Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3

Abstract

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.

FIGURE 1. Food withdrawal for 24 h decreased the phosphorylation of Akt and GSK3 in mouse brain

Food was withheld for 24 h and then protein extracts from the cerebral cortex and hippocampus (n = 4 per group) were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (A) and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (B).

FIGURE 2. Glucose administration increased the phosphorylation of Akt and GSK3 in mouse brain

Food was withdrawn overnight (14 h) and after an ip injection of 2 g/kg glucose, blood glucose and serum insulin concentrations were measured (A), and protein extracts from the cerebral cortex and hippocampus were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (B), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3αβ (C). Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from three mice per group.

FIGURE 3. Insulin transiently increases the phosphorylation of Akt and GSK3 in mouse brain

Food was withdrawn overnight and after an ip injection of 5 international units/kg insulin, blood glucose and serum insulin concentrations were measured (A), and protein extracts from the cerebral cortex and hippocampus were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (B), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (C). Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from three mice per group.

FIGURE 4. Streptozotocin administration rapidly increases the phosphorylation of Akt and GSK3 in mouse brain

Mice were treated with streptozotocin, and after 3 days, protein extracts from the cerebral cortex and hippocampus were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (A), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (B). C, control; S, streptozotocin-treated. Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from four mice per group. *, p <0.05 compared with control values.

FIGURE 5. Streptozotocin administration rapidly decreases the phosphorylation of Akt and GSK3 in mouse epididymal fat

Mice were treated with streptozotocin, and after 3 days, protein extracts from epididymal fat were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (A), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and total GSK3α/β (B). C, control; S, streptozotocin-treated.

FIGURE 6. Effects of streptozotocin treatment on activities of Akt and GSK3

Mice were treated with streptozotocin, and after 3 days the cerebral cortex and epididymal fat were obtained. A, Akt activity and levels of Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt were measured in the same mice. B, GSK3α and GSK3β activities and levels of Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, and Total GSK3α/β were measured in the same mice.

FIGURE 7. Mouse brain Akt and GSK3 are highly phosphorylated 4 weeks after streptozotocin treatment

Mice were treated with streptozotocin, and after 4 weeks, protein extracts from the cerebral cortex and hippocampus were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, Total Akt (A) and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (B). C, control; S, streptozotocin-treated. Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from five mice per group. *, p <0.05 compared with control values.

FIGURE 8. Akt and GSK3 are highly phosphorylated in db/db mouse brain

Protein extracts from the cerebral cortex and hippocampus were immunoblotted in 8-week-old db/db mice for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (A), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (B). Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from three mice per group. *, p<0.05 compared with control values.

FIGURE 9. Increased brain Akt and GSK3 phosphorylation in streptozotocin-treated mice is reversed by insulin administration

Mice were treated with streptozotocin and after 3 days, food was withdrawn overnight. After an ip injection of 5 international units/kg of insulin, blood glucose and serum insulin concentrations were measured (A). Insulin values were divided by ten to show the changes in insulin and glucose levels on the same graph. Protein extracts from the cerebral cortex and hippocampus were immunoblotted for Phospho-Thr308-Akt, Phospho-Ser473-Akt, and Total Akt (B), and Phospho-Ser21-GSK3α, Phospho-Ser9-GSK3β, Phospho-Tyr279/216-GSK3α/β, and Total GSK3α/β (C). Quantitative values were obtained by densitometric measurements of immunoblots and are means ± S.E. from three mice per group.