Ca2+/Calmodulin-Dependent Protein Kinase Kinase Is Not Involved in Hypothalamic AMP-Activated Protein Kinase Activation by Neuroglucopenia

Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK) activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca2+/Calmodulin-dependent protein kinase kinase (CaMKK), which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO), a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG)-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV) in rats 30 min before 2DG ICV injection (40 µmol) to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol) did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK

CaMKKα and CaMKKβ protein expression in hypothalamic nuclei and non-hypothalamic brain areas.

<p>Tissue lysates (40 µg protein) were subjected to Western blotting with anti-CaMKKα or anti-CaMKKβ antibodies (Santa Cruz) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036335#s4" target="_blank">Materials and Methods</a>”. Hip; hippocampus, Arc; arcuate nucleus, VMH/DMH; ventromedial hypothalamus/dorsomedial hypothalamus, PVN; paraventricular nucleus, LH; lateral hypothalamus, HB; hindbrain.</p

STO-609 does not block 2DG-induced AMPK activation in ARC and VMH/DMH.

<p>Saline or STO-609 (50 nmol) was injected ICV in fed rats 30 min prior to saline or 2DG (40µmol) ICV injection. Hypothalamic nuclei were dissected 10 min after saline or 2DG injection. α1 (A) and α2 (B) AMPK activities were measured in microdissected Arcuate and VMH/DMH nuclei. n = 9 rats per group. Data are means ± SEM. *<i>p</i><0.05 vs. saline-saline group.</p

STO-609 does not affect counter-regulatory responses to neuroglucopenia <i>in vivo</i>.

<p>Saline or STO-609 (5, 20 or 50 nmol) was injected ICV in fed rats 30 min before ICV injection of saline or 2DG (40 µmol) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036335#s4" target="_blank">Materials and Methods</a>”. Glycemia (A) and 1-h food intake (B) were measured. n = 5−9 rats per group. Data are means ± SEM. *<i>p</i><0.05 vs. saline-saline group.</p