Fructose-2, 6-Bisphosphate Associated Regulatory Enzymes Develop in Concordance in Mice Brain During Early Postnatal Life

Fructose-2, 6-bisphosphate (fru-2, 6P2), synthesized by 6-phosphofructo-2-kinase (PFK2), regulates glucose metabolism via modulating phosphofructokinase-1 (PFK1) and fructose-1, 6-bisphosphatase (FBPase1) reciprocally in mammalian tissues. How this control system develops in brain is poorly understood. This article presents the postnatal comparative profiles of fru-2, 6P2 and PFK2 & fru-2, 6P2 dependent regulation of PFK1 and FBPase1 in mice brain. Fru-2, 6P2 and PFK2 activity both attained their adult levels in concordance from day1 to 1wk age. Western blot analysis of mice liver and brain & rat liver PFK2 using anti rat liver PFK2/FBPase2 confirmed that both, mice liver and brain isoforms cross- react efficiently with this antibody. In addition, DEAE-eluted brain fractions from different postnatal ages revealed that 1day mice brain expresses a liver type enzyme (∼55 kDa) that is replaced by an adult brain type protein (∼110 kDa) from 1wk onward ages. As compared to 1day mice, significantly decreased Km values of PFK2 at 1wk-10wk ages also suggest the existence of a kinetically different isoform of this enzyme from 1wk onward ages. In vitro effects of fru-2, 6P2 on partially enriched brain PFK1 and FBPase1 suggest that fru-2, 6P2 dependent respective stimulatory and inhibitory responses of both these enzymes increase progressively from day1 to 3wk age. This is well corroborated with the postnatal age-dependent linear increase in PFK1 and decrease in FBPase1 activities in mice brain. The results suggest that fru-2, 6P2 associated regulatory components develop in concordance in mice brain during early postnatal life

Acute liver failure in rats activates glutamine-glutamate cycle but declines antioxidant enzymes to induce oxidative stress in cerebral cortex and cerebellum.

BACKGROUND AND PURPOSE: Liver dysfunction led hyperammonemia (HA) causes a nervous system disorder; hepatic encephalopathy (HE). In the brain, ammonia induced glutamate-excitotoxicity and oxidative stress are considered to play important roles in the pathogenesis of HE. The brain ammonia metabolism and antioxidant enzymes constitute the main components of this mechanism; however, need to be defined in a suitable animal model. This study was aimed to examine this aspect in the rats with acute liver failure (ALF). METHODS: ALF in the rats was induced by intraperitoneal administration of 300 mg thioacetamide/Kg. b.w up to 2 days. Glutamine synthetase (GS) and glutaminase (GA), the two brain ammonia metabolizing enzymes vis a vis ammonia and glutamate levels and profiles of all the antioxidant enzymes vis a vis oxidative stress markers were measured in the cerebral cortex and cerebellum of the control and the ALF rats. RESULTS: The ALF rats showed significantly increased levels of ammonia in the blood (HA) but little changes in the cortex and cerebellum. This was consistent with the activation of the GS-GA cycle and static levels of glutamate in these brain regions. However, significantly increased levels of lipid peroxidation and protein carbonyl contents were consistent with the reduced levels of all the antioxidant enzymes in both the brain regions of these ALF rats. CONCLUSION: ALF activates the GS-GA cycle to metabolize excess ammonia and thereby, maintains static levels of ammonia and glutamate in the cerebral cortex and cerebellum. Moreover, ALF induces oxidative stress by reducing the levels of all the antioxidant enzymes which is likely to play important role, independent of glutamate levels, in the pathogenesis of acute HE

GS and GA get activated in cerebral cortex and cerebellum of the ALF rats.

<p>Activity profiles of GS (A) and GA (B) from control and ALF rats. The values represent mean ± SD where n = 4 *p<0.05 (control vs ALF rats).</p

Changes in LFT markers and ammonia in serum of the ALF rats.

<p>The data represents Mean ± S.D, where n = 4, **p<0.01, ***p<0.001 (control vs ALF group).</p

Changes in the levels of oxidative stress markers in cerebral cortex and cerebellum of the ALF rats.

<p>The data presents Mean ± S.D, where n = 4, *p<0.05, **p<0.01, (control vs ALF group).</p

Level of active catalase declines in cerebral cortex and cerebellum of the ALF rats.

<p>Catalase activity measured in cell free extract (A) and non-denaturing PAGE pattern of active catalase (B and C). The technical details are same as described in Fig. 3 except 8% PAGE was used in case of B. *p<0.05; **p<0.01 (control vs ALF rats).</p

ALF did not induce alterations in the active level of GR in cerebral cortex and cerebellum.

<p>GR activity measured in cell free extract (A) and non-denaturing PAGE pattern of active GR (B and C). The technical details are same as described in Fig. 5.</p

Levels of ammonia and glutamate in cerebral cortex and cerebellum of the control and ALF rats.

<p>The data represents Mean ± S.D, where n = 4.</p

SOD activity shows differential pattern in cerebral cortex and cerebellum of the ALF rats.

<p>A represents SOD activity measured in cell free extracts; values are mean ± SD where n = 4 and each experiment done twice. In case of B, pooled tissue extracts from 4 rats containing 60 µg protein in each lane was electrophoresed on 12% non-denaturing PAGE followed by substrate specific development of SOD bands. The gel photograph in B is a representative of 4 PAGE repeats. In panel C, relative densitometric values of SOD band from experimental group, as % of the control lane, have been presented as mean ± SD from 4 PAGE repeats. *p<0.05; **p<0.01, ***p<0.001 (control vs ALF rats).</p