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Glucose kinetics and fatty acids in dogs on matched insulin infusion after glucose load
Glucose Kinetics and Fatty Acids in Dogs on Matched Insulin Infusion After Glucose Load By MLADEN VRANIC, PETER FONO, NADA KOVACEVIC.AND BONIFACEJ.
The endogenous insulin secretion of five conscious, depancreatized fasted dogs was replaced by intraportal insulin htfusions of 192-263 pU/kg per min to establish whether the release of extra insulin is essential during a glucose tolerance test. Infusion rate of insulin about 200 pU/kg per min was considered to be near basal since it maintained unchanged glucose turnover as well as the plasma concentrations of glucose, free fatty acids (FFA), and immunoreactive insulin @RI). The glucose tolerance tests of these dogs were then compared to those of six normal and six partially depancreatized dogs. It was found that after an intravenous injection of glucose the depancreatized dogs maintained on portal or peripheral insulin infusion had a 50% decreased exponential slope of the plasma glucose
LIN
concentration versus time (k). In contrast to the normal dogs, their plasma glucose levels were still elevated but declining 60-140 min after the glucose load. The declining glucose levels were associated with an increased disappearance rate (R,,), but there was no inhibition of the rate of glucose production. FFA decreased only slightly after the glucose load. Five days after partial pancreatectomy the dogs reacted to a glucose injection with a lesser output of insulin than normal dogs. Their homeostatic deficiencies with respect to glucose kinetics and FFA response were less marked than in depancreatized dogs maintained on matched insulin infusion. It was concluded that a prompt release of extra insulin is an essential part of the normal borneostatic mechanisms that determine a glucose tolerance curve.
HE INTRAVENOUS GLUCOSE TOLERANCE TEST is widely used to detect abnormalities of the mechanisms involved in the control of glucose homeostasis. Soskin et al.1 suggested that a normal glucose tolerance test can be obtained in depancreatized dogs provided a minimal amount of insulin is infused at a constant rate. They concluded therefore that insulin release does not play an essential role in the homeostatic mechanisms that determine a normal glucose tolerance test. In their experiments when insulin was given immediately after total pancreatectomy, general anesthesia and stress due to extensive surgery were necessary complications. In other experiments their depancreatized dogs were maintained on insulin injections until recovery. It is difficult to ascertain whether such depancreatized dogs react physiologically,
T
From the Department of Physiology, University of Toronto, Toronto, C‘nmdu. Recei,,ed for publicutiort April 7, 1971. This work MWS supported by Grnnf MA-2197 from the Medical Resecrrch Cormcil of Canada and by the Canadian Diabetic Association. MLADEN VRANIC, M.D., D.Sc.: Associate Professor, Department of Physiology, Medical Faculty, University of Toronto, Toronto, Canada. PETER FONO, B.Sc.: Student, Faculty of Toronto, Canada. NADA KOVACEVIC, P.ENG.: Research Medicine, University of Toronto, Assistant, Department of Physiology, University of Toronto, Toronto, Canada. BONIFACE J. LIN, M.D.: Assistant Professor, Department of Physiology, University of Toronto, Toronto, Canndn. 954
METABOLISM,VOL. 20, No. 10 (OCTOBER). 1971
GLUCOSE KINETICS
AND FATTY ACIDS
955
since they are exposed chronically to an unphysiologic amount and route of supply of insulin. Their techniques were therefore limited. This alone would make their conclusions doubtful. It was further shown that during glucose infusion the release of insulin is indispensable for the regulation of glucose production in the dog.” It was also reported that the rate of glucose utilization in human diabetics increases during a glucose tolerance test to a lesser extent than in normal humans.3 However, Kosaka et al.” obtained a normal glucose tolerance curve in the acutely depancreatectomized, anaesthetized dog. Their work thus appeared to corroborate the earlier conclusions of Soskin and led to the conclusion that increased insulin secretion is not indispensable for a normal glucose tolerance test. In view of the conflicting evidence, therefore, it seemed desirable that this problem should be reinvestigated using techniques that were designed to avoid general anaesthesia and the acute stress that accompanies extensive surgery. Insulin was infused, after removal of a remnant pancreatic graft, into the portal vein of conscious dogs. These dogs were at no time without insulin, and surgical stress was reduced merely to the removal of the subcutaneous graft. The plasma concentrations of FFA, IRI, and glucose and the turnover of glucose were determined before and after removal of the pancreatic graft to ascertain that the rate of insulin infusion was near the basal insulin secretion rate. Glucose tolerance tests were performed in normal dogs, partially depancreatized dogs, and dogs on constant near basal intraportal or peripheral insulin infusion. The early part of the glucose tolerance curve was analyzed by calculating the slope of the logarithm of the plasma concentration versus time. Glucose turnover was determined at 60 min after the glucose load to study at that time the deviations from the initial dynamic steady state in the three groups of dogs. MATERIALS AND METHODS
Experimental
Procedures
A glucose load (0.5 g/kg body weight, i.v.) was given to six normal, six partially depancreatized, and five totally depancreatized dogs maintained on a fixed intraportal infusion of insulin. In four dogs of the last group a glucose injection was repeated with insulin infusion switched from the portal to the cephalic vein. These 21 experiments were performed in eight dogs (Table 1). An intravenous injection of U-I%-glucose (20 &i) was given 80 min prior to, and 60 min after a glucose load to determine the rates of production (appearance, R,) and of disappearance (Rd) and the metabolic clearance (M) of 1sC-glucose using the method of successive measured injections of tracer.5 The slope of the logarithm of the concentration of plasma glucose versus time (k) was calculated at 20-60 min after the injection of the glucose load. The period from 20-60 min after the glucose load was assessed by the nontracer method to avoid intermixing problems when the concentration of unlabeled glucose is changing rapidly in the pool. The period from 60-140 min was analyzed by a tracer method. IRI and FFA response curves following the glucose load were also determined.
Animals and Surgical Procedures Adult female dogs (9-13 kg body weight) were acclimatized to the standard kennel conditions for 4 wk. All experiments were carried out on conscious dogs trained to stand or lie in a Pavlov stand. Partial pancreatectomy and exteriorization of the
956
VRANIC
ET AL.
pediculated uncinate process of the pancreas into a subcutaneous pocket as described by Rappaport et a1.Q was carried out under general anesthesia 5 days prior to the experiment. Concurrently, a vinyl tubing (Becton, Dickinson & Co., No. 6179) filled with heparin (200 U/ml) was inserted into a tributary of the splenic vein pointing toward the portal vein. The postoperative care and diet of the operated dogs has been described earlier.7 These dogs retained approximately one-third of their pancreas, which was capable of maintaining normoglycemia during fasting and also of preventing postalimentary glycosuria. In earlier work the subcutaneous graft was enclosed in a polyethylene jacket, and postoperative glycosuria was observed occasional1y.Q In the present work the ventral side of the graft was covered with a polyethylene sheet; the dorsal side received collateral blood supply from the underlying muscles to improve the viability of the graft. On the day of the experiment one catheter was inserted under local (procain) anesthesia into the vena cava (with the tip below the points of entry of the hepatic veins) via the saphenous vein. This catheter, filled with a dilute heparin solution (5 U/ml) was used for the collection of blood samples and for injections of I%- and l*C-glucose. Another catheter was inserted into one of the cephalic veins to test the effect of switching the insulin infusion from the portal to a nonsplanchnic vein. After the experiment the partially depancreatized dogs were placed in a metabolic cage until the next morning when the pancreatic graft was removed under local anesthesia and an infusion of insulin (192-263 pU/kg per min) into the portal circulation was started promptly. (The infusion solution: 40 mu/ml porcine insulin and 5 mg/ml of gelatin, Upjohn Co., Kalamazoo. in an acid-saline solution of pH = 3.9-4.0. Thus, the binding of insulin to glassware and tubing was minimized.) Equilibration for 45-60 min was allowed, during which minor adjustments in the rate of infusion of insulin were made when warranted according to the blood glucose level estimated by serial DEXTROSTIX (Ames) tests. A constant rate was then maintained. At autopsy no infection was detected in the skin, abdomen, or thorax, and all the catheters were patent in all dogs.
Processing of Blood Samples The collection and processing of blood samples have been described.7 The heparin solution was withdrawn from the portal cannula prior to the experiment. A solution of heparin (5 U/ml) was used during the experiment to keep the sampling catheter patent. The isolation of glucose with help of an ion exchange resin (BIO-RAD AG 2-X8) and determination of glucose (Huggett and Nixons) as well as counting procedures have also been described.7 Aliquots of I%-glucose standard were treated in the same way as plasma samples. Radioactive strengths were corrected for quenching using the ratio of external standard counts. Plasma FFA were measured by titration,9 and IRI by the method of Yalow and Berson.10 The reference standard used in the insulin assay was porcine insulin, and the guinea pig antiserum had been prepared against porcine insulin. The only source of plasma insulin in pancreatectomized dogs maintained on insulin was porcine insulin.
Calculations Single exponential functions were fitted to sets of concentrations of labeled glucose (dpm/unit volume, to calculate the rate of glucose disappearance) and specific activities (dpm/&g glucose, to calculate the rate of glucose appearance) with respect to time, while linear functions were fitted to glucose concentrations versus time (dc/dt). These calculations as well as statistics were carried out on a Canadian General Electric Co. time sharing computer system. The rate of appearance was calculated as R, = N,‘K’, where K is the constant of the single exponential function fitted to the specific activity of glucose with respect to time, and N, the intermixing mass (pool size) of body glucose.5J1Ja The appearance of unlabeled glucose was detected only since corrections for recycling of the label were not done. The following two different equations were used to calculate R,: R, = R, - dN/dt Assuming constancy of glucose space, dN/dt = (N,/c,).(dc/dt).
957
GLUCOSE KINETICS AND FATTY ACIDS
N,/c, = Intermixing mass of body glucose divided by glucose concentration. Both parameters are obtained by extrapolation to the time of tracer injection. R,* = K.*N,, where K* is the constant of the single exponential equation fitted to the concentrations of labeled glucose (dpm/O.l ml) with respect to time.s The mean differences between R, and Rc* were l%-2% when glucose in plasma was near dynamic steady state (DSS) at normal plasma glucose level. A 5%-8% dxerence was observed between R, and Rd* when deviations from DSS were more marked. In the depancreatized dog maintained on insulin infusion the plasma glucose levels were still declining after the glucose tolerance test when the tracer injection was given. Figure 1 shows in a representative experiment that exponential functions fitted at that time to either specific activities or concentration of tracer with respect to time had essentially the same accuracy of the fit as in the fasted dog prior to the glucose tolerance test. In all experiments except one, the plasma glucose levels were below the renal threshold for glucose at the time when the second tracer was given, therefore R, approximates the rate of glucose utilization. Metabolic clearance (M) = rate of glucose disappearance/mean plasma glucose concentration.13
RESULTS Plasma Concentrations
of Glucose, FFA,
and IRI
The effects of a glucose injection in normal, partially depancreatized, and depancreatized insulin-infused dogs are shown in Figs. 2, 3 and 4. In the period prior to glucose injection there were no significant differences in the initial concentrations of glucose, FFA, and IRI except in one dog that became hyperglycemic after removal of the pancreatic graft. These data suggest that the exogenous supply of insulin had been comparable to the endogenous supply before removal of the graft. The depancreatized dogs maintained on matched intraportal insulin infusion had a decreased tolerance for glucose. The exponential slope of the concentration of glucose in plasma (k) was reduced 37% in six partially depan-
Fig. l.-Specific activities (black circles), DPM/unit volume (black triangles), and the concentrations of plasma glucose (open circles) before (T5) and 60-140 min after glucose load (Ts) in depancreatized insulin-infused dog (7). Single exponential functions were fitted to values of specific activities, and concentration of tracer and linear functions to concentrations of glucose in plasma from 20 min following successive tracer injections. Respective values of N,, R,, Rc, and Rc* were 276 mg/kg and 2.58, 3.15, and 3.09 mg/kg per min before glucose load; after load values were 444 mg/kg, 2.75, 4.22, and 4.01 mg/kg per min, respectively. Increased R, is presumably consequence of increased plasma glucose levels.
5 s -‘, $ z Z i
r0
$ c : ’ i $, s 2 i = s
t4sIZO,oo_ so - \ I 0
, I 40
80 Minuiu
oll*r
I 40
I 0 frclcer
injection
I 80
958
.~ ---__J-~_.L
~~
200
100
UTES
L
I
0
-
-L
A-
100
L_,+
0
200
4 S
I
260 t
j
360
400 ' 4 g
Fig. 2.-Effect of sequence of i.v. glucose injections of 0.5 g/kg (Tg) in dog 5: (a) normal; (b) , 5 days after partial pancreatectomy, and (c) and (d) following day after pancreatectomy on constant insulin infusion. PEX indicates removal of pancreatic graft and beginning of intraportal infusion of canine insulin (P.I.). One glucose injection was given when dog was on intraportal insulin infusion (c), while other injection occurred during infusion into cephalic vein (d). Times of i.v. injections of i4C-glucose are shown as Ti-Tr. Computer-fitted single exponential functions to sets of values of specific activity (black circles) and time extrapolated to time of tracer injection are shown on upper scale; k in lower scale; slope of log plasma glucose cont. X (-100) in min-l.
Fig. 3.-Concentrations of FFA, IRI, and glucose in plasma following i.v. glucose injections of 0.5 g/kg at point (tg) . Dotted line crosses initial values of FFA and IRI; k in lower scale, slope of log plasma glucose cont. X ( - 100) in min-I. (Left) Six normal dogs. (Right) Six partially depancreatized dogs.
.________.[71:_.k#'~~<~ IO -
.__________a.___~_~~~____.._ %+-a
0-
i
i
I 400 L
;
i
! _I
-50
I
I
0
50
1 9
-I
I
100
I
-50 MINUTES
0
I (
~__I
-
50
100
GLUCOSE KINETICS
959
AND FATTY ACIDS
Fig. 4.-Five dogs following removal of remnant pancreatic graft on coninsulin stant infusions (shown in parentheses). In four dogs route of infusion was switched from portal to cephalic vein, with rates of infusion kept constant at 192, 205, 230, and 263 ,uU/kg per min, respectively. Fifth dog was infused with 203 ,uU/kg per min intraportally only; this dog was hyperglycemic at time of graft removal and its extremely low k is shown separately. (See Fig. 3 for meanings of symbols.)
20 -i 2
IO ,_______ L
_____________
jj ‘9* f&j __ +s_
$+f+ ___
_ __________
LL O-
500
L
400 L
300 t
200 -
100 -
O-
l INSULIN INFUSION
PORTAL
I --loo
0
t
9
100
200
I MINUTES
t
9
creatized (p < 0.01) dogs. In four dogs maintained on intraportal insulin infusion there was a further 52% decrease in k (p < O.OOl), while the fifth infused dog had a k of only 0.47 minute- l. The same conclusion was reached regardless of whether k was obtained by fitting single exponential functions to either absolute glucose concentrations or to their difference from the initial level. In normal dogs the initial glucose concentrations were regained 78 & 10 min after the load, but in partially depancreatized dogs not until 107 i. 12 min. On portal infusion the initial values were restored in two dogs at 100 and 75 min, respectively, while increased values persisted in three dogs until the end of the experiment. Two minutes after the glucose load, IRI rose fivefold in normal dogs but only 1.7-fold in the partially depancreatized dogs. In the latter, a decreased response in IRI was noted within 30 min following the glucose load. The two insulin areas obtained by integration during the O-60 min period after the glucose load were also different: 771 -e 135 JLJ and 329 & 116 ,JJ (p < 0.05) in normal and partially depancreatized dogs, respectively. The IRI values remained constant during the intraportal infusion of insulin. Figure 5 shows that the partially depancreatized dogs had also a decreased regression line of mean IRI values on mean glucose concentrations following injection of the glucose load. Similar differences were reported between genetically prediabetic males and normal subjects .14 As expected, the IRI values remained constant in depancreatized dogs during the intraportal infusion of insulin.
960
VRANIC ET AL.
Fig. !L-Correlation be40 -. tween mean glucose connormal: IRI= 0.081 G.C.+ 1.97 . centration and mean IRI in plasma 3.5-60 min follow( portiolly deponcreatized ) ing a glucose challenge 3. - -.s 0 IRI = 0.032 G.C. (0.5 g/kg i.v.) , calculated T > according to Soeldner et =t al.14 Circles indicate mean =20 serum IRI-mean blood : glucose at each of the six ; and five time intervals in normal (black circles) and 10 partially depancreatized (open circles) dogs, respectively. Correlation co0 1 I I I efficients: r = 0.976, p < 100 200 300 400 0.01 (normal); r = 0.875, p < 0.05 (partially depanMeon glucose concentration mg/lOO ml creatized)-~. Analysis of covariance”0 showed that two slopes are significantly different at 2% level. Regressions of IRI values on glucose concentrations were also calculated in individual dogs and averaged, yielding mean slopes of 0.091 and 0.016 in normal and partially depancreatized dogs, respectively.
In the partially depancreatized dog the maximum reduction in FFA was 37% at 66 min as compared to normal reduction of 63% (p < 0.001) at 42 min. A small maximum decrease of 23% was noted even in the dog on matched insulin infusion, part of this decrease (3%-5% at most) could be attributed to the dilution of plasma caused by the hypertonic injection of glucose. Table l.-Plasma Glucose Concentrations (mg/lOO ml) at Time of Tracer Injection (C,) and 80 Min Thereafter (Cs,,). Total of 21 Glucose Tolerance Tests were Performed on Eight Female Dogs Depancreatized Normal Fasted Dog Number
_ co
1
---
2 3a 3b 4a 4b
--102 106 93 92
5 6 7 8
104 _-_ --101
After c80
cot
Partially Depancreatized G’lT*
118 109 112 108
106
103
105
141
After GTT
Fasted
Cephalic
After GTT ____ co c60
After GTT
C”
cao
co
cm
.~ co
cm
co
cso
100 95 89 91
108 127 _ _
99 149 _ _
129 180 _ _
96 132 _ _
167 _ _
222 _ __
315 _ _
259 _ _
_ _
_ _
114 92
113 104 105 82
129 116 96 81
137 159 172 145
105 114 113 71
112 144 102 85
91 119 88 74
142 134 168 162
72 82 129 121
151 88 214 194
91 68 181 135
CLV -
100 106 91 90
Fasted
on Insulin
Portal
* GTT, glucose tolerance test. T C, after GTT refers to tracer injection given 60 min after i.v. injection of 0.5 g/kg glucose.
2.78 & 0.27
M
23.4 +- 1.6
Normal
27.8 + 0.8” (19)
2.92 + 0.23 (7) 3.05 Zk 0.19 (11) 3.10 -t 0.21 (11) 3.17 f 0.15 (14) -0.13 f 0.23
After
28.5 +- 1.6
0.12 2 0.17
3.01 + 0.26
3.22 -c 0.17
3.17 + 0.18
3.29 -c 0.19
30.5 + 1.3 (7)
3.52 t 0.30 (7) 4.69 + 0.01 (48) 5.03 f 0.19 (56) 4.16 + 0.29 (38) - 1.17 -c 0.33
After
Partially Depancreatized Before
$
t
5
§
4 0.39
28.2 + 1.27
-0.30
3.46 2 0.41
3.78 C 0.25
3.74 ” 0.25
After
28.6 + 1.43 (1)
3.51 * 0.34 (2) 4.80 i- 0.22 $ (28) 5.09 + 0.25 t (35) 3.84 -c 0.73 (11) -1.46 -c 0.28*
Portal infusion
3.44 c 0.41
Before
31.40 & 1.60
- 1.55 + 0.46
4.25 + 0.93
5.26 + 0.67
4.90 f 0.44
3.35 2 0.30
After
Cephalic Infusion
In parentheses: per cent changes following glucose injection. Two different equations were used to calculate rate of glucose disappearance (Rd and Rd*>. Statistical comparisons are between values before and after glucose injection for each group of animals. * p < 0.05. t p < 0.02. $ p < 0.01. F,p < 0.001. Comparisons between groups before glucose injections show a slightly higher V in partially depancreatized and insulin infused dogs when compared to normal (p < 0.05); R, of insulin infused dog was higher than in normal dog (p < 0.05).
V
+ 0.08
2.80 f 0.34
Rd*
-0.03
2.75 + 0.29
Rd
dN/dt
2.72 2 0.22
VI
Before
Table 2.Rates of Appearance (R,), Disappearance (RJ, Accumulation (dN/dt), (mg/kg/mln), Metabolic Clearance (M), (ml/kg/mln), and Volume of Distribution (V) of Glucose (% Body Weight) Before and After 0.5 g/kg Glucose Injected Intravenously in Slx Normal, Six Partially Depancreatized, Five Depancreatlzed Dogs on Intraportal and in Four Depancreatized Dogs on Cephalic Insulin Infusion
962
VRANIC
ET AL.
Glucose Turnover
The rates of glucose production, diappearance, and metabolic clearance are shown in Table 2. Concentrations of glucose in plasma at the time of tracer injection and 80 min thereafter are shown in Table 1. Glucose turnover of the partially depancreatized dog and of the depancreatized dogs maintained on insulin infusion were compared to assess how well the exogenous supply of insulin was matched to that of the previous endogenous supply. The depancreatized dogs maintained on constant insulin infusion had a slightly higher Rd and M than the partially depancreatized dogs. Since the depancreatized dogs were infused at different rates of insulin, it was interesting to find out whether changes in Rd, M, and R, were correlated to the rates of insulin infusion (Fig. 6). To make these correlations more meaningful, seven additional points from a previous publication? were added to this figure. A positive correlation was found between the increments of Rd, M, and the insulin infusion rates, and a negative correlation was found between the increments in R, and the infusion rate of insulin. Only an infusion of insulin near 200 ,,U/kg per min maintained these parameters unchanged. Thus it appears that two depancreatized dogs were given an amount of insulin that was slightly above basal. At 60-140 min after the glucose tolerance test the normal dog was near its initial dynamic steady state and its rate of glucose accumulation (dN/dt) was only -0.13 mg/kg per min. In contrast, the efliciency of the homeostatic mechanisms of glucose was impaired in the partially depancreatized and depancreatized insulin-infused dog, since they had still not attained the dynamic steady state as judged by the negative dN/dt values. This was caused by a significantly increased R,,, since there was no difference in R,, between the post- and preglucose tolerance values. Table 2 shows no differences in the postglucose tolerance values when the portal infusion of insulin was switched to the cephalic vein.
Fig. 6.-(Upper) Regression of metabolic clearance (M) of glucose on rate of insulin infusion after removal of pancreatic autograft r = 0.951, p < 0.002. Correlations between R, and rate of insulin infusion yielded essentially same correlation coefficient. (Lower) Regression of rates of production (R,) of glucose on rates of insulin infusion after removal of pancreatic autograft: r = -0.850, p < 0.002. Seven points included in graph have been calculated from Vranic and Wrensha1l.i Glucose concentrations in all experiments were below 200 mg/ 100 ml. 100 RAIES
200
OF INTRAPORTAL
INFUSION
INSULIN
MICRW/Kg-min
963
GLUCOSEKINETICSANDFATTYACIDS DISCUSSION
The purpose of these studies was to investigate the metabolic events following a glucose tolerance test in conscious depancreatized dogs maintained on a constant near basal rate of insulin infusion. The importance of intraportal supply of this hormone has previously been emphasized,15 and therefore exogenous insulin was administered by its physiologic site of entry. The pancreatic uncinate process of the dog is said to lack the glucagon producing alpha cells,16-18 so that infusion of insulin alone could compensate the sudden deprivation of pancreatic hormones following its removal. Further work is needed to ascertain whether more insuhn is secreted in normal dogs, because they produce pancreatic glucagon as well. Sampling of the total venous outflow from the uncinate process in situ showed the mean rate of insulin output to be 190-202 pU/kg per min by the mouse hemidiaphragm bioassay and the immunoassay, respective1y.l” Similar mean values were reported in normal dogs on the basis of the rate of disappearance of immunoreactive 1311-Insulin20 or cannulation of pancreatic vessels .21 Higher secretion rates of insulin, considered to be maximal, were reported in the rat using the method of neutralizing antibodies.22 It appeared that infusion rates of about 200 pU/kg per min might replace and match the endogenous secretion of the pancreatic graft. So far 34 experiments were performed in which endogenous insulin secretion was replaced by a matched exogenous portal insulin supply. These dealt with physiologic challenges such as infusions of glucose and ribose,2 glucagon,23 and exercise.“* The basal insulin infusion did not maintain homeostasis in two dogs that were hyperglycemic before remova of the graft. Of the remaining 32 dogs, the match was achieved in 88% of the experiments. The phrase “quantitative matching” is only meaningful if it can be shown that under physiologic conditions insulin is secreted continuously. That a continuous supply of insulin is normally available to the animal is suggested by the facts that changes in rates of production and utilization and the concentration of ghrcose occur promptly following the cessation of endogenous or exogenous insulin s~pp1y.r~~~ This is in keeping with a short biologic half life for insulin. To ascertain the success of matching the endogenous insulin supply it is important to determine glucose turnover, since it is known that glucose concentrations do not always reflect accurately a change of the hormonal balance. For example, prolonged fasting,2s exercise,27 chronic administration of steroids28 or growth hormone2” cause a marked change in the glucose transfer rates with only slight changes in the glucose levels. In this work small increases in insulin supply were closely related to increments in Rd and M, suggesting that a control system involving insuhn secretion adjusts glucose concentration primarily by changing the overall glucose utilization (fine tuning). The inhibitory effect of insulin on R, is less marked. In 1934 Soskin et a1.l reported a normal glucose tolerance test in depancreatized dogs when insulin was infused at a constant rate, but this suggestion has not been confirmed. In our experiments insulin infusions near 200 PU/kg per min were given intraportally to conscious depancreatized dogs that have not been diabetic. In contrast, Soskin’s peripheral infusions of insulin varied
964
VRANIC ET AL.
between half and 33 times the basal infusion rate, and in most of their experiments insulin and glucose infusions were given simultaneously as they had difficulties in maintaining glucose homeostasis by insulin infusions only. In this experiment the analysis of the glucose tolerance test proves that dogs on basal insulin infusion were unable to handle a glucose load normally. The importance of a normal release of insulin was indicated, since both partially depancreatized and depancreatized insulin-infused dogs had a significantly reduced k in the early period and they failed to regain their initial DSS in the late glucose tolerance period. At 60 min after the glucose load the plasma glucose levels were still elevated but declining. The increased glucose level were associated with an increase of Rd but there was no inhibition in the rate of glucose production at that time. This work thus extends the earlier observations that clamping of the remnant pancreatic autograft for 30-60 min promptly reduced tolerance for glucose in the conscious dogs. The restoration of the graft’s blood supply restored the initial tolerance for glucose. This effect was considered to be due to sudden cessation of insulin supply. The handling of the graft served as a sham operation for clamping or removal of the graftz5 In the present experiments replacement of the graft by an intraportal infusion of insulin induced a decrease in tolerance for glucose (Fig. 2) that was smaller than the decrease caused by complete insulin deprivation. The partially depancreatized dogs reacted to the glucose challenge with a reduced insulin response. It might be that the impaired insulin response is due not only to the reduced size of the pancreas but also to the postoperative consequences of the abdominal operation. This possibility, however, does not alter the main observation that the normal glucose tolerance test depends on a normal release of extra insulin. This conclusion finds support in the observations that 5 days after removal of two-thirds of the pancreas the insulin sensitivity of these dogs did not change. Canine insulin was infused intraportally at 1.4 mu/kg per min for 1 hr before and 5 days after partial pancreatectomy; the per cent reductions in the plasma glucose levels were 37 + 0.7 and 33 t 4.6 (mean +- SEM), respectively. (Vranic, M., Fono, P., Kovacevic, N., and Cherrington, A.: unpublished observations.) In the present experiments normal dogs were near the dynamic steady state during the sampling period of the second tracer injection. At that time the plasma glucose concentrations were still declining in the partially depancreatized and the depancreatized insulin-infused dogs; the rate of decrease was calculated by a linear regression. Method of successive measured injections of tracer has been validated for the rate of appearance of 12C-glucose in the eviscerated dog. Valid rates of glucose appearance were obtained even when the glucose concentrations were changing in p1asma.l” More recently Cowan et al.11 have achieved validation to within 10% of the rate of appearance of glucose in the normal conscious dog. With a rapid linear decrease in the rate of appearance of tracee, the tracer-determined rate did not vary significantly from those rates determined in experiments near to dynamic steady states. With a measured constant acceleration in the rate of appearance of tracee, the tracer-determined
965
GLUCOSE KINETICS AND FATTY ACIDS
rate of appearance was found to compare with the measured rate near the midpoint of the sampling period that followed. In the present experiments the concentration of plasma glucose was decreasing toward a dynamic steady state when the second injection of tracer was given (Fig. 1). The rates of change of the plasma glucose concentration were not large at that time: -0.38 & 0.10 and -0.5 1 ,+ 0.07 mg/lOO ml per min in the partially depancreatized and the depancreatized insulin-infused dogs, respectively. It is of interest that the partially depancreatized dog had the same Rd as the depancreatized insulin-infused dog, 60 min after the glucose load. R,r is a function of the interstitial insulin and glucose concentration. The partially depancreatized dog had lower plasma glucose levels at the time of the tracer injection than the depancreatized dog. This could therefore indicate that the partially depancreatized dog, which is unable to react to a glucose challenge with a rapid release of insulin, maintains an elevated rate of insulin secretion and therefore a higher concentration of the interstitial IRI than the normal dog. The concentrations of IRI in plasma at that time were not increased significantly. The decrease in FFA concentration following a glucose load was less marked in both partially depancreatized and depancreatized insulin-infused dogs than in normal dogs. It is apparent therefore that the prompt decrease of FFA following a glucose load is due to the release of insulin. It was surprising to find the same k values and turnover data following a glucose tolerance test irrespective of whether the dogs were maintained on portal or cephalic insulin infusion. It is difficult to comment on the physiologic significance of this finding because the dogs were maintained on a fixed basal insulin supply. In normal subjects a rapid increase in the rate of secretion of insulin follows a glucose injection. It is conceivable that the route of insulin supply plays a more important role during the glucose tolerance test in subjects with an intact pancreas when the rate of insulin secretion changes considerably. ACKNOWLEDGMENT The authors thank Prof. R.E. Haist for his stimulating interest in this work, Prof. J. Grayson for reading the manuscript and Miss Marie Hanright, Mrs. Ruth MacKesnon, Mrs. Mary Scott, Miss Winifreda B. Asico, and Mr. Hugh MacDonald for expert assistance. Dextrostix reagent strips were kindly supplied by the Ames Co., Canada; gelatine solution by the Upjohn Co., U.S.A.; and Festal tablets used in the maintenance of the partially depancreatized dogs by the Hoeschst Co., Canada. REFERENCES 1. Soskin, S., Allweiss, M. D., and Cohn, D. J.: Inlluence of the pancreas and the liver upon the dextrose tolerance curve. Amer. J. Physiol. 109:155, 1934. 2. Ishiwata, K., Hetenyi, G., Jr., and Vranic, M.: Effect of d-glucose or d-ribose on the turnover of glucose in pancreatectomized dogs maintained on a matched intraportal infusion of insulin. Diabetes 12:
820, 1969. 3. Forbath, N., and Hetenyi, G. Jr.: Glucose dynamics in normal subjects and diabetic patients before and after a glucose load. Diabetes 15:778, 1966. 4. Kosaka, K., Mizuno, Y., Ogata, Y., and Kuzuya, N.: Studies of glucose metabolism immediately following total pancreatectomy. Diabetes 15: 179, 1966.
966 5. Wrenshall, G. A., and Hetenyi, G., Jr.: Successive measured injections of tracer as a method for determining characteristics of accumulation and turnover in higher animals with access limited to blood. Metabolism 8:531, 1959. 6. Rappaport, A. M., Vranic, M., and Wrenshall, G. A.: A pedunculated subcutaneous autotransplant of an isolated pancreas remnant for the temporary deprivation of internal pancreatic secretion in the dog. Surgery 59:792, 1966. 7. Vranic, M., and Wrenshall, G. A.: Matched rates of insulin infusion and secretion and concurrent tracer determined rates of glucose appearance and disappearance in fasting dogs. Canad. J. Physiol. Pharmacol. 46:383, 1968. 8. Huggett, A. St. G., and Nixon, D. A.: Enzymic determination of glucose. Biochem. J. 66:12P, 1957. 9. Dole, V. P., and Meinertz, M.: Microdetermination of long chain fatty acids in plasma and tissues. J. Biol. Chem. 235: 2595, 1960. 10. Yalow, R. S., and Berson, S. A.: Immunoassay of plasma insulin. In Glick, D. (Ed.): Methods of Biochemical Analysis Vol. XII. New York, Interscience, 1964, p. 69. 11. Cowan, J. S., Schachter, D., and Hetenyi, G. Jr.: Validity of a tracer-injection method for studying glucose turnover in normal dogs. J. Nucl. Med. 10:98. 1969. 12. Wrenshall, G. A., Hetenyi, G. Jr., and Best, C. H.: The validity of rates of glucose appearance in the dog calculated by the method of successive tracer injections. Canad. J. Biochem. 39:267, 1961. 13. Riggs, C. D.: The Mathematical Approach to Physiological Problems. Baltimore, Williams and Wilkins, 1963. 14. Soeldner, J. S., Gleason, R. E., Williams, R. F., Garcia, M. J., Beardwood, D. M., and Marble, A.: Diminished serum insulin response to glucose in genetic prediabetic males with normal glucose tolerance test. Diabetes 17:17, 1968. 15. Madison, L. L.: The role of insulin in controlling carbohydrate metabolism in the liver. In Leibel, B. S., and Wrenshall, G. A. (Eds.): On the Nature and Treatment of Diabetes. Amsterdam, Excerpta Medica, 1965. 16. Bencosme, S. A., and Liepa, E.: Regional differences of the pancreatic
VRANIC
ET AL.
islet. Endocrinology 57:588, 1955. 17. Hellman, B., Wallgren, A., and Hellerstriim, C.: Two types of islet alpha cells in different parts of the pancreas of the dog. Nature (London) 194:1201. 1962. 18. Munger, B. L., Caramia, F.. and Lacy, P. E.: The ultrastructural basis for identification of cell types in the pancreatic islets. 11. Rabbits, dogs, cat and oppossum. Z. Zellforsch. 67:776. 1965. 19. Rappaport, A. M., Davidson, J. K., Kawamura, T.. Lin, B. J.. Zelin, S., Henderson, J.. and Haist. R. E.: Quantitative determination of insulin output following intravenous glucose tolerance tests in the dog. Canad. J. Physiol. Pharmacol. 46: 373, 1968. 20. Campbell. J., and Rastogi, K. S.: Actions of growth hormone: Enhancement of insulin utilization with inhibition of insulin effect on blood glucose in the dog. Metabolism 18:930, 1969. 21. Lefebvre, P. J., and Luyckx, A. S.: The regulation of insulin secretion in the dog: Effects of glucagon, theophylline and imidazole. Diabetes 18: 363, 1969. 22. Wright. P. H.. Rivera-Colimlim. L., and Malaisse. W. J.: Endogenous insulin secretion in the rat following injection of anti-insulin serum. Amer. J. Physiol. 211: 1089, 1966. 23. Cherrington, A., Vranic, M., Fono, P., and Kovacevic, N.: Effects of insulin and glucagon in dogs following removal of the pancreatic autograft. Proc. Canad. Fed. Biol. Sot. 12:64, 1969. 24. Vranic. M.. Kovacevic, N.. and Wrenshall, G. A.: Exercise and glucose turnover in the normal, partially and totally depancreatized dogs on matched intraportal insulin infusion. Fed. Proc. 29:317, 1970. 25. Wrenshall, G. A., Vranic, M., Cowan, J. S., and Rappaport, A. M.: Effects of sudden deprivation and restoration of insulin secretion on glucose metabolism in dogs. Diabetes 14:689, 1965. 26. Cowan, J. S., Vranic. M., and Wrenshall, G. A.: Effects of preceding diet and fasting on glucose turnover in normal dogs. Metabolism 18: 319, 1969. 27. Vranic, M., and Wrenshall, G. A.: Exercise, insulin and glucose turnover in dogs. Endocrinology 85: 165. 1969. 28. Ninomiya, R., Forbath, N. F., and Hetenyi, G., Jr.: Effects of adrenal steroids on glucose kinetics in normal and diabetic
GLUCOSE
KINETICS
AND FATTY
967
ACIDS
dogs. Diabetes 14:729, 1965. 29. Altszuler, N., Rathgeb, I., Winkler, B., DeBodo, R. C., and Steele, R.: The effects of growth hormone on carbohydrate and lipid metabolism in the dog. Ann.
N.Y. Acad. Sci. 148:411, 30. Snedecor, G. Methods (ed. 5). Ames, College Press, 1957.
1968. W.: Statistical Iowa, Iowa State
The endogenous insulin secretion of five conscious, depancreatized fasted dogs was replaced by intraportal insulin htfusions of 192-263 pU/kg per min to establish whether the release of extra insulin is essential during a glucose tolerance test. Infusion rate of insulin about 200 pU/kg per min was considered to be near basal since it maintained unchanged glucose turnover as well as the plasma concentrations of glucose, free fatty acids (FFA), and immunoreactive insulin @RI). The glucose tolerance tests of these dogs were then compared to those of six normal and six partially depancreatized dogs. It was found that after an intravenous injection of glucose the depancreatized dogs maintained on portal or peripheral insulin infusion had a 50% decreased exponential slope of the plasma glucose
LIN
concentration versus time (k). In contrast to the normal dogs, their plasma glucose levels were still elevated but declining 60-140 min after the glucose load. The declining glucose levels were associated with an increased disappearance rate (R,,), but there was no inhibition of the rate of glucose production. FFA decreased only slightly after the glucose load. Five days after partial pancreatectomy the dogs reacted to a glucose injection with a lesser output of insulin than normal dogs. Their homeostatic deficiencies with respect to glucose kinetics and FFA response were less marked than in depancreatized dogs maintained on matched insulin infusion. It was concluded that a prompt release of extra insulin is an essential part of the normal borneostatic mechanisms that determine a glucose tolerance curve.
HE INTRAVENOUS GLUCOSE TOLERANCE TEST is widely used to detect abnormalities of the mechanisms involved in the control of glucose homeostasis. Soskin et al.1 suggested that a normal glucose tolerance test can be obtained in depancreatized dogs provided a minimal amount of insulin is infused at a constant rate. They concluded therefore that insulin release does not play an essential role in the homeostatic mechanisms that determine a normal glucose tolerance test. In their experiments when insulin was given immediately after total pancreatectomy, general anesthesia and stress due to extensive surgery were necessary complications. In other experiments their depancreatized dogs were maintained on insulin injections until recovery. It is difficult to ascertain whether such depancreatized dogs react physiologically,
T
From the Department of Physiology, University of Toronto, Toronto, C‘nmdu. Recei,,ed for publicutiort April 7, 1971. This work MWS supported by Grnnf MA-2197 from the Medical Resecrrch Cormcil of Canada and by the Canadian Diabetic Association. MLADEN VRANIC, M.D., D.Sc.: Associate Professor, Department of Physiology, Medical Faculty, University of Toronto, Toronto, Canada. PETER FONO, B.Sc.: Student, Faculty of Toronto, Canada. NADA KOVACEVIC, P.ENG.: Research Medicine, University of Toronto, Assistant, Department of Physiology, University of Toronto, Toronto, Canada. BONIFACE J. LIN, M.D.: Assistant Professor, Department of Physiology, University of Toronto, Toronto, Canndn. 954
METABOLISM,VOL. 20, No. 10 (OCTOBER). 1971
GLUCOSE KINETICS
AND FATTY ACIDS
955
since they are exposed chronically to an unphysiologic amount and route of supply of insulin. Their techniques were therefore limited. This alone would make their conclusions doubtful. It was further shown that during glucose infusion the release of insulin is indispensable for the regulation of glucose production in the dog.” It was also reported that the rate of glucose utilization in human diabetics increases during a glucose tolerance test to a lesser extent than in normal humans.3 However, Kosaka et al.” obtained a normal glucose tolerance curve in the acutely depancreatectomized, anaesthetized dog. Their work thus appeared to corroborate the earlier conclusions of Soskin and led to the conclusion that increased insulin secretion is not indispensable for a normal glucose tolerance test. In view of the conflicting evidence, therefore, it seemed desirable that this problem should be reinvestigated using techniques that were designed to avoid general anaesthesia and the acute stress that accompanies extensive surgery. Insulin was infused, after removal of a remnant pancreatic graft, into the portal vein of conscious dogs. These dogs were at no time without insulin, and surgical stress was reduced merely to the removal of the subcutaneous graft. The plasma concentrations of FFA, IRI, and glucose and the turnover of glucose were determined before and after removal of the pancreatic graft to ascertain that the rate of insulin infusion was near the basal insulin secretion rate. Glucose tolerance tests were performed in normal dogs, partially depancreatized dogs, and dogs on constant near basal intraportal or peripheral insulin infusion. The early part of the glucose tolerance curve was analyzed by calculating the slope of the logarithm of the plasma concentration versus time. Glucose turnover was determined at 60 min after the glucose load to study at that time the deviations from the initial dynamic steady state in the three groups of dogs. MATERIALS AND METHODS
Experimental
Procedures
A glucose load (0.5 g/kg body weight, i.v.) was given to six normal, six partially depancreatized, and five totally depancreatized dogs maintained on a fixed intraportal infusion of insulin. In four dogs of the last group a glucose injection was repeated with insulin infusion switched from the portal to the cephalic vein. These 21 experiments were performed in eight dogs (Table 1). An intravenous injection of U-I%-glucose (20 &i) was given 80 min prior to, and 60 min after a glucose load to determine the rates of production (appearance, R,) and of disappearance (Rd) and the metabolic clearance (M) of 1sC-glucose using the method of successive measured injections of tracer.5 The slope of the logarithm of the concentration of plasma glucose versus time (k) was calculated at 20-60 min after the injection of the glucose load. The period from 20-60 min after the glucose load was assessed by the nontracer method to avoid intermixing problems when the concentration of unlabeled glucose is changing rapidly in the pool. The period from 60-140 min was analyzed by a tracer method. IRI and FFA response curves following the glucose load were also determined.
Animals and Surgical Procedures Adult female dogs (9-13 kg body weight) were acclimatized to the standard kennel conditions for 4 wk. All experiments were carried out on conscious dogs trained to stand or lie in a Pavlov stand. Partial pancreatectomy and exteriorization of the
956
VRANIC
ET AL.
pediculated uncinate process of the pancreas into a subcutaneous pocket as described by Rappaport et a1.Q was carried out under general anesthesia 5 days prior to the experiment. Concurrently, a vinyl tubing (Becton, Dickinson & Co., No. 6179) filled with heparin (200 U/ml) was inserted into a tributary of the splenic vein pointing toward the portal vein. The postoperative care and diet of the operated dogs has been described earlier.7 These dogs retained approximately one-third of their pancreas, which was capable of maintaining normoglycemia during fasting and also of preventing postalimentary glycosuria. In earlier work the subcutaneous graft was enclosed in a polyethylene jacket, and postoperative glycosuria was observed occasional1y.Q In the present work the ventral side of the graft was covered with a polyethylene sheet; the dorsal side received collateral blood supply from the underlying muscles to improve the viability of the graft. On the day of the experiment one catheter was inserted under local (procain) anesthesia into the vena cava (with the tip below the points of entry of the hepatic veins) via the saphenous vein. This catheter, filled with a dilute heparin solution (5 U/ml) was used for the collection of blood samples and for injections of I%- and l*C-glucose. Another catheter was inserted into one of the cephalic veins to test the effect of switching the insulin infusion from the portal to a nonsplanchnic vein. After the experiment the partially depancreatized dogs were placed in a metabolic cage until the next morning when the pancreatic graft was removed under local anesthesia and an infusion of insulin (192-263 pU/kg per min) into the portal circulation was started promptly. (The infusion solution: 40 mu/ml porcine insulin and 5 mg/ml of gelatin, Upjohn Co., Kalamazoo. in an acid-saline solution of pH = 3.9-4.0. Thus, the binding of insulin to glassware and tubing was minimized.) Equilibration for 45-60 min was allowed, during which minor adjustments in the rate of infusion of insulin were made when warranted according to the blood glucose level estimated by serial DEXTROSTIX (Ames) tests. A constant rate was then maintained. At autopsy no infection was detected in the skin, abdomen, or thorax, and all the catheters were patent in all dogs.
Processing of Blood Samples The collection and processing of blood samples have been described.7 The heparin solution was withdrawn from the portal cannula prior to the experiment. A solution of heparin (5 U/ml) was used during the experiment to keep the sampling catheter patent. The isolation of glucose with help of an ion exchange resin (BIO-RAD AG 2-X8) and determination of glucose (Huggett and Nixons) as well as counting procedures have also been described.7 Aliquots of I%-glucose standard were treated in the same way as plasma samples. Radioactive strengths were corrected for quenching using the ratio of external standard counts. Plasma FFA were measured by titration,9 and IRI by the method of Yalow and Berson.10 The reference standard used in the insulin assay was porcine insulin, and the guinea pig antiserum had been prepared against porcine insulin. The only source of plasma insulin in pancreatectomized dogs maintained on insulin was porcine insulin.
Calculations Single exponential functions were fitted to sets of concentrations of labeled glucose (dpm/unit volume, to calculate the rate of glucose disappearance) and specific activities (dpm/&g glucose, to calculate the rate of glucose appearance) with respect to time, while linear functions were fitted to glucose concentrations versus time (dc/dt). These calculations as well as statistics were carried out on a Canadian General Electric Co. time sharing computer system. The rate of appearance was calculated as R, = N,‘K’, where K is the constant of the single exponential function fitted to the specific activity of glucose with respect to time, and N, the intermixing mass (pool size) of body glucose.5J1Ja The appearance of unlabeled glucose was detected only since corrections for recycling of the label were not done. The following two different equations were used to calculate R,: R, = R, - dN/dt Assuming constancy of glucose space, dN/dt = (N,/c,).(dc/dt).
957
GLUCOSE KINETICS AND FATTY ACIDS
N,/c, = Intermixing mass of body glucose divided by glucose concentration. Both parameters are obtained by extrapolation to the time of tracer injection. R,* = K.*N,, where K* is the constant of the single exponential equation fitted to the concentrations of labeled glucose (dpm/O.l ml) with respect to time.s The mean differences between R, and Rc* were l%-2% when glucose in plasma was near dynamic steady state (DSS) at normal plasma glucose level. A 5%-8% dxerence was observed between R, and Rd* when deviations from DSS were more marked. In the depancreatized dog maintained on insulin infusion the plasma glucose levels were still declining after the glucose tolerance test when the tracer injection was given. Figure 1 shows in a representative experiment that exponential functions fitted at that time to either specific activities or concentration of tracer with respect to time had essentially the same accuracy of the fit as in the fasted dog prior to the glucose tolerance test. In all experiments except one, the plasma glucose levels were below the renal threshold for glucose at the time when the second tracer was given, therefore R, approximates the rate of glucose utilization. Metabolic clearance (M) = rate of glucose disappearance/mean plasma glucose concentration.13
RESULTS Plasma Concentrations
of Glucose, FFA,
and IRI
The effects of a glucose injection in normal, partially depancreatized, and depancreatized insulin-infused dogs are shown in Figs. 2, 3 and 4. In the period prior to glucose injection there were no significant differences in the initial concentrations of glucose, FFA, and IRI except in one dog that became hyperglycemic after removal of the pancreatic graft. These data suggest that the exogenous supply of insulin had been comparable to the endogenous supply before removal of the graft. The depancreatized dogs maintained on matched intraportal insulin infusion had a decreased tolerance for glucose. The exponential slope of the concentration of glucose in plasma (k) was reduced 37% in six partially depan-
Fig. l.-Specific activities (black circles), DPM/unit volume (black triangles), and the concentrations of plasma glucose (open circles) before (T5) and 60-140 min after glucose load (Ts) in depancreatized insulin-infused dog (7). Single exponential functions were fitted to values of specific activities, and concentration of tracer and linear functions to concentrations of glucose in plasma from 20 min following successive tracer injections. Respective values of N,, R,, Rc, and Rc* were 276 mg/kg and 2.58, 3.15, and 3.09 mg/kg per min before glucose load; after load values were 444 mg/kg, 2.75, 4.22, and 4.01 mg/kg per min, respectively. Increased R, is presumably consequence of increased plasma glucose levels.
5 s -‘, $ z Z i
r0
$ c : ’ i $, s 2 i = s
t4sIZO,oo_ so - \ I 0
, I 40
80 Minuiu
oll*r
I 40
I 0 frclcer
injection
I 80
958
.~ ---__J-~_.L
~~
200
100
UTES
L
I
0
-
-L
A-
100
L_,+
0
200
4 S
I
260 t
j
360
400 ' 4 g
Fig. 2.-Effect of sequence of i.v. glucose injections of 0.5 g/kg (Tg) in dog 5: (a) normal; (b) , 5 days after partial pancreatectomy, and (c) and (d) following day after pancreatectomy on constant insulin infusion. PEX indicates removal of pancreatic graft and beginning of intraportal infusion of canine insulin (P.I.). One glucose injection was given when dog was on intraportal insulin infusion (c), while other injection occurred during infusion into cephalic vein (d). Times of i.v. injections of i4C-glucose are shown as Ti-Tr. Computer-fitted single exponential functions to sets of values of specific activity (black circles) and time extrapolated to time of tracer injection are shown on upper scale; k in lower scale; slope of log plasma glucose cont. X (-100) in min-l.
Fig. 3.-Concentrations of FFA, IRI, and glucose in plasma following i.v. glucose injections of 0.5 g/kg at point (tg) . Dotted line crosses initial values of FFA and IRI; k in lower scale, slope of log plasma glucose cont. X ( - 100) in min-I. (Left) Six normal dogs. (Right) Six partially depancreatized dogs.
.________.[71:_.k#'~~<~ IO -
.__________a.___~_~~~____.._ %+-a
0-
i
i
I 400 L
;
i
! _I
-50
I
I
0
50
1 9
-I
I
100
I
-50 MINUTES
0
I (
~__I
-
50
100
GLUCOSE KINETICS
959
AND FATTY ACIDS
Fig. 4.-Five dogs following removal of remnant pancreatic graft on coninsulin stant infusions (shown in parentheses). In four dogs route of infusion was switched from portal to cephalic vein, with rates of infusion kept constant at 192, 205, 230, and 263 ,uU/kg per min, respectively. Fifth dog was infused with 203 ,uU/kg per min intraportally only; this dog was hyperglycemic at time of graft removal and its extremely low k is shown separately. (See Fig. 3 for meanings of symbols.)
20 -i 2
IO ,_______ L
_____________
jj ‘9* f&j __ +s_
$+f+ ___
_ __________
LL O-
500
L
400 L
300 t
200 -
100 -
O-
l INSULIN INFUSION
PORTAL
I --loo
0
t
9
100
200
I MINUTES
t
9
creatized (p < 0.01) dogs. In four dogs maintained on intraportal insulin infusion there was a further 52% decrease in k (p < O.OOl), while the fifth infused dog had a k of only 0.47 minute- l. The same conclusion was reached regardless of whether k was obtained by fitting single exponential functions to either absolute glucose concentrations or to their difference from the initial level. In normal dogs the initial glucose concentrations were regained 78 & 10 min after the load, but in partially depancreatized dogs not until 107 i. 12 min. On portal infusion the initial values were restored in two dogs at 100 and 75 min, respectively, while increased values persisted in three dogs until the end of the experiment. Two minutes after the glucose load, IRI rose fivefold in normal dogs but only 1.7-fold in the partially depancreatized dogs. In the latter, a decreased response in IRI was noted within 30 min following the glucose load. The two insulin areas obtained by integration during the O-60 min period after the glucose load were also different: 771 -e 135 JLJ and 329 & 116 ,JJ (p < 0.05) in normal and partially depancreatized dogs, respectively. The IRI values remained constant during the intraportal infusion of insulin. Figure 5 shows that the partially depancreatized dogs had also a decreased regression line of mean IRI values on mean glucose concentrations following injection of the glucose load. Similar differences were reported between genetically prediabetic males and normal subjects .14 As expected, the IRI values remained constant in depancreatized dogs during the intraportal infusion of insulin.
960
VRANIC ET AL.
Fig. !L-Correlation be40 -. tween mean glucose connormal: IRI= 0.081 G.C.+ 1.97 . centration and mean IRI in plasma 3.5-60 min follow( portiolly deponcreatized ) ing a glucose challenge 3. - -.s 0 IRI = 0.032 G.C. (0.5 g/kg i.v.) , calculated T > according to Soeldner et =t al.14 Circles indicate mean =20 serum IRI-mean blood : glucose at each of the six ; and five time intervals in normal (black circles) and 10 partially depancreatized (open circles) dogs, respectively. Correlation co0 1 I I I efficients: r = 0.976, p < 100 200 300 400 0.01 (normal); r = 0.875, p < 0.05 (partially depanMeon glucose concentration mg/lOO ml creatized)-~. Analysis of covariance”0 showed that two slopes are significantly different at 2% level. Regressions of IRI values on glucose concentrations were also calculated in individual dogs and averaged, yielding mean slopes of 0.091 and 0.016 in normal and partially depancreatized dogs, respectively.
In the partially depancreatized dog the maximum reduction in FFA was 37% at 66 min as compared to normal reduction of 63% (p < 0.001) at 42 min. A small maximum decrease of 23% was noted even in the dog on matched insulin infusion, part of this decrease (3%-5% at most) could be attributed to the dilution of plasma caused by the hypertonic injection of glucose. Table l.-Plasma Glucose Concentrations (mg/lOO ml) at Time of Tracer Injection (C,) and 80 Min Thereafter (Cs,,). Total of 21 Glucose Tolerance Tests were Performed on Eight Female Dogs Depancreatized Normal Fasted Dog Number
_ co
1
---
2 3a 3b 4a 4b
--102 106 93 92
5 6 7 8
104 _-_ --101
After c80
cot
Partially Depancreatized G’lT*
118 109 112 108
106
103
105
141
After GTT
Fasted
Cephalic
After GTT ____ co c60
After GTT
C”
cao
co
cm
.~ co
cm
co
cso
100 95 89 91
108 127 _ _
99 149 _ _
129 180 _ _
96 132 _ _
167 _ _
222 _ __
315 _ _
259 _ _
_ _
_ _
114 92
113 104 105 82
129 116 96 81
137 159 172 145
105 114 113 71
112 144 102 85
91 119 88 74
142 134 168 162
72 82 129 121
151 88 214 194
91 68 181 135
CLV -
100 106 91 90
Fasted
on Insulin
Portal
* GTT, glucose tolerance test. T C, after GTT refers to tracer injection given 60 min after i.v. injection of 0.5 g/kg glucose.
2.78 & 0.27
M
23.4 +- 1.6
Normal
27.8 + 0.8” (19)
2.92 + 0.23 (7) 3.05 Zk 0.19 (11) 3.10 -t 0.21 (11) 3.17 f 0.15 (14) -0.13 f 0.23
After
28.5 +- 1.6
0.12 2 0.17
3.01 + 0.26
3.22 -c 0.17
3.17 + 0.18
3.29 -c 0.19
30.5 + 1.3 (7)
3.52 t 0.30 (7) 4.69 + 0.01 (48) 5.03 f 0.19 (56) 4.16 + 0.29 (38) - 1.17 -c 0.33
After
Partially Depancreatized Before
$
t
5
§
4 0.39
28.2 + 1.27
-0.30
3.46 2 0.41
3.78 C 0.25
3.74 ” 0.25
After
28.6 + 1.43 (1)
3.51 * 0.34 (2) 4.80 i- 0.22 $ (28) 5.09 + 0.25 t (35) 3.84 -c 0.73 (11) -1.46 -c 0.28*
Portal infusion
3.44 c 0.41
Before
31.40 & 1.60
- 1.55 + 0.46
4.25 + 0.93
5.26 + 0.67
4.90 f 0.44
3.35 2 0.30
After
Cephalic Infusion
In parentheses: per cent changes following glucose injection. Two different equations were used to calculate rate of glucose disappearance (Rd and Rd*>. Statistical comparisons are between values before and after glucose injection for each group of animals. * p < 0.05. t p < 0.02. $ p < 0.01. F,p < 0.001. Comparisons between groups before glucose injections show a slightly higher V in partially depancreatized and insulin infused dogs when compared to normal (p < 0.05); R, of insulin infused dog was higher than in normal dog (p < 0.05).
V
+ 0.08
2.80 f 0.34
Rd*
-0.03
2.75 + 0.29
Rd
dN/dt
2.72 2 0.22
VI
Before
Table 2.Rates of Appearance (R,), Disappearance (RJ, Accumulation (dN/dt), (mg/kg/mln), Metabolic Clearance (M), (ml/kg/mln), and Volume of Distribution (V) of Glucose (% Body Weight) Before and After 0.5 g/kg Glucose Injected Intravenously in Slx Normal, Six Partially Depancreatized, Five Depancreatlzed Dogs on Intraportal and in Four Depancreatized Dogs on Cephalic Insulin Infusion
962
VRANIC
ET AL.
Glucose Turnover
The rates of glucose production, diappearance, and metabolic clearance are shown in Table 2. Concentrations of glucose in plasma at the time of tracer injection and 80 min thereafter are shown in Table 1. Glucose turnover of the partially depancreatized dog and of the depancreatized dogs maintained on insulin infusion were compared to assess how well the exogenous supply of insulin was matched to that of the previous endogenous supply. The depancreatized dogs maintained on constant insulin infusion had a slightly higher Rd and M than the partially depancreatized dogs. Since the depancreatized dogs were infused at different rates of insulin, it was interesting to find out whether changes in Rd, M, and R, were correlated to the rates of insulin infusion (Fig. 6). To make these correlations more meaningful, seven additional points from a previous publication? were added to this figure. A positive correlation was found between the increments of Rd, M, and the insulin infusion rates, and a negative correlation was found between the increments in R, and the infusion rate of insulin. Only an infusion of insulin near 200 ,,U/kg per min maintained these parameters unchanged. Thus it appears that two depancreatized dogs were given an amount of insulin that was slightly above basal. At 60-140 min after the glucose tolerance test the normal dog was near its initial dynamic steady state and its rate of glucose accumulation (dN/dt) was only -0.13 mg/kg per min. In contrast, the efliciency of the homeostatic mechanisms of glucose was impaired in the partially depancreatized and depancreatized insulin-infused dog, since they had still not attained the dynamic steady state as judged by the negative dN/dt values. This was caused by a significantly increased R,,, since there was no difference in R,, between the post- and preglucose tolerance values. Table 2 shows no differences in the postglucose tolerance values when the portal infusion of insulin was switched to the cephalic vein.
Fig. 6.-(Upper) Regression of metabolic clearance (M) of glucose on rate of insulin infusion after removal of pancreatic autograft r = 0.951, p < 0.002. Correlations between R, and rate of insulin infusion yielded essentially same correlation coefficient. (Lower) Regression of rates of production (R,) of glucose on rates of insulin infusion after removal of pancreatic autograft: r = -0.850, p < 0.002. Seven points included in graph have been calculated from Vranic and Wrensha1l.i Glucose concentrations in all experiments were below 200 mg/ 100 ml. 100 RAIES
200
OF INTRAPORTAL
INFUSION
INSULIN
MICRW/Kg-min
963
GLUCOSEKINETICSANDFATTYACIDS DISCUSSION
The purpose of these studies was to investigate the metabolic events following a glucose tolerance test in conscious depancreatized dogs maintained on a constant near basal rate of insulin infusion. The importance of intraportal supply of this hormone has previously been emphasized,15 and therefore exogenous insulin was administered by its physiologic site of entry. The pancreatic uncinate process of the dog is said to lack the glucagon producing alpha cells,16-18 so that infusion of insulin alone could compensate the sudden deprivation of pancreatic hormones following its removal. Further work is needed to ascertain whether more insuhn is secreted in normal dogs, because they produce pancreatic glucagon as well. Sampling of the total venous outflow from the uncinate process in situ showed the mean rate of insulin output to be 190-202 pU/kg per min by the mouse hemidiaphragm bioassay and the immunoassay, respective1y.l” Similar mean values were reported in normal dogs on the basis of the rate of disappearance of immunoreactive 1311-Insulin20 or cannulation of pancreatic vessels .21 Higher secretion rates of insulin, considered to be maximal, were reported in the rat using the method of neutralizing antibodies.22 It appeared that infusion rates of about 200 pU/kg per min might replace and match the endogenous secretion of the pancreatic graft. So far 34 experiments were performed in which endogenous insulin secretion was replaced by a matched exogenous portal insulin supply. These dealt with physiologic challenges such as infusions of glucose and ribose,2 glucagon,23 and exercise.“* The basal insulin infusion did not maintain homeostasis in two dogs that were hyperglycemic before remova of the graft. Of the remaining 32 dogs, the match was achieved in 88% of the experiments. The phrase “quantitative matching” is only meaningful if it can be shown that under physiologic conditions insulin is secreted continuously. That a continuous supply of insulin is normally available to the animal is suggested by the facts that changes in rates of production and utilization and the concentration of ghrcose occur promptly following the cessation of endogenous or exogenous insulin s~pp1y.r~~~ This is in keeping with a short biologic half life for insulin. To ascertain the success of matching the endogenous insulin supply it is important to determine glucose turnover, since it is known that glucose concentrations do not always reflect accurately a change of the hormonal balance. For example, prolonged fasting,2s exercise,27 chronic administration of steroids28 or growth hormone2” cause a marked change in the glucose transfer rates with only slight changes in the glucose levels. In this work small increases in insulin supply were closely related to increments in Rd and M, suggesting that a control system involving insuhn secretion adjusts glucose concentration primarily by changing the overall glucose utilization (fine tuning). The inhibitory effect of insulin on R, is less marked. In 1934 Soskin et a1.l reported a normal glucose tolerance test in depancreatized dogs when insulin was infused at a constant rate, but this suggestion has not been confirmed. In our experiments insulin infusions near 200 PU/kg per min were given intraportally to conscious depancreatized dogs that have not been diabetic. In contrast, Soskin’s peripheral infusions of insulin varied
964
VRANIC ET AL.
between half and 33 times the basal infusion rate, and in most of their experiments insulin and glucose infusions were given simultaneously as they had difficulties in maintaining glucose homeostasis by insulin infusions only. In this experiment the analysis of the glucose tolerance test proves that dogs on basal insulin infusion were unable to handle a glucose load normally. The importance of a normal release of insulin was indicated, since both partially depancreatized and depancreatized insulin-infused dogs had a significantly reduced k in the early period and they failed to regain their initial DSS in the late glucose tolerance period. At 60 min after the glucose load the plasma glucose levels were still elevated but declining. The increased glucose level were associated with an increase of Rd but there was no inhibition in the rate of glucose production at that time. This work thus extends the earlier observations that clamping of the remnant pancreatic autograft for 30-60 min promptly reduced tolerance for glucose in the conscious dogs. The restoration of the graft’s blood supply restored the initial tolerance for glucose. This effect was considered to be due to sudden cessation of insulin supply. The handling of the graft served as a sham operation for clamping or removal of the graftz5 In the present experiments replacement of the graft by an intraportal infusion of insulin induced a decrease in tolerance for glucose (Fig. 2) that was smaller than the decrease caused by complete insulin deprivation. The partially depancreatized dogs reacted to the glucose challenge with a reduced insulin response. It might be that the impaired insulin response is due not only to the reduced size of the pancreas but also to the postoperative consequences of the abdominal operation. This possibility, however, does not alter the main observation that the normal glucose tolerance test depends on a normal release of extra insulin. This conclusion finds support in the observations that 5 days after removal of two-thirds of the pancreas the insulin sensitivity of these dogs did not change. Canine insulin was infused intraportally at 1.4 mu/kg per min for 1 hr before and 5 days after partial pancreatectomy; the per cent reductions in the plasma glucose levels were 37 + 0.7 and 33 t 4.6 (mean +- SEM), respectively. (Vranic, M., Fono, P., Kovacevic, N., and Cherrington, A.: unpublished observations.) In the present experiments normal dogs were near the dynamic steady state during the sampling period of the second tracer injection. At that time the plasma glucose concentrations were still declining in the partially depancreatized and the depancreatized insulin-infused dogs; the rate of decrease was calculated by a linear regression. Method of successive measured injections of tracer has been validated for the rate of appearance of 12C-glucose in the eviscerated dog. Valid rates of glucose appearance were obtained even when the glucose concentrations were changing in p1asma.l” More recently Cowan et al.11 have achieved validation to within 10% of the rate of appearance of glucose in the normal conscious dog. With a rapid linear decrease in the rate of appearance of tracee, the tracer-determined rate did not vary significantly from those rates determined in experiments near to dynamic steady states. With a measured constant acceleration in the rate of appearance of tracee, the tracer-determined
965
GLUCOSE KINETICS AND FATTY ACIDS
rate of appearance was found to compare with the measured rate near the midpoint of the sampling period that followed. In the present experiments the concentration of plasma glucose was decreasing toward a dynamic steady state when the second injection of tracer was given (Fig. 1). The rates of change of the plasma glucose concentration were not large at that time: -0.38 & 0.10 and -0.5 1 ,+ 0.07 mg/lOO ml per min in the partially depancreatized and the depancreatized insulin-infused dogs, respectively. It is of interest that the partially depancreatized dog had the same Rd as the depancreatized insulin-infused dog, 60 min after the glucose load. R,r is a function of the interstitial insulin and glucose concentration. The partially depancreatized dog had lower plasma glucose levels at the time of the tracer injection than the depancreatized dog. This could therefore indicate that the partially depancreatized dog, which is unable to react to a glucose challenge with a rapid release of insulin, maintains an elevated rate of insulin secretion and therefore a higher concentration of the interstitial IRI than the normal dog. The concentrations of IRI in plasma at that time were not increased significantly. The decrease in FFA concentration following a glucose load was less marked in both partially depancreatized and depancreatized insulin-infused dogs than in normal dogs. It is apparent therefore that the prompt decrease of FFA following a glucose load is due to the release of insulin. It was surprising to find the same k values and turnover data following a glucose tolerance test irrespective of whether the dogs were maintained on portal or cephalic insulin infusion. It is difficult to comment on the physiologic significance of this finding because the dogs were maintained on a fixed basal insulin supply. In normal subjects a rapid increase in the rate of secretion of insulin follows a glucose injection. It is conceivable that the route of insulin supply plays a more important role during the glucose tolerance test in subjects with an intact pancreas when the rate of insulin secretion changes considerably. ACKNOWLEDGMENT The authors thank Prof. R.E. Haist for his stimulating interest in this work, Prof. J. Grayson for reading the manuscript and Miss Marie Hanright, Mrs. Ruth MacKesnon, Mrs. Mary Scott, Miss Winifreda B. Asico, and Mr. Hugh MacDonald for expert assistance. Dextrostix reagent strips were kindly supplied by the Ames Co., Canada; gelatine solution by the Upjohn Co., U.S.A.; and Festal tablets used in the maintenance of the partially depancreatized dogs by the Hoeschst Co., Canada. REFERENCES 1. Soskin, S., Allweiss, M. D., and Cohn, D. J.: Inlluence of the pancreas and the liver upon the dextrose tolerance curve. Amer. J. Physiol. 109:155, 1934. 2. Ishiwata, K., Hetenyi, G., Jr., and Vranic, M.: Effect of d-glucose or d-ribose on the turnover of glucose in pancreatectomized dogs maintained on a matched intraportal infusion of insulin. Diabetes 12:
820, 1969. 3. Forbath, N., and Hetenyi, G. Jr.: Glucose dynamics in normal subjects and diabetic patients before and after a glucose load. Diabetes 15:778, 1966. 4. Kosaka, K., Mizuno, Y., Ogata, Y., and Kuzuya, N.: Studies of glucose metabolism immediately following total pancreatectomy. Diabetes 15: 179, 1966.
966 5. Wrenshall, G. A., and Hetenyi, G., Jr.: Successive measured injections of tracer as a method for determining characteristics of accumulation and turnover in higher animals with access limited to blood. Metabolism 8:531, 1959. 6. Rappaport, A. M., Vranic, M., and Wrenshall, G. A.: A pedunculated subcutaneous autotransplant of an isolated pancreas remnant for the temporary deprivation of internal pancreatic secretion in the dog. Surgery 59:792, 1966. 7. Vranic, M., and Wrenshall, G. A.: Matched rates of insulin infusion and secretion and concurrent tracer determined rates of glucose appearance and disappearance in fasting dogs. Canad. J. Physiol. Pharmacol. 46:383, 1968. 8. Huggett, A. St. G., and Nixon, D. A.: Enzymic determination of glucose. Biochem. J. 66:12P, 1957. 9. Dole, V. P., and Meinertz, M.: Microdetermination of long chain fatty acids in plasma and tissues. J. Biol. Chem. 235: 2595, 1960. 10. Yalow, R. S., and Berson, S. A.: Immunoassay of plasma insulin. In Glick, D. (Ed.): Methods of Biochemical Analysis Vol. XII. New York, Interscience, 1964, p. 69. 11. Cowan, J. S., Schachter, D., and Hetenyi, G. Jr.: Validity of a tracer-injection method for studying glucose turnover in normal dogs. J. Nucl. Med. 10:98. 1969. 12. Wrenshall, G. A., Hetenyi, G. Jr., and Best, C. H.: The validity of rates of glucose appearance in the dog calculated by the method of successive tracer injections. Canad. J. Biochem. 39:267, 1961. 13. Riggs, C. D.: The Mathematical Approach to Physiological Problems. Baltimore, Williams and Wilkins, 1963. 14. Soeldner, J. S., Gleason, R. E., Williams, R. F., Garcia, M. J., Beardwood, D. M., and Marble, A.: Diminished serum insulin response to glucose in genetic prediabetic males with normal glucose tolerance test. Diabetes 17:17, 1968. 15. Madison, L. L.: The role of insulin in controlling carbohydrate metabolism in the liver. In Leibel, B. S., and Wrenshall, G. A. (Eds.): On the Nature and Treatment of Diabetes. Amsterdam, Excerpta Medica, 1965. 16. Bencosme, S. A., and Liepa, E.: Regional differences of the pancreatic
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islet. Endocrinology 57:588, 1955. 17. Hellman, B., Wallgren, A., and Hellerstriim, C.: Two types of islet alpha cells in different parts of the pancreas of the dog. Nature (London) 194:1201. 1962. 18. Munger, B. L., Caramia, F.. and Lacy, P. E.: The ultrastructural basis for identification of cell types in the pancreatic islets. 11. Rabbits, dogs, cat and oppossum. Z. Zellforsch. 67:776. 1965. 19. Rappaport, A. M., Davidson, J. K., Kawamura, T.. Lin, B. J.. Zelin, S., Henderson, J.. and Haist. R. E.: Quantitative determination of insulin output following intravenous glucose tolerance tests in the dog. Canad. J. Physiol. Pharmacol. 46: 373, 1968. 20. Campbell. J., and Rastogi, K. S.: Actions of growth hormone: Enhancement of insulin utilization with inhibition of insulin effect on blood glucose in the dog. Metabolism 18:930, 1969. 21. Lefebvre, P. J., and Luyckx, A. S.: The regulation of insulin secretion in the dog: Effects of glucagon, theophylline and imidazole. Diabetes 18: 363, 1969. 22. Wright. P. H.. Rivera-Colimlim. L., and Malaisse. W. J.: Endogenous insulin secretion in the rat following injection of anti-insulin serum. Amer. J. Physiol. 211: 1089, 1966. 23. Cherrington, A., Vranic, M., Fono, P., and Kovacevic, N.: Effects of insulin and glucagon in dogs following removal of the pancreatic autograft. Proc. Canad. Fed. Biol. Sot. 12:64, 1969. 24. Vranic. M.. Kovacevic, N.. and Wrenshall, G. A.: Exercise and glucose turnover in the normal, partially and totally depancreatized dogs on matched intraportal insulin infusion. Fed. Proc. 29:317, 1970. 25. Wrenshall, G. A., Vranic, M., Cowan, J. S., and Rappaport, A. M.: Effects of sudden deprivation and restoration of insulin secretion on glucose metabolism in dogs. Diabetes 14:689, 1965. 26. Cowan, J. S., Vranic. M., and Wrenshall, G. A.: Effects of preceding diet and fasting on glucose turnover in normal dogs. Metabolism 18: 319, 1969. 27. Vranic, M., and Wrenshall, G. A.: Exercise, insulin and glucose turnover in dogs. Endocrinology 85: 165. 1969. 28. Ninomiya, R., Forbath, N. F., and Hetenyi, G., Jr.: Effects of adrenal steroids on glucose kinetics in normal and diabetic
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dogs. Diabetes 14:729, 1965. 29. Altszuler, N., Rathgeb, I., Winkler, B., DeBodo, R. C., and Steele, R.: The effects of growth hormone on carbohydrate and lipid metabolism in the dog. Ann.
N.Y. Acad. Sci. 148:411, 30. Snedecor, G. Methods (ed. 5). Ames, College Press, 1957.
1968. W.: Statistical Iowa, Iowa State