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 Ca(2+)/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.

Role of the liver in glucose homeostasis in PI 3-kinase p85alpha-deficient mice.

Phosphoinositide 3-kinase (PI3K) p85alpha-deficient mice exhibit hypoglycemia as a result of increased insulin sensitivity and glucose uptake in peripheral tissues. Although PI3K is central to the metabolic actions of insulin, its mechanism of action in liver is not well understood. In the present study, we investigated hepatic insulin signaling and glucose homeostasis in p85alpha-deficient and wild-type mice. In the livers of p85alpha-deficient mice, p50alpha played a compensatory role in insulin-stimulated PI3K activation by binding to insulin receptor substrate (IRS)-1/2. In p85alpha-deficient mice, the ratio of p50alpha over p110 catalytic subunit of PI3K in the liver was higher than in the muscles. PI3K activity associated with IRS-1/2 was not affected by the lack of p85alpha in the liver. Insulin-stimulated Akt and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activities in the liver were similar in p85alpha-deficient and wild-type mice. A hyperinsulinemic-euglycemic clamp study revealed that the glucose infusion rate and the rate of disappearance were higher in p85alpha-deficient mice than in wild-type mice but that endogenous glucose production tended to be higher in p85alpha-deficient mice than in wild-type mice. Consistent with this finding, the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in livers after fasting was higher in p85alpha-deficient mice than in wild-type mice. After mice were fasted, the intrahepatic glucose-6-phosphate level was almost completely depleted in p85alpha-deficient mice. The glycogen content fell to nearly zero as a result of glycogenolysis shortly after the initiation of fasting in p85alpha-deficient mice. The absence of an increase in insulin-stimulated PI3K activation in the liver of p85alpha-deficient mice, unlike the muscles, may be associated with the molecular balance between the regulatory subunit and the catalytic subunit of PI3K. Gluconeogenesis was rather elevated in p85alpha-deficient mice, compared with in wild-type mice, and the liver seemed to partially compensate for the increase in glucose uptake in peripheral tissues.

Role of hypothalamic adenosine 5'-monophosphate-activated protein kinase in the impaired counterregulatory response induced by repetitive neuroglucopenia.

Antecedent hypoglycemia blunts counterregulatory responses that normally restore glycemia, a phenomenon known as hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to impaired counterregulatory responses are largely unknown. Hypothalamic AMP-activated protein kinase (AMPK) acts as a glucose sensor. To determine whether failure to activate AMPK could be involved in the etiology of HAAF, we developed a model of HAAF using repetitive intracerebroventricular (icv) injection of 2-deoxy-D-glucose (2DG) resulting in transient neuroglucopenia in normal rats. Ten minutes after a single icv injection of 2DG, both alpha1- and alpha2-AMPK activities were increased 30-50% in arcuate and ventromedial/dorsomedial hypothalamus but not in other hypothalamic regions, hindbrain, or cortex. Increased AMPK activity persisted in arcuate hypothalamus at 60 min after 2DG injection when serum glucagon and corticosterone levels were increased 2.5- to 3.4-fold. When 2DG was injected icv daily for 4 d, hypothalamic alpha1- and alpha2-AMPK responses were markedly blunted in arcuate hypothalamus, and alpha1-AMPK was also blunted in mediobasal hypothalamus 10 min after 2DG on d 4. Both AMPK isoforms were activated normally in arcuate hypothalamus at 60 min. Counterregulatory hormone responses were impaired by recurrent neuroglucopenia and were partially restored by icv injection of 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside, an AMPK activator, before 2DG. Glycogen content increased 2-fold in hypothalamus after recurrent neuroglucopenia, suggesting that glycogen supercompensation could be involved in down-regulating the AMPK glucose-sensing pathway in HAAF. Thus, activation of hypothalamic AMPK may be important for the full counterregulatory hormone response to neuroglucopenia. Furthermore, impaired or delayed AMPK activation in specific hypothalamic regions may play a critical role in the etiology of HAAF.