Fructose-2,6-bisphosphate is lower in copper deficient rat cerebellum despite higher content of phosphorylated AMP-activated protein kinase

Abstract

Limitation in copper (Cu) leads to pathophysiology in developing brain. Cu deficiency impairs brain mitochondria and results in high brain lactate suggesting augmented anaerobic glycolysis. AMP activated protein kinase (AMPK) is a cellular energy "master-switch" that is thought to augment glycolysis through phosphorylation and activation phosphofructokinase 2 (PFK2) resulting in increases of the glycolytic stimulator fructose-2,6-bisphosphate (F2,6BP). Previously, Cu deficiency has been shown to augment cerebellar AMPK activation. Cerebella of Cu-adequate (Cu+) and Cu-deficient (Cu-) rat pups were assessed to evaluate if AMPK activation in Cu- cerebella functioned to enhance PFK2 activation and increase F2,BP concentration. Higher levels of pAMPK were detected in Cu- cerebella. However, PFK2 activity, mRNA, and protein abundance were not affected by Cu deficiency. Surprisingly, F2,6BP levels were markedly lower in Cu- cerebella. Lower F2,6BP may be due to inhibition of PFK2 by citrate, as citrate concentration was significantly higher in Cu- cerebella. Data suggest AMPK activation in Cu- cerebellum does not augment glycolysis through a PFK2 mechanism. Furthermore, other metabolite data suggest that glycolysis may actually be blunted, since levels of glucose and glucose-6-phosphate were higher in Cu- cerebella than controls.

Figure 1

Characterization of induced Cu deficiency in P24 rat pups. Cu+ and Cu− P24 rat pups were assessed for copper status (n = 5). Body weight, a reflection of Cu deficiency, was reduced in Cu− rat pups compared to controls. Tissue Cu content in both liver and brain were also significantly lower in Cu− animals, confirming Cu deficiency. Values are means ± SEM; * P < 0.01.

Figure 2

Evaluation of cerebellar Cu status in P24 rat pups. Cu deficiency in cerebellum was evaluated by Western blot using the Cu deficiency marker CCS, which was significantly increased in Cu− (−) cerebellar homogenates compared to Cu+ controls (+). Mitochondrial impairment in Cu− cerebella was confirmed via Western blot of cerebellar homogenates showing lower CCO subunit IV (COX IV) protein content. Protein loading, 24 μg per lane, in a 15% gel was evaluated using Ponceau S stain. Values are means ± SEM; * P < 0.01.

Figure 3

AMPK is activated in Cu− P24 rat pup cerebellum. Western blots of cerebellar homogenates demonstrate that Cu− cerebella (−) of P24 rat pups have higher levels of phosphorylated AMPK (pAMPK) than Cu+ pups (+) (n = 4). Total AMPK remained unchanged by Cu deficiency. Each lane was loaded with 40 μg protein. Values are means ± SEM; * P < 0.05.

Figure 4

Cerebellar PFK2 evaluation. A) Analysis of cerebellar homogenates revealed that Cu− cerebellar total PFK2 activity (n = 4) was not altered by Cu deficiency. B) Cerebellar fructose-2,6-bisphosphate levels (F2,6BP) were evaluated in Cu+ and Cu− rats. In two independent experiments, Experiment I (n = 4) and II (n = 5), F2,6BP concentration was found to be markedly lower. C) Astocytes are thought to have a high F2,6BP content. However, astrocytic content was not different between Cu+ and Cu− cerebella, as assessed by astrocytic marker GFAP abundance (n = 4). Values are means ± SEM; * P < 0.01.

Figure 5

Heart-type and brain/placental PFK2 mRNA expression, brain/placental PFK2 protein concentration, and citrate concentration in Cu+ and Cu− cerebellum. Analysis of cerebellar total mRNA (Panel A) showed that a splice variant mRNA for an AMPK insensitive heart-type PFK2 outnumbered mRNA transcripts of the AMPK sensitive brain/placental splice variants (n = 5). Cu deficiency did not impact gene expression. Unlike superscripts are statistically different, P < 0.01, factorial ANOVA. Analysis of cerebellar homogenates revealed that Cu− cerebellar levels of brain/placental PFK2 protein (Panel B; n = 4) were not different than Cu+ controls. Analysis of cerebellar extracts revealed a significantly higher concentration of the PFK2 inhibitor citrate in Cu− cerebella (Panel C; n = 5). Values are means ± SEM; * P < 0.01.

Figure 6

Glucose metabolism in Cu− cerebellum. Cerebellar concentrations of glucose (n = 5) and glucose-6-phosphate (G6P) (n = 5) were found to be higher in Cu− rats without a corresponding increase in serum glucose (n = 5) (Panel A). Levels of cerebellar glucose transporters Glut1 and Glut3 were not altered in Cu− rats (−) compared to Cu+ controls (+) (Panel B; n = 4). Values are means ± SEM; * P < 0.05.