Glucoregulatory hormones in man at high altitude

Summary Concentrations of glucose, lactic acid, free fatty acid (FFA), insulin, cortisol and growth hormone (GH) in the blood were monitored in 15 euglycaemic men (sojourners, SJ) at sea level (SL) and while at altitudes of 3500 m and 5080 m, in acclimatised low landers (ALL) and in high altitude natives (HAN). In SJ, blood glucose and insulin concentrations showed a significant increase on the 3rd and 7th day after arrival at high altitude (HA), thereafter returning to sea level values and remaining the same during the entire period of their stay at 3500 m. Subsequently, on arrival at higher altitude (5080 m) the glucose concentrations again showed an increase over the preceding values and returned to SL values on day 41 while at 5080 m. A significant increase in cortisol concentrations was seen on day 3 after arrival at HA and the increased levels were maintained until day 21 at 3500 m. The cortisol concentrations on day 30 after arrival at 5080 m came down to SL values and remained unchanged thereafter. No appreciable change in GH and FFA was seen during the sojourn at HA. On the other hand, blood lactic acid concentration decreased significantly. There was no difference between the fasting glucose concentrations in ALL at 3500 m and in HAN at 3500 m and 4200 m compared to values of SJ at SL, whereas ALL at 4200 m had higher glucose values. Concentrations of plasma insulin and GH in ALL and HAN were higher than the values of SJ at SL, whereas cortisol values did not show any difference. These observations indicated that at HA the glucose values were high for the insulin concentration observed and might have been due to increased secretion of GH by the pituitary gland.     

Ambient temperature and ketone body plasma concentration in fasting geese

The effect on ketonemia of alternate exposure to ambient temperatures (Ta) of 25 and 5°C was investigated in fasting geese. Three experimental birds were compared to three controls continuously exposed to 25°C Ta while fasting. During the first 9 days of fasting, when both groups were exposed to 25°C, plasma concentration of -hydroxybutyrate (-OHB) increased similarly in both, from 0.10±0.02 to 6.62±0.71 mmol·L^–1. It later plateaued at 8–9 mmol·L^–1 in the control birds. When the experimental birds were exposed to 5°C Ta between the 9th and 15th day of the fast, it increased further during the first 24 h but thereafter decreased of 57%, from 8.62±1.56 to 3.73±1.24 mmol·L^–1. This decrease was reversed within the 6 days of return to 25°C Ta. In both groups, plasma acetoacetate (AcAc) concentration remained very low during the fast: 51±1 mol·L^–1. This reversible cold-induced effect on ketonemia may be used for investigating the possible role of ketone bodies in protein sparing during fasting.     

The oxidation of glucose, ketone bodies and acetate by the brain of normal and ketonaemic sheep.

1. The utilization and oxidation of glucose, acetate and ketone bodies by the brain of sheep has been determined from measurements of arteriovenous (A-V) differences and cerebral blood flow, as well as by infusing 14C-labelled metabolites. 2. The A-V difference for glucose was generally more than one sixth, on a molar basis, that of oxygen. 3. The mean rate of glucose utilization by the brain of conscious sheep (0-508 +/- 0-063 mumole/g per minute) was maintained even when the capillary glucose concentration was below 1-4 mM. 4. The amount of 14CO2 produced from [U-14C]glucose by the brain was consistent with glucose being the only energy source for the brain, even during hypoglycaemia and hyperketonaemia. 5. There was no appreciable production of lactate or pyruvate by the brain. 6. There was no significant A-V difference for acetate across the brain in normal or undernourished pregnant sheep. The small A-V differences that were measured show that less than 5% of the CO2 produced could be derived from acetate, a conclusion that is supported by experiments using [U-14C]acetate. 7. No significant A-V difference was detectable across the brain for 3-hydroxybutyrate or acetoacetate in normal fed, pregnant ketonaemic or even anaesthetized sheep infused with acetoacetate. Experiments in which [U-14C]-D(-)-3-hydroxybutyrate was infused also showed that less than 5% of CO2 was derived from ketone bodies. 8. In anaesthetized sheep infused with acetoacetate, measurements were made simultaneously across brain, heart and skeletal muscle. In contrast to the non-significant uptake of ketone bodies by the brain, uptake by heart and skeletal muscle was sufficient to account for nearly 60% of their oxygen consumption. 9. Experiments using [14C]hydroxybutyrate confirmed that during infusion of acetoacetate most of the CO2 produced by the heart, but not by the brain, was derived from ketone bodies. 10. In anaesthetized sheep ketone bodies penetrate only slowly into cerebrospinal fluid. 11. It is proposed that mechanisms for the utilization of ketones by the sheep brain have not evolved because glucose utilization by the brain is a smaller fraction of whole body glucose utilization than in man and rats.     

Depressed metabolism and low protein catabolism in fasting grey seal pups

Grey seal pups (Halichoerus grypus) were collected at the time of weaning (mid-October) and fasted for 52 days at thermoneutrality in separate cages. Body weight decreased exponentially, while metabolic rate dropped 45% from an average of 2.95 +/- 0.15 (SEM) W kg-1 at day 2 of fasting to a stable level of 1.62 +/- 0.06 (SEM) W kg-1 from day 10 to day 47 of fasting. Respiratory quotient was low, indicating extensive catabolism of triglycerides, while plasma cortisol was fairly stable at 110 +/- 8 (SEM) nmol l-1 throughout the fasting period. Daily urinary output decreased from 236 +/- 20 (SEM) ml day-1 at day 2 to a stable value of 87 +/- 6 (SEM) ml day-1 between days 8 and 50 of fasting. The urine was analysed for urea, uric acid, creatinine, ammonia, total nitrogen and osmolality. Urea was always the principal excretory end-product, amounting to between 70 and 80% of the total excreted nitrogen. The urine was moderately concentrated (range 770-1300 mosmol kg-1). Total excreted urinary nitrogen decreased by 68% from 3.7 +/- 0.7 (SEM) g day-1 to 1.2 +/- 0.4 (SEM) g day-1 between days 2 and 50. The urinary nitrogen was used to calculate the daily amount of protein being oxidized and its energy content was compared with the measured basal metabolic rate of individual animals. Approximately 6% of the energy expended by grey seal pups during the post-weaning fast is derived from oxidation of protein. It is concluded that a rapid depression of basal metabolic rate and extensive blubber catabolism enable grey seal pups to endure prolonged periods of fasting without any apparent signs of discomfort or stress.     

Effects of ketosis on glucose flux in children and adults

Sequential glucose flux studies were carried out in five normal and six epileptic children and ten adult volunteers using [6,6-2H2]glucose to determine the effect of ketosis on carbohydrate homeostasis in children and adults. All subjects were studied after 14 and 30-38 h of fasting while consuming a normal diet and the epileptic children under 14 h of fasting while consuming an isocaloric ketogenic diet (75% fat wt/wt). Glucose flux, when expressed per kilogram body weight, was inversely correlated with the degree of ketosis in children (P less than 0.001) and in adults (P less than 0.01), but not when both children and adults were considered together (r = 0.078). When glucose flux was corrected for estimated brain weight, the relationship between glucose flux and ketonemia was linearly related in children (P less than 0.001), in adults (P less than 0.02), and when all subjects were considered together (P less than 0.001). The inverse relationship between ketonemia and glucose flux corrected for estimated brain mass is consistent with a partial replacement of glucose by ketone bodies for cerebral metabolism and may provide a more rational means of expressing glucose flux data to take into account the higher brain-to-body ratio in children.     

Acetate metabolism in the isolated sheep foetus

1. Sheep foetuses over the age range 105-142 days and maintained on an extracorporeal circuit removed exogenous acetate at 2.0-4.9 mg/kg.min.2. Urinary loss of acetate was less than 12.5% of the administered amount.3. The rate of acetate removal appeared to be unaffected by the addition of glucose to the circuit. The removal of glucose also seemed to be unaffected by the presence of acetate.4. A decline in the plasma concentration of free fatty acids and ketone bodies was also observed during the perfusion.5. It is concluded that under these conditions acetate may have accounted for about one half of the oxygen consumption. The contribution of free fatty acids and ketone bodies must have been small. The relevance of these findings to the nutrition of the foetus in utero is discussed.     

Water flux and early signs of entrance into phase III of fasting in grey seal pups.

Body water loss and turnover rate were measured in fasting newly weaned grey seal (Halichoerus grypus) pups without access to water for 52 days, by use of bolus injections of deuterated water. Total body water (N) was 41.8 +/- 2.3 (SD)% of total body weight at day 3 and 44.5 +/- 5.9 (SD)% at day 51 of fasting (P greater than 0.05), while the water content of body core decreased from 73 to 61% over the same period. The average water efflux rate in the same period was 362 +/- 17 (SD) ml d-1, the reduction in N over the same period contributing 35% to this end. The biological half-time of deuterated water was 38.2 +/- 3.3 (SD) d. Plasma osmolality increased from a stable level of 329 +/- 11 (SD) mosmol kg-1 to 445 +/- 11 (SD) mosmol kg-1 after day 38 of fasting. Plasma urea was the major contributor to this increase, in spite of a steadily decreasing urine urea concentration (Nord?y et al. 1990) throughout the entire experimental period. It is concluded that grey seal pups endure 52 d of fasting without intake of water with a minor dehydration of body core, while they become hyperosmotic due to increased catabolism of protein after 38 d when sparing of the insulating blubber layer in preparation for life in cold water seems to occur.     

Glucose intolerance following chronic metabolic acidosis in man.

The effect of chronic metabolic acidosis (0.1 g/(kg . day) X 3 days) on carbohydrate metabolism was examined with the glucose-clamp technique in 16 healthy volunteers. Hyperglycemic clamp. Plasma glucose concentration is acutely raised and maintained 125 mg/dl above the basal level. Because the glucose concentration is held constant, the glucose infusion rate is an index of glucose metabolism (M). Following NH4Cl, M decreased from 8.95 +/- 1.12 to 7.35 +/- 0.76 (P less than 0.05) despite an increased plasma insulin concentration (I) 23 +/- 9%, P less than 0.05). Consequently the M/I ratio, an index of tissue sensitivity to insulin, decreased by 32 +/- 5% (P less than 0.005). Euglycemic clamp. Plasma insulin concentration is acutely raised and maintained 101 +/- 3 microU/ml above basal and plasma glucose is held constant at the fasting level by a variable glucose infusion (M). Following NH4Cl both M and M/I decreased by 15 +/- 4% (P = 0.005) and 15 +/- 5% (P = 0.01), respectively. Metabolic acidosis had no effect on basal [3-3H]glucose production or the percent of decline (91 +/- 4%) following hyperinsulinemia. Both hyperglycemic and euglycemic clamp studies indicate that impaired glucose metabolism following metabolic acidosis results from impaired tissue sensitivity to insulin.     

Insulin responses and glucose levels in plasma and cerebrospinal fluid during fasting and refeeding in the rat

The present experiments were designed to investigate the rate of penetration of insulin from the plasma into the cerebrospinal fluid (CSF) during 24 hr of fasting and refeeding in the light phase. The results show that under these conditions basal CSF-immunoreactive insulin (IRI) levels were positively correlated with plasma IRI levels. Basal plasma IRI fell during a fast but was similar to prefast control after one day of refeeding. Although CSF-IRI levels rose during glucose infusion, CSF-IRI was not elevated by glucose during a fast. During refeeding, CSF-IRI responses returned toward control, prefeeding values. This study suggests a decreased transport of insulin from plasma to CSF during fasting. The lower CSF-IRI levels achieved under these conditions may determine meal size by allowing larger meals after a fast.     

Nocturnal hypothermia in fasting Japanese quail: the effect of ambient temperature

Japanese quail were equipped with intraperitoneal transmitters for telemetric measurement of body temperature (Tb) and activity. Food deprivation at + 24 degrees C for four days induced a well-defined nocturnal hypothermic response. The normal day-night difference (about 1 degrees C) in Tb increased 1 degrees C per day, reaching 5 degrees C on the fourth night of fast. Nocturnal motor activity decreased during the fast, while daytime activity first increased and then returned to the original level by the end of the fasting period. A strong correlation between Tb and motor activity was found during scotophase (r = .91) but not during photophase (r = .02). At + 4 degrees C, where food was deprived for two days, the level of hypothermia was not different from that at + 24 degrees C, but birds fasting in the cold reached the hypothermic level more rapidly. In control birds, the decrease in Tb at the beginning of scotophase was independent of ambient temperature. In the combined data, the level of hypothermia correlated strongly with body mass loss (r = .90), which shows that quail can directly or indirectly sense the amount of body energy reserves. This is the first report of hypothermia in a fasting gallinaceous bird. The consistent level of hypothermia at varying ambient temperatures suggests that either nonenergetic costs or phylogenetic constraints prevent deeper hypothermia in cold. Accordingly, the regulation of hypothermia cannot be explained by using only energetic arguments.     

Epigenetic regulation of the glucose transporter gene Slc2a1 by β‐hydroxybutyrate underlies preferential glucose supply to the brain of fasted mice

We carried out liquid chromatography–tandem mass spectrometry analysis of metabolites in mice. Those metabolome data showed that hepatic glucose content is reduced, but that brain glucose content is unaffected, during fasting, consistent with the priority given to brain glucose consumption during fasting. The molecular mechanisms for this preferential glucose supply to the brain are not fully understood. We also showed that the fasting‐induced production of the ketone body β‐hydroxybutyrate (β‐OHB) enhances expression of the glucose transporter gene Slc2a1 (Glut1) via histone modification. Upon β‐OHB treatment, Slc2a1 expression was up‐regulated, with a concomitant increase in H3K9 acetylation at the critical cis‐regulatory region of the Slc2a1 gene in brain microvascular endothelial cells and NB2a neuronal cells, shown by quantitative PCR analysis and chromatin immunoprecipitation assay. CRISPR/Cas9‐mediated disruption of the Hdac2 gene increased Slc2a1 expression, suggesting that it is one of the responsible histone deacetylases (HDACs). These results confirm that β‐OHB is a HDAC inhibitor and show that β‐OHB plays an important role in fasting‐induced epigenetic activation of a glucose transporter gene in the brain.     

Metabolic and hormonal responses during squash

Summary The metabolic and hormonal response during squash was observed in eight normal men. Significant increases from resting were found for blood glucose, lactate, pyruvate, alanine and glycerol while total ketone bodies and plasma nonesterified fatty acids rose after play stopped. Insulin and C-peptide decreased significantly and catecholamines, ACTH, prolactin and growth hormone increased.     

心脏瓣膜置换围手术期血糖控制对血乳酸水平的影响

背景：心脏瓣膜置换后极易发生高血糖,对患者预后不利,而围术期血糖和患者的乳酸有密切的关系。 目的：观测心脏瓣膜置换患者不同时间血糖及血乳酸水平变化,并进行相关性分析。 方法：将63例心脏瓣膜置换患者随机分为两组,对照组进行传统标准治疗,血糖浓度维持于7.5-11.1 mmol/L;实验组通过胰岛素持续输注进行强化治疗,血糖浓度维持于3.6-7.5 mmol/L。 结果与结论：实验组患者体外循环中及瓣膜置换后血糖水平较对照组显著降低(P < 0.05);两组患者随着瓣膜置换时间延长血乳酸水平都明显升高,并发生高乳酸血症,实验组并发症发生率显著低于对照组(P < 0.05);两组病例都有血糖、血乳酸逐渐升高到再恢复正常的过程,尤其以置换后6 h为最高;血糖值与血乳酸值的变化趋势大致相同。通过一元线性回归分析血糖与乳酸的相关系数为0.838,单侧P < 0.01,证明两个变量间有直线相关关系。结果提示,应用胰岛素严格控制血糖,对控制心脏瓣膜置换中及瓣膜置换后血糖、血乳酸水平有利,可以降低患者置换后并发症的发生率。围术期血糖与血乳酸的水平呈正相关。     

Effects of fasting on hypoxic ventilatory responses and the contribution of histamine H1 receptors in mice

We tested the hypothesis that fasting affects hypoxic ventilatory responses through metabolic changes via histamine H1 receptors. Wild-type (WT) and histamine H1 receptor knockout (H1RKO) mice were studied in fed and fasted states. In the fed WT, hypoxic-gas exposure elicited an increase and a subsequent decline in ventilation (hypoxic ventilatory decline or HVD). HVD was influenced by fasting in breathing pattern with metabolic rate. Fasting elicited hypoglycemia, a drop in R, and increases in free fatty acid and ketone bodies in the serum. In H1RKO, HVD was blunted in the fed state, but it appeared in the fasted state. There was a minimal drop in R following fasting and a low triglyceride concentration. Thus, fasting affects HVD through a change in energy mobilization from glucose to lipid metabolism. Histamine H1 receptors are involved in HVD during fed and fasted states, resulting in adaptation to the environmental conditions.     

Cerebral Utilization of Glucose,Ketone Bodies and Oxygen in Starving Infant Rats and the Effect of Intrauterine Growth Retardation

Cerebral arteriovenous differences of acetoacetate, D-β-hydroxybutyrate, glucose, lactate and oxygen and brain DNA content was measured at 20 days of age in intrauterine growth retarded (IUGR) rats and normal littermates after 48 and 72 h of starvation. Cerebral blood flow (CBF) was measured with labeled micro-spheres in other comparable groups of IUGR and control rats. CBF was similar in IUGR and normal littermates (0.57±0.09 and 0.58±0.10 ml/min respectively). After 48 h of starvation, arterial glucose was significantly lower in IUGR than control animals but the arterial concentrations of ketone bodies were similar. After 48 h of starvation, cerebral arteriovenous difference of β-hydroxybutyrate was significantly higher in control than IUGR rats also when expressed per mg brain DNA as was the fractional uptake of D-p-hydroxybutyrate. After 72 h of starvation, arterial concentrations of ketone bodies were significantly lower in IUGR rats than controls but the fractional uptake of D-β-hydroxybutyrate was increased compared to IUGR rats starved for 48 h. The average percentage of calculated total substrate uptake (μmol/min) accounted for by ketone bodies increased in control animals from 31.1 % after 48 h of starvation to 41.0% after 72 h of starvation. In IUGR rats these percentage values were 26.5 and 25.7 respectively. After 72 h of starvation the fraction of total cerebral uptake of substrates accounted for by ketone bodies was significantly higher in control than IUGR rats. As total cerebral uptake of substrates was similar between IUGR and control animals it is concluded that IUGR rats are more dependent on glucose as a substrate for the brain during starvation.     

Plasma oxytocin,prolactin, insulin and LH after 24 h of fasting and after refeeding in lactating sows

The effects of 24 h of fasting and refeeding on the release of oxytocin, prolactin, insulin and LH in three lactating sows were investigated. The sows were starved, but supplied with water ad libitum, from 09.00 h on day 27 of lactation until 15.00 h on day 28 of lactation, when they were refed. Blood samples were collected continuously, using an automatic collection system, at a rate of 1 ml min^-1 from 09.00 to 21.00 h on day 28 (P1 = 6 h period after the 24 h fast, P2 = 6 h period after refeeding). For both P1 and P2 the mean number of nursings was 7.0 ± 1.0. Plasma insulin and glucose decreased to very low levels during fasting and increased (P < 0.001) after refeeding (insulin, 2.5 ± 0.7 vs. 28.9 ± 0.7 mU 1^-1; glucose, 2.6 ± 0.3 vs. 6.4 ± 0.3 mmol 1^-1). Following fasting, levels of prolactin were low (2.8 ± 0.1 μg 1^-1), and sucking did not induce significant release of prolactin. However, prolactin increased rapidly after refeeding (5.4 ± 0.1 μg 1^-1, P < 0.001). Neither the 24 h fast nor refeeding had a marked effect on basal levels of oxytocin, the percentage of sucklings with an oxytocin peak or the size of oxytocin peak. LH release (average and basal levels and number of pulses/6 h) during fasting was similar to that measured after refeeding. Plasma CCK increased significantly after feeding. The results indicate that the release of prolactin is also regulated by feed intake. Consequently the 24 h fast in lactating sows may alter milk production via an inhibition of prolactin release. Neither a stimulatory effect of insulin nor an inhibitory effect of prolactin on LH release was observed.     

Induction processes in blood-brain transfer of ketone bodies during starvation.

Fed and starved rats were studied on successive days during a 5-day starvation period. The ability of ketone bodies to pass the blood-brain barrier was estimated by single common carotid injections of labeled ketone bodies and water, and results were expressed as the ratio between the normalized activities of tracers in tissue and blood, the brain uptake index (BUI). BUI of D-3-hydroxybutyrate and acetoacetate decreased as their total concentrations increased in the injectate bolus: BUI of D-3-hydroxybutyrate decreased significantly from 8% at 0.2 mM to 3--4% at 20.2 mM in fed rats and from 11.5% at 0.2 mM to 6% at 20.2 mM in starved rats, indicating saturation of the uptake mechanism. The BUI of both ketone bodies increased significantly with increasing duration of starvation, indicating adaptation to ketonemia. Enzymatic kinetics explained the uptake behavior of D-3-hydroxybutyrate in both fed and starved rats and involved a rise of Km and Vmax during starvation consistent with a doubling of the transport rate at the degree of ketonemia found in starved rats. The uptake of glucose was not influenced by starvation or ketonemia.     

Improved metabolic control after supplemented fasting in overweight type II diabetic patients

Twenty obese type II diabetes patients were treated in a metabolic ward during 4 weeks with a very low calorie regimen (200 kcal/day). They were given dietary advice and reexamined 3 months after discharge. Mean body weight decreased by 10 kg during fasting, blood glucose was normalized, urinary glucose disappeared and the K-value at i.v. glucose tolerance test increased. Fasting serum insulin concentrations decreased by 54%. Serum triglycerides (TG) decreased by 65%, serum cholesterol (Chol) by 28% and high density lipoprotein (HDL) Chol by 14%. Three months later, only serum TG remained significantly decreased (-47%) while HDL Chol was significantly higher than on admission (+11%). Fasting blood glucose remained significantly lower (-25%) with a low urinary glucose excretion. Supplemented fasting appears to be a safe and useful tool in the treatment of obese type II diabetics. It causes, at least during a limited follow-up period, a significant improvement in glucose control and lipoprotein metabolism in spite of a concomitant reduction of the antidiabetic medication.     

Glucose-induced exertional fatigue in muscle phosphofructokinase deficiency.

BACKGROUND. The exercise capacity of patients with muscle phosphofructokinase deficiency is low and fluctuates from day to day. The basis of this variable exercise tolerance is unknown, but our patients with this disorder report that fatigue of active muscles is more rapid after a high-carbohydrate meal. METHODS AND RESULTS. To determine the effect of carbohydrate on exercise performance, we asked four patients with muscle phosphofructokinase deficiency to perform cycle exercise under conditions of differing availability of substrate--i.e., after an overnight fast, and during an infusion of glucose or triglyceride (with 10 U of heparin per kilogram of body weight) after an overnight fast. As compared with fasting and the infusion of triglyceride with heparin, the glucose infusion lowered plasma levels of free fatty acids and ketones, reduced maximal work capacity by 60 to 70 percent, and lowered maximal oxygen consumption by 30 to 40 percent. Glucose also increased the relative intensity of submaximal exercise, as indicated by a higher heart rate at a given workload during exercise. The maximal cardiac output (i.e., oxygen delivery) was not affected by varying substrate availability, but the maximal systemic arteriovenous oxygen difference was significantly lower during glucose infusion (mean +/- SE, 5.5 +/- 0.3 ml per deciliter) than after fasting (7.6 +/- 0.4 ml per deciliter, P less than 0.05) or during the infusion of triglyceride with heparin (8.9 +/- 1.3 ml per deciliter, P less than 0.05). CONCLUSIONS. In muscle phosphofructokinase deficiency, the oxidative capacity of muscle and the capacity for aerobic exercise vary according to the availability of blood-borne fuels. We believe that glucose infusion lowers exercise tolerance by inhibiting lipolysis and thus depriving muscle of oxidative substrate (plasma free fatty acids and ketones); this impairs the capacity of working muscle to extract oxygen and lowers maximal oxygen consumption.     

Cerebral glucose utilization in awake unstressed rats

Regional cerebral glucose utilization (rCMRGlc) was measured in awake unstressed rats by use of [2-14C]glucose autoradiography. Rats prepared by chronic catheterization of the jugular and epigastric veins were placed in specially designed chambers after surgery. The catheters were passed through the top of the chambers. Seven days after surgery, plasma glucose was normal (7.5 mM), plasma ketone bodies were low (0.13 mM), and body weight was maintained. Plasma epinephrine was 31 pg/ml; approximately 3% of the level for stressed (immobilized) rats. Injections and blood sampling through the catheters did not produce changes in heart rate, blood pressure, plasma catecholamines, or blood metabolites. Visual observations did not reveal any signs that the rats were aware of the [2-14C]glucose injections and the subsequent blood sampling. The rCMRGlc in stressed rats was significantly greater than in unstressed rats. This method provides a model for physiological studies in unstressed unanesthetized rats.