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. 2012 Jun 15;287(25):21492-500.
doi: 10.1074/jbc.M112.370379. Epub 2012 May 3.

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Yuqing Huo  1 , Xin Guo, Honggui Li, Hang Xu, Vera Halim, Weiyu Zhang, Huan Wang, Yang-Yi Fan, Kuok Teong Ong, Shih-Lung Woo, Robert S Chapkin, Douglas G Mashek, Yanming Chen, Hui Dong, Fuer Lu, Lai Wei, Chaodong Wu
Affiliations

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Yuqing Huo et al. J Biol Chem. .

Abstract

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.

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Figures

FIGURE 1.
FIGURE 1.
Selective overexpression of PFKFB3/iPFK2 in adipose tissue protects mice from diet-induced insulin resistance. A, genomic DNA was prepared from Adi-PFKFB3TG (Tg) mice and wild-type littermates and used for PCR analyses of aP2-PFKFB3 transgene. B, iPFK2 (encoded by PFKFB3) abundance in white adipose tissue (WAT), liver, skeletal muscle, and bone marrow (BM) was measured using Western blots. C, insulin tolerance tests. D, glucose tolerance tests. C and D, male Tg mice and WT mice, at 5–6 weeks of age, were fed an HFD for 12 weeks. After the feeding regimen, HFD-fed mice were fasted for 4 h and received an intraperitoneal injection of insulin (1 unit/kg) (C) or glucose (2 g/kg) (D). Data are means ± S.E., n = 6–10. †, p < 0.05; ††, p < 0.01 Tg versus WT at the same time point.
FIGURE 2.
FIGURE 2.
Overexpression of PFKFB3/iPFK2 in adipose tissue decreases diet-induced adipose tissue inflammatory response and improves insulin signaling while increasing adiposity. At 5–6 weeks of age, male Tg and WT mice were fed an HFD for 12 weeks. A, changes in fat mass. Visceral fat content was estimated from the sum of epididymal, perinephric, and mesenteric fat mass. B, changes in adiposity. C, adipose tissue histology. The sections of epididymal fat pad were stained with hematoxylin and eosin (10×). D, macrophage infiltration in adipose tissue. The sections of epididymal fat pad were immunostained for F4/80. E, fraction of adipose tissue macrophages. F, adipose tissue inflammatory signaling. The levels of NF-κB p65 and phospho-p65 (Ser-468) were examined using Western blot analyses. G, adipose tissue gene expression was quantified using real time RT-PCR. H, adipose tissue insulin signaling. Tissue samples of HFD-fed mice were collected at 5 min after a bolus injection of insulin (1 unit/kg) or PBS into the portal vein. The levels of Akt1/2 and phospho-Akt (Ser-473) were examined using Western blot analyses. A, B, E, and G, data are means ± S.E., n = 6–10. †, p < 0.05; ††, p < 0.01 Tg versus WT (B and E) for the same fat pad (A) and for the same gene (G for an increase); *, p < 0.05; **, p < 0.01 Tg versus WT for the same gene (G for a decrease).
FIGURE 3.
FIGURE 3.
Overexpression of PFKFB3/iPFK2 in adipose tissue decreases diet-induced liver inflammatory response and improves insulin signaling while increasing hepatic steatosis. At 5–6 weeks of age, male Tg and WT mice were fed an HFD for 12 weeks. A, changes in liver weight. B, liver histology. Liver sections were stained with hematoxylin and eosin (10×). C, levels of hepatic triglycerides were quantified using the biochemical assay. D, liver gene expression was quantified using real time RT-PCR. E, VLDL-triglyceride secretion. HFD-fed mice were fasted for 5 h and injected with tyloxapol (500 mg/kg, i.v.). Plasma levels of triglycerides were measured in blood samples taken at the indicated time points after the injection. F, liver macrophages/Kupffer cells. Liver sections were immunostained for F4/80. G, fraction of liver macrophages/Kupffer cells. H, liver inflammatory signaling. The levels of NF-κB p65 and phospho-p65 (Ser-468) were examined using Western blot analyses. I, liver expression of proinflammatory cytokines was quantified using real time RT-PCR. J, liver insulin signaling. Tissue samples of HFD-fed mice were collected at 5 min after a bolus injection of insulin (1 unit/kg) or PBS into the portal vein. The levels of Akt1/2 and phospho-Akt (Ser-473) were examined using Western blot analyses. A, C–E, G, and I, data are means ± S.E., n = 6–10. †, p < 0.05; ††, p < 0.01 Tg versus WT (A, C, and G) for the same gene (D and I).
FIGURE 4.
FIGURE 4.
Overexpression of PFKFB3/iPFK2 in adipose tissue alters lipid profiles in adipose, liver, and plasma. At 5–6 weeks of age, male Tg and WT mice were fed an HFD for 12 weeks. A, lipid profile in triglycerides fractionated from adipose tissue samples. B, lipid profile in fatty acids fractionated from adipose tissue samples. C, lipid profile in triglycerides fractionated from liver samples. D, lipid profile in fatty acids fractionated from liver samples. E, plasma lipid profile. A–E, data are means ± S.E., n = 4–6. †, p < 0.05; ††, p < 0.01 Tg versus WT for the same fatty acid (A–E for an increase); *, p < 0.05 Tg versus WT (C and D for a decrease).
FIGURE 5.
FIGURE 5.
PFKFB3/iPFK2 overexpression decreases adipocyte inflammatory response and improves insulin signaling while increasing fat deposition. Stable iPFK2-OX adipocytes and GFP-expressing adipocytes were established and subjected to metabolic and inflammatory assays. Each of the assays was performed at least in quadruplicate. A, amounts of iPFK2 in cells lysates were examined using Western blot analyses. B, changes in the rates of glucose incorporation into lipid. C, adipocyte gene expression was quantified using real time RT-PCR. D, production of ROS was measured using the nitro blue tetrazolium (NBT) assay. E, adipocyte inflammatory signaling. The levels of NF-κB p65 and phospho-p65 (Ser-468) were examined using Western blot analyses. F, adipocyte expression of IL-6. G, adipocyte insulin signaling. Prior to harvest, adipocytes were incubated with or without insulin (100 nm) for 30 min. Phospho-Akt (Ser-473) to Akt1/2 ratios were calculated using densitometry and expressed as fold changes. D–G, adipocytes were incubated with or without palmitate (250 μm) for 24 h (D, E, and G) or TNFα (10 ng/ml) for 6 h (F). H, lipid profile of adipocyte-conditioned medium. iPFK2-OX adipocytes and GFP-expressing 3T3-L1 were differentiated for 10 days before collection of conditioned medium. B–D, F, and H, †, p < 0.05; ††, p < 0.01 iPFK2-OX versus GFP (B) for the same gene (C) under the same condition (D and F) and for the same fatty acid (H). *, p < 0.05; **, p < 0.01 TNFα versus PBS for the same cell line in F.
FIGURE 6.
FIGURE 6.
Adipocyte factors generated in response PFKFB3/iPFK2 overexpression decrease hepatocyte inflammatory response and improve insulin signaling while increasing hepatocyte fat deposition. Wild-type mouse primary hepatocytes were treated with adipocyte-CM in M199 medium at a 1:1 ratio for 48 h in the presence or absence of supplemental palmitate (250 μm) for the last 24 h. Each of the assays was performed at least in quadruplicate. A, adipocyte-CM-treated mouse primary hepatocytes were stained with Oil Red O for 1 h (left four panels) and/or subjected to quantification of triglyceride content (right panel). AU, arbitrary unit. B, hepatocyte ROS production. C, hepatocyte levels of NF-κB p65 and phospho-p65 (Ser-468). D, hepatocyte gene expression. E, hepatocyte insulin signaling. A, B, and D, numeric data are means ± S.E. †, p < 0.05; ††, p < 0.01 iPFK2-OX-CM versus GFP-CM under the same condition (palmitate or BSA in A and B) and for the same gene (D). E, hepatocytes were incubated with or without insulin (100 nm) for 30 min prior to harvest. Phospho-Akt (Ser-473) to Akt1/2 ratios were calculated using densitometry and expressed as fold changes. Pal, palmitate.
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