Cholinergic Involvement in Vascular and Glucoregulatory Actions of Insulin in Rats
http://www.100md.com
糖尿病学杂志 2006年第2期
1 Lipid Research Unit, Centre Hospitalier de l’Universitee Laval (CHUL) Research Center, Centre Hospitalier Universitaire de Queebec (CHUQ), Ste-Foy, Quebec, Canada
2 Diabetes Research Unit, CHUL Research Center, CHUQ, Ste-Foy, Quebec, Canada
Key Words: EDL, extensor digitorum longus
ABSTRACT
This study was designed to test the glucose metabolic and vasodilator actions of insulin in rats and its relation to cholinergic systemeCdependent mechanisms. The first group of rats had pulsed Doppler flow probes and intravascular catheters implanted to determine blood pressure, heart rate, and regional blood flows. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp technique carried out in the absence or presence of atropine. The second group of rats was used to determine the cholinergic contribution to in vivo insulin-mediated glucose utilization in individual muscles. Glucose uptake was examined by using [3H]2-deoxy-D-glucose. Muscarinic cholinergic blockade was found to significantly (P = 0.002) reduce insulin sensitivity and to completely abrogate the renal (P = 0.008) and hindquarter (P = 0.02) vasodilator responses to euglycemic infusion of insulin. A significant reduction in insulin-stimulated in vivo glucose uptake was also noted in soleus (P = 0.006), quadriceps (P = 0.03), gastrocnemius (P = 0.02), and extensor digitorum longus (EDL) (P = 0.001) muscles, when insulin was infused at a rate of 4 mU · kgeC1 · mineC1, whereas at the rate of 16 mU · kgeC1 · mineC1, a significant reduction in glucose uptake was only observed in EDL (P = 0.03) and quadriceps (P = 0.01) muscles. Together, these results demonstrate a potential role for cholinergic involvement with physiological insulin actions in glucose clearance and blood flow regulation in rats.
Insulin is well recognized for its ability to stimulate glucose uptake in skeletal muscles, heart, and adipose tissues. On a quantitative basis, skeletal muscle has been identified as the predominant site of insulin-stimulated glucose disposal and as a major tissue responsible for postprandial hyperglycemia in insulin-resistant states (1,2). In addition to its effects on glucose metabolism, insulin was shown to increase skeletal muscle blood flow and to decrease vascular resistance in humans (3). The hemodynamic responses to insulin have attracted a great deal of attention over the last few years, particularly in relation to its glucose lowering action, and studies in both experimental animal models and humans have confirmed the vasodilating action of insulin over a range of physiological and pharmacological insulin concentrations and by using different techniques (4eC7). The vascular effect of insulin has been proposed as an important physiological mechanism that amplifies the overall action of insulin to promote the disposal of glucose and other substrates (8). Particularly relevant to this is the demonstration that when the action of insulin on total blood flow (9) or capillary recruitment (10) is prevented in vivo, it concomitantly induces an acute state of insulin resistance. Among the most likely mechanism responsible for the insulin-hemodynamic effects, there is evidence indicating a role for endothelium-derived nitric oxide (NO). Abrogation of NO release by the NO synthase inhibitor NG-monomethyl-L-arginine was shown to prevent the vasodilating action of insulin in skeletal muscle (11,12), whereas a concomitant reduction in insulin sensitivity was also reported (8).
The mechanisms underlying insulin stimulation of NO release remain to be fully elucidated. Among the possible mechanisms, insulin could conceivably stimulate NO release by a direct, local effect at the vascular endothelium, as previously suggested (13), or indirectly through the activation of a cholinergically mediated stimulation of NO release, as proposed in liver (14). Evidence for cholinergic neurogenic vasodilation in skeletal muscle has been provided in many animal species (15eC18), and interruption of nerve supply to skeletal muscle was shown to result in the development of insulin resistance in the affected muscle (19). Furthermore, applying atropine during the infusion of insulin in anesthetized cats and rats was reported to reduce the effect of insulin on glucose uptake in skeletal muscle (14,20). Therefore, in light of these previous findings, we hypothesized that part of the mechanism underlying the glucose metabolic and vasodilator actions of insulin involves a cholinergic component. Thus, in continuity with our previous work indicating a blood flow regulatory action of insulin in rats (5,21) and its paradoxical effect in insulin-resistant rats (5,22), we undertook the present study to examine the involvement of cholinergic systemeCdependent mechanisms in vascular and glucoregulatory actions of insulin in rats. In this study, rats were chronically instrumented with intravascular catheters and pulsed Doppler flow probes to assess the effect of an acute treatment with atropine on vascular responses to euglycemic infusion of insulin and to determine insulin sensitivity in conscious, unrestrained rats. Furthermore, in the present study, the influence of cholinergic mechanisms on insulin action on glucose uptake in muscles was examined in vivo in conscious and undisturbed rats.
RESEARCH DESIGN AND METHODS
All experimental procedures were approved by the animal care and handling committee of Laval University and conformed to the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Male Sprague-Dawley rats (Charles River, St. Constant, Canada) weighing 250eC300 g were housed in stainless steel cages. They were randomly divided into two groups. One group (n = 78) was used for euglycemic-hyperinsulinemic clamp studies and hemodynamic measurements in conscious rats. In this group of rats, there were two surgical procedures undertaken in all animals. The first surgical procedure was to place the flow probes around specific arteries, and 7 days later, the second surgical procedure was to place the vascular catheters. Then, a euglycemic-hyperinsulinemic clamp study was conducted 72 h after the second surgical step. The second group of rats (n = 37) was used to determine in vivo glucose utilization in individual muscles. In this group of rats, only one surgical procedure was undertaken to place the vascular catheters.
Surgical preparation.
The rats were anesthetized with a mixture of ketamine and xylazine (100 and 10 mg/kg i.p., respectively) and had pulsed Doppler flow probes implanted around the left renal and superior mesenteric arteries and the lower abdominal aorta to monitor renal, mesenteric, and hindquarter blood flows, according to Gardiner and Bennett (23) and as previously described (5). After surgery, the rats were given subcutaneous injections of buprenorphine (0.05 mg/kg), returned to their cages, and allowed to recover for at least 7 days. After the recovery period, the rats were reanesthetized (as above), and the leads of the implanted probes were soldered to a microconnector (Microtech). Three separate catheters were implanted in the right jugular vein (for intravenous infusions) and one catheter in left femoral artery (for blood pressure and heart rate measurements). The catheters were tunneled subcutaneously to emerge at the same point as the probe wires. The rats were given subcutaneous injections of buprenorphine (0.05 mg/kg) and returned to their cages. Recovery after surgery was deemed satisfactory by the resumption of growth and normalization of 24-h food intake the day after this second surgical procedure. Experiments began 72 h after this last surgical step.
Euglycemic-hyperinsulinemic clamp studies.
The rats were deprived of food for 10eC12 h before the glucose clamp study. Before each experiment, blood glucose and plasma insulin were determined, and the resting heart rate, blood pressure, and regional blood flows were recorded over 15 min in quiet, unrestrained rats. After that time, the first group of rats (n = 50) received a continuous intravenous infusion of saline (0.9% NaCl), and the second group (n = 28) received atropine (1 mg/kg, administered intravenously as a bolus, followed by a continuous intravenous infusion of 1 mg · kgeC1 · heC1). The rate of infusion was 0.3 ml/h. The two groups of rats were further divided in three subgroups each. The euglycemic-hyperinsulinemic clamp was performed in two subgroups receiving saline only (n = 34) and in two other subgroups receiving atropine (n = 18). The other subgroups of saline-treated (n = 16) or atropine-treated (n = 10) rats were infused with saline/BSA 0.2%, instead of insulin and dextrose, to approximately match the saline load delivered during the clamp studies. Sixty minutes after the beginning of intravenous infusion of atropine or saline, a second blood sample (0.3 ml) was collected for blood glucose and plasma insulin determinations. The euglycemic-hyperinsulinemic clamp was then carried out over 120 min in the designated groups as previously described (5). Insulin was infused at a rate of 4 or 16 mU · kgeC1 · mineC1 in the four subgroups of rats receiving atropine or saline (only one dose of insulin was tested per subgroup). Heart rate, blood pressure, and regional blood flows were continuously measured.
Effect of atropine on in vivo [3H]2-deoxy-D-glucose uptake in individual muscles.
The second group of rats (n = 37) was used for measurement of steady-state insulin-stimulated glucose uptake in individual muscles, during a euglycemic infusion of insulin (at 4 or 16 mU · kgeC1 · mineC1) carried out in the absence or presence of atropine, as described above. This was performed by measuring the incorporation of [3H]2-deoxy-D-glucose according to Kreagen et al. (24) and as previously described (21). Briefly, a bolus injection of 250 e藽i/kg [3H]2-deoxy-D-glucose and 25 e藽i/kg [14C]sucrose in a 0.5-ml saline solution were injected during the last 25 min of a euglycemic clamp performed at the doses of 4 or 16 mU · kgeC1 · mineC1 insulin and in the absence or presence of atropine. Blood samples were taken at regular intervals (2.5, 5, 7.5, 10, 15, and 20 min) after the bolus injection for determination of plasma glucose, [3H]2-deoxy-D-glucose, and [14C]sucrose. Twenty-five minutes after the bolus, the animals were rapidly killed by decapitation, and hindlimb muscles (soleus, extensor digitorum longus [EDL], gastrocnemius, and quadriceps) were rapidly excised, frozen into liquid nitrogen, and stored at eC80°C. Subsequently, tissue samples (30eC50 mg) were dissolved in 1 ml 0.5 mol/l ammonium hydroxide at 55°C for 16 h. Thereafter, hydrogen peroxide (30%) was added for 60 min at 55°C to decrease quenching, followed by the addition of 10 ml scintillation fluid (Amersham, Mississauga, Ontario, Canada). The samples and plasma (20 e蘬) were then counted in a liquid scintillation counter (Wallach 1409). The accumulation of [3H]2-deoxy-D-glucose in muscle, corrected for the extracellular space with [14C]sucrose, was used as an index of glucose uptake rates (25).
Drugs.
The drugs used were atropine (Sigma Aldrich, Oakville, Ontario, Canada), dextrose (Sigma Aldrich), BSA (Sigma Aldrich), insulin (Humulin R; Eli Lilly, Toronto, Ontario, Canada), [3H]2-deoxy-D-glucose (Perkin Elmer, Woodbridge, Ontario, Canada), [14C]sucrose (Mandel-NEN Lifesciences, Guelph, Ontario, Canada), buprenorphine (Schering-Plough, Welwyn Garden City, Hertfordshire, U.K.), ketamine (Rogar/STB, London, Ontario, Canada), and xylazine (Bayer, Toronto, Ontario, Canada).
Analytic methods.
Blood for plasma glucose and insulin determinations in the basal state and during insulin infusion was drawn, put in untreated polypropylene tubes, and centrifuged with an Eppendorf microcentrifuge (Minimax; International Equipment). Plasma was stored at eC20°C until assay. The glucose concentration of the supernatant was measured by the glucose oxidase method (26) using a glucose analyzer (Technicon RA-XT), and plasma insulin level was measured by radioimmunoassay using porcine insulin standards and polyethylene glycol for separation (27).
Data analysis.
Data are means ± SE; n is the number of observations. Data were analyzed for statistical significance by an ANOVA for repeated measurements. Post hoc comparisons were made using Fisher’s test. A P value <0.05 was taken to indicate a significant difference.
RESULTS
Table 1 illustrates the effects of continuous intravenous infusion of atropine on plasma levels of insulin and glucose and baseline values of heart rate, mean arterial blood pressure, regional blood flows, and vascular conductances in conscious, unrestrained rats. Thus, the continuous intravenous infusion of atropine had no effect on plasma glucose or insulin concentrations. However, the infusion of atropine was found to slightly but significantly increase basal heart rate and mean arterial blood pressure values and to decrease basal superior mesenteric blood flow and vascular conductance values, whereas no changes in resting values of renal or hindquarter blood flows or vascular conductances were noted.
Hemodynamic responses to insulin during the euglycemic-hyperinsulinemic clamp.
Figure 1A and B shows that insulin infusion at a rate of 4 or 16 mU · kgeC1 · mineC1 caused significant increases in renal and hindquarter blood flows but had no effect on heart rate or mean arterial blood pressure compared with measurements after a control infusion of vehicle (saline/0.2% BSA). Furthermore, a significant decrease in superior mesenteric flow was observed at the highest dose of insulin tested (Fig. 1B). These cardiovascular responses were associated with increases in renal (Fig. 2A and B) and hindquarter vascular conductances (Fig. 2B) and decreases in superior mesenteric vascular conductance (Fig. 2B) when compared with the effects of control infusion of vehicle.
In rats receiving atropine, the same infusions of insulin were found to cause slight but significant increases in heart rate (Fig. 1B) and mean blood pressure (Fig. 1A and B), when compared with the effects of control infusion of vehicle in atropine-treated rats. These responses did not differ from those observed in untreated rats, although in those animals, the heart rate and blood pressure responses to insulin did not reach the level of significance. However, the increases in renal and hindquarter flows and vascular conductances previously noted in untreated rats were completely abolished by the treatment with atropine (Figs. 1 and 2). A marked and long-lasting hindquarter vasoconstriction was observed at the dose of 4 mU · kgeC1 · mineC1 insulin (Fig. 2A). Moreover, there were significant decreases in superior mesenteric blood flows and vascular conductances (Figs. 1 and 2), which did not differ from those observed in rats receiving insulin alone.
Responses during euglycemic-hyperinsulinemic clamp.
During the euglycemic-hyperinsulinemic clamp, which was performed at an insulin infusion rate of 4 or 16 mU · kgeC1 · mineC1 and in the presence or absence of atropine, we found that for each dose of insulin tested, fasting plasma insulin levels rose acutely and achieved similar plateaus, according to the dose of insulin, whereas normal plasma glucose levels were maintained in every group of rats (Table 2). However, the average glucose infusion rate required to maintain euglycemia during the last hour of the clamp, the conditions of which closely approximated a steady-state insulin concentration and which represented the whole-body glucose utilization, was significantly smaller in the atropine-treated group than in the group receiving only insulin at the dose of 4 mU · kgeC1 · mineC1 (Table 2). This difference was not observed when insulin was infused at a rate of 16 mU · kgeC1 · mineC1 in both experimental groups.
Effect of atropine on in vivo [3H]2-deoxy-D-glucose uptake in individual muscles.
Figure 3 shows that in untreated rats, the euglycemic infusion of insulin at a rate of 4 or 16 mU · kgeC1 · mineC1 elicited significant increases in glucose uptake in soleus, quadriceps, gastrocnemius, and EDL muscles when compared with that measured after a control infusion of vehicle. However, atropine treatment was found to markedly and significantly reduce (50%) in vivo insulin-stimulated glucose uptake in every studied muscle, when insulin was infused at a rate of 4 mU · kgeC1 · mineC1. However, at the rate of 16 mU · kgeC1 · mineC1, a slight but significant reduction in glucose uptake was only observed in EDL and quadriceps muscles. In the absence of insulin infusion, no effect of atropine was noted on basal glucose uptake in any muscles, when compared with that measured after a control infusion of vehicle only.
DISCUSSION
Atropine and insulin sensitivity.
The present study indicates that at the whole-animal level acute treatment with atropine significantly reduces insulin sensitivity, as determined using a euglycemic-hyperinsulinemic clamp performed in conscious rats at a low dose of insulin. Thus, at the dose of 4 mU · kgeC1 · mineC1 insulin, we found that the glucose infusion rate required to maintain euglycemia in the face of physiological hyperinsulinemia was significantly lower (25%) in the group of rats receiving atropine than in the control group. However, atropine had no effect on basal plasma glucose or insulin concentrations, suggesting that muscarinic cholinergic antagonism has no net systemic glucoregulatory effect in terms of the plasma glucose concentration. Moreover, during the euglycemic infusion of insulin, atropine had no effect on steady-state plasma insulin concentrations and variability of plasma glucose concentrations. However, the reduction in insulin sensitivity was no longer observed at supra-physiological levels of insulin. Thus, it is likely that the apparent insulin resistance noted at a low dose of insulin was overcome at higher insulin levels (16 mU · kgeC1 · mineC1) (5). These latter findings contrast with those of Xie and Lautt (28), who used a higher dose of insulin than us and showed a reduction in insulin sensitivity in fed anesthetized rats intravenously treated with atropine. Part of the discrepancy could be explained by differences in the prandial status of the animals, because fed rats would be anticipated to be more responsive to insulin action and to have a higher degree of basal parasympathetic tone than overnight-fasted rats (29).
Our results are compatible with the involvement of cholinergic input on the effect of insulin on whole-body clearance of glucose. This contention is supported by several pieces of evidence implicating the parasympathetic nerves on glucoregulation and indicating that parasympathetic neuropathy could be contributory to insulin resistance. Insulin resistance is commonly observed in clinical conditions that are associated with autonomic dysfunction, such as hypertension (30), diabetes (31), and obesity (32). Furthermore, electrical stimulation of the lateral hypothalamic nucleus (parasympathetic area) was shown to reduce blood glucose level in rabbits (33), and vagal nerve stimulation (34) and selective stimulation of hepatic efferent parasympathetic nerves (35,36) were found to decrease hepatic glucose output and increase hepatic glucose uptake and glycogen synthesis. In recently published studies, and using a modified euglycemic technique to quantify insulin sensitivity, Lautt (14) described a novel neurohormonal mechanism by which postprandial elevation in insulin levels activates a hepatic parasympathetic reflex release of a putative hepatic insulineCsensitizing substance, which sensitizes skeletal muscle to the effects of insulin. The reflex is mediated by acetylcholine, which binds to muscarinic receptors and stimulates the production of NO within the liver (14). Surgical denervation of the liver, cholinergic blockade by using atropine, or prevention of hepatic NO liberation by the use of NO synthase antagonists were all found to cause severe insulin resistance that was attributed to the blockade of hepatic insulineCsensitizing substance release (14). Although the present findings are unable to support or deny the hepatic autonomic reflex control of peripheral insulin action, as proposed by Lautt (14), our results agreed, however, with the presence of a cholinergic mediation of the action of insulin on glucose metabolism. Furthermore, in the experiments in which we have combined atropine treatment with in vivo administration of [3H]2-deoxy-D-glucose during insulin clamps, which permits maintenance of insulin at a steady-state level during investigation of its effect on glucose uptake in muscles, we found a marked reduction (50%) in insulin-mediated glucose uptake by a physiological dose (4 mU · kgeC1 · mineC1) in every tested muscle. At the dose of 16 mU · kgeC1 · mineC1 insulin, a significant reduction in glucose uptake in the presence of atropine was only noted in EDL and quadriceps muscles. These results highly suggest the existence of a cholinergic mediation of the actions of insulin on glucose uptake and blood flow regulation (see below) at the skeletal muscle level. Furthermore, the demonstration here that atropine treatment alone (i.e., in the absence of insulin) did not affect basal glucose uptake in any tested muscles excluded the possibility that glucose uptake may have already been substantially inhibited, before insulin addition (i.e., as a result from a redistribution of blood flow in the leg muscles to the detriment of glucose uptake).
Atropine and insulin-mediated hemodynamic responses.
In untreated rats, we found that the euglycemic infusion of insulin elicited vasodilations in renal and hindquarter vascular beds and a transient vasoconstriction in the superior mesenteric vascular bed but no changes in mean arterial blood pressure or heart rate. These hemodynamic responses are consistent with those we previously reported in normal Wistar, Wistar-Kyoto, and Sprague-Dawley rats (5,21). In contrast, in the group of rats receiving atropine, we found that the physiological effects of insulin to vasodilate skeletal muscle and renal vasculatures were completely abolished, whereas the superior mesenteric vasoconstrictor response to insulin was not affected by the treatment. These observations highly suggest that cholinergic vasodilation could play an important role in the mediation of the vascular response to insulin, particularly at the skeletal muscle level. Our results are consistent with those of Mahajan et al. (37), showing that local methacholine enhances insulin-mediated muscle glucose uptake in vivo by augmenting capillary recruitment in anesthetized rats, but our results contrast with those of Randin et al. (38), demonstrating an absence of effect of atropine on the vascular response to euglycemic hyperinsulinemia in lean healthy humans. The reason for the conflicting results is not readily apparent. Differences in the experimental design, such as the dose of insulin, amount of atropine infused, and the site and method used to measure blood flow, may have contributed to these seemingly disparate results. Furthermore, we must consider that the extrapolation of the data in rats to humans may be difficult, because evidence for cholinergic vasodilator nerves in human skeletal muscle (in contrast to many animal species) has not been established (although the presence of local non-neuronal cholinergic mechanisms is not excluded) (39). It appears, therefore, that differences between rats and humans, in the effects of atropine on insulin stimulation of whole-body glucose uptake and muscle blood flow could be related to anatomical rather than to methodological differences.
Previous studies have clearly demonstrated that the insulin-mediated skeletal muscle vasodilation is mediated by NO, because this effect is abolished by NG-monomethyl-L-arginine (11,12), and by inhibition of tetrahydrobiopterin synthesis, a cofactor essential for NO synthesis (40). It was suggested that insulin stimulates NO release by a direct, local effect at the vascular endothelium (13). However, the present findings raise the possibility that a cholinergically mediated stimulation of NO-dependent vasodilatory mechanisms could contribute, at least in part, to the endothelium-dependent vasodilator response to insulin. Alternatively, our results could also indicate that part of the vasodilator response to insulin is related to inhibition of adrenergic neurotransmission by stimulation of inhibitory prejunctional cholinergic receptors located on vascular adrenergic nerve endings (41). Thus, in the presence of atropine, the vasodilator response to euglycemic infusion of insulin might have been limited by stimulation of sympathetic vasoconstrictor outflow. This contention is supported by previous studies carried out in humans and animals and demonstrating that insulin administration stimulates sympathetic nerve activity as determined by measurements of plasma catecholamine levels or by direct microneurographic recordings of sympathetic nerve action potentials targeted specifically at the skeletal musculature (42eC45). Thus, in the presence of atropine, it is likely that the endothelium-dependent vasodilator response to insulin was overcome by unopposed sympathetic vasoconstrictor influences.
Impairment in the vascular responses to insulin is thought to contribute to deficient uptake of glucose by peripheral tissues (presumably because of less delivery of glucose) (3). Therefore, according to that concept, it is likely that cholinergic blockade during the infusion of insulin (at a rate of 4 mU · kgeC1 · mineC1) has contributed to reduce insulin sensitivity by altering the hemodynamic responses to insulin, thus restricting insulin and glucose distribution through insulin-sensitive tissues and then reducing insulin action on glucose extraction at the level of skeletal muscle. Furthermore, the present findings raise the possibility that interruption of the cholinergic contribution to the blood flow response to insulin may have contributed, at least in part, to the state of insulin resistance previously reported by Buse and Buse (19) in denervated skeletal muscles, although this is not supported by a recent study using a different approach and technique to measure insulin sensitivity and blood flow in anesthetized rats (46). At the highest dose of insulin tested (16 mU · kgeC1 · mineC1), we found that the reduction in insulin sensitivity previously noted at the low dose of insulin was no more observed, despite a persistent defect in the vascular responses to insulin in the atropine-treated rats and a slight but significant reduction in glucose uptake in some but not all muscles in vivo (EDL and quadriceps only). The failure of atropine to achieve the same outcome at a higher dose of insulin might suggest that at a supra-physiological concentration of insulin (2,700 pmol/l), the "atropine-sensitive" insulin blood flow effect is not rate-limiting on glucose uptake, as previously described for physiological insulin and that steps in insulin action beyond any effect on blood flow are important determinants of muscle glucose uptake.
In conclusion, the overall concept presented in this study is novel and original. Taken together, the results of our experiments carried out in vivo strongly suggest that in rats, a high percentage of insulin-mediated muscle glucose uptake by a physiological dose involves a cholinergic mechanism. This atropine-sensitive effect is largely attributable to the vascular action of insulin in rats.
ACKNOWLEDGMENTS
H.B. has received grants from the Heart and Stroke Foundation of Quebec and the Canadian Institutes of Health Research.
We thank Marie Tremblay for her expert assistance.
REFERENCES
Baron AD, Brechtel G, Wallace P, Edelman SV: Rates and tissue sites of non-insulin and insulin mediated glucose uptake in humans. Am J Physiol 255:E769eCE774, 1988
Capaldo B, Lembo G, Napoli R, Rendina V, Albano G, Sacca L, Trimarco B: Skeletal muscle is a primarily site of insulin resistance in essential hypertension. Metabolism 40:1320eC1322, 1991
Baron AD: Hemodynamic actions of insulin. Am J Physiol 267:E187eCE202, 1994
Liang CS, Doherty JU, Faillace R, Maekawa K, Arnold S, Gavras H, Hood WB: Insulin infusion in conscious dogs: effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J Clin Invest 69:1321eC1336, 1982
Pitre M, Nadeau A, Bachelard H: Insulin sensitivity and hemodynamic responses to insulin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol 271:E658eCE668, 1996
Cleland SJ, Petrie JR, Ueda S, Elliott HL, Connell JMC: Insulin-mediated vasodilation and glucose uptake are functionally linked in humans. Hypertension 33:554eC558, 1999
Clark ADH, Barrett EJ, Rattigan S, Wallis MG, Clark MG: Insulin stimulated laser Doppler signal by rat muscle in vivo, consistent with nutritive flow recruitment. Clin Sci 100:283eC290, 2001
Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G: Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest 96:786eC792, 1995
Baron AD, Clark MG: Role of blood flow in the regulation of muscle glucose uptake. Annu Rev Nutr 17:487eC499, 1997
Rattigan S, Clark MG, Barrett EJ: Acute vasoconstriction-induced insulin resistance in rat muscle in vivo. Diabetes 48:564eC569, 1999
Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod P: Nitric oxide release accounts for insulin’s vascular effects in humans. J Clin Invest 94:2511eC2515, 1994
Steinberg HO, Brechtel G, Johnson A, Fineberg N, Baron AD: Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent: a novel action of insulin to increase nitric oxide release. J Clin Invest 94:1172eC1179, 1994
Sartori C, Trueb L, Nicod P, Scherrer U: Effects of sympathectomy and nitric oxide synthase inhibition on vascular actions of insulin in humans. Hypertension 34:586eC589, 1999
Lautt WW: The HISS story overview: a novel hepatic neurohumoral regulation of peripheral insulin sensitivity in health and diabetes. Can J Physiol Pharmacol 77:553eC562, 1999
Folkow B, Haeger K, Uvns B: Cholinergic vasodilator nerves in the sympathetic outflow to the muscles of the hind limbs of the cat. Acta Physiol Scand 15:401eC411, 1948
Loke KE, Sobey CG, Dusting GJ, Woodman OL: Cholinergic neurogenic vasodilation is mediated by nitric oxide in the dog hindlimb. Cardiovasc Res 28:542eC547, 1994
Uvns B: Cholinergic vasodilator nerves. Fed Proc 25:1618eC1622, 1966
Bolme P, Fuxe K: Adrenergic and cholinergic nerve terminals in skeletal muscle vessels. Acta Physiol Scand 78:52eC59, 1970
Buse MG, Buse J: Glucose uptake and the response to insulin in the isolated rat diaphragm: the effect of denervation. Diabetes 8:218eC225, 1959
Xie H, Lautt WW: Induction of insulin resistance by cholinergic blockade with atropine in the cat. J Auton Pharmacol 15:361eC369, 1995
Gaudreault N, Santuree M, Pitre M, Nadeau A, Marette A, Bachelard H: Effects of insulin on regional blood flow and glucose uptake in Wistar and Sprague Dawley rats. Metabolism 50:65eC73, 2001
Santuree M, Pitre M, Marette A, Deshaies Y, Lemieux C, Larivieere R, Nadeau A, Bachelard H: Induction of insulin resistance by high-sucrose feeding does not raise mean arterial blood pressure but impairs hemodynamic responses to insulin in rats. Br J Pharmacol 137:185eC196, 2002
Gardiner SM, Bennett T: Regional haemodynamic responses to adrenoceptor antagonism in conscious rats. Am J Physiol 255:H813eCH824, 1988
Kraegen EW, James DE, Jenkins AB, Chisolm DJ: Dose-response curves for in vivo insulin sensitivity in individual tissues in rats. Am J Physiol 248:E353eCE362, 1985
Roy D, Perreault M, Marette A: Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am J Physiol 274:E692eCE699, 1998
Richterich R, Dauwalder H: Zur bertimmung der plasmaglucokonzentration mit der hexokinase-glucose-6-phosphat-dehydrogenase methode. Schweiz Med Wochenschr 101:615eC618, 1971
Desbuquois B, Aurbach GD: Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocr Metab 37:732eC738, 1971
Xie H, Lautt WW: Insulin resistance caused by hepatic cholinergic interruption and reversed by acetylcholine administration. Am J Physiol 271:E587eCE592, 1996
Lautt WW, Macedo MP, Sadri P, Takayama S, Duarte-Ramos F, Legare DJ: Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting. Am J Physiol 281:G29eCG36, 2001
Julius S: Autonomic nervous dysfunction in essential hypertension. Diabetes Care 14:249eC259, 1991
Valensi P, Huard JP, Giroux C, Attali JR: Factors involved in cardiac autonomic neuropathy in diabetic patients. J Diabetes Complications 11:180eC187, 1997
Richter WO, Geib HC, Aleksic S, Schwandt P: Cardiac autonomic nerve function and insulin sensitivity in obese subjects. Int J Obes Relat Metab Disord 20:966eC969, 1996
Shimazu T, Fukuda A, Ban T: Reciprocal influence of the ventromedial and lateral hypothalamic nuclei on blood glucose level and liver glycogen content. Nature 210:1178eC1179, 1966
Shimazu T: Regulation of glycogen metabolism in liver by the autonomic nervous system: activation of glycogen synthetase by vagal stimulation. Biochim Biophys Acta 252:28eC38, 1971
Gardemann A, Jungermann K: Control of glucose balance in the perfused rat liver by the parasympathetic innervation. Biol Chem Hoppe-Seyler 367:559eC566, 1986
Lautt WW, Wong C: Hepatic parasympathetic neural effect on glucose balance in the intact liver. Can J Physiol Pharmacol 56:679eC682, 1978
Mahajan H, Richards SM, Rattigan S, Clark MG: Local methacholine but not bradykinin potentiates insulin-mediated glucose uptake in muscle in vivo by augmenting capillary recruitment. Diabetologia 47:2226eC2234, 2004
Randin D, Vollenweider P, Tappy L, Jequier E, Nicod P, Scherrer U: Effects of adrenergic and cholinergic blockade on insulin induced stimulation of calf blood flow in humans. Am J Physiol 266:R809eCR816, 1994
Joyner MJ, Dietz NM: Sympathetic vasodilation in human muscle. Acta Physiol Scand 177:329eC336, 2003
Verma S, Arikawa E, Yao L, Laher I, McNeil JH: Insulin-induced vasodilation is dependent on tetrahydrobiopterin synthesis. Metabolism 47:1037eC1039, 1998
Shepherd JT: Circulation to skeletal muscle. In Handbook of Physiology. The Cardiovascular System: Peripheral Circulation and Organ Blood Flow. Shepherd JT, Abboud FM, Eds. Bethesda, MD, American Physiological Society, 1983, p.319eC370
Rowe JW, Young JB, Minaker KL, Stevens AL, Pallotta J, Landsberg L: Effect of insulin and glucose infusions on sympathetic nervous system activity in normal man. Diabetes 30:219eC225, 1981
Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL: Hyperinsulinemia produces both sympathetic neural activation and vasodilatation in normal humans. J Clin Invest 87:2246eC2252, 1991
Berne C, Fagius J, Pollare L, Hjemdahl P: The sympathetic response to euglycemic hyperinsulinaemia: evidence from microelectrode nerve recordings in healthy subjects. Diabetologia 35:873eC879, 1992
Vollenweider P, Tappy L, Owlya R, Jeequier E, Nicod P, Scherrer U: Insulin-induced sympathetic activation and vasodilation in skeletal muscle: effect of insulin resistance in lean subjects. Diabetes 44:641eC645, 1995
Turinsky J, Damrau-Abney A, Loegering DJ: Blood flow and glucose uptake in denervated, insulin-resistant muscles. Am J Physiol 274:R311eCR317, 1998(Martin Leevesque, Marta S)
2 Diabetes Research Unit, CHUL Research Center, CHUQ, Ste-Foy, Quebec, Canada
Key Words: EDL, extensor digitorum longus
ABSTRACT
This study was designed to test the glucose metabolic and vasodilator actions of insulin in rats and its relation to cholinergic systemeCdependent mechanisms. The first group of rats had pulsed Doppler flow probes and intravascular catheters implanted to determine blood pressure, heart rate, and regional blood flows. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp technique carried out in the absence or presence of atropine. The second group of rats was used to determine the cholinergic contribution to in vivo insulin-mediated glucose utilization in individual muscles. Glucose uptake was examined by using [3H]2-deoxy-D-glucose. Muscarinic cholinergic blockade was found to significantly (P = 0.002) reduce insulin sensitivity and to completely abrogate the renal (P = 0.008) and hindquarter (P = 0.02) vasodilator responses to euglycemic infusion of insulin. A significant reduction in insulin-stimulated in vivo glucose uptake was also noted in soleus (P = 0.006), quadriceps (P = 0.03), gastrocnemius (P = 0.02), and extensor digitorum longus (EDL) (P = 0.001) muscles, when insulin was infused at a rate of 4 mU · kgeC1 · mineC1, whereas at the rate of 16 mU · kgeC1 · mineC1, a significant reduction in glucose uptake was only observed in EDL (P = 0.03) and quadriceps (P = 0.01) muscles. Together, these results demonstrate a potential role for cholinergic involvement with physiological insulin actions in glucose clearance and blood flow regulation in rats.
Insulin is well recognized for its ability to stimulate glucose uptake in skeletal muscles, heart, and adipose tissues. On a quantitative basis, skeletal muscle has been identified as the predominant site of insulin-stimulated glucose disposal and as a major tissue responsible for postprandial hyperglycemia in insulin-resistant states (1,2). In addition to its effects on glucose metabolism, insulin was shown to increase skeletal muscle blood flow and to decrease vascular resistance in humans (3). The hemodynamic responses to insulin have attracted a great deal of attention over the last few years, particularly in relation to its glucose lowering action, and studies in both experimental animal models and humans have confirmed the vasodilating action of insulin over a range of physiological and pharmacological insulin concentrations and by using different techniques (4eC7). The vascular effect of insulin has been proposed as an important physiological mechanism that amplifies the overall action of insulin to promote the disposal of glucose and other substrates (8). Particularly relevant to this is the demonstration that when the action of insulin on total blood flow (9) or capillary recruitment (10) is prevented in vivo, it concomitantly induces an acute state of insulin resistance. Among the most likely mechanism responsible for the insulin-hemodynamic effects, there is evidence indicating a role for endothelium-derived nitric oxide (NO). Abrogation of NO release by the NO synthase inhibitor NG-monomethyl-L-arginine was shown to prevent the vasodilating action of insulin in skeletal muscle (11,12), whereas a concomitant reduction in insulin sensitivity was also reported (8).
The mechanisms underlying insulin stimulation of NO release remain to be fully elucidated. Among the possible mechanisms, insulin could conceivably stimulate NO release by a direct, local effect at the vascular endothelium, as previously suggested (13), or indirectly through the activation of a cholinergically mediated stimulation of NO release, as proposed in liver (14). Evidence for cholinergic neurogenic vasodilation in skeletal muscle has been provided in many animal species (15eC18), and interruption of nerve supply to skeletal muscle was shown to result in the development of insulin resistance in the affected muscle (19). Furthermore, applying atropine during the infusion of insulin in anesthetized cats and rats was reported to reduce the effect of insulin on glucose uptake in skeletal muscle (14,20). Therefore, in light of these previous findings, we hypothesized that part of the mechanism underlying the glucose metabolic and vasodilator actions of insulin involves a cholinergic component. Thus, in continuity with our previous work indicating a blood flow regulatory action of insulin in rats (5,21) and its paradoxical effect in insulin-resistant rats (5,22), we undertook the present study to examine the involvement of cholinergic systemeCdependent mechanisms in vascular and glucoregulatory actions of insulin in rats. In this study, rats were chronically instrumented with intravascular catheters and pulsed Doppler flow probes to assess the effect of an acute treatment with atropine on vascular responses to euglycemic infusion of insulin and to determine insulin sensitivity in conscious, unrestrained rats. Furthermore, in the present study, the influence of cholinergic mechanisms on insulin action on glucose uptake in muscles was examined in vivo in conscious and undisturbed rats.
RESEARCH DESIGN AND METHODS
All experimental procedures were approved by the animal care and handling committee of Laval University and conformed to the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health. Male Sprague-Dawley rats (Charles River, St. Constant, Canada) weighing 250eC300 g were housed in stainless steel cages. They were randomly divided into two groups. One group (n = 78) was used for euglycemic-hyperinsulinemic clamp studies and hemodynamic measurements in conscious rats. In this group of rats, there were two surgical procedures undertaken in all animals. The first surgical procedure was to place the flow probes around specific arteries, and 7 days later, the second surgical procedure was to place the vascular catheters. Then, a euglycemic-hyperinsulinemic clamp study was conducted 72 h after the second surgical step. The second group of rats (n = 37) was used to determine in vivo glucose utilization in individual muscles. In this group of rats, only one surgical procedure was undertaken to place the vascular catheters.
Surgical preparation.
The rats were anesthetized with a mixture of ketamine and xylazine (100 and 10 mg/kg i.p., respectively) and had pulsed Doppler flow probes implanted around the left renal and superior mesenteric arteries and the lower abdominal aorta to monitor renal, mesenteric, and hindquarter blood flows, according to Gardiner and Bennett (23) and as previously described (5). After surgery, the rats were given subcutaneous injections of buprenorphine (0.05 mg/kg), returned to their cages, and allowed to recover for at least 7 days. After the recovery period, the rats were reanesthetized (as above), and the leads of the implanted probes were soldered to a microconnector (Microtech). Three separate catheters were implanted in the right jugular vein (for intravenous infusions) and one catheter in left femoral artery (for blood pressure and heart rate measurements). The catheters were tunneled subcutaneously to emerge at the same point as the probe wires. The rats were given subcutaneous injections of buprenorphine (0.05 mg/kg) and returned to their cages. Recovery after surgery was deemed satisfactory by the resumption of growth and normalization of 24-h food intake the day after this second surgical procedure. Experiments began 72 h after this last surgical step.
Euglycemic-hyperinsulinemic clamp studies.
The rats were deprived of food for 10eC12 h before the glucose clamp study. Before each experiment, blood glucose and plasma insulin were determined, and the resting heart rate, blood pressure, and regional blood flows were recorded over 15 min in quiet, unrestrained rats. After that time, the first group of rats (n = 50) received a continuous intravenous infusion of saline (0.9% NaCl), and the second group (n = 28) received atropine (1 mg/kg, administered intravenously as a bolus, followed by a continuous intravenous infusion of 1 mg · kgeC1 · heC1). The rate of infusion was 0.3 ml/h. The two groups of rats were further divided in three subgroups each. The euglycemic-hyperinsulinemic clamp was performed in two subgroups receiving saline only (n = 34) and in two other subgroups receiving atropine (n = 18). The other subgroups of saline-treated (n = 16) or atropine-treated (n = 10) rats were infused with saline/BSA 0.2%, instead of insulin and dextrose, to approximately match the saline load delivered during the clamp studies. Sixty minutes after the beginning of intravenous infusion of atropine or saline, a second blood sample (0.3 ml) was collected for blood glucose and plasma insulin determinations. The euglycemic-hyperinsulinemic clamp was then carried out over 120 min in the designated groups as previously described (5). Insulin was infused at a rate of 4 or 16 mU · kgeC1 · mineC1 in the four subgroups of rats receiving atropine or saline (only one dose of insulin was tested per subgroup). Heart rate, blood pressure, and regional blood flows were continuously measured.
Effect of atropine on in vivo [3H]2-deoxy-D-glucose uptake in individual muscles.
The second group of rats (n = 37) was used for measurement of steady-state insulin-stimulated glucose uptake in individual muscles, during a euglycemic infusion of insulin (at 4 or 16 mU · kgeC1 · mineC1) carried out in the absence or presence of atropine, as described above. This was performed by measuring the incorporation of [3H]2-deoxy-D-glucose according to Kreagen et al. (24) and as previously described (21). Briefly, a bolus injection of 250 e藽i/kg [3H]2-deoxy-D-glucose and 25 e藽i/kg [14C]sucrose in a 0.5-ml saline solution were injected during the last 25 min of a euglycemic clamp performed at the doses of 4 or 16 mU · kgeC1 · mineC1 insulin and in the absence or presence of atropine. Blood samples were taken at regular intervals (2.5, 5, 7.5, 10, 15, and 20 min) after the bolus injection for determination of plasma glucose, [3H]2-deoxy-D-glucose, and [14C]sucrose. Twenty-five minutes after the bolus, the animals were rapidly killed by decapitation, and hindlimb muscles (soleus, extensor digitorum longus [EDL], gastrocnemius, and quadriceps) were rapidly excised, frozen into liquid nitrogen, and stored at eC80°C. Subsequently, tissue samples (30eC50 mg) were dissolved in 1 ml 0.5 mol/l ammonium hydroxide at 55°C for 16 h. Thereafter, hydrogen peroxide (30%) was added for 60 min at 55°C to decrease quenching, followed by the addition of 10 ml scintillation fluid (Amersham, Mississauga, Ontario, Canada). The samples and plasma (20 e蘬) were then counted in a liquid scintillation counter (Wallach 1409). The accumulation of [3H]2-deoxy-D-glucose in muscle, corrected for the extracellular space with [14C]sucrose, was used as an index of glucose uptake rates (25).
Drugs.
The drugs used were atropine (Sigma Aldrich, Oakville, Ontario, Canada), dextrose (Sigma Aldrich), BSA (Sigma Aldrich), insulin (Humulin R; Eli Lilly, Toronto, Ontario, Canada), [3H]2-deoxy-D-glucose (Perkin Elmer, Woodbridge, Ontario, Canada), [14C]sucrose (Mandel-NEN Lifesciences, Guelph, Ontario, Canada), buprenorphine (Schering-Plough, Welwyn Garden City, Hertfordshire, U.K.), ketamine (Rogar/STB, London, Ontario, Canada), and xylazine (Bayer, Toronto, Ontario, Canada).
Analytic methods.
Blood for plasma glucose and insulin determinations in the basal state and during insulin infusion was drawn, put in untreated polypropylene tubes, and centrifuged with an Eppendorf microcentrifuge (Minimax; International Equipment). Plasma was stored at eC20°C until assay. The glucose concentration of the supernatant was measured by the glucose oxidase method (26) using a glucose analyzer (Technicon RA-XT), and plasma insulin level was measured by radioimmunoassay using porcine insulin standards and polyethylene glycol for separation (27).
Data analysis.
Data are means ± SE; n is the number of observations. Data were analyzed for statistical significance by an ANOVA for repeated measurements. Post hoc comparisons were made using Fisher’s test. A P value <0.05 was taken to indicate a significant difference.
RESULTS
Table 1 illustrates the effects of continuous intravenous infusion of atropine on plasma levels of insulin and glucose and baseline values of heart rate, mean arterial blood pressure, regional blood flows, and vascular conductances in conscious, unrestrained rats. Thus, the continuous intravenous infusion of atropine had no effect on plasma glucose or insulin concentrations. However, the infusion of atropine was found to slightly but significantly increase basal heart rate and mean arterial blood pressure values and to decrease basal superior mesenteric blood flow and vascular conductance values, whereas no changes in resting values of renal or hindquarter blood flows or vascular conductances were noted.
Hemodynamic responses to insulin during the euglycemic-hyperinsulinemic clamp.
Figure 1A and B shows that insulin infusion at a rate of 4 or 16 mU · kgeC1 · mineC1 caused significant increases in renal and hindquarter blood flows but had no effect on heart rate or mean arterial blood pressure compared with measurements after a control infusion of vehicle (saline/0.2% BSA). Furthermore, a significant decrease in superior mesenteric flow was observed at the highest dose of insulin tested (Fig. 1B). These cardiovascular responses were associated with increases in renal (Fig. 2A and B) and hindquarter vascular conductances (Fig. 2B) and decreases in superior mesenteric vascular conductance (Fig. 2B) when compared with the effects of control infusion of vehicle.
In rats receiving atropine, the same infusions of insulin were found to cause slight but significant increases in heart rate (Fig. 1B) and mean blood pressure (Fig. 1A and B), when compared with the effects of control infusion of vehicle in atropine-treated rats. These responses did not differ from those observed in untreated rats, although in those animals, the heart rate and blood pressure responses to insulin did not reach the level of significance. However, the increases in renal and hindquarter flows and vascular conductances previously noted in untreated rats were completely abolished by the treatment with atropine (Figs. 1 and 2). A marked and long-lasting hindquarter vasoconstriction was observed at the dose of 4 mU · kgeC1 · mineC1 insulin (Fig. 2A). Moreover, there were significant decreases in superior mesenteric blood flows and vascular conductances (Figs. 1 and 2), which did not differ from those observed in rats receiving insulin alone.
Responses during euglycemic-hyperinsulinemic clamp.
During the euglycemic-hyperinsulinemic clamp, which was performed at an insulin infusion rate of 4 or 16 mU · kgeC1 · mineC1 and in the presence or absence of atropine, we found that for each dose of insulin tested, fasting plasma insulin levels rose acutely and achieved similar plateaus, according to the dose of insulin, whereas normal plasma glucose levels were maintained in every group of rats (Table 2). However, the average glucose infusion rate required to maintain euglycemia during the last hour of the clamp, the conditions of which closely approximated a steady-state insulin concentration and which represented the whole-body glucose utilization, was significantly smaller in the atropine-treated group than in the group receiving only insulin at the dose of 4 mU · kgeC1 · mineC1 (Table 2). This difference was not observed when insulin was infused at a rate of 16 mU · kgeC1 · mineC1 in both experimental groups.
Effect of atropine on in vivo [3H]2-deoxy-D-glucose uptake in individual muscles.
Figure 3 shows that in untreated rats, the euglycemic infusion of insulin at a rate of 4 or 16 mU · kgeC1 · mineC1 elicited significant increases in glucose uptake in soleus, quadriceps, gastrocnemius, and EDL muscles when compared with that measured after a control infusion of vehicle. However, atropine treatment was found to markedly and significantly reduce (50%) in vivo insulin-stimulated glucose uptake in every studied muscle, when insulin was infused at a rate of 4 mU · kgeC1 · mineC1. However, at the rate of 16 mU · kgeC1 · mineC1, a slight but significant reduction in glucose uptake was only observed in EDL and quadriceps muscles. In the absence of insulin infusion, no effect of atropine was noted on basal glucose uptake in any muscles, when compared with that measured after a control infusion of vehicle only.
DISCUSSION
Atropine and insulin sensitivity.
The present study indicates that at the whole-animal level acute treatment with atropine significantly reduces insulin sensitivity, as determined using a euglycemic-hyperinsulinemic clamp performed in conscious rats at a low dose of insulin. Thus, at the dose of 4 mU · kgeC1 · mineC1 insulin, we found that the glucose infusion rate required to maintain euglycemia in the face of physiological hyperinsulinemia was significantly lower (25%) in the group of rats receiving atropine than in the control group. However, atropine had no effect on basal plasma glucose or insulin concentrations, suggesting that muscarinic cholinergic antagonism has no net systemic glucoregulatory effect in terms of the plasma glucose concentration. Moreover, during the euglycemic infusion of insulin, atropine had no effect on steady-state plasma insulin concentrations and variability of plasma glucose concentrations. However, the reduction in insulin sensitivity was no longer observed at supra-physiological levels of insulin. Thus, it is likely that the apparent insulin resistance noted at a low dose of insulin was overcome at higher insulin levels (16 mU · kgeC1 · mineC1) (5). These latter findings contrast with those of Xie and Lautt (28), who used a higher dose of insulin than us and showed a reduction in insulin sensitivity in fed anesthetized rats intravenously treated with atropine. Part of the discrepancy could be explained by differences in the prandial status of the animals, because fed rats would be anticipated to be more responsive to insulin action and to have a higher degree of basal parasympathetic tone than overnight-fasted rats (29).
Our results are compatible with the involvement of cholinergic input on the effect of insulin on whole-body clearance of glucose. This contention is supported by several pieces of evidence implicating the parasympathetic nerves on glucoregulation and indicating that parasympathetic neuropathy could be contributory to insulin resistance. Insulin resistance is commonly observed in clinical conditions that are associated with autonomic dysfunction, such as hypertension (30), diabetes (31), and obesity (32). Furthermore, electrical stimulation of the lateral hypothalamic nucleus (parasympathetic area) was shown to reduce blood glucose level in rabbits (33), and vagal nerve stimulation (34) and selective stimulation of hepatic efferent parasympathetic nerves (35,36) were found to decrease hepatic glucose output and increase hepatic glucose uptake and glycogen synthesis. In recently published studies, and using a modified euglycemic technique to quantify insulin sensitivity, Lautt (14) described a novel neurohormonal mechanism by which postprandial elevation in insulin levels activates a hepatic parasympathetic reflex release of a putative hepatic insulineCsensitizing substance, which sensitizes skeletal muscle to the effects of insulin. The reflex is mediated by acetylcholine, which binds to muscarinic receptors and stimulates the production of NO within the liver (14). Surgical denervation of the liver, cholinergic blockade by using atropine, or prevention of hepatic NO liberation by the use of NO synthase antagonists were all found to cause severe insulin resistance that was attributed to the blockade of hepatic insulineCsensitizing substance release (14). Although the present findings are unable to support or deny the hepatic autonomic reflex control of peripheral insulin action, as proposed by Lautt (14), our results agreed, however, with the presence of a cholinergic mediation of the action of insulin on glucose metabolism. Furthermore, in the experiments in which we have combined atropine treatment with in vivo administration of [3H]2-deoxy-D-glucose during insulin clamps, which permits maintenance of insulin at a steady-state level during investigation of its effect on glucose uptake in muscles, we found a marked reduction (50%) in insulin-mediated glucose uptake by a physiological dose (4 mU · kgeC1 · mineC1) in every tested muscle. At the dose of 16 mU · kgeC1 · mineC1 insulin, a significant reduction in glucose uptake in the presence of atropine was only noted in EDL and quadriceps muscles. These results highly suggest the existence of a cholinergic mediation of the actions of insulin on glucose uptake and blood flow regulation (see below) at the skeletal muscle level. Furthermore, the demonstration here that atropine treatment alone (i.e., in the absence of insulin) did not affect basal glucose uptake in any tested muscles excluded the possibility that glucose uptake may have already been substantially inhibited, before insulin addition (i.e., as a result from a redistribution of blood flow in the leg muscles to the detriment of glucose uptake).
Atropine and insulin-mediated hemodynamic responses.
In untreated rats, we found that the euglycemic infusion of insulin elicited vasodilations in renal and hindquarter vascular beds and a transient vasoconstriction in the superior mesenteric vascular bed but no changes in mean arterial blood pressure or heart rate. These hemodynamic responses are consistent with those we previously reported in normal Wistar, Wistar-Kyoto, and Sprague-Dawley rats (5,21). In contrast, in the group of rats receiving atropine, we found that the physiological effects of insulin to vasodilate skeletal muscle and renal vasculatures were completely abolished, whereas the superior mesenteric vasoconstrictor response to insulin was not affected by the treatment. These observations highly suggest that cholinergic vasodilation could play an important role in the mediation of the vascular response to insulin, particularly at the skeletal muscle level. Our results are consistent with those of Mahajan et al. (37), showing that local methacholine enhances insulin-mediated muscle glucose uptake in vivo by augmenting capillary recruitment in anesthetized rats, but our results contrast with those of Randin et al. (38), demonstrating an absence of effect of atropine on the vascular response to euglycemic hyperinsulinemia in lean healthy humans. The reason for the conflicting results is not readily apparent. Differences in the experimental design, such as the dose of insulin, amount of atropine infused, and the site and method used to measure blood flow, may have contributed to these seemingly disparate results. Furthermore, we must consider that the extrapolation of the data in rats to humans may be difficult, because evidence for cholinergic vasodilator nerves in human skeletal muscle (in contrast to many animal species) has not been established (although the presence of local non-neuronal cholinergic mechanisms is not excluded) (39). It appears, therefore, that differences between rats and humans, in the effects of atropine on insulin stimulation of whole-body glucose uptake and muscle blood flow could be related to anatomical rather than to methodological differences.
Previous studies have clearly demonstrated that the insulin-mediated skeletal muscle vasodilation is mediated by NO, because this effect is abolished by NG-monomethyl-L-arginine (11,12), and by inhibition of tetrahydrobiopterin synthesis, a cofactor essential for NO synthesis (40). It was suggested that insulin stimulates NO release by a direct, local effect at the vascular endothelium (13). However, the present findings raise the possibility that a cholinergically mediated stimulation of NO-dependent vasodilatory mechanisms could contribute, at least in part, to the endothelium-dependent vasodilator response to insulin. Alternatively, our results could also indicate that part of the vasodilator response to insulin is related to inhibition of adrenergic neurotransmission by stimulation of inhibitory prejunctional cholinergic receptors located on vascular adrenergic nerve endings (41). Thus, in the presence of atropine, the vasodilator response to euglycemic infusion of insulin might have been limited by stimulation of sympathetic vasoconstrictor outflow. This contention is supported by previous studies carried out in humans and animals and demonstrating that insulin administration stimulates sympathetic nerve activity as determined by measurements of plasma catecholamine levels or by direct microneurographic recordings of sympathetic nerve action potentials targeted specifically at the skeletal musculature (42eC45). Thus, in the presence of atropine, it is likely that the endothelium-dependent vasodilator response to insulin was overcome by unopposed sympathetic vasoconstrictor influences.
Impairment in the vascular responses to insulin is thought to contribute to deficient uptake of glucose by peripheral tissues (presumably because of less delivery of glucose) (3). Therefore, according to that concept, it is likely that cholinergic blockade during the infusion of insulin (at a rate of 4 mU · kgeC1 · mineC1) has contributed to reduce insulin sensitivity by altering the hemodynamic responses to insulin, thus restricting insulin and glucose distribution through insulin-sensitive tissues and then reducing insulin action on glucose extraction at the level of skeletal muscle. Furthermore, the present findings raise the possibility that interruption of the cholinergic contribution to the blood flow response to insulin may have contributed, at least in part, to the state of insulin resistance previously reported by Buse and Buse (19) in denervated skeletal muscles, although this is not supported by a recent study using a different approach and technique to measure insulin sensitivity and blood flow in anesthetized rats (46). At the highest dose of insulin tested (16 mU · kgeC1 · mineC1), we found that the reduction in insulin sensitivity previously noted at the low dose of insulin was no more observed, despite a persistent defect in the vascular responses to insulin in the atropine-treated rats and a slight but significant reduction in glucose uptake in some but not all muscles in vivo (EDL and quadriceps only). The failure of atropine to achieve the same outcome at a higher dose of insulin might suggest that at a supra-physiological concentration of insulin (2,700 pmol/l), the "atropine-sensitive" insulin blood flow effect is not rate-limiting on glucose uptake, as previously described for physiological insulin and that steps in insulin action beyond any effect on blood flow are important determinants of muscle glucose uptake.
In conclusion, the overall concept presented in this study is novel and original. Taken together, the results of our experiments carried out in vivo strongly suggest that in rats, a high percentage of insulin-mediated muscle glucose uptake by a physiological dose involves a cholinergic mechanism. This atropine-sensitive effect is largely attributable to the vascular action of insulin in rats.
ACKNOWLEDGMENTS
H.B. has received grants from the Heart and Stroke Foundation of Quebec and the Canadian Institutes of Health Research.
We thank Marie Tremblay for her expert assistance.
REFERENCES
Baron AD, Brechtel G, Wallace P, Edelman SV: Rates and tissue sites of non-insulin and insulin mediated glucose uptake in humans. Am J Physiol 255:E769eCE774, 1988
Capaldo B, Lembo G, Napoli R, Rendina V, Albano G, Sacca L, Trimarco B: Skeletal muscle is a primarily site of insulin resistance in essential hypertension. Metabolism 40:1320eC1322, 1991
Baron AD: Hemodynamic actions of insulin. Am J Physiol 267:E187eCE202, 1994
Liang CS, Doherty JU, Faillace R, Maekawa K, Arnold S, Gavras H, Hood WB: Insulin infusion in conscious dogs: effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J Clin Invest 69:1321eC1336, 1982
Pitre M, Nadeau A, Bachelard H: Insulin sensitivity and hemodynamic responses to insulin in Wistar-Kyoto and spontaneously hypertensive rats. Am J Physiol 271:E658eCE668, 1996
Cleland SJ, Petrie JR, Ueda S, Elliott HL, Connell JMC: Insulin-mediated vasodilation and glucose uptake are functionally linked in humans. Hypertension 33:554eC558, 1999
Clark ADH, Barrett EJ, Rattigan S, Wallis MG, Clark MG: Insulin stimulated laser Doppler signal by rat muscle in vivo, consistent with nutritive flow recruitment. Clin Sci 100:283eC290, 2001
Baron AD, Steinberg HO, Chaker H, Leaming R, Johnson A, Brechtel G: Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans. J Clin Invest 96:786eC792, 1995
Baron AD, Clark MG: Role of blood flow in the regulation of muscle glucose uptake. Annu Rev Nutr 17:487eC499, 1997
Rattigan S, Clark MG, Barrett EJ: Acute vasoconstriction-induced insulin resistance in rat muscle in vivo. Diabetes 48:564eC569, 1999
Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod P: Nitric oxide release accounts for insulin’s vascular effects in humans. J Clin Invest 94:2511eC2515, 1994
Steinberg HO, Brechtel G, Johnson A, Fineberg N, Baron AD: Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent: a novel action of insulin to increase nitric oxide release. J Clin Invest 94:1172eC1179, 1994
Sartori C, Trueb L, Nicod P, Scherrer U: Effects of sympathectomy and nitric oxide synthase inhibition on vascular actions of insulin in humans. Hypertension 34:586eC589, 1999
Lautt WW: The HISS story overview: a novel hepatic neurohumoral regulation of peripheral insulin sensitivity in health and diabetes. Can J Physiol Pharmacol 77:553eC562, 1999
Folkow B, Haeger K, Uvns B: Cholinergic vasodilator nerves in the sympathetic outflow to the muscles of the hind limbs of the cat. Acta Physiol Scand 15:401eC411, 1948
Loke KE, Sobey CG, Dusting GJ, Woodman OL: Cholinergic neurogenic vasodilation is mediated by nitric oxide in the dog hindlimb. Cardiovasc Res 28:542eC547, 1994
Uvns B: Cholinergic vasodilator nerves. Fed Proc 25:1618eC1622, 1966
Bolme P, Fuxe K: Adrenergic and cholinergic nerve terminals in skeletal muscle vessels. Acta Physiol Scand 78:52eC59, 1970
Buse MG, Buse J: Glucose uptake and the response to insulin in the isolated rat diaphragm: the effect of denervation. Diabetes 8:218eC225, 1959
Xie H, Lautt WW: Induction of insulin resistance by cholinergic blockade with atropine in the cat. J Auton Pharmacol 15:361eC369, 1995
Gaudreault N, Santuree M, Pitre M, Nadeau A, Marette A, Bachelard H: Effects of insulin on regional blood flow and glucose uptake in Wistar and Sprague Dawley rats. Metabolism 50:65eC73, 2001
Santuree M, Pitre M, Marette A, Deshaies Y, Lemieux C, Larivieere R, Nadeau A, Bachelard H: Induction of insulin resistance by high-sucrose feeding does not raise mean arterial blood pressure but impairs hemodynamic responses to insulin in rats. Br J Pharmacol 137:185eC196, 2002
Gardiner SM, Bennett T: Regional haemodynamic responses to adrenoceptor antagonism in conscious rats. Am J Physiol 255:H813eCH824, 1988
Kraegen EW, James DE, Jenkins AB, Chisolm DJ: Dose-response curves for in vivo insulin sensitivity in individual tissues in rats. Am J Physiol 248:E353eCE362, 1985
Roy D, Perreault M, Marette A: Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am J Physiol 274:E692eCE699, 1998
Richterich R, Dauwalder H: Zur bertimmung der plasmaglucokonzentration mit der hexokinase-glucose-6-phosphat-dehydrogenase methode. Schweiz Med Wochenschr 101:615eC618, 1971
Desbuquois B, Aurbach GD: Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocr Metab 37:732eC738, 1971
Xie H, Lautt WW: Insulin resistance caused by hepatic cholinergic interruption and reversed by acetylcholine administration. Am J Physiol 271:E587eCE592, 1996
Lautt WW, Macedo MP, Sadri P, Takayama S, Duarte-Ramos F, Legare DJ: Hepatic parasympathetic (HISS) control of insulin sensitivity determined by feeding and fasting. Am J Physiol 281:G29eCG36, 2001
Julius S: Autonomic nervous dysfunction in essential hypertension. Diabetes Care 14:249eC259, 1991
Valensi P, Huard JP, Giroux C, Attali JR: Factors involved in cardiac autonomic neuropathy in diabetic patients. J Diabetes Complications 11:180eC187, 1997
Richter WO, Geib HC, Aleksic S, Schwandt P: Cardiac autonomic nerve function and insulin sensitivity in obese subjects. Int J Obes Relat Metab Disord 20:966eC969, 1996
Shimazu T, Fukuda A, Ban T: Reciprocal influence of the ventromedial and lateral hypothalamic nuclei on blood glucose level and liver glycogen content. Nature 210:1178eC1179, 1966
Shimazu T: Regulation of glycogen metabolism in liver by the autonomic nervous system: activation of glycogen synthetase by vagal stimulation. Biochim Biophys Acta 252:28eC38, 1971
Gardemann A, Jungermann K: Control of glucose balance in the perfused rat liver by the parasympathetic innervation. Biol Chem Hoppe-Seyler 367:559eC566, 1986
Lautt WW, Wong C: Hepatic parasympathetic neural effect on glucose balance in the intact liver. Can J Physiol Pharmacol 56:679eC682, 1978
Mahajan H, Richards SM, Rattigan S, Clark MG: Local methacholine but not bradykinin potentiates insulin-mediated glucose uptake in muscle in vivo by augmenting capillary recruitment. Diabetologia 47:2226eC2234, 2004
Randin D, Vollenweider P, Tappy L, Jequier E, Nicod P, Scherrer U: Effects of adrenergic and cholinergic blockade on insulin induced stimulation of calf blood flow in humans. Am J Physiol 266:R809eCR816, 1994
Joyner MJ, Dietz NM: Sympathetic vasodilation in human muscle. Acta Physiol Scand 177:329eC336, 2003
Verma S, Arikawa E, Yao L, Laher I, McNeil JH: Insulin-induced vasodilation is dependent on tetrahydrobiopterin synthesis. Metabolism 47:1037eC1039, 1998
Shepherd JT: Circulation to skeletal muscle. In Handbook of Physiology. The Cardiovascular System: Peripheral Circulation and Organ Blood Flow. Shepherd JT, Abboud FM, Eds. Bethesda, MD, American Physiological Society, 1983, p.319eC370
Rowe JW, Young JB, Minaker KL, Stevens AL, Pallotta J, Landsberg L: Effect of insulin and glucose infusions on sympathetic nervous system activity in normal man. Diabetes 30:219eC225, 1981
Anderson EA, Hoffman RP, Balon TW, Sinkey CA, Mark AL: Hyperinsulinemia produces both sympathetic neural activation and vasodilatation in normal humans. J Clin Invest 87:2246eC2252, 1991
Berne C, Fagius J, Pollare L, Hjemdahl P: The sympathetic response to euglycemic hyperinsulinaemia: evidence from microelectrode nerve recordings in healthy subjects. Diabetologia 35:873eC879, 1992
Vollenweider P, Tappy L, Owlya R, Jeequier E, Nicod P, Scherrer U: Insulin-induced sympathetic activation and vasodilation in skeletal muscle: effect of insulin resistance in lean subjects. Diabetes 44:641eC645, 1995
Turinsky J, Damrau-Abney A, Loegering DJ: Blood flow and glucose uptake in denervated, insulin-resistant muscles. Am J Physiol 274:R311eCR317, 1998(Martin Leevesque, Marta S)