Type II diabetes, characterized by elevated glucose levels in the context of insulin resistance and relative insulin deficiency, is an increasingly prevalent health problem in our society. When fully manifested, type II diabetes can be considered a chronic disease, which at present can only be managed but not be cured. Hence, type II diabetes is often recognized at an advanced stage by physicians when significant vascular complications have already occurred 1. Recent experimental evidence has been provided suggesting that vascular defects, in particular at the level of the endothelium, may not only be a consequence of type II diabetes, but may also contribute to the development of it by impairing insulin action 2,3. Detection of a vulnerable endothelium may, therefore, have added value for early diagnosis and treatment of people at risk for type II diabetes. The endothelial glycocalyx, which shields the vascular wall from direct exposure to the flowing blood, has, since the last decade, been indicated to play an important role in the protection of the endothelium against atherogenic insults 4,5, whereas we recently provided evidence suggesting that this compartment may also be involved in the regulation of insulin sensitivity. In anesthetized rats, we showed that insulin, by diminishing the barrier properties of the glycocalyx and increasing its accessibility for the flowing blood, rapidly increased microvascular blood volume in muscles 6, an action that has been indicated in studies by Rattigan and colleagues 7–9 to contribute to insulin-mediated glucose disposal. Indeed, we observed that acute enzymatic degradation of the glycocalyx in the rats by intravenous administration of hyaluronidase was associated with an acute decrease in whole-body insulin-mediated glucose disposal during an intravenous insulin tolerance test 6. The physiological relevance of these latter observations for glucose regulation may, however, be questioned, as the insulin tolerance test was performed during isoflurane anesthesia, requiring a supraphysiological dose of insulin to be administered. In the current study we, therefore, tested in conscious rats whether acute glycocalyx degradation would reduce insulin sensitivity as well, and whether this would affect glucose tolerance. In this study, intravenous glucose tolerance tests (IVGTTs) and intravenous insulin tolerance tests (IVITTs) were performed in chronically instrumented rats, and comparisons were made between control rats and those intravenously injected with hyaluronidase, an enzyme which has been demonstrated in several experimental studies to result in a loss of the glycocalyx 10–13.
Animals and instrumentation
Experiments were performed in male Wistar rats (350–450 g; n=29) that received standard chow and water ad libitum. All procedures were performed in accordance with the requirements of the Animal Ethics Care and Use committee of Maastricht University. After provision by the external supplier (Harlan, Horst, the Netherlands) the animals were housed at the animal facility of Maastricht University and allowed to acclimatize for at least 1 week before surgery. On the day of the surgery, rats received the narcotic analgesic buprenorphine (0.03 mg/ml temgesic; Schering-Plough, Haarlem, the Netherlands) at 0.1 mg/kg subcutaneously; 30–45 min later the animals were put under isoflurane anesthesia (2%) and their femoral artery and vein were cannulated with a polyurethane catheter (1.02 mm outer diameter, 0.61 mm inner diameter; Charles River Wiga GmbH, Sulzfeld, Germany). The cannulae were tunneled subcutaneously, attached to the skin by sutures, and closed with an iron plug. After surgery the cannulae were filled with heparinized glycerol and closed. The cannulae were flushed every 3 days with heparinized saline. At least 1 week later, an IVGTT or IVITT was performed after an overnight fast (10–12 h). Both the cannula in the femoral artery and that in the vein were connected with a blunt 30 G needle to a polyethylene tube of ∼20 cm length (1.45 mm outer diameter, 0.75 mm inner diameter), allowing the rat to move freely in the cage. The cannula in the femoral artery was connected to a data acquisition system (IdeeQ 1.70; Instrument Development Engineering and Evaluation, Maastricht University, Maastricht, the Netherlands) to measure blood pressure and heart rate. The rats were handled regularly (at least three times a week) during the acclimatization period, so that they got used to this connection within 15–30 min, as evidenced by stabilization of blood pressure and heart rate and the absence of protest movement. Thereafter, IVGTT or IVITT was performed.
Intravenous glucose tolerance test
To determine the effect of acute glycocalyx degradation on glucose tolerance, IVGTTs were performed in the first group of rats. All animals underwent two IVGTTs: the first test (run 1) was considered as a reference test, whereas the second test (run 2) was the experimental test with the rats receiving either a bolus of hyaluronidase or a control bolus of saline before the run. During the IVGTT, 1 g/kg glucose was infused into the femoral vein, and blood samples were collected to measure blood glucose and plasma insulin levels. The arterial line was used for the withdrawal of these blood samples. Blood glucose level was measured using a glucose meter (Ascensia contour, Bayer, Mijdrecht, the Netherlands) at t=−5 and −3 min before glucose infusion and t=2, 4, 6, 8, 10, 12, 15, 20, 30, 60, and 90 min after glucose infusion. Plasma insulin concentrations were measured using an ELISA kit (ALPCO Diagnostics, Salem, New Hampshire, USA) in samples taken at t=−5 and −3 min before glucose infusion and t=6, 10, 15, 30, 60, and 90 min after glucose infusion (Flowchart 1). After the measurements, the animals were disconnected from the sampling and infusion lines, the cannulae were flushed, and the rats were returned to the housing facility. One week later, the second IVGTT was performed in the rats: one group of rats received a bolus of 1 ml of saline through the femoral vein (n=8) and the other a bolus of 1 ml of hyaluronidase (500 U/ml; bovine testes, type IV-S; Sigma-Aldrich, Zwijndrecht, the Netherlands; n=7) 1 h before the start of the second IVGTT. Because the initial experiments indicated that insulin levels were increased after hyaluronidase treatment, C-peptide levels were determined as a reference for insulin secretion in the later experiments as well. The C-peptide levels were measured (ELISA; ALPCO Diagnostics) in the plasma samples taken for insulin determination in three animals of the control group and four animals of the experimental group during the first and second IVGTTs.
Intravenous insulin tolerance test
To verify whether acute glycocalyx degradation was associated with an acute reduction in insulin sensitivity, similar to our previous study in the anesthetized rats, whole-body insulin sensitivity was tested in a second group of conscious rats through IVITTs. Again, two runs were performed in the rats: a reference test and an experimental test. First, endogenous insulin and glucagon production by the pancreas was blocked by constant infusion of somatostatin (300 µg/kg/h; 100 µg/ml), which was started 30 min before the run. Thereafter, to prevent hypoglycemia, a bolus of 1 g/kg glucose (0.5 g/ml) was administered through the venous line and after 10 min, a bolus of 0.1 U/kg insulin (0.1 U/ml) was administered. Blood glucose levels (±0.6 µl blood) were measured using a glucose meter, by sampling blood from the cannula in the femoral artery, at t=−30, −20, −10 and −0 min before glucose infusion and t=2, 4, 6, 8, and 10 min after glucose infusion; thereafter, the bolus of insulin was administered at t=11 min and blood glucose levels were further measured at t=13, 15, 17, 19, 21, 26, 31, 41, and 61 min. Plasma insulin levels (±60 µl blood) were measured by ELISA t=−30 and 0 min before glucose infusion and t=2, 6, 10, 13, 17, 21, 26, 41, and 61 min after glucose infusion (Flowchart 2). After the measurements, the animals were disconnected from the sampling and infusion lines, the cannulae were flushed, and the rats were returned to the housing facility. As in the IVGTTs, 1 week later a second IVITT was performed in the control (n=7) and experimental rats (n=7). In the control group, a bolus of saline was infused through the femoral vein 1 h before the start of the IVITT, and in the experimental group, a bolus of 1 ml hyaluronidase was administered.
Intravenous glucose tolerance test
As a reflection of the circulating levels of glucose, insulin, and C-peptide during the IVGTTs, we calculated the incremental area under the curve (AUC) of their concentrations, corrected for baseline, versus time after glucose infusion using the linear trapezoidal rule 14.
Intravenous insulin tolerance test
As a measure of the insulin sensitivity, the decline in the blood glucose level between 2 and 15 min after insulin infusion was determined, and the glucose disposal rate (Kitt) was calculated from the slope of the linear regression line of the logarithm of blood glucose against time multiplied by 100 15.
The data are presented as mean±SEM. Differences between the first and second IVGTTs and IVITTs were tested using paired t-tests. A P-value of less than 0.05 was considered statistically significant.
Baseline characteristics are listed in Table 1. There were no significant differences in baseline glucose and insulin levels, body weight, heart rate, and blood pressure between the first and second runs in control rats, as well as in hyaluronidase-treated rats. Somatostatin infusion resulted in significant decreases in glucose and insulin levels in all groups.
Intravenous glucose tolerance test
To determine the effect of glycocalyx degradation on glucose tolerance and glucose-stimulated insulin secretion, IVGTTs were performed in conscious rats. The mean glucose and insulin curves after intravenous glucose infusion are shown in Figs 1a and 2a. The AUC of glucose did not change between the first and second runs in control rats (112±8% of run 1), as well as in hyaluronidase-treated rats (99±9% of run 1; Fig. 1b). In control rats, the AUC of insulin was not different between the first and second run (100±11% of run 1, Fig. 2b). In contrast, the AUC of insulin was significantly increased (152±16% of run 1, Fig. 1b) after enzymatic treatment of the glycocalyx with hyaluronidase (P<0.05). For reference we also measured C-peptide levels during both runs in three animals from the control group and four animals from the reference group. The AUC of C-peptide levels (220±52% of run 1; Fig. 3) tended to increase after hyaluronidase treatment; however, this was not the case in the control group (94±34% of run 1; Fig. 3; P=0.08).
Intravenous insulin tolerance test
To determine the effect of glycocalyx degradation on whole-body insulin-mediated glucose disposal, IVITTs were performed in conscious rats, and the glucose disposal rate was monitored. The Kitt, which is the slope of the linear regression line of the logarithm of blood glucose, is shown for each individual experiment, together with the mean Kitt of all experiments, in Fig. 4. There was no change in Kitt after a bolus of saline (102±6% of run 1); however, after treatment with hyaluronidase the Kitt decreased significantly (to 81±6% of run 1, P<0.05).
When plasma glucose levels rise by an exogenous glucose load, insulin is released by the pancreas, causing hepatic glucose production to decrease and glucose to be taken up by insulin-dependent tissues, mainly skeletal muscles 16. In an insulin-resistant state the ability of insulin to inhibit hepatic glucose release and promote glucose uptake is impaired. In a previous study 6, we showed in anesthetized rats that acute enzymatic glycocalyx degradation is associated with a decrease in insulin-mediated glucose disposal, suggesting a role for the glycocalyx in regulation of insulin sensitivity. In the current study, we similarly demonstrated this role in conscious rats and showed that the evoked insulin resistance did not affect glucose tolerance because of increased pancreatic insulin release in response to an exogenous glucose load. These data suggest that glycocalyx degradation, which is regarded as an early marker of vascular vulnerability in humans at risk for cardiovascular disease, is associated with an acute impairment in insulin sensitivity. However, this defect does not directly manifest itself as glucose intolerance because of a compensatory insulin response.
Role of the glycocalyx in regulation of muscle insulin sensitivity
Insulin-mediated glucose uptake in muscles has been coupled with the ability of insulin to rapidly increase capillary blood volume, and it has been suggested that this enables insulin to be efficiently delivered to the myocytes 8,9. Although traditionally the insulin-mediated increase in capillary blood volume was explained by an increase in the number of perfused capillaries (capillary recruitment) 7, we recently showed in anesthetized rats that insulin can also increase blood volume within already perfused microvessels by increasing blood accessibility into the glycocalyx, and that this vascular action of insulin is impaired after enzymatic glycocalyx degradation. Further, it was suggested that this impaired insulin-mediated microvascular blood volume increase in muscles underlay the ∼30% reduction in the glucose disposal rate in the IVITT after hyaluronidase treatment in the rats 6. To check whether the reduction in glucose disposal from the blood was due to diminished insulin action in skeletal muscle, Akt phosphorylation (expressed as the ratio of phosphorylated Akt to total Akt) was measured in the soleus muscle after the IVITT in a few of these anesthetized rats (n=5, unpaired measurements). The amount of phosphorylated Akt was 0.16±0.03 in control rats versus 0.09±0.02 in hyaluronidase-treated rats (unpublished observations). These data suggest that the amount of insulin that was delivered to the muscle and activating the myocytes was decreased. In contrast, in the liver there seemed to be no difference in Akt phosphorylation between the control rats and the hyaluronidase-treated rats (0.46±0.11 in control rats vs. 0.54±0.11 in hyaluronidase-treated rats; unpublished observations). These data indicated a novel role for the endothelial glycocalyx in the regulation of insulin sensitivity.
Insulin tolerance tests in our previous study were performed without the use of somatostatin and during isoflurane anesthesia 6, and it has been shown previously that isoflurane anesthesia affects glucose utilization 17,18. Therefore, in the current study, we measured glucose and insulin tolerance in conscious rats 1 week after cannulation. The animals recovered well from the surgery after 1 week, as observed from comparable post surgery and presurgery body weights, and physiological blood pressures and heart rates, as well as blood glucose and plasma insulin levels at baseline. Further, these parameters were comparable between the first and second tests in the control group, indicating that this model is suitable for performing repeated measurements. In contrast to the IVITTs performed during anesthesia, in which glucose-mediated insulin release was already greatly inhibited by the isoflurane anesthesia 19, in IVITTs performed in conscious animals, somatostatin was used to block endogenous insulin and glucagon production. The effectiveness of somatostatin was illustrated by the significant decrease in glucose and insulin levels after 30 min of insulin infusion in all groups (Table 1). Further, in the anesthetized rats the bolus of insulin infused was supraphysiological (1 U/kg), whereas in the current study a more physiological bolus of insulin (0.1 U/kg) was infused. The reduction in insulin sensitivity after hyaluronidase treatment was confirmed in the present study, in which we found a comparable (i.e. ∼25%) reduction in the glucose disposal rate in conscious rats after hyaluronidase treatment, indicating that the decrease in insulin-mediated glucose disposal after glycocalyx damage was not greatly influenced by isoflurane anesthesia.
The reduction in insulin sensitivity was not accompanied by impairment of glucose tolerance (Fig. 1). However, it appeared that about 1.5-fold more insulin was needed to dispose of the intravenously infused glucose load in the rats that had received hyaluronidase before the second test (Fig. 2). The increase in insulin response could be explained primarily by an increase in the release of insulin by the pancreas, as observed from the elevated C-peptide levels after hyaluronidase treatment (Fig. 3). C-peptide is cosecreted in equimolar amounts when insulin is released from the pancreas; however, C-peptide is not cleared by the liver and the clearance of C-peptide from circulation is slower than insulin clearance 20,21. In addition to increased insulin release by the β-cells, it cannot be ruled out that diminished clearance of insulin from the blood as a result of compromised insulin delivery to the target tissues may have contributed to the increased insulin response to glucose infusion after hyaluronidase treatment. The current data does not allow us to determine whether the increased insulin release after hyaluronidase treatment was a result of a direct effect of hyaluronidase on the β-cells or a result of the rapid feedback in response to the decrease in peripheral insulin sensitivity. Although there is currently a distinct lack of information on the contribution of hyaluronan or the glycocalyx to the regulation of insulin release by the pancreas, it has been well recognized in rodent, dog, and human studies that a healthy pancreas is readily able to compensate for a reduction in insulin sensitivity 16,22. The precise mechanisms by which a normal organism detects insulin resistance and compensates with hyperinsulinemia remain unknown at present. The data from the current study indicate that the pancreas is able to compensate immediately for the reduced insulin sensitivity induced by acute glycocalyx degradation.
Whereas in the current study glycocalyx degradation was induced by a bolus of the enzyme hyaluronidase, a treatment that was shown to effectively reduce glycocalyx thickness and barrier properties in previous animal studies 10,11, additional studies have shown that short-term hyperglycemia and acute hyperlipidemic conditions also rapidly initiate glycocalyx loss 23–25. As a result, it has been suggested that glycocalyx loss may be an early event in the development of endothelial dysfunction during exposure to traditional risk factors 26. Given the indicated relationship between endothelial dysfunction and insulin resistance, our finding that insulin sensitivity is decreased after acute degradation of the glycocalyx may indicate that glycocalyx loss is a common target linking endothelial dysfunction to insulin resistance, for example, during the metabolic syndrome. Our results show that the decrease in insulin sensitivity induced by glycocalyx loss is not readily translated into glucose intolerance because of a compensatory insulin response, thereby resembling an early stage of insulin resistance in the progression to type II diabetes. When unrecognized for a prolonged period, this stage may be followed by dysfunction and loss of β-cells, and finally progression into overt type II diabetes. Early detection of glycocalyx loss, which is now possible using sublingual Sidestream Dark Field imaging 27, may, in the future, facilitate identification of humans at risk for cardiovascular and associated metabolic diseases.
The study was supported by the Dutch Diabetes Research Foundation (grant number 2006.00.027), the Netherlands Heart Foundation (grant number 2005T037), and the Center for Translational Molecular Medicine (work package 01C-104-04-PREDICCT).
Conflicts of interest
There are no conflicts of interest.
1. Paneni F, Beckman JA, Creager MA, Cosentino F.Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I.Eur Heart J2013;34:2436–2443.
2. Clark MG.Impaired microvascular perfusion: a consequence of vascular dysfunction and a potential cause of insulin resistance in muscle.Am J Physiol Endocrinol Metab2008;295:E732–E750.
3. Rattigan S, Richards SM, Keske MA.Microvascular contributions to insulin resistance.Diabetes2013;62:343–345.
4. Bergman RN.Orchestration of glucose homeostasis: from a small acorn to the California oak.Diabetes2007;56:1489–1501.
5. Richards OC, Raines SM, Attie AD.The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action.Endocr Rev2010;31:343–363.
6. Eskens BJ, Mooij HL, Cleutjens JP, Roos JM, Cobelens JE, Vink H, Vanteeffelen JW.Rapid insulin-mediated increase in microvascular glycocalyx
accessibility in skeletal muscle may contribute to insulin-mediated glucose disposal in rats.PLoS One2013;8:e55399.
7. Rattigan S, Wheatley C, Richards SM, Barrett EJ, Clark MG.Exercise and insulin-mediated capillary recruitment in muscle.Exerc Sport Sci Rev2005;33:43–48.
8. Vincent MA, Clerk LH, Lindner JR, Klibanov AL, Clark MG, Rattigan S, Barrett EJ.Microvascular recruitment is an early insulin effect that regulates skeletal muscle glucose uptake in vivo.Diabetes2004;53:1418–1423.
9. Vincent MA, Clerk LH, Rattigan S, Clark MG, Barrett EJ.Active role for the vasculature in the delivery of insulin to skeletal muscle.Clin Exp Pharmacol Physiol2005;32:302–307.
10. Cabrales P, Vazquez BY, Tsai AG, Intaglietta M.Microvascular and capillary perfusion following glycocalyx
degradation.J Appl Physiol2007;102:2251–2259.
11. Henry CB, Duling BR.Permeation of the luminal capillary glycocalyx
is determined by hyaluronan.Am J Physiol1999;277:H508–H514.
12. Van den Berg BM, Vink H, Spaan JA.The endothelial glycocalyx
protects against myocardial edema.Circ Res2003;92:592–594.
13. VanTeeffelen JW, Brands J, Janssen BJ, Vink H.Effect of acute hyaluronidase treatment of the glycocalyx
on tracer-based whole body vascular volume estimates in mice.J Appl Physiol2013;114:1132–1140.
14. Jazet IM, Pijl H, Frolich M, Schoemaker RC, Meinders AE.Factors predicting the blood glucose lowering effect of a 30-day very low calorie diet in obese type 2 diabetic patients.Diabet Med2005;22:52–55.
15. Borghouts LB, Backx K, Mensink MF, Keizer HA.Effect of training intensity on insulin sensitivity
as evaluated by insulin tolerance test.Eur J Appl Physiol Occup Physiol1999;80:461–466.
16. Defronzo RA.Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.Diabetes2009;58:773–795.
17. Diltoer M, Camu F.Glucose homeostasis and insulin secretion during isoflurane anesthesia in humans.Anesthesiology1988;68:880–886.
18. Tanaka K, Kawano T, Tomino T, Kawano H, Okada T, Oshita S, et al..Mechanisms of impaired glucose tolerance
and insulin secretion during isoflurane anesthesia.Anesthesiology2009;111:1044–1051.
19. Zuurbier CJ, Keijzers PJ, Koeman A, Van Wezel HB, Hollmann MW.Anesthesia’s effects on plasma glucose and insulin and cardiac hexokinase at similar hemodynamics and without major surgical stress in fed rats.Anesth Analg2008;106:135–142.
20. Cobelli C, Toffolo GM, Dalla Man C, Campioni M, Denti P, Caumo A, et al..Assessment of beta-cell function in humans, simultaneously with insulin sensitivity
and hepatic extraction, from intravenous and oral glucose tests.Am J Physiol Endocrinol Metab2007;293:E1–E15.
21. Frangioudakis G, Gyte AC, Loxham SJ, Poucher SM.The intravenous glucose tolerance
test in cannulated Wistar rats: a robust method for the in vivo assessment of glucose-stimulated insulin secretion.J Pharmacol Toxicol Methods2008;57:106–113.
22. Bergman RN, Ader M, Huecking K, Van Citters G.Accurate assessment of beta-cell function: the hyperbolic correction.Diabetes2002;51Suppl 1S212–S220.
23. Van den Berg BM, Spaan JA, Vink H.Impaired glycocalyx
barrier properties contribute to enhanced intimal low-density lipoprotein accumulation at the carotid artery bifurcation in mice.Pflugers Arch2009;457:1199–1206.
24. Vink H, Constantinescu AA, Spaan JA.Oxidized lipoproteins degrade the endothelial surface layer: implications for platelet-endothelial cell adhesion.Circulation2000;101:1500–1502.
25. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C.Short-term hyperglycemia increases endothelial glycocalyx
permeability and acutely decreases lineal density of capillaries with flowing red blood cells.J Appl Physiol2005;99:1471–1476.
26. Nieuwdorp M, Meuwese MC, Vink H, Hoekstra JB, Kastelein JJ, Stroes ES.The endothelial glycocalyx
: a potential barrier between health and vascular disease.Curr Opin Lipidol2005;16:507–511.
27. Broekhuizen LN, Mooij HL, Kastelein JJ, Stroes ES, Vink H, Nieuwdorp M.Endothelial glycocalyx
as potential diagnostic and therapeutic target in cardiovascular disease.Curr Opin Lipidol2009;20:57–62.