Ileum) samples. Western blots had been performed as previously described [30,31]. The levels of iPFK2, c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183), NF-B p65 and phospho-p65 (Ser468) have been analyzed.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript2.5. Measurement of microbiota composition Ahead of and in the course of therapy with rosiglitazone or PBS, fecal samples of HFD-fed mice were collected, pooled and homogenized. Total genomic DNA was isolated and subjected to realtime PCR working with primers specific to Bifidobacterium [32] and Lactobacillus [33]. 2.six. Evaluation of systemic insulin sensitivity Plasma levels of glucose and insulin were measured as previously described [26] and applied to calculate homeostasis model assessment of insulin resistance (HOMA-IR), an indicator of systemic insulin resistance, utilizing the following equation: HOMA-IR=basal glucose (mmol/ L) asal insulin (mU/L)/22.5.J Nutr Biochem. Author manuscript; offered in PMC 2013 May perhaps 01.Guo et al.Page2.7. Statistical solutions Numeric information are presented as indicates tandard error (S.E.). Statistical significance was assessed by unpaired, two-tailed evaluation of variance or Student’s t test. Variations had been deemed substantial at the two-tailed P.05.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript3. Results3.1. PFKFB3/iPFK2 is expressed at higher abundance in intestine The quantity of iPFK2 was determined in several tissues in wild-type mice. Amongst the important tissues which are involved inside the regulation of systemic insulin sensitivity and metabolic homeostasis, iPFK2 was at high abundance in intestine and white adipose tissue but at really low abundance in the liver, skeletal muscle and brown adipose tissue (Fig. 1). 3.two. HFD feeding stimulates intestine PFKFB3/iPFK2 expression and induces intestine inflammatory response HFD feeding induces inflammation in intestine [16]. To address dietary responses of intestine PFKFB3/iPFK2 expression in relation to inflammatory responses in intestine, the level of intestine iPFK2 in LFD- and/or HFD-fed wild-type C57BL/6J mice was examined. Compared with LFD-fed mice, HFD-fed mice displayed an increase in intestine iPFK2 amount (Fig. 2A). This enhance in intestine PFKFB3/iPFK2 expression appears to be a defensive response, given a crucial role for PFKFB3/iPFK2 in defending against dietinduced intestine inflammatory response (see below in Fig. 4). Subsequent, intestine inflammatory response was examined. Compared with controls, the phosphorylation of JNK1 in intestine of HFD-fed mice was improved (Fig. 2B), although the phosphorylation of NF-B was undetectable in mice on either an HFD or LFD (information not shown). Also, in HFD-fed mice, intestine mRNA levels of TNF and IL-6 had been substantially higher than their respective levels in controls (Fig.Formula of 5-Bromo-3-fluoro-2-nitropyridine 2C).4-Bromoisoxazol-3-amine structure These final results demonstrate a rise in intestine inflammatory response in HFD-fed mice.PMID:23671446 three.three. PFKFB3/iPFK2 disruption blunts dietary response of intestine iPFK2 The response of intestine PFKFB3/iPFK2 to HFD feeding was examined in PFKFB3+/- mice. Heterozygous PFKFB3 disruption was confirmed making use of PCR analyses of genomic DNA (Fig. 3A). On an LFD, intestine iPFK2 amount in PFKFB3+/- mice was reduce than in wild-type littermates (C57BL/6J background) (Fig. 3B), further demonstrating PFKFB3/ iPFK2 disruption. Upon feeding an HFD, PFKFB3+/- mice didn’t exhibit an increase in intestine iPFK2 amount as did wild-type mice. Therefore, intact PFKFB3/iPFK2 appears to become requi.