RT info:eu-repo/semantics/doctoralThesis
T1 Hepatic insulin-degrading enzyme regulation and its role on glucagon signaling
A1 González Casimiro, Carlos Manuel
A2 Universidad de Valladolid. Escuela de Doctorado
K1 Diabetes
K1 Endocrinology
K1 Insulina hepática
K1 Hepatic insulin
K1 Homeostasis de la glucosa
K1 Glucose homeostasis
K1 Metabolismo
K1 Metabolism
K1 2411.04 Fisiología Endocrina
AB Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed Zn2+-metalloendopeptidase that degrades insulin and glucagon among other substrates. By decades, its main function has been attributed to hepatic insulin clearance, a process that regulates availability of circulating insulin levels, but recent studies performed by our group indicate a more important role of this protein regulating hepatic insulin sensitivity and glucose homeostasis. However, its regulation in response to nutritional state and the fasting-to-postprandial transition is poorly understood and much less attention has been dedicated to its role on regulating glucagon signal transduction and mitochondrial function in hepatocytes. In this thesis, we studied the regulation of IDE mRNA and protein levels as well as its proteolytic activity in the liver under fasting (18 h) and refeeding (30 min and 3 h) conditions, in mice fed a standard (SD) or high-fat (HFD) diets. Likewise, we aim to elucidate the role of IDE on glucagon signaling and its impact on energy metabolism in hepatocytes using a loss-of-function approach. Livers from L-IDE-KO and WT mice were used to obtain tissue extracts and primary hepatocytes for culture. The mouse hepatocyte cell line (AML12) was transduced with an shRNA targeting Ide mRNA by means of a lentiviral vector and obtaining a stable line (AML12-shRNA-IDE). L-IDE-KO primary hepatocytes and AML12-shRNA-IDE with their respective controls were stimulated with glucagon and the signaling pathway was analyzed by western blot and ELISA. Mitochondrial function and energy metabolism of AML12-shRNA-IDE and control cells were assessed by Seahorse XFe24 Analyzer with a Mito Stress Assay. In the liver of mice fed a HFD, fasting reduced IDE protein levels (~30%); whereas refeeding increased its activity (~45%) in both mice fed an SD and HFD. Circulating lactate concentrations directly correlated with hepatic IDE activity and protein levels. Of note, L-lactate in liver lysates augmented IDE activity in a dose-dependent manner. Additionally, IDE protein levels in liver, but not its activity, inversely correlated (R2 = 0.3734, p < 0.01) with a surrogate marker of insulin resistance (HOMA index). Liver extracts and primary hepatocytes from L-IDE-KO mice, compared to WT, showed decreased expression of glucagon receptor (~60%), CREB protein (~40%), and diminished phosphorylation of CREB (~50%) upon glucagon stimulation. Ide expression and IDE protein levels were reduced by ~50% in AML12-shRNA-IDE cells. At basal state, glucagon receptor, FoxO1 and CREB protein were significantly lower in AML12-shRNA-IDE cells than in control cells, (~40%, ~75% and ~75%, respectively). Glucagon stimulation resulted in less (~30%) cAMP levels and changes in the kinetic of glucagon-mediated phosphorylation of CREB and other PKA substrates in AML12-shRNA-IDE. Seahorse analyses showed that both oxygen consumption and extracellular acidification rates increased 2-fold in AML12-shRNA-IDE with a 2-fold increment of mitochondrial and glycolytic adenosine triphosphate (ATP) production. Finally, we generated, using homology modeling by satisfaction of spatial restrains technique, complete 3D structures of human and murine IDE isoforms with 15a and 15b exons. Our results highlight that the nutritional regulation of IDE in liver is more complex than previously expected in mice. Reduced IDE expression in mouse hepatocytes has a deleterious effect on glucagon signaling, affecting this intracellular pathway in parallel with a shift to a more energetic phenotype. These findings suggest that IDE is necessary for proper glucagon signal transduction and regulation of the energy production in hepatocytes.
YR 2021
FD 2021
LK https://uvadoc.uva.es/handle/10324/60284
UL https://uvadoc.uva.es/handle/10324/60284
LA eng
NO Escuela de Doctorado
DS UVaDOC
RD 18-dic-2024