RESUMO
Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.
Assuntos
Metabolismo Energético , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Gluconeogênese , Glicólise , Hepatócitos/metabolismo , Modelos Biológicos , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Frutose-Bifosfatase/química , Frutose-Bifosfatase/genética , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Células HeLa , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microscopia Confocal , Transporte Proteico , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate-limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT-PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT-PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly-ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility.
Assuntos
Células Germinativas/citologia , Células Germinativas/metabolismo , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Isoformas de Proteínas/metabolismo , Testículo/citologia , Testículo/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Glicogênio Sintase/genética , Immunoblotting , Masculino , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Testículo/enzimologiaRESUMO
In primary cultured hepatocytes, fructose-1,6-bisphosphatase (FBPase) localization is modulated by glucose, dihydroxyacetone (DHA) and insulin. In the absence of these substrates, FBPase was present in the cytoplasm, but the addition of glucose or DHA induced its translocation to the nucleus. As expected, we observed the opposite effect of glucose on glucokinase localization. The addition of insulin in the absence of glucose largely increased the amount of nuclear FBPase. Moreover, at high concentrations of glucose or DHA, FBPase shifted from the cytosol to the cell periphery and co-localized with GS. Interestingly, the synthesis of Glu-6-P and glycogen induced by DHA was not inhibited by insulin. These results indicate that FBPase is involved in glycogen synthesis from gluconeogenic precursors. Overall, these findings show that translocation may be a new integrative mechanism for gluconeogenesis and glyconeogenesis.