Final Report Summary - PRIMARY HEPATOCYTES (Primary mouse hepatocytes as a model system to study polarized endocytic trafficking)
During the second year of my Marie Curie Fellowship I focused on dissecting the phenotype of Rab5 knockdown in mouse liver in vivo and primary hepatocytes in vitro. Knockdown of Rab5, the master regulator of the endosomal machinery, led to a dramatic reduction in early endosomes, late endosomes and lysosomes in the liver. I found that this was accompanied by reduced kinetics of LDL endocytosis in primary hepatocytes in vitro: an assay, which I developed during the first funding year. This result was in agreement with an increase in LDL in the serum of the Rab5KD animals, further strengthening the observed block of endocytosis. Using my newly established interferin knockdown technique in primary cultures, I was able to show that the loss of the endosomal organelles was not due to the reduction of membrane flux, since blocking membrane input by knocking down dynamin-2 had no affect on the number of endosomes. In the liver, I demonstrated that loss of endosomes also caused failure to deliver apical proteins, such as the bile acid transporter BSEP, to the bile canaliculi, suggesting a requirement of early endosomes for polarised cargo sorting. Interestingly, failure to deliver BSEP to the bile canaliculi resulted in impairment in bile acid metabolism, which I confirmed by an increase in total bile acids in the serum of these animals. In addition, knockdown of Rab5 caused induction of hypoglycaemia, which I defined as a complete inhibition of major gluconeogenic genes, glucose-6-phosphatase and PEPCK. I am currently further characterising this phenotype and hope to reveal new connection between glucose metabolism and endocytosis. Therefore, I expect my work to give rise to significant contributions of our understanding how endocytosis contributes to the regulation of cell polarity and liver physiology