Atorvastatin Improves Survival in Septic Rats: Effect on Tissue Inflammatory Pathway and on Insulin Signaling

The aim of the present study was to investigate whether the survival-improving effect of atorvastatin in sepsis is accompanied by a reduction in tissue activation of inflammatory pathways and, in parallel, an improvement in tissue insulin signaling in rats. Diffuse sepsis was induced by cecal ligation and puncture surgery (CLP) in male Wistar rats. Serum glucose and inflammatory cytokines levels were assessed 24 h after CLP. The effect of atorvastatin on survival of septic animals was investigated in parallel with insulin signaling and its modulators in liver, muscle and adipose tissue. Atorvastatin improves survival in septic rats and this improvement is accompanied by a marked improvement in insulin sensitivity, characterized by an increase in glucose disappearance rate during the insulin tolerance test. Sepsis induced an increase in the expression/activation of TLR4 and its downstream signaling JNK and IKK/NF-κB activation, and blunted insulin-induced insulin signaling in liver, muscle and adipose tissue; atorvastatin reversed all these alterations in parallel with a decrease in circulating levels of TNF-α and IL-6. In summary, this study demonstrates that atorvastatin treatment increased survival, with a significant effect upon insulin sensitivity, improving insulin signaling in peripheral tissues of rats during peritoneal-induced sepsis. The effect of atorvastatin on the suppression of the TLR-dependent inflammatory pathway may play a central role in regulation of insulin signaling and survival in sepsis insult

Effect of rosuvastatin and dapagliflozin on gut microbiota and insulin sensitivity of C57BL6/J mice fed a high-fat diet

Orientador: Mario Jose Abdalla SaadTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A obesidade é um problema crescente e atinge um número cada vez maior de indivíduos em todo o mundo. Ela está correlacionada ao desenvolvimento de diversos distúrbios metabólicos, como resistência à insulina, diabetes tipo 2, dislipidemias e doenças cardiovasculares. Nos últimos anos, a microbiota intestinal tem sido amplamente correlacionada a esses distúrbios metabólicos. Dentre os fármacos muito utilizados na prática clínica, para tratar comorbidades relacionadas à obesidade, estão a dapagliflozina e a rosuvastatina. Sendo assim, o principal objetivo deste trabalho foi avaliar os efeitos da administração destes fármacos sobre a microbiota intestinal e os parâmetros metabólicos de camundongos submetidos à dieta hiperlipídica. Para tanto, camundongos C57BL6/J machos foram divididos em quatro grupos: controle (C); controle tratado com dapagliflozina (C+DP) ou rosuvastatina (C+RS); dieta (DH) e dieta tratado com dapagliflozina (DH+DP) ou rosuvastatina (DH+RS). Os fármacos foram administrados por via oral, através de gavagem, durante 30 dias. O tratamento com dapagliflozina induziu uma melhora na tolerância à glicose e resistência à insulina, nos animais em dieta hiperlipídica, além de uma melhora na sinalização da insulina em fígado, músculo e tecido adiposo. Da mesma forma, a dapagliflozina alterou a composição da microbiota intestinal, promovendo uma redução na proporção de Bacteroidetes e um aumento de Proteobacteria no grupo controle e, de maneira menos pronunciada, no grupo em dieta hiperlipídica. Observou-se, também, um aumento relativo na prevalência de Akkermansia, Faecalibacterium, Bilophila e Ruminococcus nos animais em dieta hiperlipídica que receberam tratamento. E uma redução do gênero Allobaculum neste mesmo grupo. O transplante da microbiota intestinal dos animais em dieta tratados com dapagliflozina não produziu o mesmo efeito do fármaco em animais obesos. O tratamento com rosuvastatina reduziu a glicemia de jejum, a tolerância à glicose e a resistência à insulina, além de aumentar a fosforilação da Akt em fígado e músculo dos animais em dieta hiperlipídica. Além disso, produziu alterações na microbiota intestinal, reduzindo a proporção de Bacteroidetes e promovendo um aumento de Firmicutes, nos animais controle. Por outro lado, nos animais em dieta, aumentou a prevalência de Bacteroidetes, enquanto a proporção de Firmicutes se manteve inalterada. Verificou-se também um aumento do gênero Bilophila e uma redução de Allobaculum nos animais em dieta hiperlipídica que receberam tratamento com esse fármaco. O transplante da microbiota intestinal dos animais em dieta tratados com rosuvastatina não reproduziu o efeito deste fármaco em animais obesos. Assim, concluímos que ambas, dapagliflozina e rosuvastatina, são capazes de modificar a microbiota intestinal dos animais com obesidade induzida por dieta. Ambas também produzem efeitos positivos sobre o metabolismo e a homeostase da glicose nesses animais. Entretanto, as alterações da microbiota não parecem ter correlação com seus efeitos metabólicosAbstract: Obesity is a growing problem and affects an increasing number of individuals all over the world. It correlates with the development of several metabolic disorders, including insulin resistance, type 2 diabetes, dyslipidemia and cardiovascular diseases. In recent years, gut microbiota has been largely related to these metabolic disorders. Among the drugs most used in clinical practice to treat obesity-related comorbidities are dapagliflozin and rosuvastatin. Thus, the main objective of this study was to evaluate the effects of dapagliflozin and rosuvastatin administration on gut microbiota and metabolic parameters of mice fed a high-fat diet. Therefore, C57BL6/J male mice were divided into four groups: control group; control group plus dapagliflozin or rosuvastatin; high-fat diet group and high-fat diet plus dapagliflozin or rosuvastatin. Drugs were administered orally by gavage for 30 days. Dapagliflozin treatment induced an improvement in glucose tolerance and insulin resistance on high-fat diet mice. In addition, there was an improvement in insulin signaling in liver, muscle and adipose tissue. Likewise, dapagliflozin altered the composition of the gut microbiota, promoting a reduction in the proportion of Bacteroidetes and an increase of Proteobacteria in the control group and, in a less pronounced way, in the high-fat diet group. There was also a relative increase in the prevalence of Akkermansia, Faecalibacterium, Bilophila, and Ruminococcus in high-fat diet mice treated with this drug. And, also, a reduction of Allobaculum gender in the same group. The gut microbiota transplantation from high-fat fed mice treated with dapagliflozin did not reproduce the same effect of drug administration in obese animals. Rosuvastatin treatment reduced fasting glucose, glucose tolerance and insulin resistance, and improved Akt phosphorylation in liver and muscle of high-fat fed mice. In addition, it produced alterations in gut microbiota, reducing the proportion of Bacteroidetes and promoting an increase of Firmicutes, in the control animals. On the other hand, in animals on high-fat diet, the prevalence of Bacteroidetes increased, while the proportion of Firmicutes remained unchanged. There was also an increase of the genus Bilophila and and a reduction of Allobaculum in high-fat fed mice that received treatment with this drug. The gut microbiota transplantation from high-fat fed mice treated with rosuvastatin did not reproduce the effect of drug administration in obese animals. Thus, we conclude that both, dapagliflozin and rosuvastatin, are able to modify the gut microbiota of diet induced obese mice. Both, also, have positive effects on glucose metabolism and homeostasis of these animals. However, the changes on gut microbiota do not seem to correlate with their metabolic effectsDoutoradoFisiopatologia MédicaDoutora em CiênciasCAPE

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