Superinduction of leptin mRNA in mouse hypothalamic neurons.

In marked contrast to several other species, including rats and humans, leptin gene expression is undetectable in mouse brain. This unexpected finding may reflect unique energy regulation pathways in the mouse. We investigated possible mechanisms by which leptin (ob) gene expression is suppressed in mouse brain: (a) the possibility that ob mRNA levels might be detectable in vitro through the superinduction of gene expression following protein synthesis inhibition and (b) whether chromatin modification of the ob gene was responsible for this repression. Experiments were conducted on mouse hypothalamic neurons in vitro. Cells were treated with (a) protein synthesis inhibitors: cycloheximide (CHX; 25 µg/ml); puromycin (50 µg/ml); anisomycin (5 µM); (b) trichostatin A (histone deacetylase inhibitor; 500 nM); and (c) 5-aza-2'-deoxycytidine (DNA methylation inhibitor; 5 µM). Following the incubations, cells were harvested for the preparation of RNA and ob mRNA was detected using real-time reverse transcription PCR. Protein synthesis inhibitors induced a rapid increase in ob mRNA levels in mouse hypothalamic neurons in vitro. For example CHX stimulation of ob mRNA was detectable at 60 min after treatment and reached a maximum between 4 and 6 h. A dose-response analysis, with concentrations of CHX of 1, 2, 10, 25, and 50 µg/ml, indicated that CHX was already effective at 1.0 µg/ml, with a maximal effect by 25 µg/ml. In contrast, incubation with trichostatin A and 5-aza-2'-deoxycytidine had no effect and ob mRNA remained undetectable. These data show that leptin gene expression is superinduced in ob-negative mouse hypothalamic neurons following inhibition of protein synthesis. They confirm that the previously reported absence of leptin mRNA in mouse brain is probably because of an active repressive mechanism, although this may not involve chromatin modification.

Resistin differentially modulates neuropeptide gene expression and AMP-activated protein kinase activity in N-1 hypothalamic neurons.

Intraventricular resistin is known to reduce food intake, modify hypothalamic gene expression (e.g. NPY, POMC) and influence the activity of novel metabolic enzymes (e.g. 5'AMP-activated protein kinase; AMPK) in the rodent brain. Previously we demonstrated that the hypothalamus, and the N-1 hypothalamic neuronal cell line, also expressed several adipokines, including resistin and adiponectin (ADPN). These data suggested that they might also impact brain function and metabolism. We used the N-1 hypothalamic neuronal cell line to examine NPY, AgRP, POMC, and ADPN expression following acute resistin treatment (45 min; 100 ng/mL and 1000 ng/mL). The total and phosphorylated levels of AMPKalpha and acetyl-CoA carboxylase (ACC) were subsequently assessed using Western blot analysis. Parallel investigations were also conducted following a) resistin overexpression, or b) after the RNAi-mediated attenuation of resistin mRNA in N-1 neurons. Resistin overexpression lowered POMC (-35%, p<0.01), ADPN (-23%, p<0.05) and NPY (-36%, p<0.05) mRNA as evaluated using realtime RT-PCR, although AgRP remained unchanged, and significant increases in pAMPKalpha and pACC were detected (+47% and +34% respectively, p<0.001). In contrast recombinant resistin only significantly increased the level of pAMPKalpha (+31%; p<0.05), but failed to significantly modify gene expression, in N-1 neurons. Conversely the RNAi-mediated silencing of resistin expression increased AgRP (+37%, p<0.05), POMC (+66%, p<0.0001), ADPN (+87%, p<0.0001), whereas NPY was reduced (-22%, p<0.01) along with pAMPKalpha and pACC (-43% and -35% respectively, p<0.001). In summary, these in vitro data suggest that endogenous resistin might be capable of fine-tuning the expression and enzymatic activity of various hypothalamic targets previously implicated in the delicate homeostatic control of food intake. As such, resistin may be part of an autocrine/paracrine loop, which may in turn contribute to some of the reported effects of resistin on energy metabolism.