Representative blots show the JNK phosphorylation in liver (A), muscle (B) and adipose tissue (C) of sham and septic rats (upper panels).

<p>Total protein expression of JNK (A–C, lower panels). Phosphorylation of c-jun in liver (D), muscle (E) and adipose tissue (F) of sham and septic rats. Serine 307 Phosphorylation of IRS1 in liver (G), muscle (H) and adipose tissue (I) of sham and septic rats (upper panels). Total protein expression of IRS-1 (G–I, lower panels). Data are presented as means ± S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.</p

To evaluate the association of TLR4 with MyD88, immunoprecipitations were performed with MyD88 antibody followed by immunoblotting with TLR4 specific antibody.

<p>Representative blots show TLR4 activation (upper panels) and expression (lower panels) in liver (A), muscle (B) and adipose tissue (C) of sham and septic rats. IKKβ phosphorylation in liver (D), muscle (E) and adipose (F) of sham and septic rats. Total protein expression of IKKβ (D–F, lower panels). Phosphorylation of IκBα in liver (G), muscle (H) and adipose (I) of sham and septic rats. Data are presented as means ±S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all other groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.</p

Effects of atorvastatin treatment on insulin signaling in the CLP rat.

<p>Representative blots show insulin-induced tyrosine phosphorylation of Insulin Receptor β (IRβ) in liver (A), muscle (B) and adipose (C) of sham and septic rats. Total protein expression of IRβ (A–C, lower panels). Insulin-induced tyrosine phosphorylation of Insulin Receptor Substrate 1 (IRS1) in liver (D), muscle (E) and adipose tissue (F) of sham and septic rats. Total protein expression of IRS1 (D–F, lower panels). Insulin-induced serine phosphorylation of Akt in liver (G), muscle (H) and adipose (I) of sham and septic rats. Insulin-induced threonine phosphorylation and total protein expression of Akt (G–I, lower panels). In this case, blots were stripped and reprobed with β-actin (A–I, lower panels) to confirm equal loading of proteins. Data are presented as means +/− S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.</p

Representative blots show the NFkB activation in nuclear fractions of liver (A), muscle (B) and adipose tissue (C) of sham and septic rats.

<p>In this case blots were stripped and reprobed with actin (A–C, lower panels) to confirm equal loading of proteins. Tissue levels of iNOS (D–F) and IL-6 (G–I) expression in liver, muscle and adipose tissue of sham and septic rats. Data are presented as means ± S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.</p

Effect of atorvastatin on survival in CLP sepsis model.

<p>Male Wistars rats, 8 weeks old, were given saline (Sepsis/Sal, n = 20) or atorvastatin 10 mg/kg (Sepsis/Ator, n = 20), 3 h and once a day after CLP. Survival of the rats was monitored at intervals of 12 h for 15 days. The overall difference in survival rate between the groups with and without atorvastatin was significant (P<0.0001) (A). Fasting blood glucose (B). Fasting insulin levels (C). Glucose disappearance rate (D). HOMA-IR index (E). Serum levels of TNF-α (F) and IL-6 (G). Data are presented as means and S.E. of six to eight rats per group. *P<0.05 (Sepsis saline vs. all others groups).</p

Kinin B₁ Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

<div><h3>Background</h3><p>Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B₁ receptor knockout mice (B₁^−/−) are leaner and exhibit improved insulin sensitivity.</p> <h3>Methodology/Principal Findings</h3><p>Here we show that kinin B₁ receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B₁ receptors. In these cells, treatment with the B₁ receptor agonist des-Arg⁹-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B₁^−/− mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B₁ receptor was limited to cells of the adipose tissue (aP2-B₁/B₁^−/−). Similarly to B₁^−/− mice, aP2-B₁/B₁^−/− mice were leaner than wild type controls. However, exclusive expression of the kinin B₁ receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B₁^−/− mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B₁ receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B₁/B₁^−/− when compared to B₁^−/− mice. When subjected to high fat diet, aP2-B₁/B₁^−/− mice gained more weight than B₁^−/− littermates, becoming as obese as the wild types.</p> <h3>Conclusions/Significance</h3><p>Thus, kinin B₁ receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.</p> </div

Kinin B₁R modulates insulin action in mouse adipocytes.

<p>A: AKT and ERK phosphorylation upon stimulation of differentiating 3T3-L1 preadipocytes with 10 µg/mL insulin (in the presence of 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 10% FBS). *, <i>P</i><0.05; <i>vs.</i> non-stimulated; ^#, <i>P</i><0.05: <i>vs.</i> insulin only. The blots and quantification are one representative experiment out of three performed independently. B: 3T3-L1 adipocytes were incubated with or without 1 µM DBK for 90 minutes prior to 10 min stimulation with 1 µg/mL insulin. Cells were harvested for immunoprecipitation (IP) followed by immunoblotting (IB) or C: membrane fractionation for assessment of GLUT4 translocation by western blotting. *, <i>P</i><0.05; <i>vs.</i> non-stimulated; ^#, <i>P</i><0.05: <i>vs.</i> insulin only. The blots and quantification are one representative experiment out of two performed independently. D: Isolated epididymal adipocytes of WT mice were incubated with 5 mM glucose media with or without 1 µM DBK for 90 minutes and [³H]-2-deoxy-glucose accumulation was measured under basal conditions or after 15 min stimulation with 10 nM insulin. Data represent mean ± SEM of at least 8 independent samples obtained from an adipocyte pool of 15 animals. *, <i>P</i><0.05 <i>vs.</i> respective control. E: Effect of 90 minutes incubation with 1 µM DBK on the insulin-induced extracellular acidification rate of 3T3-L1 adipocytes measured by <i>Cytosensor</i> system (*, <i>P</i><0.05; <i>n</i> = 4 per group). Data represent mean ± SEM of extracellular acidification. F: Random fed WT or B₁^−/− mice (<i>n</i> = 4 per group) were injected in the vena cava with either saline or a 10 U insulin bolus. Epididymal fat was harvested after 10 minutes for assessment of AKT phosphorylation by western blotting or G: GLUT4 translocation. H: Glucose uptake was assessed in isolated epididymal adipocytes of WT and B₁^−/− cells under basal conditions or after 15 min stimulation with 10 nM insulin. Data represent mean ± SEM of at least 8 independent samples obtained from an adipocyte pool of 15 animals. *, <i>P</i><0.05; **, <i>P</i><0.001 <i>vs.</i> respective control. I: Fasted mice received 0.2 U human insulin per kg, 0.083 mCi [³H]-2-deoxy-glucose per kg was subcutaneously injected, mice were sacrificed and tissue collected. Data show mean ± SEM of 5 animals per genotype. *, <i>P</i><0.05.</p

Rescue of kinin B₁R expression in adipocytes of kinin B₁R knockout mice (B₁^−/−) partially rescues GLUT4 expression and function in adipose tissue.

<p>A: Expression of GLUT4 mRNA in epididymal fat of random fed mice measured by realtime PCR. B: GLUT4 translocation in epididymal fat at basal states or after insulin stimulation (10 U intravenous bolus for 10 min). The blots and quantification are one representative experiment out of two performed independently. C: Basal glucose uptake in isolated adipocytes. [³H]-2-deoxy-glucose accumulation was measured during 5 minutes. Data represent mean ± SEM of six animals per group. D: Basal AKT phosphorylation in the epididymal fat of random fed mice. The blots and quantification are one representative experiment (pool of 4–5 mice each) out of two performed independently. *, P<0.05; **, P<0.001.</p

aP2-B₁/−B₁^−/− mice are lean.

<p>A: Body weight. B: Body composition. *, <i>P</i><0.05 vs. B₁^−/− and aP2-B₁/B₁^−/−, fat mass comparison; #, <i>P</i><0.05 vs. WT, lean mass comparison. C: Organ weight. D: Histology of adipose tissue (H&E staining). Representative pictures of at least five animals per group. E: Adipocyte volume. F: Adipocyte number. G: Basal and maximum (insulin-stimulated) lipogenesis in isolated adipocytes. H: Basal and isoproterenol-induced lipolysis as determined by glycerol production by isolated adipocytes. Values are means ± SEM of six animals per group. *, <i>P</i><0.05; **, <i>P</i><0.001.</p

The kinin B₁R is constitutively expressed in WAT and downregulated with obesity.

<p>A: Kinin B₁R mRNA expression was quantified in different organs of male C57Bl/6 mice (<i>n</i> = 5) using TaqMan real-time PCR. Data are expressed as mean±SEM of the 2^−ΔCt parameter, which represents the relative expression of B₁R mRNA in relation to β-actin mRNA. WAT, white adipose tissue; ND, non-detectable; a.u., arbitrary units. B: Effects of acute treatment with the B₁R agonist des-Arg⁹-bradykinin (DBK), the B₂ receptor agonist bradykinin (BK) or DBK in the presence of the B₁R antagonist [Leu⁸]-DBK on the extracellular acidification rate of isolated adipocytes from wild type (WT) and B₁ knockout (B₁^−/−) mice (<i>n</i> = 5 animals per group) measured using the <i>Cytosensor</i> system. C: Representative RT-PCR showing expression of components of the kallikrein kinin system kallikrein in WAT, lung, and heart of C57Bl/6 male (<i>n</i> = 3). KK, tissue kallikrein; CPM, carboxypeptidase M; B₂, kinin B₂ receptor. C-, negative control. D: C57BL/6 mice were submitted to HFD for 9 weeks (<i>n</i> = 5 per group). Kinin B₁R expression was quantified in WAT by real time PCR. Results are mean ± SEM. *, <i>P</i><0.05.</p