles (27, 28). At this stage, however, it is not known whether these dysregulations are the result of altered methionine metabolite levels, abnormal mitochondria functions, or oxidative stress in these db/db diabetic livers. Also, it is not clear whether these associations play any direct or indirect roles on glucose overproduction in the diabetic liver. Apart from phosphorylation, keratin transglutamination occurs under both physiological and pathological status (45). The TTG is a ubiquitous enzyme that cross-links glutamine residues with lysine residues, resulting in protein polymerization, cross-linking of dissimilar proteins, and incorporation of diamines and polyamines into proteins (46). This modification helps to provide a compact protective structure. Notably, we have found that the levels of tissue transglutaminase were dramatically decreased in db/db mice. Nevertheless, whether there is a direct link between the reduced keratin levels and the declined transglutamination reactions needs to be further investigated. Our results also suggest that some of the expression changes of these cytoskeletal proteins might be secondary to the altered systematic energy metabolism.

In summary, this study has established a systematic proteomic differential protein profile associated with the livers from db/db obese/diabetic mice. Treatment with ANGPTL4 significantly restored most of the protein changes to the levels of lean mice. We conclude that this hormone is able to act on the liver tissue and especially to modulate the methionine metabolic cycle and mitochondria functions, which might represent the potential mechanisms underlying its antidiabetic activities. In addition, this study also provided a liver proteomic map that could be useful in the screening of hypoglycemic agents for T2DM in db/db mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents, Chemicals, and Animals
All 2D-DIGE reagents and equipment for isoelectric focusing (IEF), including IPGphor, Immobiline DryStrip Kit, Immobiline Drystrips (24 cm, pH 4–7 Linear), and IPG buffer 3–10, and the Ettan DALT system were purchased from GE Healthcare (Uppsala, Sweden). Iodoacetamide, dithioerythritol, {alpha}-cyano-4-hydroxy cinnamic acid, urea, thiourea, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, SAM, SAH, and homocysteine were from Sigma (St. Louis, MO). Sequencing grade trypsin was from Promega Corp. (Madison, WI). Male C57BL/KsJ-lepr(db)/lepr(db) and age-matched nondiabetic littermates db/+ mice (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) between 8 and 10 wk of age were used for this study. The mice were housed in a room under controlled temperature (23 ± 1 C), with free access to water and standard mouse chow. All the experiments were conducted under our institutional guidelines for the humane treatment of laboratory animals.

Construction and Production of Adenoviral Vector for Expression of ANGPTL4, and Animal Treatment
The adenovirus expression vector that encodes FLAG-tagged ANGPTL4 was generated using the Adeno-X Expression System (BD Biosciences CLONTECH, Palo Alto, CA) as described previously (15). The recombinant virus was packaged and amplified in human embryonic kidney 293 cells, and purified by CsCl density gradient centrifugation. The recombinant adenovirus that encodes luciferase was kindly provided by Dr. Christopher Rhodes (47). Recombinant adenoviruses (5 x 109 plaque-forming units) were introduced into the mice through tail vein injection. After 2 wk, the mice were killed and tissue collected for subsequent processing.

Preparation of Liver Lysates and Labeling
Livers were collected from four individual mice in each treatment group (lean mice treated with adenovirus that encodes luciferase or ANGPTL4, and db/db mice treated with adenovirus that encodes luciferase or ANGPTL4). The apical liver lobes were weighed and homogenized with a Teflon homogenizer (five strokes at 400 rpm) in the lysis buffer (7 M urea; 2 M thiourea; 4% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; and 30 mM Tris-HCl, pH 8.5; at 4 C). To remove cell debris, the homogenates were centrifuged at 15,000 x g for 15 min. The supernatant was removed, aliquoted, and frozen at –80 C. For sample labeling, 50 µg of the lysate was labeled with 400 pmol of cyanine dyes, Cy3 or Cy5, according to the standard protocols. Cy2 was used for labeling of the internal standard that includes equal amounts of liver lysates from all samples. The labeling was terminated by the addition of 1 µl of 10 mM lysine.

2D-DIGE and Image Analysis
The labeled samples were randomly mixed to allow every gel to contain 50 µg each of Cy2-labeled internal standard and Cy3- and Cy5-labeled samples, respectively. For the first-dimension separation, the labeling mixture was applied to six Immobiline DryStrips (24 cm, pH 4–7 linear) by cup loading with a total running time of 55 kV/·h of IEF. The second dimension was carried out with six 12.5% SDS-PAGE gels, and gel images were subsequently acquired at the recommended wavelengths by using a Typhoon 9410 high-performance gel and blot imager (GE Healthcare). Four independent replicates (individually processed liver samples) were analyzed in each group to minimize the biological variations. Eight gels were run to obtain four images (2 x Cy3- and 2 x Cy5-randomly labeled) for each treatment group. A total of 16 images in total with good separation qualities were analyzed using the DeCyder differential in-gel analysis and biological variation analysis software programs (GE Healthcare). The internal standards (8 x Cy-2 images) were included in the analysis procedure to eliminate the technical variations. Statistical data were drawn from four replicated gels for each group. Spots with a value of P < 0.05 (Student’s t test) and an average change greater than 1.5-fold were considered statistically significant regulated spots.

Protein Identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and QSTAR Quandrupole TOF Tandem Mass Spectrometry
For protein identification, the cyanine dye-labeled gels were subsequently stained with modified silver staining as described previously (48). Selected differentially expressed spots from the DeCyder analysis of the DIGE images were excised from the gels. In-gel trypsin digestion was performed manually. In brief, the gel plugs were washed with 50 mM ammonium bicarbonate, and then 50% (vol/vol) acetonitrile in water followed by 100% (vol/vol) acetonitrile for dehydration. After overnight digestion with trypsin (Promega) in 50 mM ammonium bicarbonate (pH 8.0) at room temperature, peptides were extracted using sequential steps of 0.2% (vol/vol) trifluoroacetic acid (TFA) in water, followed by 50% (vol/vol) acetonitrile in 0.1% (vol/vol) TFA. Some of the peptide extracts were desalted using ZipTips C18 (Millipore Corp., Bedford, MA) according to the manufacturer’s protocol. The peptides were eluted with 5 µl of 50% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA. The peptide extracts were subsequently used for MALDI-TOF and/or nano-flow LC/MS/MS analysis. For MALDI-TOF peptide mass fingerprinting protein identification, MALDI mass spectra were recorded with Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) that was operated in the positive ion reflectron mode at 20 kV accelerating voltage. {alpha}-Cyano-4-hydroxy cinnamic acid [10 mg/ml in 50% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA] was used as matrix. Spectra were externally calibrated using Calmix standard (Applied Biosystems). The peptide masses were searched against the National Center for Biotechnology Information nonredundant mouse database (http:www.ncbi.nlm.hih.gov) using the MASCOT programs (http://www.matrixscience.com/search_form_select.html). One missed cleavage per peptide was allowed, and an initial mass tolerance of 50 ppm was used in all searches. Partial carbamidomethylation for cysteine and oxidation for methionine were assumed. Nano-flow liquid chromatography electrospray tandem mass spectrometry analysis (nano-LC/MS/MS) was performed using a QSTAR hybrid quadrupole/orthogonal acceleration TOF spectrometer (Applied Biosystems) interfaced to an Agilent 1100 chromatography system (Agilent Technologies, Palo Alto, CA). Samples were dissolved in aqueous formic acid and injected onto a 300-µm, 150-mm Vydac C18 column and eluted with an acetonitrile/0.1% (vol/vol) formic acid gradient. The MS/MS spectra were used to search the mouse subset of the National Center for Biotechnology Information nonredundant database with the ProID search engine (Applied Biosystems).

Measurement of SAM and SAH
Mouse liver tissue (100 mg) was homogenized in 3 vol 0.4 M HClO4. After centrifugation at 10,000 x g for 15 min at 4 C, the supernatant was filtered through a 0.2-µm polypropylene syringe filter. Acid extract (25 µl) was applied directly onto the HPLC system (Agilent). Separation was carried out with a Jupiter C18 column (5 µm, 250 x 2.00 mm) from Phenomenex (Torrance, CA). The mobile phase consisted of 50 mM sodium acetate and 10 mM heptane sulfonate in acetonitrile-water (7:93, vol/vol) solution. The pH of the solution was adjusted to 4.4 with acetic acid. The flow rate was set at 0.2 ml/min, and the UV detection was at 260 nm. The calibration standard was dissolved in water and then processed in the same way as the liver samples. The metabolite concentrations were calculated from the computer-integrated areas of the peaks in the sample chromatogram in relation to the areas obtained from standard solutions.

Western Blotting Analysis
Proteins derived from liver lysates were separated by electrophoresis on 12% polyacrylamide gels under denaturing conditions and transferred to nylon membranes. Blots were incubated with antibodies to murine cytokeratins (Santa Cruz Biotechnology, Santa Cruz, CA), GRP78 (Sigma), cytoplasmic actin (Sigma), TTG (Upstate Biotechnology, Lake Placid, NY), glutathione S-transferase and 14-3-3 (Invitrogen, Carlsbad, CA). To assure lane to lane equivalency of total protein content, these same membranes were also probed for the enzyme glyceraldehyde-3-phosphate dehydrogenase. Antigen-antibody complexes on the membranes were detected by the Amersham enhanced chemiluminescence detection system (GE Healthcare) and quantified by densitometry.

Preparation of Mitochondria from Liver Tissues and Determination of Enzyme Activities
The fresh liver tissues derived from lean mice and db/db mice treated with adenovirus-encoding ANGPTL4 or luciferase were homogenized in 9 vol of cold medium (0.25 M sucrose; 10 mM Trizma; 1 mM EDTA buffer, pH 7.4) using a glass-glass homogenizer. Mitochondria were prepared by the method of Pedersen et al. (49). The protein concentrations of the purified mitochondria solution were determined by the BCA method (Pierce Chemical Co., Rockford, IL).

MRC enzyme activities were measured as described previously (50). Briefly, the NADH:Q oxidoreductase (complex I) activity was evaluated by observing the NADH absorbance decrease at 340 nm and 30 C, as a result of rotenone-sensitive rate of NADH oxidation. The activity of the enzyme was determined as the difference of absorbance in the absence and in the presence of rotenone. Succinate cytochrome c reductase (complexes II–III) activity was measured as the antimycin-A-sensitive rate of cytochrome c reduction (at 550 nm and 30 C) using succinate as the substrate. The cytochrome c oxidase activity (complex IV) was measured using the assay kit from Sigma, which was based on the decrease in absorbance at 550 nm during the oxidation of ferrocytochrome c by cytochrome c oxidase. ATPase hydrolytic activity (complex V) was measured in a ATP-regenerating system as described in Ref. 51 . The reaction was started by the addition of 1 mM ATP and the rate of NADH oxidation, equimolar to ATP hydrolysis, was monitored as the decrease in absorbance at 340 nm. Enzyme activities were expressed as nanomoles substrate used per minute per milligram of mitochondrial protein. Cytochrome oxidase activity was given as milliunits per microgram. All results were derived from three independent experiments.

Isolation and Recombinant Adenovirus Transduction of Primary Hepatocytes
Hepatocytes were isolated from male C57 mice by in situ perfusion of the liver with collagenase as we described previously (15). After centrifugation to separate from liver nonparenchymal cells, hepatocytes were seeded onto 12-well plastic dishes coated with type 1 collagen (Sigma) at a density of 5 x 105 cells per well. The cells were allowed to adhere to the cell culture dishes for 8 h before being infected with recombinant adenovirus encoding ANGPTL4 or luciferase at the concentration of 50 plaque-forming units per cell. At 24 h after infection, cells were harvested for Western blotting and real time PCR analysis. In addition, the MRC enzyme complexes activities were also measured from the mitochondria isolated from these cell cultures as described above.

Real-Time PCR
Total RNA was isolated from mouse liver tissues and primary hepatocytes using the RNeasy kit (QIAGEN, Chatsworth, CA). Subsequently, mRNA was reverse transcribed into cDNA using the oligo-dT primer (Roche). The relative gene abundance was quantified by Taqman real-time PCR using the predeveloped assay kits (Applied Biosystems). Signals from each sample were normalized to values obtained for the 18S rRNA gene, which was run as a housekeeping gene simultaneously with the experimental samples. The reactions were performed on the ABI 7000 sequence detection system.

Statistical Analysis
Data are presented as means ± SD. Means were compared using Student’s unpaired t test, paired t test, or one-way ANOVA as appropriate. P < 0.05 was taken to indicate a significant difference.


   FOOTNOTES
 
This work was supported by a grant from the Seeding Fund for Basic Research of the University of Hong Kong (to Y.W.) and Grant HKU 7609/05M from the Hong Kong Research Grant Council (to A.X.).

The authors have nothing to disclose.

First Published Online January 9, 2007

Abbreviations: ANGPTL4, Angiopoietin-like protein 4; 2D-DIGE, two-dimensional fluorescence difference gel electrophoresis; DMDH, dimethylglycine dehydrogenase; FTHFD, formyltetrahydrofolate dehydrogenase; GRP78, 78-kDa glucose-regulated protein; GST, gluthione-S-transferase; 2HPCL, 2-hydroxyphytanoyl-coenzyme A lyase; IDPc, cytosolic NADP(+)-dependent isocitrate dehydrogenase; IEF, isoelectric focusing; LC/MS/MS, liquid chromatography/tandem mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MAT1A, methionine adenosyltransferase I {alpha}; MRC, mitochondria respiratory chain; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrose; SAM, S-adenosylmethionine; SDH, sarcosine dehydrogenase; T2DM, Type 2 diabetes mellitus; TFA, trifluroacetic acid; TTG, tissue transglutaminase.

Received for publication June 19, 2006. Accepted for publication January 5, 2007.


   REFERENCES
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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