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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Angiopoietin-Related Growth Factor Suppresses Gluconeogenesis through the Akt/Forkhead Box Class O1-Dependent Pathway in Hepatocytes

Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., Tsukuba, Japan (M.K., S.H.); Departments of Pharmaceutical Science (M.K., T.H.) and Pharmaceutical Molecular Biology (Y.O.), Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; and Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan (Y.O.)

Received June 21, 2007; accepted September 4, 2007.

	Abstract

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Angiopoietin-related growth factor (AGF; or Angptl6) is a liver-derived, circulating factor and is considered to be a regulator of metabolic homeostasis. AGF is capable of counteracting both obesity and obesity-related insulin resistance. However, the target tissues and the molecular mechanisms underlying the antiobesity and antidiabetic actions of AGF have not been completely defined. Using rat hepatoma H4IIEc3 cells or primary hepatocytes, we demonstrate that AGF suppresses glucose production in a concentration-dependent manner through reduced expression of a key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), at both transcriptional and translational levels. The action of AGF on glucose production was inhibited by pretreatment of the cells with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a phosphoinositide 3-kinase (PI3K) inhibitor, and Akt (protein kinase B) inhibitors. AGF increased the phosphorylation of Akt and its substrates, glycogen synthase kinase 3 and forkhead box class O1 (FoxO1), a key transcription factor for G6Pase expression. Furthermore, an immunohistochemical approach with anti-FoxO1 antibody demonstrated that AGF stimulation promoted translocation of FoxO1 from the nucleus to the cytoplasm in the cells. These results suggest that in hepatocytes, AGF suppresses gluconeogenesis via reduced transcriptional activity of FoxO1 resulting from the activation of PI3K/Akt signaling cascades.

AGF is a liver-derived circulating factor (Oike et al., 2003) that counteracts high-fat-induced obesity and related insulin resistance through increased energy expenditure (Oike et al., 2005). Both visceral and s.c. fat depots were significantly increased in AGF-deficient mice, and sections of white adipose tissue from these mice showed increased adipocyte size relative to those from wild-type mice. Furthermore, a large amount of lipid accumulation in liver, skeletal muscle, and brown adipose tissue was observed in AGF-deficient mice. These mice also showed significant decreases in whole-body oxygen consumption rates compared with wild-type mice, whereas no increase was observed in daily food intake in AGF-deficient mice compared with wild-type mice, indicating that inactivation of AGF in vivo leads to decreased energy expenditure and obesity (Oike et al., 2005). Thus, AGF is a potential target for pharmacological interventions counter-acting obesity and related metabolic diseases. However, the target tissues and the molecular mechanisms by which AGF counteracts obesity and related insulin resistance are not well defined.

View larger version (14K): [in this window] [in a new window]   Fig. 1. AGF decreases glucose production through reduced expression of G6Pase but not PEPCK in H4IIEc3 cells. A, H4IIEc3 cells were treated with or without AGF for 21 h and then fed glucose-free medium for 3 h. Collected media were assessed for glucose production as described under Materials and Methods. B and C, cells were cultured in the absence or presence of 1 µg/ml AGF for the indicated times (for measurement of mRNA expression) or 24 h (for measurement of protein expression) (n = 6). C, expression of G6pc and Pck mRNA was measured by real-time PCR. mRNA levels were normalized using -actin as the housekeeping gene (n = 3). B, expression of G6Pase and PEPCK protein was measured by Western blot analysis. Blots were then stripped and reprobed with anti--actin antibody, respectively, to verify that equal amounts of proteins were present in each sample. Values represent the means ± S.E.M. *, P < 0.05; **, P < 0.01 compared with PBS control.

	Materials and Methods

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Cell Culture. The rat hepatoma cell line H4IIE was cultured in minimal essential medium containing 10% fetal bovine serum, 0.1 mM nonessential amino acid solution, sodium pyruvate solution, and 100 U/ml penicillin/100 µg/ml streptomycin and maintained at 37°C in 5% CO₂. Primary rat hepatocytes were isolated as described previously (Shih and Towle, 1999). In brief, primary hepatocytes were isolated from male Wistar rats (6W-9W) using the collagenase perfusion method. Hepatocytes were plated on collagen-coated 3.5-cm dishes at 1 x 10⁶ cells/dish in HepatoZYME-SFM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and 100 U/ml penicillin/100 µg/ml streptomycin and maintained at 37°C in 5% CO₂.

Purification of Recombinant AGF Protein. Purification of recombinant AGF protein was described by Oike et al. (2004). In brief, the conditioned medium from transfected human embryonic kidney 293 cells was collected and passed through a 0.22-µm-pore size filter (Millipore, Bedford, MA). To purify AGF protein, the filtered conditioned medium was transferred to an anti-FLAG antibody (M2) affinity gel (Sigma-Aldrich, St. Louis, MO), and only AGF protein was trapped in the gel. After the gel was washed with phosphate-buffered saline (PBS), protein was eluted by adding Gly-HCl, pH 3.0, and immediately neutralized with Tris, pH 8.0. AGF protein was dialyzed in PBS overnight at 4°C. Protein concentration was measured with the BCA protein assay kit (Pierce Chemical, Rockford, IL).

Glucose Production Assay. Cells were cultured on 48-well plates at a density of 2 x 10⁵ (H4IIEc3 cells) or 1 x 10⁵ (primary hepatocytes) cells/well. After a 6-h attachment period, cells were treated with or without AGF in serum-free medium for 21 h. These cells were washed twice with PBS to remove glucose and then incubated for 3 h in 100 µl of glucose production medium [glucose- and phenol red-free Dulbecco's modified Eagle's medium containing sodium pyruvate (2 mM)]. Fifty microliters of medium was sampled for measurement of glucose concentration using an Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen). Glucose concentrations were normalized to cellular protein concentrations measured by the BCA protein assay kit. Student's t test was used for statistical analysis.

Preparation of Total RNA and Quantitative PCR. Cells were plated on 35-mm dishes at a density of 2 x 10⁶ (H4IIEc3 cells) or 1 x 10⁶ (primary hepatocytes) cells/dish. After a 6-h attachment period, cells were treated with or without AGF in serum-free medium for 24 h, and then total RNA was isolated using the QIAGEN RNeasy mini kit (QIAGEN, Valencia, CA). Quantitative PCR was performed with ABI Prism 7700 and SYBR Green Reagent (Applied Biosystems, Foster City, CA). cDNA was synthesized from 0.5 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen). Absolute cDNA abundance was calculated using the standard curve obtained from rat genomic DNAs. PCR conditions were as follows: 10 min at 95° C, then 50 cycles of 15 s at 94°C, 30 s at 60°C, and 1 min at 72°C. Levels of mRNA were normalized using -actin as the housekeeping gene. The following oligonucleotide DNA primers were synthesized: for rat G6pc, the forward primer was 5'-TGAAACTTTCAGCCACATCCG-3', and the reverse primer was 5'-GCAGGTAAAATCCAAGTGCGAA-3'; for rat Pck1, the forward primer was 5'-TCCCATTGGCTACGTCCCTAA-3', and the reverse primer was 5'-CCACCTCCTTCTCCCAGAATTC-3'; and for rat -actin, the forward primer was 5'-GGTCGTACCACTGGCATTGTG-3', and the reverse primer was 5'-GCTCGGTCAGGATCTTCATGAG-3'.

Isolation of Whole-Cell, Nuclear, and Cytoplasmic Extracts and Western Blot Analyses. Cells were plated on 35-mm dishes at a density of 2 x 10⁶ (H4IIEc3 cells) or 1 x 10⁶ (primary hepatocytes) cells/dish. After a 6-h attachment period, cells were treated with or without AGF in serum-free medium for the indicated time. These cells were washed twice with ice-cold PBS and treated with lysis buffer [1 mM EDTA, 1% SDS, Complete phosphatase inhibitors cocktail (Sigma-Aldrich), 10 mM HEPES-HCl, pH 7.5] The cell lysates were boiled for 10 min and centrifuged to obtain the supernatants as cell extracts. Cellular protein concentrations were measured with the BCA protein assay kit (Pierce Chemical). Cytoplasmic or nuclear proteins from H4IIEc3 cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer's instruction (Pierce Chemical). SDS sample buffer (2x) was added to the cell lysates. After boiling for 5 min, the samples were separated by 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using a semidry apparatus. After soaking in 5% nonfat milk for 1 h at room temperature, the membrane was incubated with primary antibodies for 16 to 20 h at 4°C. The specific signals were amplified by addition of horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence system.

Immunohistochemistry. Cells were plated at 5 x 10⁴ cells/dish. After a 6-h attachment period, cells were treated with or without AGF in serum-free medium for the indicated time and fixed with 3.7% formaldehyde in PBS and permeabilized with 0.5% Triton X-100. FoxO1 was visualized using anti-FoxO1 polyclonal antibody (1 µg/ml, final concentration) and fluorescein isothiocyanate-conjugated Affinity Purified donkey anti-rabbit IgG (20 µg/ml, final concentration). Slides were examined using an Eclipse TE 300 fluorescence microscope (Nikon, Tokyo, Japan).

	Results

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AGF Suppressed Gluconeogenesis via Decreased Expression of G6Pase in Hepatocytes. In both type 1 and type 2 diabetes, excessive hepatic glucose production is a major contributor of both fasting and postprandial hyperglycemia. To examine the influence of AGF on hepatic glucose metabolism, we measured glucose production in H4IIEc3 cells. Treatment of the cells with AGF induced a concentration-dependent reduction in the amount of glucose produced (Fig. 1A). The rate of gluconeogenesis is controlled principally by the activities of unidirectional enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase. PEPCK catalyzes one of the rate limiting steps of gluconeogenesis, the conversion of oxaloacetate to phosphoenolpyruvate, whereas G6Pase catalyzes the final step of gluconeogenesis, the production of free glucose from glucose 6-phosphate. The genes of these gluconeogenic enzymes are controlled at the transcriptional level by hormones, mainly insulin, glucagon, and glucocorticoid. Insulin inhibits gluconeogenesis by suppressing the expression of PEPCK and G6Pase, whereas glucagon and glucocorticoids stimulate hepatic glucose production by inducing these genes (O'Brien and Granner, 1996). To examine the influence of AGF on the expression of these enzymes, we measured the levels of Pck and G6pc mRNAs using quantitative PCR in AGF-treated H4IIEc3 cells. Decreased expression of G6pc mRNA was observed after 6 h of treatment with 1 µg/ml AGF and remained depressed for at least 24 h (Fig. 1C). Likewise, expression of G6pase protein was also decreased by 1 µg/ml AGF after 24-h treatment (Fig. 1). In contrast, PEPCK was not affected by AGF at either the mRNA or protein levels (Fig. 1B). We also assessed the effects of AGF on glucose production and expression of G6Pase in primary rat hepatocytes. AGF reduced glucose output by 30% in a concentration-dependent manner (Fig. 2A). At 1 µg/ml, AGF decreased expression of G6Pase mRNA (Fig. 2B) and protein (Fig. 2C) in cultured rat hepatocytes.

View larger version (12K): [in this window] [in a new window]   Fig. 2. AGF decreases glucose production and expression of G6Pase in primary rat hepatocytes. A, AGF inhibited glucose production in primary rat hepatocytes. The glucose production assay was performed as described in the legend of Fig. 1A. B, expression of G6pc mRNA by cells treated with or without 1 µg/ml AGF for 12 h was measured by real-time PCR as described in Fig. 1B. C, expression of G6Pase and PEPCK proteins by cells treated with or without 1 µg/ml AGF for 24 h was measured by Western blot analysis. 14-3-3 Protein was used as loading control. Blots were then stripped and reprobed with anti-14-3-3 antibody, respectively, to verify that an equal amount of protein was present in each sample. Values represent the means ± S.E.M. (n = 6). *, P < 0.05; **, P < 0.01 compared with PBS control.

To determine whether Akt actually plays a role in the AGF-mediated reduction of glucose production, we used a variety of PI3K-Akt signaling inhibitors and monitored glucose production. The reduction of glucose production mediated by 1 µg/ml AGF was blocked by pretreatment of the cells with 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl]-3-O-octadecylcarbonate (Akt inhibitor I) (Hu et al., 2000), Triciribine (Akt inhibitor V) (Kim et al., 2005), and LY294002, a specific PI3K inhibitor, in a concentration-dependent manner (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3. AGF inhibited glucose production through stimulation of PI3K-Akt signaling cascades in H4IIEc3 cells. AGF-induced reduction of glucose production was abolished by pretreatment with PI3K and Akt inhibitors. Cells were pretreated with 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl]-3-O-octadecylcarbonate (Akt inhibitor I) (I), Triciribine (Akt inhibitor V) (V), or LY294002 for 15 min at the indicated concentration and then treated with 1 µg/ml AGF for 21 h. The glucose production assay was performed as described in the legend of Fig. 1A. Values represent the means ± S.E.M. (n = 6), **, P < 0.01 compared with PBS control; #, P < 0.05; ##, P < 0.01 compared with AGF-treated.

AGF-Induced Phosphorylation of Akt, GSK-3, and FoxO1. Treatment of H4IIEc3 hepatoma cells with AGF increased the phosphorylation of Akt (Fig. 4A) in a concentration-dependent manner. AGF-mediated enhancement of Akt phosphorylation reached a maximal level 10 min after AGF addition (Fig. 4B), followed by subsequent phosphorylation of GSK-3 and FoxO1 (Guo et al., 1999) (Fig. 4, A and B).

View larger version (22K): [in this window] [in a new window]   Fig. 4. AGF induced Akt, GSK-3, and FoxO1 phosphorylation in a concentration-dependent manner. A, cells were treated with AGF for 10 min at the indicated concentration. B, AGF-induced GSK-3 and FoxO1 phosphorylation following Akt phosphorylation. Cells were treated with 100 ng/ml AGF for the indicated times. Western blot analyses were performed using anti-phospho-Akt (Ser473), anti-phospho-GSK-3, or anti-phospho-Fxo1 antibodies. Blots were then stripped and reprobed with anti-Akt, anti-GSK-3, or anti-FoxO1 antibodies to verify that each lane contained equal amounts of proteins.

View larger version (31K): [in this window] [in a new window]   Fig. 5. AGF induced translocation of FoxO1 from nucleus to cytoplasm in H4IIEc3 cells. A, subnuclear FoxO1 was reduced by treatment with AGF. Cells were treated with or without 1 µg/ml AGF for the indicated time and then fractionated to cytoplasmic and nuclear fractions as described under Materials and Methods. Immunoblotting was performed with anti-FoxO1 antibody (left). The graph represents the ratio of band densities, comparing cytoplasmic with nuclear FoxO1 protein (right). B, cells were treated with or without 1 µg/ml AGF for 3 h and then fixed with 3.7% formaldehyde. Immunohistochemistry was performed with anti-FoxO1 antibody and fluorescein isothiocyanate-conjugated secondly antibody. Scale bar, 30 µm.

	Discussion

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G6Pase and PEPCK are such proteins whose expression is regulated by insulin, whereas expression of PEPCK was not affected by AGF. The insulin receptor is a tyrosine kinase that, when activated by insulin, phosphorylates members of the family of insulin receptor substrates. This leads to the recruitment of PI 3-kinase to the plasma membrane, where it is involved in the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, the second messenger of the insulin receptor, as well as a variety of growth factor receptors. Phosphatidylinositol 3,4,5-trisphosphate indirectly activates Akt. Activation of Akt in hepatoma cells is sufficient to repress the glucocorticoid- and cAMP-promoted induction of PEPCK gene transcription (Agati et al., 1998; Liao et al., 1998), and the overexpression of the gene for Akt in cultured hepatocytes decreases the levels of mRNA for PEPCK and G6Pase (Schmoll et al., 2000). This suggests that the downstream targets of Akt, transcription factors such as FoxO1, hepatocyte nuclear factor-3, and cAMP response element binding, are involved in the effect of insulin on the transcription of the genes for these two gluconeogenic enzymes. However, Kotani et al. (1999) have shown that a dominant negative form of Akt does not prevent insulin's inhibition of transcription for the PEPCK gene promoter in hepatoma cells. Furthermore, overexpression of Foxo1 markedly increased the expression of the catalytic subunit of G6Pase. In contrast, both basal and dexamethasone/cAMP-induced levels of PEPCK mRNA were unaffected by Foxo1-overexpression (Barthel et al., 2001). These observations suggested that suppression of PEPCK expression by insulin was not only regulated by Akt-FoxO1 signaling cascades, but the other cascades coregulate this function. Moreover, AGF mediated the adhesion and migration cellular events through interaction with Arg-Gly-Asp (RGD)-binding integrins (Zhang et al., 2006). The integrin signaling via RGD peptides induced an increase of phospho-Akt (Pinkse et al., 2005). Taking into consideration these reports, we assumed that AGF activates Akt using the different signaling cascades from insulin. Although activation of Akt occurs, AGF does not affect PEPCK expression.

The most important finding of the present study is that AGF stimulates nuclear export of FoxO1. K14-AGF transgenic mice (in which expression of AGF is forced in epidermal cells) show keratinocyte proliferation and increased cutaneous wound healing, suggesting that AGF promotes keratinocyte growth (Oike et al., 2003). In addition, significant immunoreactivity is detected in the epidermis of K14-AGF mice by using an antibody directed against phospho-Ser473 of Akt compared with that of controls, suggesting that Akt signaling is involved in epidermal proliferation induced by AGF (Oike et al., 2003). Furthermore, K14-AGF mice exhibit increased vascular permeability and increased numbers of blood vessels, suggesting that AGF has angiogenic activity like other angiopoietins and angiopoietin-related proteins, such as Ang-1, angiopoietin-related protein 2, and ANGPTL3 (Oike et al., 2004). In addition, inhibition of FoxO1 transcriptional activities by nuclear export or gene silencing promotes endothelial cell proliferation (Fosbrink et al., 2006) and angiogenesis and postnatal neovascularization (Potente et al., 2005). Taken together with these reports, FoxO1 may be a key molecule in the function of AGF in glucose metabolism, cell proliferation, and angiogenesis.

In summary, we found that AGF induced PI3K/Akt/FoxO1 signaling cascades and thereby reduced G6Pase protein expression in hepatocytes. These results suggest that AGF plays a crucial role in regulating glucose metabolism and in the pathogenesis of T2DM. The elucidation of the molecular and cellular mechanisms underlying AGF-induced modulation of metabolic gene expression via FoxO1-dependent transcription may enable us to develop new therapies for diabetes.

	Acknowledgements

 
We thank K. Yasunaga for providing AGF-transfected human embryonic kidney 293 and technical advice and Tomoko Kojima for technical assistance.

	Footnotes

 
This work was performed as a part of a research and development project of the Industrial Science and Technology Program supported by the New Energy and Industrial Technology Development Organization.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.127530.

ABBREVIATIONS: T2DM, type 2 diabetes mellitus; AGF, angiopoietin-related growth factor; G6Pase, glucose-6-phosphatase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; FoxO1, forkhead box class O1; PEPCK, phosphoenolpyruvate carboxykinase; GSK, glycogen synthase kinase; PI3K, phosphoinositide 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one.

Address correspondence to: Masashi Kitazawa, Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. E-mail: masashi.kitazawa{at}jp.astellas.com

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.127530v1 323/3/787    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kitazawa, M. Articles by Hashimoto, S. Search for Related Content PubMed PubMed Citation Articles by Kitazawa, M. Articles by Hashimoto, S.