RESUMO
Type 1 diabetic patients develop severe secondary complications because insulin treatment does not guarantee normoglycemia. Thus, efficient regulation of glucose homeostasis is a major challenge in diabetes therapy. Skeletal muscle is the most important tissue for glucose disposal after a meal. However, the lack of insulin during diabetes impairs glucose uptake. To increase glucose removal from blood, skeletal muscle of transgenic mice was engineered both to produce basal levels of insulin and to express the liver enzyme glucokinase. After streptozotozin (STZ) administration of double-transgenic mice, a synergic action in skeletal muscle between the insulin produced and the increased glucose phosphorylation by glucokinase was established, preventing hyperglycemia and metabolic alterations. These findings suggested that insulin and glucokinase might be expressed in skeletal muscle, using adeno-associated viral 1 (AAV1) vectors as a new gene therapy approach for diabetes. AAV1-Ins+GK-treated diabetic mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Furthermore, these mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake. Therefore, the joint action of basal insulin production and glucokinase activity may generate a "glucose sensor" in skeletal muscle that allows proper regulation of glycemia in diabetic animals and thus prevents secondary complications.
Assuntos
Diabetes Mellitus Tipo 1/terapia , Glucoquinase/genética , Insulina/genética , Músculo Esquelético/metabolismo , Animais , Glicemia/análise , Northern Blotting , Western Blotting , Dependovirus/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Expressão Gênica , Vetores Genéticos/genética , Glucoquinase/metabolismo , Hiperglicemia/genética , Hiperglicemia/patologia , Hiperglicemia/terapia , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , RadioimunoensaioRESUMO
A permissive site for insertion of heterologous peptide sequences has been identified in the capsid proteins of AAV2. While attempting to use this site for insertion of a nuclear localization sequence, we have observed a drastic reduction in the yield of DNA-containing particles. ELISA analysis showed that capsid assembly was modestly affected, whereas genome encapsidation was more profoundly altered, a phenomenon we did not observed when a RGD peptide was inserted at the same location. Furthermore, the NLS viruses displayed poor transduction efficiency on HeLa and 293 cells. Altogether, these results indicate that the nature of the peptide inserted at position 587 in the capsid may have important consequences on both particle formation and infectivity.
