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Angiopoietin-related Growth Factor (AGF) /ANGIOPOIETIN-LIKE 6 (ANGPTL6)
A new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance

Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration

We report here the identification of an angiopoietin-related growth factor (AGF). To examine the biological function of AGF in vivo, we created transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF mice exhibited swollen and reddish ears, nose and eyelids. Histological analyses of K14-AGF mice revealed significantly thickened epidermis and a marked increase in proliferating epidermal cells as well as vascular cells in the skin compared with nontransgenic controls. In addition, we found rapid wound closure in the healing process and an unusual closure of holes punched in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in platelets and mast cells, and detected at wounded skin, whereas there was no expression of AGF detected in normal skin tissues, suggesting that AGF derived from these infiltrated cells affects epidermal proliferation and thereby plays a role in the wound healing process. These findings demonstrate that biological functions of AGF in epidermal keratinocytes could lead to novel therapeutic strategies for wound care and epidermal regenerative medicine.
Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9


Angiopoietin-related growth factor (AGF) promotes angiogenesis
We report here the identification of angiopoietin-related growth factor (AGF) as a positive mediator for angiogenesis. To investigate the biologic function of AGF in angiogenesis, we analyzed the vasculature in the dermis of transgenic mice expressing AGF in mouse epidermal keratinocytes (K14-AGF). K14-AGF transgenic mice were grossly red, especially in the ears and snout, suggesting that hypervascularization had occurred in their skin. Histologic examination of ear skin from K14-AGF transgenic mice revealed increased numbers of microvessels in the dermis, whereas the expression of several angiogenic factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGFs), and angiopoietin-1 (Ang-1), was decreased. We showed that AGF is a secreted protein and does not bind to tyrosine kinase with immunoglobulin and EGF-homology domain (Tie1) or Tie2 receptors. An in vitro chamber assay revealed that AGF directly promotes chemotactic activity of vascular endothelial cells. Both mouse corneal and matrigel plug assays showed that AGF induces neovascularization in vivo. Furthermore, we found that plasma leakage occurred after direct injection of AGF into the mouse dermis, suggesting that AGF directly induces a permeability change in the local vasculature. On the basis of these observations, we propose that AGF is a novel angiogenic factor and that handling of its biologic functions could lead to novel therapeutic strategies for control of angiogenesis.
Oike Y, et al. Blood. 2004 May 15;103(10):3760-5

Angiopoietin-related growth factor antagonizes obesity and insulin resistance
Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet-induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance.
Oike Y, et al. Nat Med. 2005 Apr;11(4):400-8. Epub 2005 Mar 20

AGF
AGF
Obesity in Angptl6-/- mice on a normal diet.
(a) Gross appearance of Angptl6-/- mice and wild-type control mice. (b) Body weight of each genotype (n = 8). (c-g) Abdominal cavity (c), CT findings at a level of 8 mm above the top of the iliac bone (d), visceral fat (n = 5) and subcutaneous fat (n = 5) weight/body weight, and histological analysis (e) and distribution of cell size (f) of WAT of Angptl6-/- mice and wild-type mice. (g) Triglyceride levels in liver (n = 5) and gastrocnemius muscle (n = 5), and hematoxylin and eosin-stained sections of BAT of Angptl6-/- and wild-type mice. Data are mean s.d. Bars in histological sections indicate 50 m. * P < 0.05, ** P < 0.01, between the two genotypes indicated. Female mice 8 months after birth were used for all experiments. Oike Y, et al. Nat Med. 2005 Apr;11(4):400-8. Epub 2005 Mar 20
Obesity in Angptl6-/- mice on a normal diet.
(a) Gross appearance of Angptl6-/- mice and wild-type control mice. (b) Body weight of each genotype (n = 8). (c-g) Abdominal cavity (c), CT findings at a level of 8 mm above the top of the iliac bone (d), visceral fat (n = 5) and subcutaneous fat (n = 5) weight/body weight, and histological analysis (e) and distribution of cell size (f) of WAT of Angptl6-/- mice and wild-type mice. (g) Triglyceride levels in liver (n = 5) and gastrocnemius muscle (n = 5), and hematoxylin and eosin-stained sections of BAT of Angptl6-/- and wild-type mice. Data are mean s.d. Bars in histological sections indicate 50 m. * P < 0.05, ** P < 0.01, between the two genotypes indicated. Female mice 8 months after birth were used for all experiments. Oike Y, et al. Nat Med. 2005 Apr;11(4):400-8. Epub 2005 Mar 20
(a) Western blotting analysis for serum AGF in Angptl6 transgenic (TG) and nontransgenic control (NTG) mice at 4 months of age. The ratio for the control is set as 100%. (b-g) Body weight (b), and gross appearance of visceral adipocyte (c) in TG and NTG mice at 4 months of age. (d) Comparison of visceral fat and subcutaneous fat weight/body weight between TG and NTG mice at 5 months of age. Tissue weight/body weight in TG and NTG mice at 4 months of age (e). n = 10-15 in each group. Histological analysis (f) and distribution of cell size (g) of WAT from TG and NTG mice at 4 months of age. Scale bars, 50 m. Data are mean s.d. *P < 0.05, **P < 0.01, between the two genotypes indicated. N.S. indicates no significant difference compared with nontransgenic wild-type mice. Female mice were used for all experiments. Oike Y, et al. Nat Med. 2005 Apr;11(4):400-8. Epub 2005 Mar 20
Resistance to high-fat diet-induced obesity and related metabolic disorders seen in Angptl6 transgenic mice.
(a) Representative gross appearance of AGF-transgenic (TG) and nontransgenic control (NTG) female mice after 3-months high-fat feeding. (b) Change in body weight in TG and NTG male and female mice after 3-months high-fat feeding (n = 10). (c) CT findings, which were shown at a level of 8 mm above the top of the iliac bone in a. (d) Visceral fat (n = 10) and subcutaneous fat (n = 10) weight/body weight. (e) Hematoxylin and eosin-stained sections and distribution of cell size of WAT, and hematoxylin and eosin-stained sections of BAT from TG and NTG mice after 3 months of high-fat feeding. (f) Triglyceride content in liver (n = 5) and gastrocnemius muscle (n = 5) from TG and NTG mice after 1 month (left) and 3 months (right) of high-fat feeding. (g) Blood glucose, plasma insulin, serum cholesterol, triglyceride, and FFA concentrations in TG and NTG mice after 3-months of high-fat feeding. (n = 10 in each group). Scale bars in e, 50 m. Data are mean s.d. * P < 0.05, ** P < 0.01, between the two genotypes indicated.
AGF decreased body weight and increased insulin sensitivity in high-fat fed-induced obese mice.
(a) The relative ratio of serum concentrations of AGF in Ad-AGF injected and Ad-GFP injected mice on day 20 relative to each mouse on day 0. The value of serum AGF concentrations on day 0 is set at 100% (n = 5-8 in each group). (b) Alteration in body weight of high-fat fed-induced obese female mice after Ad-AGF and Ad-GFP injections (n = 8 in each group). (c-g) Comparison of food intake/lean body weight (c), fasting blood glucose (d), random fed blood glucose (e), glucose tolerance test (f) and insulin tolerance test (g) between Ad-AGF injected and Ad-GFP injected mice (n = 5-8 in each group). Data are mean s.d. * P < 0.05, ** P < 0.01, between the two groups. N.S. indicates no significant difference compared with Ad-GFP-injected mice.
Sequence and expression analyses of AGF. (A) Deduced amino acid sequences of human and mouse AGF. Open and filled arrows indicate the limits of the coiled-coil and fibrinogen-like domains, respectively. (B) The evolutionary relationship of AGF (red) to the angiopoietin superfamily was derived by using DNASIS FOR WINDOWS V. 2.1 (Hitachi Software, Tokyo). The length of each horizontal line is proportional to the degree of amino acid sequence divergence. (C) Western blot analysis of various mouse tissues by using the anti-AGF (Upper) and anti-actin (Lower) antibodies. (D) Immunoreaction using the anti-AGF antibody shows AGF specifically expressed in hepatic parenchymal cells, not in the Glisson region. (Scale bar = 100 m.) (E) Analysis of AGF mRNA expression in hematopoietic cells from adult bone marrow or BMMCs by RT-PCR. CD4+/CD8+, T cell; B220, B cell; Mac-1, macrophage and monocyte; Gr-1, granulocyte; Ter119, erythrocyte; CD41, megakaryocyte/platelet; CD45+Lin+, mature HCs; CD45+Lin-, immature HCs; c-Kit+Sca-1+Lin-, hematopoietic stem cell-enriched population. A mixture of anti-Mac-1, -Gr-1, -B220, -CD4, -CD8, and -Ly-6 antibodies was used as a lineage marker (Lin). GAPDH mRNA served as a loading control. All RNA without RT treatment (RT-) show no transcript by PCR. BMMCs (RT-) is one representative data. Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9
Markedly thickened epidermal layers in K14-AGF mice. (A) Schematic representation of the transgene used to generate K14-AGF mice. K14, intron, and pA indicate the human K14 promoter, rabbit -globin intron, and a polyadenylation signal derived from the K14 gene, respectively. (B and C) Expression of the transgene was detected in the whole skin of F1 mice (TG) 3 days after birth by Northern (B) and Western (C) blotting analysis. No expression of the transgene was detected in controls (C). Blotting analysis for GAPDH was performed as an internal control experiment. (D and E) Comparison of the mRNA (D) and protein (E) level of AGF from skin and liver between K14-AGF mice and controls. Arrow in D indicates the transcription of the transgene. Open and filled arrowheads in D indicate 1.8- and 4.0-kb endogeneous AGF transcripts, respectively. Five micrograms of protein was loaded in each lane in E.(F and G) Immunohistochemical analysis of AGF detects expression of the transgene in the epidermis of skin from the ears of F1 K14-AGF mice (F) and their controls (G). (Scale bar = 100 m.) (H and I) Front view of the K14-AGF mouse and controls. Swelling of the eyelid (arrows in H), ears, and nose, and wavy whiskers (open arrowheads in H) were detected in K14-AGF mice. (J and K) Hematoxylin/eosin histology of swollen ear of the K14-AGF mouse (J) and control (K). (Scale bar = 100 m.) (L) Photographs of ears injected intravenously with Evans blue dye to visualize plasma leakage. The ear of a K14-AGF mouse was strongly blue, whereas the control ear was not changed. One representative experiment is shown. Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9
Actively cycling expression of keratin proteins in epidermal cells of K14-AGF mice. (A–F) Comparison of levels of DNA synthesis in the epidermis from K14-AGF mice and controls. Skin sections from both mice were stained immunohistochemically for BrdUrd (A and B) and anti-phospho-histone H3 (C and D)by using peroxidase-based detection. Sections were counterstained with hematoxylin. Arrows and arrowheads indicate examples (brown-stained nuclei) of BrdUrd-positive and phospho-histone H3-positive cells, respectively. (E and F) The average numbers of labeled cells with BrdUrd and anti-phospho-histone H3 immunoreactivity from five sections each from three mice, respectively. (G and H) Immunoreactivity against anti-phospho-Akt antibody was seen in the thickened epidermis from the K14-AGF mouse (G), whereas no immunoreactivity was seen in epidermis from controls (H). (I–N)K5(J) and K14 (L) were detected in the basal layer of the epidermis in controls. Similar sections obtained from K14-AGF mice show positive staining in the suprabasal layer of the epidermis as well(I and K). K1 staining was similar for both the K14-AGF mouse (M) and controls (N). (Scale bar = 50 m.) (O) Surface levels of 1-integrins in basal keratinocytes of K14-AGF (red line) and controls (black line). Three peaks for intensity of 1-integrin expression are detected in K14-AGF, whereas one peak with high intensity is seen in controls. Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9
Reepithelialization of wounds in K14-AGF mice. (A–D) Representative photograph ear wound healing. K14-AGF and control ears were punched in the center creating a 2-mm open hole and followed for 28 days. (A and B) One can see the progression of hole closure from day 1 (A) to day 28 (B). Open arrowhead in B indicates shortened hole in K14-AGF. (E–K) Wounding was accomplished by ear segment excisions. Shown are representative data of skin sections with staining for anti-K14 antibody from the exposed portion of the remaining ear of K14-AGF mice (E–G) and control littermates (H–K)at1,2,3, and 5 days after the initial wounds. All sections were photographed at the same magnification. (Scale bar = 100 m.) Arrowheads indicate migrating and proliferating epidermal keratinocytes, indicating that keratinocytes overlapped the injury site rapidly in K14-AGF mice. (L) Analysis of frequencies of wound closure by histological examination at 1, 2, 3, and 5 days after the initial wounding. Filled (controls) and open (K14-AGF mice) columns represent the number of mice in which the wound was completely covered with keratinocytes. Ten mice were examined on each day of the experiment. Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9
Expression of AGF mRNA in normal and wounded skin. The total RNA (10 g) from normal and wounded ear skin was analyzed by Northern blotting analysis with cDNA probes for AGF and KGF. The relative amount of each mRNA was quantified with normalization to 28S rRNA levels. The time after injury is indicated on top of each lane: 1, 2, 3, 5, and 8 days. Oike Y, et al. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9494-9
AGF and TIE receptors. AGF does not bind to tyrosine kinase with immunoglobulin and EGF-homology domain (Tie) receptors. (A) Schematic representation of the plasmid construction used to generate FLAG-AGF protein. CMV-p and SS indicate the cytomegalovirus promoter and a signal sequence, respectively. (B) After transfection of HEK293 cells with a mouse AGF cDNA with a 5'-terminal extension encoding a FLAG-tag (panel A), mouse AGF-FLAG fusion protein was detected in culture supernatants by Western blot analysis with an anti-FLAG antibody (left) and an antimouse AGF antibody (right), with or without 2-mercaptoethanol (2-ME). Lanes contain approximately 10 ng purified protein. Arrows indicate the monomer of FLAG-AGF protein. (C) Ties are not receptors for AGF. BIAcore binding assay of AGF (200 ng) to the Tie1 and Tie2 receptors. As a positive control, human Ang-2-6xHis-tagged protein (200 ng) specifically bound to the Tie2-Fc protein (460 ng), but not to the Tie1-Fc (460 ng). Error bar represents mean SD. Blood. 2004 May 15;103(10):3760-5. Oike Y, et al. Blood. 2004 May 15;103(10):3760-5
Increased number of microvessels in K14-AGF transgenic mice. (A) Gross appearance of the K14-AGF mouse and a control showing that the skin of ears and snout of K14-AGF mice are red compared with controls. (B) Immunohistochemical analysis with anti–PECAM-1 antibody of ear skin from K14-AGF mouse and a control. Increased PECAM-1+ microvessels (purple) are detected in the dermis and subcutaneous layers of K14-AGF mouse. Bar indicates 100 m. (C) Electron microscopic analysis (original magnification, x 1700) shows that increased vessels are capillary-sized (arrows) in the K14-AGF mouse. BC indicates epidermal basal cells. (D-E) Representative photograph of blood vessels in the ear from the K14-AGF mouse (D) and controls (E) stained with fluorescein-labeled Lycopersicon esculentum lectin. Abundant capillary-sized vessels in the K14-AGF mouse are detected. (F-G) Quantitative analysis for the number of vessels shown in panels D and E. Length of vessels (F) and number of vessel joints (G) in K14-AGF transgenic mice () relative to controls () are shown as percentages. Columns represent mean values + SD (n = 5). Oike Y, et al. Blood. 2004 May 15;103(10):3760-5

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