Research projects

Extracellular vesicles (EVs) have emerged as key mediators in non-alcoholic fatty liver disease (NAFLD) through their intra and extra-hepatic effects. As part of this project and due to the close relationship between NAFLD and type 2 diabetes, we analyzed the effects of lipotoxic hepatocyte-derived EVs in the pancreas focusing on inflammation and activation of pancreatic stellate cells (PSCs).

Methods: C57BL/6J male mice were fed a chow (CHD) or high-fat diet (HFD) for 12-14 weeks. EVs were isolated from: 1) primary hepatocytes from CHD-fed mice without (EVC) or with treatment with palmitic acid (PA) (EVPA) or PA plus TGFβ (EVP+T), 2) hepatocytes from (HFD)-fed mice (EVHFD). EVs (EVC and EVP, 50 μg) were injected intravenously (i.v.) into lean C57BL/6J male mice (2 injections/week during 3 weeks). At the end of the treatment, pancreatic immune cells were isolated and analyzed by flow cytometry. EVP+T were used to treat rat primary PSCs and activation features were analyzed. 

Results: The percentage of CD45+ cells was significantly increased in the pancreas of mice injected EVP compared to mice receiving EVC. Although the percentage of pancreatic resident macrophages (F4/80+/CC11b+) was similar in the pancreas of the 2 groups, mice receiving EVP showed less M2-polarized macrophages (CD206+). Regarding lymphocytes, the percentage of CD3+ cells was significantly elevated in the pancreas of mice injected lipotoxic EVP, an effect corroborated by immunofluorescence analysis of pancreatic sections. On the other hand, primary rat PSCs were able to respond to TGFβ (as control for activation), EVP and EVP+T by increasing αSMA and Col1A1 levels. During this year, we also completed the study of the macrophage-hepatocyte interactome mediated by hepatocyte-derived lipotoxic EVs by treating peritoneal macrophages or Kupffer cells (KCs) from WT and TLR4-KO mice with EVC and EVPA. Our results revealed attenuated inflammatory responses in TLR4-KO macrophages/KCs receiving EVPA.

 Conclusion:  Hepatocyte-derived lipotoxic EVs target the pancreas and induce inflammation in the endocrine pancreas, as well as PSCs activation.