Journal of Cerebral Blood Flow and Metabolism
10:774-780 © 1990 Raven Press, Ltd., New York
Regional Blood-Brain Glucose Transfer and Glucose Utilization in Chronically Hyperglycemic, Diabetic Rats Following Acute Glycemic Normalization
D. A. Pelligrino, M. D. Lipa, and R. F. Albrecht Department of Anesthesiology, Michael Reese Hospital and Medical Center and University of Illinois, Chicago, Illinois, U.S.A.
Summary: Regional rates of brain glucose utilization
to acute glycemic normalization, rCMRglc values in CHD rats were either unchanged or moderately lower than con tro!. These findings indicate that no blood-brain barrier glucose transport repression is present in CHD rats. In fact, the results suggest an increased transport capacity. The increased rCMRgic observed in the acutely normal ized CHD rats may be a manifestation of the "hypogly cemic symptoms" observed in chronically hyperglycemic patients following acute glycemic reductions to the nor mal range. The present results imply that these symptoms are not related to the presence of a relative cerebral glu copenia, as others have suggested. Key Words: Blood brain barrier-Glucose influx-Cerebral metabolism Brain glucose utilization-Brain glucose-Brain lactate Chronic hyperglycemia-Diabetes.
(rCMRglc) and glucose influx (rJin), along with regional brain tissue glucose concentrations, were measured in chronically hyperglycemic diabetic (CHD) rats following acute glycemic normalization. These results were com pared to those obtained in nondiabetic normoglycemic controls. The diabetic rats were evaluated at 6-8 weeks following i.p. streptozotocin injection. All rats were N20 (70%) sedated, paralyzed, and artificially ventilated for study. Acutely normoglycemic (plasma glucose 7.9 f,Lmol/ml) CHD rats, compared to control (plasma glucose 8.5 f,Lmollml), demonstrated significantly higher (p < 0.05) rCMRgic and rJin values in 8 of the II regions ana lyzed. Tissue/plasma glucose concentration ratios were significantly greater than control in 9 of II regions. Prior =
=
Earlier studies suggested the development of blood-brain barrier (BBB) glucose transport repres sion in the chronically hyperglycemic diabetic rat (Gjedde and Crone, 1981; McCall et aI., 1982). In a situation of repressed BBB glucose transport, it can be speculated that acute normalization of blood glu cose would result in lower brain tissue glucose lev els than those observed in normoglycemic nondia betics. The creation of a relative cerebral glucope nia may be beneficial in the ischemic patient. Furthermore, this mechanism has been used as a possible explanation for the development of hypo glycemic symptoms in diabetic patients following acute glycemic reductions to normoglycemic levels (Lilavivathra et aI., 1979; DeFronzo et aI., 1980). However, recent studies have questioned the pres ence of a repression of BBB glucose transport in the chronically hyperglycemic diabetic rat (Duckrow, 1988; Harik and LaManna, 1988). In the present study, we employed the method ology of Cunningham and Cremer (1981) to measure
The avoidance of hyperglycemia is a desirable goal in patients at risk for cerebral ischemia. Hy perglycemia and the resultant elevation in brain tis sue glucose levels can exacerbate the brain damage accompanying cerebral ischemia, presumably via an enhanced tissue lactic acidosis (Kalimo et aI., 1981). Thus, in the clinical management of the hy perglycemic diabetic, a proper course of action ap pears to be to reduce blood glucose to normoglyce mic levels prior to any procedure carrying a risk for cerebral ischemia (see Pulsinelli et aI., 1983). Received October 24, 1989; revised April 30, 1990; accepted May 2, 1990. Address correspondence and reprint requests to Dr. D. A. Pel ligrino at Department of Anesthesiology, Michael Reese Hospi tal and Medical Center, Lake Shore Drive at 31st Street, Chi cago, IL 60616 U.S.A. Abbreviations used: BBB, blood-brain barrier; CHD, chroni cally hyperglycemic diabetic; 2-DG, 2-deoxyglucose; DGP, 2deoxyglucose 6-phosphate; MANOVA, multiway analysis of variance; MAP, mean arterial pressure; rCMRg1c, regional
CMRglc; rJin, regional glucose influx; STZ, streptozotocin.
774
BRAIN GLUCOSE TRANSPORT IN DIABETES regional BBB glucose influx (rJin), regional glucose utilization (rCMRg\c), and tissue glucose in concen trations, principally in chronically hyperglycemic diabetic rats following acute glycemic normaliza tion. These results were compared to those ob tained in nondiabetic normoglycemic controls. The rJin measurement was used to assess whether the diabetic rats exhibited a reduced maximal BBB transport velocity for glucose (TmaX> , as others have suggested (Gjedde and Crone, 1981; McCall et aI. 1982). This analysis was based on relationships pro vided by the Michaelis-Menten equation and as sumed no change in transporter affinity (Kt) for glu cose and identical plasma glucose levels in the groups being compared. In addition, previous stud ies have demonstrated that BBB glucose transport varies directly with acute changes in CMRglc (Cre mer et aI., 1983, 1988; see also Hawkins et aI., 1983), although to a quantitatively lesser degree. Thus, the rCMRglc assessment was employed to in vestigate the possibility that rJin (Tmax ) differences between diabetic and nondiabetic normoglycemic rats, should they exist, may be related, in part, to differences in cerebral metabolic activity. METHODS
The experimental protocol was approved by the Insti tutional Animal Care and Use Committee. Male Sprague Dawley rats (250-350 g) were employed for study. The surgical preparation was initiated by anesthetic induction with halothane followed by a tracheostomy. The rats were then paralyzed (with curare) and placed on a rodent respirator. Two femoral arterial, two femoral venous, and one subclavian venous catheter were inserted, with anes thetic maintenance on 0.7% halothane170% N20/30% 02' Following surgery, the wound sites were infiltrated with a local anesthetic (bupivacaine) and the halothane was dis continued. The rats were quickly placed into an acrylic tube designed for use in brain microwave fixation. The tube was then positioned in a microwave apparatus (Toshiba). The catheters, tracheostomy tube, and a rectal thermister were passed to the exterior of the microwave chamber. The rats were reconnected to the respirator and ventilation was maintained on 70% N20/30% 02' The en tire transfer procedure was accomplished in < 20 s. Rectal temperature was maintained at 37°C throughout the ex periments and mean arterial pressure (MAP) was contin uously recorded. The rats were studied at 60-90 min fol lowing halothane discontinuation. We used a sequential, double-label, 2-deoxyglucose (2DG) procedure (Cunningham and Cremer, 198 1) for mea surement of rJin and rCMRglc• The method also permits one to obtain regional brain tissue glucose (and lactate) concentrations. The method employed a 5 min evaluation period. Bolus i.v. injections of 25 j.LCi of [U-14C]2-DG (Amersham) and 30 j.LCi of [l,2,_3H]2-DG (New England Nuclear) were made at 5 and 3 min, respectively, prior to termination of the study with brain fixation via focused
775
microwave irradiation 00 kW, 0.6-0.8 s). Ten arterial samples were drawn during the 5 min period for analysis of plasma glucose and plasma 14C and 3H activities. Fol lowing fixation, the brain was removed, frozen in freon 22, and 11 regions dissected out. The tissue was weighed, extracted in 0.3 M perchlorate, and neutralized with KOH. The extract was divided into two portions. One was used for analysis of free 2-DG and 2-deoxyglucose 6-phosphate (DGP) 14C and 3H activities. The other was taken for analysis of tissue glucose and lactate concen trations. Separation of 2-DG and DGP was accomplished by injecting 250 j.Ll of the extracts into an anion exchange column (Carbopak-Dionex) in a HPLC system (Waters). A flow rate of 1.5 ml/min was used for the mobile phase. This consisted of a 15 mM NaOH solution for a period of 5 min, during which time 100% of the 2-DG was eluted and collected into scintillation vials. The mobile phase was abruptly switched at 5 min to a 200 mM sodium ac etate solution. All of the DGP eluted within 5 min of the changeover. This eluate was also collected into scintilla tion vials. The injections of standards, consisting of a mixture of eH]2-DG and [14C]DGP (from ARC), were performed prior to and following all tissue extract sepa rations on a given day. In every case, these standard de terminations demonstrated a 100% recovery and com plete separation of labeled 2-DG and DGP. Brain tissue 2-DG activities and glucose concentrations were cor rected for plasma contamination. The brain plasma vol ume values used in this correction were determined in preliminary studies (three in each experimental group) and were based on the eHlinulin space (see Duckrow, 1988). The regional values varied from 12-27 j.Ll/g, with no appreciable differences when comparing the three exper imental groups. The calculations for rJin and rCMRglc were made according to the procedure described by Cun ningham and Cremer ( 198 1). We used the phosphoryla tion constant (0.37) and 2-DG/glucose transfer coefficient (1.4) given by these authors in our calculations. The experimental groups were (a) nondiabetic normo glycemic control (n 7); (b) chronically hyperglycemic diabetic (n 7); and (c) acutely normoglycemic, chron ically hyperglycemic diabetic (n 6). Diabetic rats were studied at 6-8 weeks following an intraperitoneal injec tion of streptozotocin (STZ) (60 mg/kg in pH 4.5 citrate buffer). Plasma samples for glucose analysis were ob tained one to three times per week in the STZ-treated rats via a percutaneous subclavian venipuncture under light halothane anesthesia. Both control and acutely normo glycemic diabetic rats were fasted overnight prior to use. Acute glycemic reductions were induced via an i.v. insu lin injection (3 U/kg, regular pork) prior to surgery fol lowed by 5 U/kg insulin i.v. postsurgically. Arterial P02, Pc02, and pH were measured with an Instrumentation Laboratories blood gas/pH analyzer. Plasma glucose was analyzed using a Beckman glucose analyzer 2. Brain tissue glucose and lactate concentra tions were assayed employing enzymatic fluorometric procedures (Lowry and Passonneau, 1972). Radioactivity was measured using a Searle Analytic 8 1 liquid scintil lation counter with quench corrections obtained via an external standard. Statistical analyses for regional evalu ations were performed using a multiway analysis of vari ance (MANOVA, Systat Series). Other statistical com parisons were done employing a nonparametric Mann Whitney U test. =
=
=
J Cereb Blood Flow Metab, Vol,
10,
No.6,
1990
776
D. A. PELL/GRING ET AL. TABLE 1. Arterial blood variables Experimental group
Normoglycemic control (n = 7) Acutely normoglycemic diabetic (n
=
Chronically hyperglycemic diabetic (n
6) =
7)
P02 (mm Hg)
Peo2 (mm Hg)
pH
132 ± 32 127 ± 27
37.5 ± 3. 2 36.9 ± 2. 4
7.381 ± 0.037 7.379 ± 0.022
1\9 ± 26
36. 1 ± 1.8
7.310 ± 0.050a
MAP (mm Hg)
Plasma glucose (fLmol/ml)
112 ± 13 119 ± 5 115 ± 11
8.5 ± 2.9 7.9 ± 2.0 33.1 ± 4.2a
All values are means ± SD. Individual values are taken as the average plasma glucose level for the 10 arterial samples taken over the 5 min evaluation period. a p < 0.05 vs. control.
RESULTS Blood variables
In the STZ-treated rats, hyperglycemia was evi dent within 24 h, with plasma glucose levels in most rats reaching 20 fLmollml at 3-4 days post-STZ and exceeding 25 fLmol/ml by 2-3 weeks post-STZ. In the diabetic rats subjected to acute glycemic nor malization, the plasma glucose concentration prior to the overnight fast was 27. 1 ± 2.2 fLmollml. This fell to 22.4 ± 1.7 fLmol/ml following the fast. In response to the postsurgical insulin injection, the plasma glucose reached 8-9 fLmollml within 30-45 min. With no further insulin, the plasma glucose remained close to this range for an additional 45-60 min, achieving an average value of 7. 9 ± 2. 0 fLmol/ml prior to experimental termination (Table 1). The plasma glucose concentration in the dia betic, chronically hyperglycemic group was 33. 1 ± 4.2 fLmoliml. All plasma glucose concentrations in Table 1 were derived from an average of the 10 timed arterial samples drawn during the terminal 5 min evaluation period. In all rats studied, the max-
imum variation between the highest and lowest plasma glucose value did not exceed 5%. No differ ences in arterial Po2, Pco2, and mean pressure were observed when comparing the experimental groups (Table 1). However, arterial pH in the chronically hy perglycemic group was lower than control (P < 0.05). Regional glucose utilization
When comparing rCMRgJc values in chronically hyperglycemic diabetic rats to those obtained in nondiabetic normoglycemic controls (Fig. 1), few significant differences were found. Thus, signifi cantly lower (p < 0.05) rCMRgJc values were ob served in the diabetic rats in the sensorimotor cor tex (76% control) and the cerebellum (58% control). Following acute glycemic normalization (Fig. 2), a significantly higher rCMRgJc was found in the dia betic group in 8 of the II regions analyzed (p < 0.05, compared to control). In those eight regions, the rCMRgJc values averaged 162% of control. Regional BBB glucose influx
The rJin values for control and acutely normo glycemic diabetic rats are given in Fig. 3. Higher rJin values were observed in the diabetic group in
1.100 1.000
"I
c
E "I
0.900
•
0.700
'"
0.600
0 E
0.500
-3
0.400
-"! '" '" ::;
0.300
'2
1.600
0.800
"I
.
c
E "I
•
0 E
0.200
-3 u
FC
SMC
CP
HT
AM
TH
HC
CE
IC
SC
PO
Brain region FIG. 1. Regional CMRglc (rCMRg1c) values in nondiabetic nor moglycemic controls vs. chronically hyperglycemic diabetic rats. The abbreviations for the different brain regions are defined as follows: FC = frontal cortex, SMC = sensorimo tor cortex, CP = caudate-putamen, HT = hypothalamus, AM = amygdala, TH = thalamus, HC = hippocampus, CE = cerebellum, IC = inferior colliculus, SC = superior collicu Ius, PO = pons. Values are expressed as means ± SD. 'p < 0.05 compared to control. White bars, diabetic chronic hy perglycemic; cross-hatched bars, normoglycemic controls.
J Cereb Blood Flow Metab, Vol.
10,
No.6,
1990
•
• •
.
1.200
. .
1.000
.
'"
0.100 0.000
1.400
'" '" ::;
'2
0.800 0.600 DADO 0.200 0.000
FC
SMC
CP
HT
AM
TH
HC
CE
IC
SC
PO
Brain region FIG. 2. A comparison of rCMRgic values in nondiabetic nor moglycemic control rats and in diabetic acutely normoglyce mic rats. Abbreviations and key are defined in the legend to Fig. 1. Values are means ± SD. 'p < 0.05 compared to con trol. See the text for additional information. White bars, dia betic, acute normoglycemia; cross-hatched bars, normogly cemic controls.
777
BRAIN GLUCOSE TRANSPORT IN DIABETES TABLE 2. Regional brain tissue lactate levels in nondiabetic normoglycemic rats and in acutely normalized diabetic rats
7 6
"I
c
'E "I
5
Diabetic,
4
Region
'"
0 E
2,
3 2
c
�
Brain region FIG. 3. Regional BBB glucose influx rates (rJin) in the same
two groups and 1 1 brain regions represented in Fig. 2. Values are means ± SD. *p < 0.05 compared to control. Abbrevia tions and key in legends to Figs. 1 and 2. See the text.
all regions analyzed, with statistical significance obtained in eight regions (the average in these eight regions was 219% of control). Seven of the re gions were those exhibiting significantly elevated rCMRglc values (with the exception of the pons see Fig. 2). Regional brain/plasma glucose concentration ratios
These results in normoglycemic controls vs. acutely normoglycemic diabetic rats are presented in Fig. 4. A significantly higher ratio in the diabetic group was seen in nine regions (p < 0.05). These included all of the regions exhibiting elevated rCMRglc values (see Fig. 2). Regional lactate concentrations
The tissue lactate levels for the 11 reglOns ana lyzed in the control and diabetic, acutely normogly cemic groups are presented in Table 2. No signifi cant differences were found for any region when comparing the two groups. 0.600
� � 0 u �
0> 0 E � .2
0.500
•
•
•
•
0.400
•
•
�
�
• •
•
0.300
a.
"c
'0
0.200
m 0.100
0.000
�
�
�
�
�
�
�
cr
�
Brain region FIG. 4. Regional brain tissue/plasma glucose ratios in non diabetic normoglycemic control and diabetic, acutely normo glycemic rats. See the legends to Figs. 1-3 and the text for additional details.
Control
acute glycemic normalization
Frontal cortex
0.994 ± 0.317
Sensorimotor cortex Caudate-putamen Hypothalamus Amygdala Thalamus Hippocampus Cerebellum Inferior colliculus Superior colliculus Pons
0.738 ± 0.439
0.718 ± 0.309 0.814 ± 0.211
0.925 ± 0.317
0.843 ± 0.360
0.830 ± 0.294 0.807 ± 0.222
0.937 0.742 0.741 0.536 0.696 1.03
0.932 0.876 0.677 0.639
0.333 0.312 ± 0.188 ± 0.442 ± ±
0.816 ± 0.402 0.856 ± 0.357
± ± ± ± ± ±
0.443 0.372 0.105 0.122 0.345 0.17
1.08 ± 0.69 0.917 ± 0.353
All values are means ± SO. Units are fLmol/g of wet weight.
DISCUSSION
The production of a cerebral hypermetabolic state following acute glycemic normalization in chronically hyperglycemic diabetic rats was an un expected finding. This result cannot be ascribed to conditions existing prior to the glycemic reduction (see Fig. 1). The higher rCMRglc values appeared to be a function of increased glucose oxidation and not simply an increase in glycolysis, as evidenced by no change in regional tissue lactate concentrations. An elevated CMRglc may be a manifestation of the "hy poglycemic symptoms" others have reported to ac company acute glycemic reductions to the normo glycemic range in diabetic patients (Lilavivathra et aI., 1979; DeFronzo et aI., 1980). These symptoms, to a large degree, were related to a sympathoadre nal activation. This type of physiologic response has been associated with enhanced cerebral meta bolic activity in other studies (see Bryan et aI., 1983). At any rate, the present results do not sup port the speculation by Gjedde and Crone (1981) that these symptoms are the result of a BBB glucose transport repression producing a relative cerebral glucopenia when plasma glucose is acutely normal ized. We found the brain tissue glucose levels to be higher in the acutely normoglycemic diabetic rats than in normoglycemic controls. As will be dis cussed later, this finding appears to be more con sistent with an enhanced BBB glucose transport ca pacity in the chronically hyperglycemic diabetic than a transport repression. Blood-brain barrier glucose transport has been shown to vary directly with the rate of cerebral glu cose utilization (Cremer et aI., 1983,1988; Hawkins et aI., 1983). Therefore, in a situation where the
J Cereb Blood Flow Metab, Vol.
10,
No.6,
1990
778
D. A. PELL/GRINO ET AL.
glucose utilization rate is acutely elevated, and plasma glucose levels are unchanged, one might predict some increase in the BBB glucose influx rate. This Jin change is most likely a result of an enhanced maximum transport velocity (TmaX> for glucose (see Cremer et aL 1988). However, the lit erature indicates that rCMRglc increases should be accompanied by lesser magnitude increases in rJin (Tmax) ' Hawkins et aL (1983) described a relation ship between Tmax and rCMRglc, based on measured brain glucose influx and utilization rates, that would predict an average 30-35% increase in rJin (or TmaX> in those eight regions in the present study exhibiting significant rCMRglc increases (mean increase 62%). Further evidence for the above can be taken from euglycemic comparisons of brain tissue glu cose concentrations between control rats and rats exhibiting elevated rCMRglc values. Thus, tissue glucose levels invariably have been shown to be significantly reduced under such conditions (Fol bergrova et aI., 1981; Cremer et aI., 1988). A falling brain tissue glucose in the face of an unchanged plasma glucose concentration is best explained by the concept that when the glucose utilization rate increases, the glucose influx rate increases to a lesser extent. In the eight regions of the diabetic, acute normo glycemic group demonstrating rCMRglc values sig nificantly higher than control, we measured rJin val ues that, when averaged, were more than double those obtained in control rats. This rJin change far exceeded that which one would predict based upon an rCMRglc change alone (see the previous para graph). It is tempting to ascribe the elevated rJin values in these acutely normoglycemic diabetic rats, at least in part, to an increased BBB glucose transport capacity. However, Cremer et aL (1988) presented evidence suggesting that the double label, 2-DG method may overestimate the actual rJin when cerebral glucose utilization is substan tially increased. They speculated that under hyper metabolic conditions, this error is created either as a result of inadequate mixing of 2-DG with the na tive brain glucose pool (metabolic compartmenta tion) or because of an increasing transport asymme try between the luminal and abluminal cerebral cap illary endothelial cell membranes. Whatever the cause, in hypermetabolic regions, the influx rate in creases from the control that Cremer et aL calcu lated, employing either of two double-membrane ki netic models for BBB glucose transfer (see also Cunningham et aI., 1986), were less than those ob tained using the double-label 2-DG method. We ap plied the same type of analysis to the data obtained in the present study. The analysis was based upon =
J Cereb Blood Flow Metab, Vol. 10, No.6,
1990
the simpler of the two transport models only (type I) (Cunningham et aI., 1986). This should be adequate for detecting the presence of the kind of error de scribed by Cremer et al. that may accompany con ditions of increased brain glucose metabolism, i.e., the influx rate changes from control estimated from the two transport models in the Cremer et al. (1988) report were virtually the same. Our calculations of rJin were performed using an algorithm, taken from Cremer et al. (1988), that al lowed for the calculation of Tmax (which is equiva lent to one-half the value of the maximum transport velocity of the two endothelial cell membranes) and employed measured values for tissue glucose (in mM) , arterial plasma glucose, and rCMRglc and a transport constant (Kt) of 6 mM. The arterial plasma concentration was corrected to a mean cerebral capillary concentration according to Cremer et al. (1988). This procedure utilized cerebral plasma flow values, obtained in a preliminary study (Pelligrino et aI., 1990). Flow values in acutely normalized di abetic rats were similar to control in subcortical and hindbrain structures and 20-25% higher than con trol in the cortex. The results of the model dependent rJin calculations are presented in Fig. 5, along with the values obtained using the 2-DG method. The eight regions that showed significantly higher "measured" rJin values (compared to con trol-see Fig. 3) are represented in the figure. These results demonstrated a fairly good agreement be tween measured and calculated rJin values not only in control animals, but also in acutely normoglyce mic diabetic rats as well. Furthermore, the relation
.,c E .,
ry
4
1
t
T
T
I
1
0 E
"" c
� 12
12
12
12
12
12
12
1 2
FC
SMC
CP
HT
AM
HC
SC
PO
FIG. 5. Regional glucose influx rates (rJin) in normoglycemic controls (cross-hatched bars) and diabetic, acute hypergly cemics (open plus cross-hatched bars) estimated from the sequential double-label 2-0G procedure (1) and from a sim ple double membrane (type I) transport scheme (2). The latter calculation employed an algorithm described by Cunning ham et al. (1986) and Cremer et al. (1988). Results are given for the eight regions demonstrating significantly higher (compared to control) rJin values derived from the 2-0G method (see Fig. 3). Additional information is provided in the text. All values are means ± SO.
BRAIN GLUCOSE TRANSPORT IN DIABETES ships between nondiabetic and diabetic rats were similar for both methods of rJin estimation. Based on rJin values averaged over the eight regions rep resented in Fig. 5, we found the influx rate to be 2.2-fold greater than control with rJin values deter mined via the 2-DG method and twofold greater with calculations based upon type I kinetics. Al though these findings do not verify the accuracy of the results summarized in Fig. 3, they do provide support for our conclusion of a higher BBB glucose transport rate in acutely normalized diabetic rats compared to normoglycemic controls. As discussed earlier, results from previous stud ies have shown that brain tissue glucose levels fall when brain glucose metabolism increases (un changed plasma glucose). The present finding of re gional tissue glucose concentrations in acutely nor moglycemic diabetic rats that were higher than con trol in the presence of elevated rCMRglc values also appears to support the existence of an increased BBB glucose transport capacity in the diabetic rat. However, one must consider the possibility that the comparatively higher tissue glucose levels in the acutely normalized diabetics may be, at least in part, a function of too short a period to achieve a steady-state relationship for glucose between brain and blood. Preliminary findings in three diabetic rats studied at 18--24 h of glycemic normalization (not shown) have indicated a sustained elevation in regional tissue/plasma glucose ratios. This suggests that the tissue/plasma glucose results of Fig. 4 are not attributable to incomplete equilibration of glu cose between tissue and plasma. The apparent in crease in BBB glucose passage cannot be explained at this time. It is seemingly related to factors asso ciated with the diabetic state and maybe chronic hyperglycemia-related changes in BBB function (in cluding possible changes in metabolismitransport coupling). One possibility is an increased availabil ity of glucose transporters. This is not an unprece dented assertion. Harik et al. (1988) reported an increased density of glucose transporters in brain microvessels harvested from diabetic rats. Choi et aI. (1989) provided evidence indicating increased levels of mRNA for the principal BBB glucose transporter in chronically hyperglycemic rats. An increased glucose transporter synthesis in response to hyperglycemia has been suggested for other tis sues as well (Csaky, 1988). In conclusion, the present results demonstrate that chronic hyperglycemic diabetes in rats is not associated with a BBB glucose transport repres sion. In fact, the opposite appears to be true. Fur thermore, acute glycemic normalization in these an imals resulted in an increased cerebral metabolism.
779
There is a suggestion that the degree of brain dam age accompanying cerebral ischemia may be di rectly related to the level of cerebral metabolic ac tivity at the time of ischemic induction (Busto et aI., 1987). The presence of a hypermetabolic state in addition to higher than normal tissue glucose levels could lead to a greater degree of neuronal damage than one might otherwise predict. Thus, current presurgical clinical management of the diabetic pa tient-i.e., to normalize blood glucose quickly may fail to diminish risk. Longer periods of preop erative normoglycemia are probably required. Acknowledgment: The authors wish to thank Anthony Sharp and Jerome Galloway for expert technical assis tance and Lois Boler for typing the manuscript. This work was supported by the Juvenile Diabetes Foundation International.
REFERENCES Bryan RM. Hawkins RA. Mans AM. Davis DW. Page RG (1983) Cerebral glucose utilization in awake unstressed rats. Am J Physiol 244:C27O-C275 Busto R. Dietrich WD, Globus MYT, Valdes I, Scheinberg P, Ginsberg MD (1987) Small differences in intra-ischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 7:729-738 Choi TB, Boado RJ, Pardridge WM (1989) Blood-brain barrier glucose transporter mRNA is increased in experimental di abetes mellitus. Biochem Biophys Res Commun 164:375-380 Cremer JE, Cunningham VJ, Seville MP (1983) Relationships between extraction and metabolism of glucose, blood flow, and tissue blood volume in regions of rat brain. J Cereb Blood Flow Metab 3:291-302 Cremer JE, Seville MP, Cunningham VJ (1988) Tracer 2deoxyglucose kinetics in brain regions of rats given kainic acid. J Cereb Blood Flow Metab 8:244-253 Csaky TZ (1988) Membrane transport of sugars in diabetes mel litus. In: Membrane Biophysics III: Biological Transport (Dinno MA, Armstrong WMCD, eds), New York, Alan R. Liss Inc., pp 37-42 Cunningham VJ, Cremer JE (1981) A method for the simulta neous estimation of regional rates of glucose influx and phosphorylation in rat brain using radiolabelled 2-de oxyglucose. Brain Res 221:319-330 Cunningham VJ, Hargreaves RJ, Pelling D, Moorhouse SR (1986) Regional blood-brain glucose transfer in the rat: a novel double-membrane kinetic analysis. J Cereb Blood Flow Metab 6:305-314 DeFronzo RA, Hendler R, Christensen N (1980) Stimulation of counter-regulatory hormonal responses in diabetic man by a fall in glucose concentration. Diabetes29:125-131 Duckrow RB (1988) Glucose transfer into rat brain during acute and chronic hyperglycemia. Metab Brain Dis 3:201-210 Folbergrova J, Ingvar M, Siesjo BK (1981) Metabolic changes in cerebral cortex, hippocampus, and cerebellum during sus tained bicuculline-induced seizures. J Neurochem 37:12281238 Gjedde A, Crone C (1981) Blood-brain glucose transfer: repres sion in chronic hyperglycemia. Science 214:456-457 Harik SI, Gravina SA, Kalaria RN (1988) Glucose transporter of the blood-brain barrier and brain in chronic hyperglycemia. J Neurochem 51:1930-1934 Harik SI, LaManna JL (1988) Vascular perfusion and blood brain glucose transport in acute and chronic hyperglycemia. J Neurochem 51:1924-1929 Hawkins RA, Mans AM, Davis DW, Hibbard LS, Lu DM (1983)
J Cereb Blood Flow Metab, Vol. /0, No.6,
1990
780
D. A. PELL/GRINO ET AL.
Glucose availability to individual cerebral structures is cor related to glucose metabolism. J Neurochem 40:1013-1018 Kalimo H, Rehncrona S, Soderfeldt B, Olsson Y, Siesjo BK (1981) Brain lactate acidosis and ischemic cell damage: 2. Histopathology. J Cereb Blood Flow Metab1:313-327 Lilavivathra U, Brodows RG, Woolf PD, Campbell RG (1979) Counter-regulatory hormonal responses to rapid glucose lowering in diabetic man. Diabetes28:873-877 Lowry OH, Passonneau JV 1 ( 972) A Flexible System of Enzy matic Analysis, New York, Academic Press
J
Cereb Blood Flow Metab, Vol. 10, No.6, 1990
McCall AL, Millington WR, Wurtman RJ (1982) Metabolic fuel and amino-acid transport in the brain in experimental diabe tes mellitus. Proc Natl Acad Sci USA 79:540&-5410 Pelligrino DA, Sharp AJ, Albrecht RF (1990) Diminished CBF response to hypoglycemia in the diabetic rat. J Neurosurg Anesthesiol2:S7 Pulsinelli WA, Levy DE, Sigsbee B, Scherer P, Plum F 1 ( 983) Increased damage after ischemic stroke in patients with hy perglycemia with or without established diabetes mellitus. Am J Med74:540--544
10:774-780 © 1990 Raven Press, Ltd., New York
Regional Blood-Brain Glucose Transfer and Glucose Utilization in Chronically Hyperglycemic, Diabetic Rats Following Acute Glycemic Normalization
D. A. Pelligrino, M. D. Lipa, and R. F. Albrecht Department of Anesthesiology, Michael Reese Hospital and Medical Center and University of Illinois, Chicago, Illinois, U.S.A.
Summary: Regional rates of brain glucose utilization
to acute glycemic normalization, rCMRglc values in CHD rats were either unchanged or moderately lower than con tro!. These findings indicate that no blood-brain barrier glucose transport repression is present in CHD rats. In fact, the results suggest an increased transport capacity. The increased rCMRgic observed in the acutely normal ized CHD rats may be a manifestation of the "hypogly cemic symptoms" observed in chronically hyperglycemic patients following acute glycemic reductions to the nor mal range. The present results imply that these symptoms are not related to the presence of a relative cerebral glu copenia, as others have suggested. Key Words: Blood brain barrier-Glucose influx-Cerebral metabolism Brain glucose utilization-Brain glucose-Brain lactate Chronic hyperglycemia-Diabetes.
(rCMRglc) and glucose influx (rJin), along with regional brain tissue glucose concentrations, were measured in chronically hyperglycemic diabetic (CHD) rats following acute glycemic normalization. These results were com pared to those obtained in nondiabetic normoglycemic controls. The diabetic rats were evaluated at 6-8 weeks following i.p. streptozotocin injection. All rats were N20 (70%) sedated, paralyzed, and artificially ventilated for study. Acutely normoglycemic (plasma glucose 7.9 f,Lmol/ml) CHD rats, compared to control (plasma glucose 8.5 f,Lmollml), demonstrated significantly higher (p < 0.05) rCMRgic and rJin values in 8 of the II regions ana lyzed. Tissue/plasma glucose concentration ratios were significantly greater than control in 9 of II regions. Prior =
=
Earlier studies suggested the development of blood-brain barrier (BBB) glucose transport repres sion in the chronically hyperglycemic diabetic rat (Gjedde and Crone, 1981; McCall et aI., 1982). In a situation of repressed BBB glucose transport, it can be speculated that acute normalization of blood glu cose would result in lower brain tissue glucose lev els than those observed in normoglycemic nondia betics. The creation of a relative cerebral glucope nia may be beneficial in the ischemic patient. Furthermore, this mechanism has been used as a possible explanation for the development of hypo glycemic symptoms in diabetic patients following acute glycemic reductions to normoglycemic levels (Lilavivathra et aI., 1979; DeFronzo et aI., 1980). However, recent studies have questioned the pres ence of a repression of BBB glucose transport in the chronically hyperglycemic diabetic rat (Duckrow, 1988; Harik and LaManna, 1988). In the present study, we employed the method ology of Cunningham and Cremer (1981) to measure
The avoidance of hyperglycemia is a desirable goal in patients at risk for cerebral ischemia. Hy perglycemia and the resultant elevation in brain tis sue glucose levels can exacerbate the brain damage accompanying cerebral ischemia, presumably via an enhanced tissue lactic acidosis (Kalimo et aI., 1981). Thus, in the clinical management of the hy perglycemic diabetic, a proper course of action ap pears to be to reduce blood glucose to normoglyce mic levels prior to any procedure carrying a risk for cerebral ischemia (see Pulsinelli et aI., 1983). Received October 24, 1989; revised April 30, 1990; accepted May 2, 1990. Address correspondence and reprint requests to Dr. D. A. Pel ligrino at Department of Anesthesiology, Michael Reese Hospi tal and Medical Center, Lake Shore Drive at 31st Street, Chi cago, IL 60616 U.S.A. Abbreviations used: BBB, blood-brain barrier; CHD, chroni cally hyperglycemic diabetic; 2-DG, 2-deoxyglucose; DGP, 2deoxyglucose 6-phosphate; MANOVA, multiway analysis of variance; MAP, mean arterial pressure; rCMRg1c, regional
CMRglc; rJin, regional glucose influx; STZ, streptozotocin.
774
BRAIN GLUCOSE TRANSPORT IN DIABETES regional BBB glucose influx (rJin), regional glucose utilization (rCMRg\c), and tissue glucose in concen trations, principally in chronically hyperglycemic diabetic rats following acute glycemic normaliza tion. These results were compared to those ob tained in nondiabetic normoglycemic controls. The rJin measurement was used to assess whether the diabetic rats exhibited a reduced maximal BBB transport velocity for glucose (TmaX> , as others have suggested (Gjedde and Crone, 1981; McCall et aI. 1982). This analysis was based on relationships pro vided by the Michaelis-Menten equation and as sumed no change in transporter affinity (Kt) for glu cose and identical plasma glucose levels in the groups being compared. In addition, previous stud ies have demonstrated that BBB glucose transport varies directly with acute changes in CMRglc (Cre mer et aI., 1983, 1988; see also Hawkins et aI., 1983), although to a quantitatively lesser degree. Thus, the rCMRglc assessment was employed to in vestigate the possibility that rJin (Tmax ) differences between diabetic and nondiabetic normoglycemic rats, should they exist, may be related, in part, to differences in cerebral metabolic activity. METHODS
The experimental protocol was approved by the Insti tutional Animal Care and Use Committee. Male Sprague Dawley rats (250-350 g) were employed for study. The surgical preparation was initiated by anesthetic induction with halothane followed by a tracheostomy. The rats were then paralyzed (with curare) and placed on a rodent respirator. Two femoral arterial, two femoral venous, and one subclavian venous catheter were inserted, with anes thetic maintenance on 0.7% halothane170% N20/30% 02' Following surgery, the wound sites were infiltrated with a local anesthetic (bupivacaine) and the halothane was dis continued. The rats were quickly placed into an acrylic tube designed for use in brain microwave fixation. The tube was then positioned in a microwave apparatus (Toshiba). The catheters, tracheostomy tube, and a rectal thermister were passed to the exterior of the microwave chamber. The rats were reconnected to the respirator and ventilation was maintained on 70% N20/30% 02' The en tire transfer procedure was accomplished in < 20 s. Rectal temperature was maintained at 37°C throughout the ex periments and mean arterial pressure (MAP) was contin uously recorded. The rats were studied at 60-90 min fol lowing halothane discontinuation. We used a sequential, double-label, 2-deoxyglucose (2DG) procedure (Cunningham and Cremer, 198 1) for mea surement of rJin and rCMRglc• The method also permits one to obtain regional brain tissue glucose (and lactate) concentrations. The method employed a 5 min evaluation period. Bolus i.v. injections of 25 j.LCi of [U-14C]2-DG (Amersham) and 30 j.LCi of [l,2,_3H]2-DG (New England Nuclear) were made at 5 and 3 min, respectively, prior to termination of the study with brain fixation via focused
775
microwave irradiation 00 kW, 0.6-0.8 s). Ten arterial samples were drawn during the 5 min period for analysis of plasma glucose and plasma 14C and 3H activities. Fol lowing fixation, the brain was removed, frozen in freon 22, and 11 regions dissected out. The tissue was weighed, extracted in 0.3 M perchlorate, and neutralized with KOH. The extract was divided into two portions. One was used for analysis of free 2-DG and 2-deoxyglucose 6-phosphate (DGP) 14C and 3H activities. The other was taken for analysis of tissue glucose and lactate concen trations. Separation of 2-DG and DGP was accomplished by injecting 250 j.Ll of the extracts into an anion exchange column (Carbopak-Dionex) in a HPLC system (Waters). A flow rate of 1.5 ml/min was used for the mobile phase. This consisted of a 15 mM NaOH solution for a period of 5 min, during which time 100% of the 2-DG was eluted and collected into scintillation vials. The mobile phase was abruptly switched at 5 min to a 200 mM sodium ac etate solution. All of the DGP eluted within 5 min of the changeover. This eluate was also collected into scintilla tion vials. The injections of standards, consisting of a mixture of eH]2-DG and [14C]DGP (from ARC), were performed prior to and following all tissue extract sepa rations on a given day. In every case, these standard de terminations demonstrated a 100% recovery and com plete separation of labeled 2-DG and DGP. Brain tissue 2-DG activities and glucose concentrations were cor rected for plasma contamination. The brain plasma vol ume values used in this correction were determined in preliminary studies (three in each experimental group) and were based on the eHlinulin space (see Duckrow, 1988). The regional values varied from 12-27 j.Ll/g, with no appreciable differences when comparing the three exper imental groups. The calculations for rJin and rCMRglc were made according to the procedure described by Cun ningham and Cremer ( 198 1). We used the phosphoryla tion constant (0.37) and 2-DG/glucose transfer coefficient (1.4) given by these authors in our calculations. The experimental groups were (a) nondiabetic normo glycemic control (n 7); (b) chronically hyperglycemic diabetic (n 7); and (c) acutely normoglycemic, chron ically hyperglycemic diabetic (n 6). Diabetic rats were studied at 6-8 weeks following an intraperitoneal injec tion of streptozotocin (STZ) (60 mg/kg in pH 4.5 citrate buffer). Plasma samples for glucose analysis were ob tained one to three times per week in the STZ-treated rats via a percutaneous subclavian venipuncture under light halothane anesthesia. Both control and acutely normo glycemic diabetic rats were fasted overnight prior to use. Acute glycemic reductions were induced via an i.v. insu lin injection (3 U/kg, regular pork) prior to surgery fol lowed by 5 U/kg insulin i.v. postsurgically. Arterial P02, Pc02, and pH were measured with an Instrumentation Laboratories blood gas/pH analyzer. Plasma glucose was analyzed using a Beckman glucose analyzer 2. Brain tissue glucose and lactate concentra tions were assayed employing enzymatic fluorometric procedures (Lowry and Passonneau, 1972). Radioactivity was measured using a Searle Analytic 8 1 liquid scintil lation counter with quench corrections obtained via an external standard. Statistical analyses for regional evalu ations were performed using a multiway analysis of vari ance (MANOVA, Systat Series). Other statistical com parisons were done employing a nonparametric Mann Whitney U test. =
=
=
J Cereb Blood Flow Metab, Vol,
10,
No.6,
1990
776
D. A. PELL/GRING ET AL. TABLE 1. Arterial blood variables Experimental group
Normoglycemic control (n = 7) Acutely normoglycemic diabetic (n
=
Chronically hyperglycemic diabetic (n
6) =
7)
P02 (mm Hg)
Peo2 (mm Hg)
pH
132 ± 32 127 ± 27
37.5 ± 3. 2 36.9 ± 2. 4
7.381 ± 0.037 7.379 ± 0.022
1\9 ± 26
36. 1 ± 1.8
7.310 ± 0.050a
MAP (mm Hg)
Plasma glucose (fLmol/ml)
112 ± 13 119 ± 5 115 ± 11
8.5 ± 2.9 7.9 ± 2.0 33.1 ± 4.2a
All values are means ± SD. Individual values are taken as the average plasma glucose level for the 10 arterial samples taken over the 5 min evaluation period. a p < 0.05 vs. control.
RESULTS Blood variables
In the STZ-treated rats, hyperglycemia was evi dent within 24 h, with plasma glucose levels in most rats reaching 20 fLmollml at 3-4 days post-STZ and exceeding 25 fLmol/ml by 2-3 weeks post-STZ. In the diabetic rats subjected to acute glycemic nor malization, the plasma glucose concentration prior to the overnight fast was 27. 1 ± 2.2 fLmollml. This fell to 22.4 ± 1.7 fLmol/ml following the fast. In response to the postsurgical insulin injection, the plasma glucose reached 8-9 fLmollml within 30-45 min. With no further insulin, the plasma glucose remained close to this range for an additional 45-60 min, achieving an average value of 7. 9 ± 2. 0 fLmol/ml prior to experimental termination (Table 1). The plasma glucose concentration in the dia betic, chronically hyperglycemic group was 33. 1 ± 4.2 fLmoliml. All plasma glucose concentrations in Table 1 were derived from an average of the 10 timed arterial samples drawn during the terminal 5 min evaluation period. In all rats studied, the max-
imum variation between the highest and lowest plasma glucose value did not exceed 5%. No differ ences in arterial Po2, Pco2, and mean pressure were observed when comparing the experimental groups (Table 1). However, arterial pH in the chronically hy perglycemic group was lower than control (P < 0.05). Regional glucose utilization
When comparing rCMRgJc values in chronically hyperglycemic diabetic rats to those obtained in nondiabetic normoglycemic controls (Fig. 1), few significant differences were found. Thus, signifi cantly lower (p < 0.05) rCMRgJc values were ob served in the diabetic rats in the sensorimotor cor tex (76% control) and the cerebellum (58% control). Following acute glycemic normalization (Fig. 2), a significantly higher rCMRgJc was found in the dia betic group in 8 of the II regions analyzed (p < 0.05, compared to control). In those eight regions, the rCMRgJc values averaged 162% of control. Regional BBB glucose influx
The rJin values for control and acutely normo glycemic diabetic rats are given in Fig. 3. Higher rJin values were observed in the diabetic group in
1.100 1.000
"I
c
E "I
0.900
•
0.700
'"
0.600
0 E
0.500
-3
0.400
-"! '" '" ::;
0.300
'2
1.600
0.800
"I
.
c
E "I
•
0 E
0.200
-3 u
FC
SMC
CP
HT
AM
TH
HC
CE
IC
SC
PO
Brain region FIG. 1. Regional CMRglc (rCMRg1c) values in nondiabetic nor moglycemic controls vs. chronically hyperglycemic diabetic rats. The abbreviations for the different brain regions are defined as follows: FC = frontal cortex, SMC = sensorimo tor cortex, CP = caudate-putamen, HT = hypothalamus, AM = amygdala, TH = thalamus, HC = hippocampus, CE = cerebellum, IC = inferior colliculus, SC = superior collicu Ius, PO = pons. Values are expressed as means ± SD. 'p < 0.05 compared to control. White bars, diabetic chronic hy perglycemic; cross-hatched bars, normoglycemic controls.
J Cereb Blood Flow Metab, Vol.
10,
No.6,
1990
•
• •
.
1.200
. .
1.000
.
'"
0.100 0.000
1.400
'" '" ::;
'2
0.800 0.600 DADO 0.200 0.000
FC
SMC
CP
HT
AM
TH
HC
CE
IC
SC
PO
Brain region FIG. 2. A comparison of rCMRgic values in nondiabetic nor moglycemic control rats and in diabetic acutely normoglyce mic rats. Abbreviations and key are defined in the legend to Fig. 1. Values are means ± SD. 'p < 0.05 compared to con trol. See the text for additional information. White bars, dia betic, acute normoglycemia; cross-hatched bars, normogly cemic controls.
777
BRAIN GLUCOSE TRANSPORT IN DIABETES TABLE 2. Regional brain tissue lactate levels in nondiabetic normoglycemic rats and in acutely normalized diabetic rats
7 6
"I
c
'E "I
5
Diabetic,
4
Region
'"
0 E
2,
3 2
c
�
Brain region FIG. 3. Regional BBB glucose influx rates (rJin) in the same
two groups and 1 1 brain regions represented in Fig. 2. Values are means ± SD. *p < 0.05 compared to control. Abbrevia tions and key in legends to Figs. 1 and 2. See the text.
all regions analyzed, with statistical significance obtained in eight regions (the average in these eight regions was 219% of control). Seven of the re gions were those exhibiting significantly elevated rCMRglc values (with the exception of the pons see Fig. 2). Regional brain/plasma glucose concentration ratios
These results in normoglycemic controls vs. acutely normoglycemic diabetic rats are presented in Fig. 4. A significantly higher ratio in the diabetic group was seen in nine regions (p < 0.05). These included all of the regions exhibiting elevated rCMRglc values (see Fig. 2). Regional lactate concentrations
The tissue lactate levels for the 11 reglOns ana lyzed in the control and diabetic, acutely normogly cemic groups are presented in Table 2. No signifi cant differences were found for any region when comparing the two groups. 0.600
� � 0 u �
0> 0 E � .2
0.500
•
•
•
•
0.400
•
•
�
�
• •
•
0.300
a.
"c
'0
0.200
m 0.100
0.000
�
�
�
�
�
�
�
cr
�
Brain region FIG. 4. Regional brain tissue/plasma glucose ratios in non diabetic normoglycemic control and diabetic, acutely normo glycemic rats. See the legends to Figs. 1-3 and the text for additional details.
Control
acute glycemic normalization
Frontal cortex
0.994 ± 0.317
Sensorimotor cortex Caudate-putamen Hypothalamus Amygdala Thalamus Hippocampus Cerebellum Inferior colliculus Superior colliculus Pons
0.738 ± 0.439
0.718 ± 0.309 0.814 ± 0.211
0.925 ± 0.317
0.843 ± 0.360
0.830 ± 0.294 0.807 ± 0.222
0.937 0.742 0.741 0.536 0.696 1.03
0.932 0.876 0.677 0.639
0.333 0.312 ± 0.188 ± 0.442 ± ±
0.816 ± 0.402 0.856 ± 0.357
± ± ± ± ± ±
0.443 0.372 0.105 0.122 0.345 0.17
1.08 ± 0.69 0.917 ± 0.353
All values are means ± SO. Units are fLmol/g of wet weight.
DISCUSSION
The production of a cerebral hypermetabolic state following acute glycemic normalization in chronically hyperglycemic diabetic rats was an un expected finding. This result cannot be ascribed to conditions existing prior to the glycemic reduction (see Fig. 1). The higher rCMRglc values appeared to be a function of increased glucose oxidation and not simply an increase in glycolysis, as evidenced by no change in regional tissue lactate concentrations. An elevated CMRglc may be a manifestation of the "hy poglycemic symptoms" others have reported to ac company acute glycemic reductions to the normo glycemic range in diabetic patients (Lilavivathra et aI., 1979; DeFronzo et aI., 1980). These symptoms, to a large degree, were related to a sympathoadre nal activation. This type of physiologic response has been associated with enhanced cerebral meta bolic activity in other studies (see Bryan et aI., 1983). At any rate, the present results do not sup port the speculation by Gjedde and Crone (1981) that these symptoms are the result of a BBB glucose transport repression producing a relative cerebral glucopenia when plasma glucose is acutely normal ized. We found the brain tissue glucose levels to be higher in the acutely normoglycemic diabetic rats than in normoglycemic controls. As will be dis cussed later, this finding appears to be more con sistent with an enhanced BBB glucose transport ca pacity in the chronically hyperglycemic diabetic than a transport repression. Blood-brain barrier glucose transport has been shown to vary directly with the rate of cerebral glu cose utilization (Cremer et aI., 1983,1988; Hawkins et aI., 1983). Therefore, in a situation where the
J Cereb Blood Flow Metab, Vol.
10,
No.6,
1990
778
D. A. PELL/GRINO ET AL.
glucose utilization rate is acutely elevated, and plasma glucose levels are unchanged, one might predict some increase in the BBB glucose influx rate. This Jin change is most likely a result of an enhanced maximum transport velocity (TmaX> for glucose (see Cremer et aL 1988). However, the lit erature indicates that rCMRglc increases should be accompanied by lesser magnitude increases in rJin (Tmax) ' Hawkins et aL (1983) described a relation ship between Tmax and rCMRglc, based on measured brain glucose influx and utilization rates, that would predict an average 30-35% increase in rJin (or TmaX> in those eight regions in the present study exhibiting significant rCMRglc increases (mean increase 62%). Further evidence for the above can be taken from euglycemic comparisons of brain tissue glu cose concentrations between control rats and rats exhibiting elevated rCMRglc values. Thus, tissue glucose levels invariably have been shown to be significantly reduced under such conditions (Fol bergrova et aI., 1981; Cremer et aI., 1988). A falling brain tissue glucose in the face of an unchanged plasma glucose concentration is best explained by the concept that when the glucose utilization rate increases, the glucose influx rate increases to a lesser extent. In the eight regions of the diabetic, acute normo glycemic group demonstrating rCMRglc values sig nificantly higher than control, we measured rJin val ues that, when averaged, were more than double those obtained in control rats. This rJin change far exceeded that which one would predict based upon an rCMRglc change alone (see the previous para graph). It is tempting to ascribe the elevated rJin values in these acutely normoglycemic diabetic rats, at least in part, to an increased BBB glucose transport capacity. However, Cremer et aL (1988) presented evidence suggesting that the double label, 2-DG method may overestimate the actual rJin when cerebral glucose utilization is substan tially increased. They speculated that under hyper metabolic conditions, this error is created either as a result of inadequate mixing of 2-DG with the na tive brain glucose pool (metabolic compartmenta tion) or because of an increasing transport asymme try between the luminal and abluminal cerebral cap illary endothelial cell membranes. Whatever the cause, in hypermetabolic regions, the influx rate in creases from the control that Cremer et aL calcu lated, employing either of two double-membrane ki netic models for BBB glucose transfer (see also Cunningham et aI., 1986), were less than those ob tained using the double-label 2-DG method. We ap plied the same type of analysis to the data obtained in the present study. The analysis was based upon =
J Cereb Blood Flow Metab, Vol. 10, No.6,
1990
the simpler of the two transport models only (type I) (Cunningham et aI., 1986). This should be adequate for detecting the presence of the kind of error de scribed by Cremer et al. that may accompany con ditions of increased brain glucose metabolism, i.e., the influx rate changes from control estimated from the two transport models in the Cremer et al. (1988) report were virtually the same. Our calculations of rJin were performed using an algorithm, taken from Cremer et al. (1988), that al lowed for the calculation of Tmax (which is equiva lent to one-half the value of the maximum transport velocity of the two endothelial cell membranes) and employed measured values for tissue glucose (in mM) , arterial plasma glucose, and rCMRglc and a transport constant (Kt) of 6 mM. The arterial plasma concentration was corrected to a mean cerebral capillary concentration according to Cremer et al. (1988). This procedure utilized cerebral plasma flow values, obtained in a preliminary study (Pelligrino et aI., 1990). Flow values in acutely normalized di abetic rats were similar to control in subcortical and hindbrain structures and 20-25% higher than con trol in the cortex. The results of the model dependent rJin calculations are presented in Fig. 5, along with the values obtained using the 2-DG method. The eight regions that showed significantly higher "measured" rJin values (compared to con trol-see Fig. 3) are represented in the figure. These results demonstrated a fairly good agreement be tween measured and calculated rJin values not only in control animals, but also in acutely normoglyce mic diabetic rats as well. Furthermore, the relation
.,c E .,
ry
4
1
t
T
T
I
1
0 E
"" c
� 12
12
12
12
12
12
12
1 2
FC
SMC
CP
HT
AM
HC
SC
PO
FIG. 5. Regional glucose influx rates (rJin) in normoglycemic controls (cross-hatched bars) and diabetic, acute hypergly cemics (open plus cross-hatched bars) estimated from the sequential double-label 2-0G procedure (1) and from a sim ple double membrane (type I) transport scheme (2). The latter calculation employed an algorithm described by Cunning ham et al. (1986) and Cremer et al. (1988). Results are given for the eight regions demonstrating significantly higher (compared to control) rJin values derived from the 2-0G method (see Fig. 3). Additional information is provided in the text. All values are means ± SO.
BRAIN GLUCOSE TRANSPORT IN DIABETES ships between nondiabetic and diabetic rats were similar for both methods of rJin estimation. Based on rJin values averaged over the eight regions rep resented in Fig. 5, we found the influx rate to be 2.2-fold greater than control with rJin values deter mined via the 2-DG method and twofold greater with calculations based upon type I kinetics. Al though these findings do not verify the accuracy of the results summarized in Fig. 3, they do provide support for our conclusion of a higher BBB glucose transport rate in acutely normalized diabetic rats compared to normoglycemic controls. As discussed earlier, results from previous stud ies have shown that brain tissue glucose levels fall when brain glucose metabolism increases (un changed plasma glucose). The present finding of re gional tissue glucose concentrations in acutely nor moglycemic diabetic rats that were higher than con trol in the presence of elevated rCMRglc values also appears to support the existence of an increased BBB glucose transport capacity in the diabetic rat. However, one must consider the possibility that the comparatively higher tissue glucose levels in the acutely normalized diabetics may be, at least in part, a function of too short a period to achieve a steady-state relationship for glucose between brain and blood. Preliminary findings in three diabetic rats studied at 18--24 h of glycemic normalization (not shown) have indicated a sustained elevation in regional tissue/plasma glucose ratios. This suggests that the tissue/plasma glucose results of Fig. 4 are not attributable to incomplete equilibration of glu cose between tissue and plasma. The apparent in crease in BBB glucose passage cannot be explained at this time. It is seemingly related to factors asso ciated with the diabetic state and maybe chronic hyperglycemia-related changes in BBB function (in cluding possible changes in metabolismitransport coupling). One possibility is an increased availabil ity of glucose transporters. This is not an unprece dented assertion. Harik et al. (1988) reported an increased density of glucose transporters in brain microvessels harvested from diabetic rats. Choi et aI. (1989) provided evidence indicating increased levels of mRNA for the principal BBB glucose transporter in chronically hyperglycemic rats. An increased glucose transporter synthesis in response to hyperglycemia has been suggested for other tis sues as well (Csaky, 1988). In conclusion, the present results demonstrate that chronic hyperglycemic diabetes in rats is not associated with a BBB glucose transport repres sion. In fact, the opposite appears to be true. Fur thermore, acute glycemic normalization in these an imals resulted in an increased cerebral metabolism.
779
There is a suggestion that the degree of brain dam age accompanying cerebral ischemia may be di rectly related to the level of cerebral metabolic ac tivity at the time of ischemic induction (Busto et aI., 1987). The presence of a hypermetabolic state in addition to higher than normal tissue glucose levels could lead to a greater degree of neuronal damage than one might otherwise predict. Thus, current presurgical clinical management of the diabetic pa tient-i.e., to normalize blood glucose quickly may fail to diminish risk. Longer periods of preop erative normoglycemia are probably required. Acknowledgment: The authors wish to thank Anthony Sharp and Jerome Galloway for expert technical assis tance and Lois Boler for typing the manuscript. This work was supported by the Juvenile Diabetes Foundation International.
REFERENCES Bryan RM. Hawkins RA. Mans AM. Davis DW. Page RG (1983) Cerebral glucose utilization in awake unstressed rats. Am J Physiol 244:C27O-C275 Busto R. Dietrich WD, Globus MYT, Valdes I, Scheinberg P, Ginsberg MD (1987) Small differences in intra-ischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 7:729-738 Choi TB, Boado RJ, Pardridge WM (1989) Blood-brain barrier glucose transporter mRNA is increased in experimental di abetes mellitus. Biochem Biophys Res Commun 164:375-380 Cremer JE, Cunningham VJ, Seville MP (1983) Relationships between extraction and metabolism of glucose, blood flow, and tissue blood volume in regions of rat brain. J Cereb Blood Flow Metab 3:291-302 Cremer JE, Seville MP, Cunningham VJ (1988) Tracer 2deoxyglucose kinetics in brain regions of rats given kainic acid. J Cereb Blood Flow Metab 8:244-253 Csaky TZ (1988) Membrane transport of sugars in diabetes mel litus. In: Membrane Biophysics III: Biological Transport (Dinno MA, Armstrong WMCD, eds), New York, Alan R. Liss Inc., pp 37-42 Cunningham VJ, Cremer JE (1981) A method for the simulta neous estimation of regional rates of glucose influx and phosphorylation in rat brain using radiolabelled 2-de oxyglucose. Brain Res 221:319-330 Cunningham VJ, Hargreaves RJ, Pelling D, Moorhouse SR (1986) Regional blood-brain glucose transfer in the rat: a novel double-membrane kinetic analysis. J Cereb Blood Flow Metab 6:305-314 DeFronzo RA, Hendler R, Christensen N (1980) Stimulation of counter-regulatory hormonal responses in diabetic man by a fall in glucose concentration. Diabetes29:125-131 Duckrow RB (1988) Glucose transfer into rat brain during acute and chronic hyperglycemia. Metab Brain Dis 3:201-210 Folbergrova J, Ingvar M, Siesjo BK (1981) Metabolic changes in cerebral cortex, hippocampus, and cerebellum during sus tained bicuculline-induced seizures. J Neurochem 37:12281238 Gjedde A, Crone C (1981) Blood-brain glucose transfer: repres sion in chronic hyperglycemia. Science 214:456-457 Harik SI, Gravina SA, Kalaria RN (1988) Glucose transporter of the blood-brain barrier and brain in chronic hyperglycemia. J Neurochem 51:1930-1934 Harik SI, LaManna JL (1988) Vascular perfusion and blood brain glucose transport in acute and chronic hyperglycemia. J Neurochem 51:1924-1929 Hawkins RA, Mans AM, Davis DW, Hibbard LS, Lu DM (1983)
J Cereb Blood Flow Metab, Vol. /0, No.6,
1990
780
D. A. PELL/GRINO ET AL.
Glucose availability to individual cerebral structures is cor related to glucose metabolism. J Neurochem 40:1013-1018 Kalimo H, Rehncrona S, Soderfeldt B, Olsson Y, Siesjo BK (1981) Brain lactate acidosis and ischemic cell damage: 2. Histopathology. J Cereb Blood Flow Metab1:313-327 Lilavivathra U, Brodows RG, Woolf PD, Campbell RG (1979) Counter-regulatory hormonal responses to rapid glucose lowering in diabetic man. Diabetes28:873-877 Lowry OH, Passonneau JV 1 ( 972) A Flexible System of Enzy matic Analysis, New York, Academic Press
J
Cereb Blood Flow Metab, Vol. 10, No.6, 1990
McCall AL, Millington WR, Wurtman RJ (1982) Metabolic fuel and amino-acid transport in the brain in experimental diabe tes mellitus. Proc Natl Acad Sci USA 79:540&-5410 Pelligrino DA, Sharp AJ, Albrecht RF (1990) Diminished CBF response to hypoglycemia in the diabetic rat. J Neurosurg Anesthesiol2:S7 Pulsinelli WA, Levy DE, Sigsbee B, Scherer P, Plum F 1 ( 983) Increased damage after ischemic stroke in patients with hy perglycemia with or without established diabetes mellitus. Am J Med74:540--544
