Role of the energy sensor AMP-activated protein kinase (AMPK) and its downstream effector uncoupling protein 2 (UCP2) in the orexigenic effect of endogenous ghrelin

Background. Ghrelin released from the stomach, stimulates food intake through stimulation of neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the hypothalamus. Several studies proposed a pivotal role for the energy sensor AMPK and UCP2 in ghrelin's effects on NPY/AgRP expression and food intake stimulation, although most of these studies focused on the effects of exogenous ghrelin. Aim. To investigate whether a rise in endogenous ghrelin levels is able to influence NPY/AgRP expression via activation of AMPK activity and UCP2 expression. Methods. Endogenous ghrelin levels were increased in wildtype (GHS-R+/+) and ghrelin receptor knockout (GHS-R-/-) mice by fasting (24h) or by induction of streptozotocin (STZ)-diabetes (15 days). Plasma octanoylated ghrelin levels were determined by radioimmunoassay. The mRNA expression of AgRP, NPY and UCP2 in the hypothalamus was measured by real-time PCR. Hypothalamic AMPK activity in tissue lysates was measured with an immunoprecipitation kinase assay. Results.Octanoylated ghrelin levels peaked (4.5fold, P<0.01) after 24h of fasting and declined thereafter due to a decrease (1.7-fold, P<0.01) in the mRNA expression of ghrelin-O-acyl transferase (GOAT). GHS-R+/+ mice showed a significant increase in AgRP mRNA (from 0.33±0.03 to 1.03±0.09; P<0.001) and NPY mRNA (from 0.54 ± 0.08 to 0.88 ± 0.05; P<0.01) expression after 24h-fasting. In GHS-R-/mice no significant increase in AgRP and NPY mRNA expression was observed. Fasting did not affect AMPK activity in both genotypes but increased UCP2 mRNA expression (GHS-R+/+: from 0.35±0.05 to 0.50±0.04; P<0.05 and GHS-R-/-: from 0.41±0.04 to 0.55±0.08; P=0.058). The hyperghrelinemia associated with the induction of STZ-diabetes was accompanied by a significant increase in the expression of NPY and AgRP in GHS-R+/+ mice compared to nondiabetic controls (AgRP: from 0.22±0.12 to 2.74±0.45; P<0.001 and NPY: from 0.73±0.06 to 2.23±0.64; P<0.05) but not in GHS-R-/mice. AMPK activity in the hypothalamus of GHSR+/+ mice after induction of diabetes (20.2±1.6 pmol/min/mg) was decreased (P<0.05) compared to non-diabetic littermates (16.3±1.3 pmol/min/mg) and there was no genotypic difference. Similarly, UCP2 mRNA levels were decreased after the induction of STZ-diabetes compared to control mice in both genotypes (GHS-R+/+: from 0.68±0.02 to 0.26±0.03; P<0.001 and GHS-R-/-: from 0.61±0.05 to 0.28±0.05; P<0.01). Conclusion. Increasing endogenous ghrelin levels by fasting and by induction of diabetes stimulates the expression of AgRP and NPY in the hypothalamus via interaction with the GHS-R. The change in AMPK activity and its downstream effector, UCP2, accompanying these changes in endogenous ghrelin levels occur independently from the GHS-R suggesting that AMPK and UCP2 do not play a major role in the orexigenic effect of endogenous ghrelin.