Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 7;17(5):768-78.
doi: 10.1016/j.cmet.2013.04.012.

Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production

Haiming Cao  1 , Motohiro Sekiya, Meric Erikci Ertunc, M Furkan Burak, Jared R Mayers, Ariel White, Karen Inouye, Lisa M Rickey, Baris C Ercal, Masato Furuhashi, Gürol Tuncman, Gökhan S Hotamisligil
Affiliations

Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production

Haiming Cao et al. Cell Metab. .

Abstract

Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Secretion of aP2 in vitro from cultured cells
a, aP2 secretion in adipocytes. Whole cell lysate (WCL) and conditioned medium (CM) from differentiated WT (+/+) or AM−/− (−/−) adipocytes lacking both aP2 and mal1 were blotted using anti-aP2, adiponectin (ACDC), mal1, or AKT antibodies. These cell lines were developed in-house. b, aP2 secretion in HEK 293 cells. Whole cell lysates (WCL) or immunoprecipated conditioned medium (CM) from HEK 293 cells transfected with Flag-AKT, Flag-GFP-aP2 or Flag-GFP plasmids were used to detect aP2 by western blotting with an anti-Flag antibody. c, CMV promoter driven aP2 expression and its regulated secretion studied in aP2−/− adipocytes. Flag-aP2 was transfected into aP2−/− cells and its appearance in the CM was examined under basal and forskolin (FSK) stimulated condition (20μM for 1h). CM samples were immunoprecipitated with an anti-Flag antibody and blotted with anti-aP2. Cell lysate was probed with aP2 and β-tubulin (β-tub) antibodies. d, Quantitation of data shown in panel c determined by using ImageJ program. * p < 0.05 in student’s t test. e, Pulse chase analysis of aP2 secretion. Cultured adipocytes were metabolically labeled and then treated with vehicle, brefeldin A (BrA, 10 μg/ml) or monensin (Mon, 5 μM), and samples were taken at indicated time points. Proteins in CM were immunoprecipitated using anti-aP2 or adiponectin (ACDC) antibodies. After electrophoresis, radiolabeled proteins were subjected to autoradiography. An aP2−/− cell lysate was used as negative control. Data are presented as means ± SEM.
Figure 2
Figure 2. Nutritional status and lipolysis regulates aP2 secretion
a, Plasma aP2 in mice fasted for 24 hours, refed for 4 hours after a 24-hour fast, or fed ad libitum. Below the graphs are corresponding aP2 western blots in WAT lysates. b, Plasma aP2 levels in mice injected with saline (Control), CL 316243 (CL, 0.1mg/kg) or isoproterenol (ISO, 1mg/kg) to induce lipolysis. At least 6 male mice were used in each experiment. c, aP2 in conditioned medium (CM) or whole cell lysates (WCL) of adipose tissue explants treated with forskolin (FSK, 20μM) or isobutylmethylxanthine (IBMX, 1mM). Below the graphs are corresponding aP2 western blots in CM and WCL. d, aP2 in CM or WCL of adipocytes treated with isobutylmethylxanthine and dibutryl cyclic adenosine monophosphate (IBMX/db-cAMP; I/C, 1mM) and insulin (Ins, 100ng/ml). Below the graphs are corresponding aP2 western blots in CM and WCL. e–f, aP2 in CM or WCL of primary human omental (O) or subcutaneous (SC) adipocytes treated with FSK, db-cAMP, or IBMX. Below the graphs are corresponding aP2 western blots in CM and WCL blotted with anti-aP2 antibody. Beta-tubulin is shown as a loading control. Data are presented as means ± SEM. * p < 0.05 in student’s t test.
Figure 3
Figure 3. Regulation of aP2 secretion in vivo
a, Plasma aP2 levels in WT, mal1−/− (M−/−), aP2−/− (A−/−), and aP2-mal1−/− (AM−/−) mice, determined by ELISA. b, Plasma aP2 levels of lean mice (WT regular diet, RD), or mice with dietary (WT high fat diet, HFD) or genetic obesity (ob/ob). c, Serum aP2 levels in human subjects, female (n=910) and male (n=904), as a function of body mass index (BMI). d, aP2 secretion from weight-matched adipose tissue explants of lean and obese mice. Adipose tissue explants were collected from WT mice maintained on regular diet or ob/ob mice and were thoroughly washed with PBS. Fresh medium was added and incubated overnight and collected for western blot analysis using anti-aP2 or adiponectin (ACDC) antibodies. e, Quantitative measurement of the relative secretion of aP2 and adiponectin from adipose tissue explants from WT mice maintained on regular diet or ob/ob mice is graphed. f, Plasma aP2 in mice that have undergone bone marrow transplantation. Bone marrow transplantation was performed between WT and aP2-mal1−/− (AM−/−) mice (as donors and recipients) and plasma aP2 levels were determined by aP2 ELISA in the resulting 4 groups of chimeras. Statistical analysis was performed by student’s t test. Data are presented as means ± SEM. * p < 0.05.
Figure 4
Figure 4. Regulation of glucose homeostasis by recombinant soluble aP2
a, Glucose production from primary mouse hepatocytes treated with glucagon (3 μM) and aP2 (10 μg/ml) compared to vehicle (Veh). b, Expression of phosphoenolpyruvate carboxykinase 1 (Pck1) in primary mouse hepatocytes treated with aP2 (10 μg/ml). c, Enzymatic activity of Pck1 in primary mouse hepatocytes treated with aP2 (50 μg/ml). d, Pck1 mRNA expression in primary hepatocytes treated with 10 (WT10 or BM10) or 50 (WT50 or BM50) μg/ml recombinant WT or lipid binding mutant (BM) protein. e, Basal hepatic glucose production rates (b-HGP) in WT mice (5h fasted) after 3 hour infusion with control or recombinant aP2 protein prior to hyperinsulinemic-euglycemic clamp studies with aP2 infusion. f, Glucose infusion rates in the same animals, g, Hepatic glucose production during hyperinsulinemic-euglycemic clamp period (c-HGP). h, Expression of gluconeogenic genes Pck1 and G6pc in liver samples from mice infused with control or recombinant aP2 protein. Statistical analysis was performed by student’s t test. Data are presented as means ± SEM. * p < 0.05 and ** p < 0.01.
Figure 5
Figure 5. Effect of aP2 on hepatic glucose production in pancreatic clamp studies
a, Reconstitution of serum aP2 in the aP2−/− mice at levels comparable to obese mice were achieved by recombinant aP2 infusion at a dose of 8μg/kg/min, in lean, 10-week-old, male mice. Plasma mal1 (b), adiponectin (c), and glucagon (d) levels in aP2−/− mice infused with either control or recombinant aP2 protein. e, clamp-hepatic glucose production (c-HGP), f, glucose infusion rates (GIR) and g, rate of whole body glucose disposal (RD) in aP2−/− mice that were infused with recombinant aP2 during the pancreatic clamp study. h, Phosphoenolpyruvate carboxykinase 1 (Pck1) activity in liver samples harvested from aP2−/− mice following infusion with recombinant aP2 protein. Statistical analysis was performed by student’s t test. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, n=8 in aP2 infusion and n=6 in control group.
Figure 6
Figure 6. Improved glucose homeostasis in obese mice after aP2 neutralization
a, Plasma aP2 levels in mice with dietary obesity before and after administration of control pre-immune (PI-IgG) or an anti-aP2 antibody for two weeks, determined by aP2 ELISA. b, aP2 protein expression in total protein extracts of white adipose tissue (WAT) from obese mice after treatment with PI-IgG or anti-aP2 antibody for two weeks. c, Body weight and d, glucose levels in obese mice treated with control PI-IgG or anti-aP2 antibody for two weeks. Body weight measurements and blood glucose levels were determined after 6 hours of daytime food withdrawal. e, Free fatty acid levels in mice before and after antibody treatment f, Glucose tolerance test (GTT) in obese mice after aP2 neutralization for two weeks (1g/kg glucose). g, Graph depicting the area under the curve (AUC) calculations for GTT shown in panel f. Statistical analysis was performed by student’s t test. Data are presented as means ± SEM. * p<0.05, ** p<0.01.
Figure 7
Figure 7. aP2 neutralization affects hepatic glucose production
a, Basal hepatic glucose production (b-HGP) in WT, high fat diet-fed, obese mice during hyperinsulinemic-euglycemic clamps following aP2 neutralization. b, Hepatic glucose production during the clamp period (c-HGP) in obese mice after aP2 neutralization. c, Glucose infusion rate (GIR) in obese mice following aP2 neutralization. d, Rate of whole body glucose disposal (RD) during the clamp period in obese mice following aP2 neutralization. e, Expression of gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) following neutralization of aP2. At least 10 male mice were used in each experiment. Statistical analysis was performed by student’s t test. Data are presented as means ± SEM. * p < 0.05. f, Model of aP2 action following secretion from adipocytes. aP2 is a fasting-regulated hormone responsive to β-adrenergic stimuli and lipolytic signals, and acts on liver to stimulate glucose production. Other potential target organs and cells remain unknown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Boden G. Effects of free fatty acids on gluconeogenesis and glycogenolysis. Life Sci. 2003;72:977–988. - PubMed
    1. Cabre A, Babio N, Lazaro I, Bullo M, Garcia-Arellano A, Masana L, Salas-Salvado J. FABP4 predicts atherogenic dyslipidemia development. The PREDIMED study. Atherosclerosis. 2012;222:229–234. - PubMed
    1. Cahill GF., Jr Fuel metabolism in starvation. Annu Rev Nutr. 2006;26:1–22. - PubMed
    1. Cao H, Gerhold K, Mayers JR, Wiest MM, Watkins SM, Hotamisligil GS. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism. Cell. 2008;134:933–944. - PMC - PubMed
    1. Cao H, Maeda K, Gorgun CZ, Kim HJ, Park SY, Shulman GI, Kim JK, Hotamisligil GS. Regulation of metabolic responses by adipocyte/macrophage Fatty Acid-binding proteins in leptin-deficient mice. Diabetes. 2006;55:1915–1922. - PubMed

Publication types