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. 2010 Feb 5;285(6):3713-3721.
doi: 10.1074/jbc.M109.058446. Epub 2009 Nov 30.

Disruption of inducible 6-phosphofructo-2-kinase ameliorates diet-induced adiposity but exacerbates systemic insulin resistance and adipose tissue inflammatory response

Yuqing Huo  1 , Xin Guo  2 , Honggui Li  2 , Huan Wang  3 , Weiyu Zhang  3 , Ying Wang  4 , Huaijun Zhou  4 , Zhanguo Gao  5 , Sucheta Telang  6 , Jason Chesney  6 , Y Eugene Chen  7 , Jianping Ye  5 , Robert S Chapkin  2 , Chaodong Wu  8
Affiliations

Disruption of inducible 6-phosphofructo-2-kinase ameliorates diet-induced adiposity but exacerbates systemic insulin resistance and adipose tissue inflammatory response

Yuqing Huo et al. J Biol Chem. .

Abstract

Adiposity is commonly associated with adipose tissue dysfunction and many overnutrition-related metabolic diseases including type 2 diabetes. Much attention has been paid to reducing adiposity as a way to improve adipose tissue function and systemic insulin sensitivity. PFKFB3/iPFK2 is a master regulator of adipocyte nutrient metabolism. Using PFKFB3(+/-) mice, the present study investigated the role of PFKFB3/iPFK2 in regulating diet-induced adiposity and systemic insulin resistance. On a high-fat diet (HFD), PFKFB3(+/-) mice gained much less body weight than did wild-type littermates. This was attributed to a smaller increase in adiposity in PFKFB3(+/-) mice than in wild-type controls. However, HFD-induced systemic insulin resistance was more severe in PFKFB3(+/-) mice than in wild-type littermates. Compared with wild-type littermates, PFKFB3(+/-) mice exhibited increased severity of HFD-induced adipose tissue dysfunction, as evidenced by increased adipose tissue lipolysis, inappropriate adipokine expression, and decreased insulin signaling, as well as increased levels of proinflammatory cytokines in both isolated adipose tissue macrophages and adipocytes. In an in vitro system, knockdown of PFKFB3/iPFK2 in 3T3-L1 adipocytes caused a decrease in the rate of glucose incorporation into lipid but an increase in the production of reactive oxygen species. Furthermore, knockdown of PFKFB3/iPFK2 in 3T3-L1 adipocytes inappropriately altered the expression of adipokines, decreased insulin signaling, increased the phosphorylation states of JNK and NFkappaB p65, and enhanced the production of proinflammatory cytokines. Together, these data suggest that PFKFB3/iPFK2, although contributing to adiposity, protects against diet-induced insulin resistance and adipose tissue inflammatory response.

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Figures

FIGURE 1.
FIGURE 1.
Disruption of PFKFB3/iPFK2 decreases iPFK2 expression and activity in adipose tissue. A, wild-type C57BL/6J mice were used for analyses. PFKFB3 is abundantly expressed in epididymal (Epi) and inguinal (Ing) white adipose tissue. BAT, brown adipose tissue. Data are means ± S.E., n = 4. ††, p < 0.01 versus liver. B, PCR analyses of mouse genomic DNA using an exon 2-specific primer with a neomycin-specific primer (+/−, heterozygous) or an exon 3-specific primer (+/+, wild-type). For C and E, data are means ± S.E., n = 4 - 6. **, p < 0.01 PFKFB3+/− versus wild-type. C, levels of PFKFB3 mRNA in epididymal adipose tissue were quantified using real-time RT-PCR. D, amount of iPFK2 in both epididymal adipose tissue and brown adipose tissue was measured using Western blot. E, levels of F26P2 in epididymal adipose tissue were determined using the 6PFK1 activation method. F, amount of iPFK2 in the liver was determined using Western blot.
FIGURE 2.
FIGURE 2.
Disruption of PFKFB3/iPFK2 blunts HFD-induced adiposity. At the age of 5–6 weeks, PFKFB3+/− mice and wild-type littermates were fed an HFD or LFD for 12 weeks. For A, C, and D, data are means ± S.E., n = 6. *, p < 0.05 and **, p < 0.01 HFD-PFKFB3+/− versus HFD-wild-type; †, p < 0.05 LFD-PFKFB3+/− versus LFD-wild-type. A, changes in body weight. B–D, changes in visceral fat content. The sum of epididymal, mesenteric, and perinephric fat mass was estimated as visceral fat content. E, adipose tissue histology. The sections of epididymal fat pad were stained with hematoxylin and eosin.
FIGURE 3.
FIGURE 3.
Disruption of PFKFB3/iPFK2 exacerbates HFD-induced adipose tissue dysfunction and systemic insulin resistance. At the age of 5–6 weeks, PFKFB3+/− mice and wild-type littermates were fed an HFD for 12 weeks. For A, B, D, and E, data are means ± S.E., n = 6. *, p < 0.05 and **, p < 0.01 HFD-PFKFB3+/− versus HFD-wild-type. A, rates of adipose tissue lipolysis were measured under both basal and isoproterenol-stimulated conditions. B, changes in the mRNA levels of adipose tissue resistin and adiponectin. C, adipose tissue insulin signaling. After anesthesia by an intraperitoneal (i.p.) injection of pentobarbital (50 mg/kg body weight), mice were injected with insulin (1 units/kg) or PBS into the inferior vena cava (i.v.) and epididymal adipose tissue samples were collected 5-min later. For D and E, mice were fasted for 4 h and received an intraperitoneal injection of insulin (1 units/kg) (D) or d-glucose (2 g/kg) (E).
FIGURE 4.
FIGURE 4.
Disruption of PFKFB3/iPFK2 exacerbates HFD-induced adipose tissue inflammatory response and increases adipose expression of genes involved in fatty acid oxidation. At the age of 5–6 weeks, PFKFB3+/− mice and wild-type littermates were fed an HFD for 12 weeks. A, macrophage infiltration in adipose tissue. The sections of epididymal fat pad were immunostained for F4/80. For B–G, data are means ± S.E., n = 4 - 6. *, p < 0.05 and **, p < 0.01 HFD-PFKFB3+/− versus HFD-wild-type (in B and D–G) or PFKFB3+/− versus wild-type on the same diet (in C); †, p < 0.05 and ††, p < 0.01 HFD versus LFD for the same genotype (in C). B, fraction of adipose tissue macrophages. C–G, mRNA levels of inflammatory markers and genes involved in fatty acid oxidation were quantified using real-time RT-PCR. C, mRNA levels of F4/80 in adipose tissue. D–F, mRNA levels of TNFα and IL-6 in adipose tissue (D), as well as in macrophages (E) and adipocytes (F) isolated from adipose tissue. G, mRNA levels of genes involved in fatty acid oxidation in adipose tissue.
FIGURE 5.
FIGURE 5.
Knockdown of PFKFB3/iPFK2 suppresses adipocyte lipid accumulation, impairs adipocyte function, and increases the adipocyte inflammatory response. Predifferentiated 3T3-L1 cells were transfected with the plasmid containing shRNA against PFKFB3/iPFK2 (iPFK2-KD) or shRNA vector (iPFK2-Ctrl) for 24 h and induced for differentiation for 6–8 days. Untransfected cells were differentiated similarly and used as the untreated control. Some transfected 3T3-L1 cells were selected by puromycin (5 μg/ml) to establish stable iPFK2-KD or iPFK2-Ctrl cell lines. A, Western blot for iPFK2. For B–D, F, G, and I, data are means ± S.E. n = 4. *, p < 0.05 and **, p < 0.01 iPFK2-KD versus iPFK2-Ctrl in B–D, and I, and in F and G for the same treatment (palmitate or vehicle in F and palmitate or vehicle with or without etomoxir or Nac in G); †, p < 0.05 and ††, p < 0.01 palmitate versus vehicle in F and palmitate in the presence of etomoxir or Nac versus palmitate alone in G under the same condition (iPFK2-KD or iPFK2-Ctrl). B, changes in the rates of glucose incorporation into lipid. C, changes in adipocyte lipid accumulation (arbitrary unit). D, changes in the mRNA levels of adipokines were quantified using real-time RT-PCR. E, adipocyte insulin signaling was analyzed using Western blot. Before harvest, the cells were incubated with or without insulin (100 nm) for 30 min. F and G, the production of ROS was measured using the NBT assay. F, after differentiation, iPFK2-KD and iPFK2-Ctrl adipocytes were treated with or without palmitate (250 μm) for 24 h. G, after differentiation, the stable cell lines were treated with or without palmitate (250 μm) for 24 h. In the last hour of treatment, 100 μm etomoxir (an inhibitor of carnitine palmitoyltransferase-1, CPT1) or 10 mm N-acetyl-l-cysteine (an antioxidant) was added. H, adipocytes were incubated with or without LPS (100 ng/ml) for 1 h prior to harvest. The levels and phosphorylation states of JNK and NFκB p65 were determined using Western blots. I, mRNA levels of proinflammatory cytokines were quantified using real-time RT-PCR.
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