Neuregulin reduces glycaemia by targeting liver metabolism

Abstract

Neuregulin (NRG), which structurally belongs to an EGF family of growth factors, activated in acute manner signaling cascades that lead to changes in whole-body glucose metabolism in fasted mice. Besides previously observed effect in glucose tolerance test (GTT), NRG also lowered basal glycaemia and lead mice to severe hypoglycaemia within 135 minutes. The latter is most probably caused by inhibiting hepatic glucose production (HGP), because during pyruvate tolerance test (PTT) NRG not only completely blocked pyruvate conversion to glucose, but lowered glycaemia even more reaching levels that were comparable to what was seen in its effect on basal glycaemia. These results suggested that liver could be the main targeted tissue through what neuregulin regulates glycaemia. Blockage of NRG binding to liver neuregulin receptor, ErbB3, by specific antibodies supported that conclusion due to completely abolishing NRG hypoglycaemic effect under GTT. Our results from analyzing liver signaling showed that in vivo neuregulin treatment increased ErbB3 tyrosine phosphorylation. It lead to an increased ErbB3 interaction with PI3K regulatory subunit, which is also an important component of classical insulin signaling pathway. It also appeared that neuregulin counteracted with insulin signaling by reducing PI3K binding to IRS proteins and it occurred most likely due to increased serine phosphorylation. The emerged situation after NRG administration induced Akt phosphorylation and the activation of downstream signaling pathways, including FoxO1, GSK3-beta and p70S6K, which regulate gluconeogenesis, glycogen synthesis and IRSs serine phosphorylation, respectively. Although NRG inhibited transcription factor FoxO1, its effect in 30 minutes to gluconeogenic gene expression was rather mild, suggesting that pyruvate conversion to glucose was blocked in another step or by regulating enzyme activities. Besides mimicking insulin signaling, we also observed that NRG targeted glucokinase for degradation. Moreover, also the glucokinase activity in liver extract was reduced. This effect was especially clear in insulin-deficient mice and could be an explanation why in this model during GTT, instead of lowering glycaemia, neuregulin treatment prevented glucose to be taken up from the circulation. Administration of blocking ErbB3 antibodies fully prevented liver ErbB3 tyrosine phosphorylation upon neuregulin stimulation and abrogated neuregulin effect on downstream signaling cascade, including PI3K interaction with IRSs and ErbB3, Akt, FoxO1, GSK3-beta and p70S6K as well as degradation of glucokinase. Hepatic ErbB1 and ErbB3 expression in type 2 diabetic db/db mice was remarkably reduced. Contrary to that, endogenous hepatic neuregulin protein expression was up-regulated. In this situation, neuregulin caused weaker activation of signaling cascade in db/db mice compared to controls, but, unexpectedly, it had greater effect on lowering whole-body glycaemia in absolute values. Contrary to control animals, in db/db mice NRG failed to inhibit pyruvate conversion to glucose. Under pyruvate tolerance test neuregulin lowered glycaemia by same extent as under basal fasted condition, suggesting that inhibition of the other component of HGP, glycogenolysis, was not affected. Monitoring endogenous neuregulin concentration in plasma of insulin-deficient mice during a 24-hour study indicated that NRG increased sharply by the end of daily resting and fasting period and decreased sharply after re-feeding. It coincided with a moment when animals usually were experiencing lowest glycaemia and insulinemias. However in control and db/db mice the pattern was not that clear, suggesting that insulin and neurgulin interact on regulating glycaemia in order to sustain energetic metabolism in the tissues. As a whole, neuregulin is emerging as an important regulator of glycaemia by targeting liver metabolism. However, to fully understand the physiological relevance of neuregulin, further studies will have to be addressed.

La neuregulina regulava l'homeòstasi de la glucosa de manera aguda. En 30 minuts activava cascades de senyalització en el fetge i reduïa la glucèmia tant en estat basal com absortiu. El tractament in vivo amb neuregulina incrementava la fosforilació en tirosines d’ErbB3 al fetge, induint la interacció d’ErbB3 amb PI3K i activant vies de senyalització que inclouen Akt, FoxO1, GSK3-beta i p70S6K. Alhora, la neuregulina reduïa la interacció de PI3K amb IRS1 i IRS2, probablement per una augmentada fosforilació en serines d’aquests. A banda, el tractament agut amb neuregulina causava una reducció de l'activitat glucoquinasa hepàtica, en paral•lel a una pèrdua de proteïna, en ratolins control i insulino-deficients. La neuregulina inhibia la producció hepàtica de glucosa, blocant la conversió de piruvat a glucosa i provocant una reducció dels nivells de glucèmia que portava als ratolins dejunats a manifestar una hipoglucèmia severa però sense provocar la mort, com hagués fet la insulina. El blocatge d’ErbB3 va abolir l’efecte hipoglucemiant de la neuregulina així com l’acció sobre l’activació d’ErbB3 i la seva cascada de senyalització. L’expressió hepàtica d’ErbB1 i ErbB3 en ratolins db/db es va reduir notablement mentre l’expressió de neuregulina endògena va incrementar. En aquesta situació, la neuregulina va induir dèbilment la seva cascada de senyalització, però, sorprenentment, va tenir un major efecte en la reducció de la glucèmia. En tests de tolerància al piruvat, la neuregulina no va poder inhibir la conversió de piruvat a glucosa, provocant una baixada en la glucèmia equiparable a la observada en condicions basals. El seguiment durant 24 hores de la concentració de neuregulina endògena en el plasma de ratolins amb deficiència d'insulina va indicar que la neuregulina augmentava al final del període diari de descans i dejuni, coincidint amb els nivells més baixos de glucèmia i insulinemia, i disminuïa després de la realimentació. Els patrons observats en els ratolins control i db / db no eren tan clars, el que suggeria que en una situació de deficiència d'insulina la neuregulina tindria un paper més important en la regulació del metabolisme.