Anaesthesia generates neuronal insulin resistance by inducing hypothermia
© Holscher et al; licensee BioMed Central Ltd. 2008
Received: 16 May 2008
Accepted: 09 October 2008
Published: 09 October 2008
Anaesthesia is commonly employed prior to surgical investigations and to permit icv injections in rodents. Indeed it is standard practise in many studies examining the subsequent actions of hormones and growth factors on the brain. Recent evidence that the basal activity of specific intracellular signalling proteins can be affected by anaesthesia prompted us to examine the effect of anaesthesia not only on the basal activity but also the insulin sensitivity of the major insulin signalling pathways.
We find that urethane- and ketamine-induced anaesthesia results in rapid activation of the phosphatidylinositol (PI) 3-kinase-protein kinase B (PKB) signalling pathway in the brain, increases tau phosphorylation while at the same time reducing basal activity of the Ras-ERK pathway. Subsequent injection of insulin does not alter the activity of either the PI 3-kinase or ERK signalling pathways, indicating a degree of neuronal molecular insulin resistance. However, if body temperature is maintained during anaesthesia then there is no alteration in the basal activity of these signalling molecules. Subsequent response of both pathways to insulin injection is restored.
The data is consistent with a hypothermia related alteration in neuronal signalling following anaesthesia, and emphasises the importance of maintaining the body temperature of rodents when monitoring insulin (or growth factor/neurotrophic agent) action in the brain of anesthetised rodents.
Insulin is produced by pancreatic β-cells, in response to rising plasma glucose levels, and initiates multiple metabolic changes to restore glucose homeostasis. A specific membrane glycoprotein acts as a high affinity sensor for insulin (insulin receptor (IR)) in many tissues (primarily liver, fat, muscle). The IR is expressed in many regions of the brain, including the hypothalamus, cortex, and hippocampus. Neuron specific deletion of the IR makes the animal more sensitive to diet induced obesity , implicating neuronal IR in the satiety response. Consistent with this, administration of insulin to the arcuate nucleus in the hypothalamus has significant effects on feeding and body weight [2–4].
Epidemiological evidence suggests that whole body insulin resistance, related to obesity, increases the risk of Alzheimer's disease, as well as vascular dementia [5–7]. In addition, there are molecular links between the development of Type 2 diabetes (an insulin resistant state) and Alzheimers's disease . For example, GSK3β activity is increased in the muscle of Type 2 diabetics  and the brain of Alzheimer's patients . This enzyme is inhibited by insulin treatment of cells , and is known to be a major tau kinase, phosphorylating residues that become hyperphosphorylated in Alzheimer's disease . Therefore, insulin resistance in the brain may contribute to a major component of Alzheimer's disease pathology.
Binding of insulin to the IR activates the intrinsic tyrosine kinase domain of the IR [13, 14]. Insulin receptor substrate (IRS) proteins are recruited to the activated IR, become phosphorylated on tyrosine residues , thereby recruiting PI 3-kinase, which converts phosphoinositol 4,5 bisphosphate (PIP2) to phosphoinositol 3,4,5 trisphosphate (PIP3) . This second messenger then brings pleckstrin homology (PH) domain containing proteins to the membrane, activating protein kinase cascades. The best characterised of these is the phosphoinositide dependent protein kinase (PDK1) pathway. PDK1 regulates many protein kinases, including protein kinase B (PKB, also known as Akt), PKC, p90 RSK, p70S6K and SGK . These protein kinases phosphorylate and regulate a wide variety of proteins involved in growth and metabolism. For example, PKB phosphorylates and inactivates GSK3 , and FOXO transcription factors . These pathways are important for the proper regulation of hepatic gene transcription by insulin [18, 19].
The second major pathway downstream of IRSs is the Ras-ERK pathway. Grb2/mSOS is a protein complex that interacts with phosphorylated IRSs (at distinct residues to those that recruit PI 3-kinase). Once bound, mSOS exchanges GDP for GTP on the small G-protein Ras, thereby activating Ras . This promotes activation of c-Raf, which phosphorylates and activates MAP/ERK kinase (MEK), which in turn phosphorylates and activates ERK1/2 [20–22]. ERK1/2 has multiple substrates, most of which are related to cell growth, hence this pathway is generally considered to be important in insulin regulation of growth.
Most studies examining insulin action in the brain utilise primary neurons, slice culture or transgenic animals. However, direct application of insulin to the brain, by intracerebroventricular (icv) injection, can be used to study acute and chronic effects of insulin in vivo. Recent evidence suggests that some of the signalling molecules described above are affected by anaesthesia [23, 24], which is commonly used prior to icv injection. In this report we examined the two major insulin signalling pathways in the brain of anaesthetised rodents, and find that anaesthesia induced hypothermia generates insulin resistance in the brain.
Anaesthesia and ICV injections
Male C57/Bl6 mice 3 months of age were obtained from Harlan, UK. All studies were performed under the regulations permitted by UK home office licence no. PPL2603b. Animals were fasted overnight and anesthetised by ip injection of either urethane (750 mg/kg dose in 0.2 ml) or ketamine/Xylazine (80–100 mg/kg + 10 mg/kg).
For some studies (as indicated in figure legends) anesthetised animals were kept at 37°C by use of a temperature controlled heating pad (Harvard apparatus, rectal probe). For icv injection of insulin (3 mU in 2 μl), animals were injected with ketamine/Xylazine, put into a stereotaxic frame (TSE systems, Germany), the scalp was removed, and a 0.7 mm hole was drilled to permit injection into the lateral ventricle using a 5 μl Hamilton syringe (coordinates from bregma: AP = 0.2, ML = 1.2, D = 2.5). Animals were injected immediately with sterile saline solution (control) or insulin. The injection was given slowly over 2 minutes. Body temperature was approximately 34°C by the time of the icv injection.
After 30 min, animals were decapitated, the brains removed and dissected into different brain sections. Brain sections were then snap frozen in dry ice and stored at -40 until analysis. Non-anesthetised controls were injected ip with saline and were decapitated after 30 min without any surgery, and the brains removed and snap frozen.
Antibodies to phospho-PKB (Thr308), phospho-PKB (Ser473), phospho-ERK (Thr202/Tyr204), were from Cell Signaling Technology (Beverly, MA, U.S.A.), antibodies to total PKB, ERK1/2 and GSK3α/β were from Upstate Biotechnologies (Lake Placid, NY, U.S.A.). Anti-β-Actin was purchased from Sigma-Aldrich, Inc. (St Louis, MO, U.S.A.). The phospho-tau (AT8) and tau-5 (total tau) antibodies were purchased from Innogenetics (Gent, Belgium) and Chemicon, respectively.
Tissue Homogenisation and Immunoblot
Frozen tissues were homogenised using a 1 ml glass Dounce homogenizer in lysis buffer containing 1% (v/v) Triton X-100, 50 mM Tris-HCl, pH 7.5, 0.27 M sucrose, 1 mM sodium orthovanadate, 0.1% (v/v) β-mercaptoethanol and Complete protease inhibitor tablets (Roche, Lewes, UK) (4°C). Following centrifugation, supernatants were collected and protein concentrations determined . Lysates (typically 2.5–30 μg) were subjected to SDS-PAGE on 4–12% NuPAGE polyacrylamide gels, then transferred to nitrocellulose membrane. Membranes were incubated with primary antibodies (diluted 1/1000 in 1% (w/v) skimmed milk or 5% (w/v) BSA in TTBS overnight at 4°C), appropriate HRP-linked secondary antibodies, then visualised using the ECL reagent (GE Healthcare) and exposed to autorad film (GE Healthcare).
Analysis of data
Densitometry was performed using Aida computer software (Raytest, Straubenhardt, Germany). Phosphorylation of ERK, Akt and tau were calculated as the ratio of the phosphospecific versus total protein antibody signal, while phosphorylation of GSK3 was a ratio of the P-GSK3 to β-actin (loading control). Mean values were calculated for each sample and the control value was set at 1. Data is presented as mean ± standard deviation, and statistical significance (p-value) between two conditions was obtained using an unpaired students t-test.
Results and discussion
Anaesthesia induces changes in the major insulin regulated protein kinase cascades resulting in loss of subsequent response to insulin
The previous reports of abnormal signalling processes in anaesthesia had indicated that the induction of hypothermia contributed to the defective signalling. Indeed, simply lowering body temperature could alter ERK and GSK3 signalling . The body temperature of the animals injected with either agent was substantially and reproducibly lowered within the timeframe of the experiment (Fig. 1C). Although it is quite likely that many other physiological processes (including hypoxia) will be induced by anaesthesia, we investigated whether maintenance of body temperature of the anesthetised animals prevented the defective response of PKB, GSK3 or ERK1/2 phosphorylation to insulin.
Maintenance of body temperature during anaesthesia is required to permit intracellular responses to insulin in the brain
This is of particular importance since prolonged insulin resistance in the brain is likely to underlie the increased risk of dementia in people with Type 2 diabetes (for review see ). The insulin resistance generated by anaesthesia-associated hypothermia may provide a model system to study the neurodegenerative effects of insulin resistance and/or the neuroprotective effects of insulin, in vivo. Post-operative cognitive defects have been widely reported, particularly in the elderly, and these may be related to general anaesthesia [28, 29]. Meanwhile, obesity induced insulin resistance leads to cognitive impairment in rodents [30, 31]. Therefore insulin resistance in the brain during anaesthesia may play an important role in postoperative cognitive decline, although it is unlikely that body temperature falls below 30°C in clinical practice, therefore further study is required to establish the exact point where hypothermia is problematic.
The data presented shows the importance of maintaining body temperature in rodents undergoing anaesthesia prior to studies investigating hormonal or growth factor regulation of cognition. The signalling molecules affected by hypothermia are not only key to the action of insulin in the brain, but also the action of neurotrophic agents and other circulating hormones (e.g. Leptin) .
The phosphorylation of tau at Ser202/Thr205 is induced by anaesthesia but remains sensitive to insulin
Anaesthesia induces abnormal signalling in the brain, but this can be prevented by maintaining body temperature. The abnormal signalling produces a form of molecular insulin resistance, although this is not equally severe in all insulin signalling pathways. Anaesthesia/hypothermia has potential as an in vivo model for the study of the effect of insulin resistance in the brain. Meanwhile great care must be taken to maintain body temperature when using anesthetised rodents to study neuronal signalling.
The project was funded by the Alzheimer's Research Trust (Cooperation Grant ART/NCG2007/A1, and project grant ART/PG/2005/1). CS is the recipient of the Diabetes UK Senior Fellowship (BDA:RD02/0002473).
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