A~KI~AN

JOURNAL

0F PmwoLocV

Vol. 228, No. 5, May 1975.

&k&d

in U.S.A.

Effect of infusion hepatic

extraction

of insulin

into

of insulin

portal

in anesthetized

PHILIP E. HARDING, GAIL BLOOM, AND JAMES B. FIELD Clinical Research Unit and Ilepartment of Medh’ne, University of Pittsburgh School Pittsburgh, Pennsylvania 15261

HARDING, PHILIP E., GAIL BLOOM, AND JAMES B. FIELD. Effeet of infusion of insulin into portal vein on hepatic extraction of insulin in me&e&ad dogs. Am. J. Physiol. 228(5) : 1580-l 588. 1975.Hepatic extraction of insulin was examined in anesthetized dogs before and after constant infusion of insulin (20 and 50 mU/min) with use of samples from the portal vein, mesenteric vein, left common hepatic vein, and the femoral artery. In 19 dogs, measurement of portal vein insulin concentration indicated an overall recovery of I IO Cl0 of the insulin infused. The range varied from 9 to 3037& indicating the potential for serious error in sampling arterial insulin concentrations were the portal vein. Equilibrium achieved 20 min after starting the infusion. Prior to insulin infusion, hepatic extraction of insulin averaged 4.56 + 0.43 mU/min, representing an extraction coefficient of 0.42 of the insulin presented to the liver. The proportion of insulin extracted by the liver did not change significantly during insulin infusion despite a IO-fold increase in portal vein insulin concentrations. During the infusion of insulin, a significant proportion of the extrahepatic clearance of insulin occurred in the mesenteric circulation. Infusion of insulin was associated with a significant increase in insulin extraction by tissues other than the liver and splanchnic beds. Initially, hepatic glucose output averaged 36 + 3 mg/min; by 20 min after insulin infusion, it was 16 + 5 mg/min. Despite continuation of insulin infusion, hepatic glucose output returned to control values even though arterial glucose concentration continued to fall. Hepatic glucose output increased with termination of insulin infusion. hepatic insulin metabolism; hepatic carbohydrate mesenteric bed insulin extraction; systemic insulin

metabolism; extraction

( 10, 12, 14, 19) have confirn~ed that 50 % of the pancreatic output of insulin is extracted by the liver in the basal state. However, there is conflicting evidence as to whether, in response to physiological alterations in insulin secretion, there occurs a change in insulin extraction, which would suggest a role for the liver in modulating changes in peripheral insulin levels. Glucose-induced increases in insulin secretion have been variously reported as decreasing (10) or increasing (24) the percent hepatic insulin extraction. A previous report from our laboratory (7) indicated that, following glucose administration into the duodenum of the anesthetized dog, insulin extraction may increase during the first 60 min of insulin secretion. After this time, however, extraction returned to control values although insulin secretion continued to increase. In vitro studies using rat SEVERAL

vein

INVESTIGATORS

approximately

on dogs

of Medicine,

liver perfused with insulin (13) indicated that extraction exhibits first-order kinetics so that the percent extraction remained constant while the insulin level in the medium fell. Samols and Ryder (19) also reported no change in hepatic insulin extraction from a control value of 41 c/o when exogenous insulin was infused into their human subjects who had portal-systemic shunts. The accurate measurement of hepatic insulin extraction depends upon obtaining fully representative samples of blood entering and leaving the liver. Experience in our laboratory and elsewhere has shown that differential distribution of insulin may occur within the portal vein (5) and in the various hepatic veins (9). The present study attempts to quantitatively assess and correct for these sampling errors and to measure hepatic insulin extraction in the anesthetized dog during basal conditions and steady-state infusion into the portal vein. The results during insulin infusion indicate that, when equilibrium is reached, the proportion of insulin removed is not difierent from that observed in the basal state. The effects of insulin infusion on hepatic glucose output have also been examined. MATERIALS

AND

METHODS

Mongrel dogs (mean weight 2 1.6 kg) were fasted overnight and anesthetized with barbital (30 mg/kg). The and placement of electromagnetic surgical preparation Aow probes on the portal vein and hepatic artery was as described by us previously (7), with the following modifications: a) hepatic venous blood was sampled from a catheter placed in the left common hepatic vein (LCHV) via the abdominal approach as described by Shoemaker et al. (20). In three preliminary experiments, two additional sampling catheters were placed in the interior vena cava (IVC) which was ligated immediately caudad to the hepatic veins so that it would contain only mixed-hepatic venous effluent. One of these catheters was placed, under direct vision, immediately opposite the entry of the hepatic veins, and the other, 2-5 cm cephalad and beyond a choker designed to constrict the IVC to 50 % of its diameter and thus create turbulent mixing of the blood passing through to the upper catheter. The pressure gradient across this constriction was < 1 cmHz0 and hepatic blood flow was not reduced. Insulin concentrations in samples from these two catheters, termed high IVC and low IVC, were then compared with simultaneous LCHV samples. b) The portal

1580

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HEPATIC

INSULIN

EXTRACTION

DURING

INSULIN

1581

INFUSION

vein flow probe was placed immediately caudad to the The portal vein gastroduodenal vein, which was ligated. sampling catheter, inserted via a peripheral mesenteric vein, was passed through the flow probe so that its multiplehole sampling tip lay immediately below the portal vein bifurcation. The flow probe was recalibrated in vitro for use under these geometric conditions and still gave an accurate linear response which was unaffected by changes in the hematocrit over the r ange 26-48 %. c) Catheters vein tributary were also placed in a peripher al mesenteric for sampling of mesenteric venous blood, and at the root of the cranial mesenteric vein, the principal tributary of the portal vein, for the infusion of insulin. The tip of the 10 cm cauda d to the infusion catheter lav approximately care was taken to portal vein sampling catheter. Although site the mesenteric sampling catheter well away from the pancreatic venous drainage, mesenteric vein insulin concentrations during the control period may be spuriously high because of som .e contamina ,tion with pancre atic venous drainage. Blood representative of that in the hepatic artery was sampled from a catheter in the femoral artery. The placement of all the catheters and flow probes in relation to the liver and the pancreatic venous drainage is illustrated in Fig. 1. At the end of each study, the catheter placements were checked and in no instance had displacement occurred. experiments, transit time of insulin In two preliminary through the liver was estimated by collecting hepatic vein samples every 10 s following the rapid injection of a measinsulin. As a result of these ured quantity of 12V-labeled ve in samples were experiments, detailed below, hepatic drawn 30 s after the simultaneous collection of all the other samples. Nineteen dogs were studied according to the following protocol: after an equilibration period of 60 min following completion of the surgery, samples and flow measurements were taken 30, 20, 10, 5, and 1 min before insulin was infused into the portal vein at a rate of approximately Samples and measurements were then ob20 mU/min. tained after 2, 5, IO, 20, 30, 40, and 50 min; the infusion was then increased to approximately 50 mU/min and samples were obtained at similar intervals. The infusion was-then stopped and samples were obtained 2, 5, 10, 20, and 30 min later. In 5 of the 19 dogs (&s no. 25, 27, 32, 33, and 34) the infusion periods were of 30 min duration only. Since insulin and glucose were measured in plasma, the blood flow measurements were corrected to plasma flow (Q) by hematocrits obtained every 30 min during each study+ The flux (F) of insulin (mU/min) or glucose (mg/ min) was then determined for each vessel at each time point by multiplying Q ( m l/ min) by plasma concentration (mu or mg/ml). The hepatic vein plasma flow (QhV) was taken as the sum of the hepatic artery (aa) and portal. differences in vein (Q1,) plasma Aows. No significant hematocrit existed between arterial, portal venous, and Hepatic insulin extraction (I&) hepatic venous blood. and clearance (ml/min) and hepatic glucose balance (mg/min) were derived from these data as described prethat in this study the coviously (7), with the exception

GALL

BLADDER-

HEPATIC

FLOW

- -

VEINS--

-

-GASTRODUODENAL ARTERY (LIGATED)

PROBES

,J SPLENIC PORTAL

VEIN

VEIN - - -. PANCREAS PREHEPATIC

PANCREATICO-

FIG.

sampling catheter

DUODENAL

1. Anatomic and tips.

infusion

CATHETERS

PORTAL VEIN (PI “--.s INFUSlON - MESENTERIC VEIN ------- FEMORAL ARTERY

Jocalioll catheters.

of

electromagnetic Solid circles

flow represent

probes position

(M) (A) and of

eficient of extraction (E) is expressed as a proportion of unity rather than a percentage. The symbols and subscripts for plasma flows, fluxes, and extraction coefhcients are as expressed in the APPENDIX and illustrated in Fig. 3. Extraction of insulin across the mesenteric vascular bed (&) was calculated from the arterial (a) and peripheral bv, the mesenteric venous (m) insulin concentrations formula (a - m)/a. If the mesenteric vein sample is contaminated with pancreatic venous drainage, this will tend to diminish I& The results, therefore, will be a minimum estimate of mesenteric extraction. The diflerence between the plasma insulin concentrations in the portal vein catheter (p) and the mesenteric vein catheter (m), multiplied by QI,, yields FpVIII (mU/min) which is a direct measure of the rate of entry of insulin into the portal vein between the two catheters. During the basal state, this represents all endogenous insulin secretion subject to the limitations mentioned above in regard to the mesenteric vein insulin concentration (Fig. 1); during the insulin infused into the infusion period, it represents the cranial mcsenteric vein plus the endogenous insulin secretionFI,+,, should, therefore, be equal to the known rate of insulin infusion in milliunits per minute when corrected for endogenous insulin secretion. Porcine insulin (Eli Lilly and Company), at a concentration of 0.1 U/ml in physiological saline containing 1 mg/ml bovine serum albumin (Nutritional Biochemicals Corporation), was infused by a Harvard infusion pump at rates 0.2 and 0.5 ml/ min. A portion of the insulin infusate from each study was retained and its insulin content was measured in the same assay as the experimental samples. Plasma insulin was measured by radioimmunoassay with dextrancoated charcoal, and plasma glucose, by the glucose oxidase method (Glucostat, Worthington Biochemical Corporation). Pooled data are expressed as means & standard errors of the mean unless otherwise indicated. RESULTS

Following the injection of a He/x& insulin transit time. bolus of 12%labeled insulin into the portal vein, peak hepatic vein radioactivity was found at 20 and 30 s, respec-

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1582

HARDING,

tively, in the two experiments performed. In each case the peak sample contained more than double the radioactivity of any other sample, suggesting a rapid and relatively homogenous passage of insulin through the liver. A second smaller peak was observed at 70 s, possibly representing the first recirculation of the injected, labeled insulin. Since the hepatic blood flow was measured and the volume of blood sampled from the LCHV was known, an estimate could be made of the amount of injected radioactivity recovered in the hepatic vein during the fn-st 60 s. This was 51 and 23 %, respectively, in the two studies. Assessment of portal vein sampling error. FPsm, representing pancreatic insulin secretion, averaged 5.1 & 0.8 mU/min (0.24 mu/kg per min) during the control period in the whole group of 19 dogs. This is very similar to previous estimates of basal pancreatic insulin secretion in anesthetized dogs ((1 l-13) and unpublished observations). During the period lo-30 min after the cessation of insulin infusion, while arterial plasma glucose was still depressed, pancreatic insulin secretion was 3.4 & 0,9 mU/min. As this change of 1.7 mU/min in pancreatic insulin secretion was small in relation to the amounts infused, the basal insulin secretion value in each experiment was deducted from the values of F,-, obtained during insulin infusion, so that the corrected values would reflect only the infused insulin. These corrected values were then integrated with respect to time to yield the term C Fp--In in milliunits which is a recovery figure for the total amount of insulin infused. Table 1 compares the value of z F,-, with the measured amount of insulin infused in each of the 19 dogs. The mean c IF,-, was 3,317 mU and the mean insulin infused was 3,297 mu. The mean recovery in the 19 dogs was 110 %. TABLE

1.

amount

Rahtionshi'

insulin

of

22 F,-,,

25*t 27* t 32* 33* t 34*t 35 36 37 38 39t got 41 48 43 44 45 46t 49t m

c

Fp-,

to known

infused mU

2,626 i ,781 5,822 2,149 1,738 427 1,600 9,450 1,622 3,485 4,502 1,857 3,708 1,749 5,659 5,635 3,105 3,626 2,490

Infused Insulin, mU

1,955 2,400 1,920 1,929 1,947 4,917 4,216 3,322 5,126 4,095 3,712 3,420 2,851 3,222 3,563 3,831 3,500 3,486 3,239

Observed - Expected

y. Recovery of Infused Insulin

671 -619 3,902 220 - 209 -4,490 -2,616 6,128 -3,504 -610 790 -1,563 857 - 1,473 2,096 1,804 - 395 140 - 749

134 74 303 111 89 9 38 285 32 85 121 54 130 54 159 147 89 104 77

Mean 3,317 3,297 20 110 SD, SE &2,142, 491 &957, 219 &2,446, 561 ~76, 17 ~_ * Infusion periods of 30 min duration. The infusion periods in the remaining dogs were 50 min. t Dogs with percent recovery of infused insulin between 65-135 70 (n = lo), whose portal vein insulin concentrations are plotted in Fig. 2.

BLOOM,

AND

FIELD

However, the standard deviation of the differences between the observed and expected figures in all the individual dogs was &2,447 mU and the percent recovery ranged from 9 to 303 %, indicating a large random error in measuring the portal vein insulin concentration. Assessment of he@& vein sampling error. In three dogs in which the vena cava was ligated below the liver, insulin concentrations in the left common hepatic vein (LCHV) were compared with those found via catheters placed opposite the entry of the hepatic veins (Fig. 1, LOW IVC) and above this point (Fig. 1, HIGH IVC). The results of these observations during steady-state insulin infusion are depicted in Table 2. The LCHV values correspond closely to the high IVC, whereas the relationship of the low IVC to both other vessels is inconstant. In addition, the coeficients of variation for plasma insulin concentration in the LCHV and high IVC are 5.6 and 5.7 %, respectively, whereas for the low IVC it is 9.4 %. Hepatic insulin extraction. The large portal vein sampling error found during insulin infusion precluded the use of these measurements in calculating Eh. During infusion, therefore, the term representing the amount of insulin delivered to the liver by the portal vein was derived as F, + known infusion rate, in milliunits per minute (see APPENDIX). Four dogs were excluded from this calculation. In two (dogs 32 and 38), the values obtained for hepatic insulin extraction were negative throughout the control period, giving no valid basis for comparison. In a third (dog 43), hepatic vein insulin levels rose higher than either the measured or predicted portal vein insulin levels during infusion. The fourth dog (dog 36) showed no rise in hepatic vein insulin despite a rise in arterial levels. The values of Eh in the remaining 15 dogs are given in Table 3, together with the plasma flows, the insulin values in the femoral artery, the portal, hepatic, and mesenteric veins, and the absolute rate of insulin uptake by the liver in milliunits per minute. The changes in arterial insulin concentration and Eh during insulin infusion are also shown in Fig. 2; included for illustration are portal vein insulin concentrations for 10 of the dogs in which the sampling error was not large, as reflected by portal vein insulin-recovery figures between 65 and 135 %. Eh values for these 10 dogs did not differ from the group as a whole. During the control period, basal insulin secretion for the group of 15 dogs was 5.5 III 0.8 mU/min (0.25 mu/kg per min); arterial insulin, 16 & 1 &J/ml; and mean Eh was 0.42 =t 0.2. During the first 50min period of insulin infusion, 20.8 & 1.4 mU/min were infused, and after 20 min the arterial insulin level had TABLE 2. Comparison of hepatic vein sampling methods during shady-state insulin infusion

--.- ~--

“N”,“* .

--

30 31 47 Values intervals. vein.

IVC

(high)

70 zt 122& 237 zt are IVC

2.9 7.4 16

-~ .--_--_-~

--

Insulin IVC

Concentration,

(low)

49 It 1.4 161 ~~25 298 zt 29

pU/ml

LCHV

59 zt 2.6 121 dx 7.6 231 + 14

icav -(high)-

0.83 0.99 0.97

IVC

(Iow)

0.69 1.32 1.27

means I+= SE of eight samples obtained at lo-min = inferior vena cava, JXHV = left common hepatic

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HEPATIC

INSULIN

EXTRACTION

DURING

TABLE 3. Hepuhc insulin extraction during insulin hf usion Plasma Min

.Femoral artery

Insulin,

INSULIN

(EJ

flu/ml --

Portal vein

1583

INFUSION

Ekpatic vein

yztvein

Hepatic artery

Portal vein

Eh

700

Uptake, mU/min

o-o c w I

600 7

COYdYOl 17 It3 16 =t3 16 *3 15 Et3 17 zt3

-30 -20 -10 -5 -1

54 AZ13 49 *7 50 A7 54

3~8 47 zt7

23 It4 23 =t4 25 +4 23 *4 23 zt4

12 zt2 13 +2 13 32 13

*2 14 zt3 First

21 &4 31

2 5

4~6 40 =t7 52 zt8 56 *9 47

10 20 30 40

93 45

50

zt6

110 *22 140 *27 169 *21 191 +24 220

40 *8 57

9~8 72 zt8 79 It9 78 zt8 74

zt27 271 zt49 309 zt103

AZ10 75 &9

103 &14 107 +14 103 *12 101 All 107 Ztll

161

zt8 363 ztz8 151 It7 148 &8 153 Zt8

-43 zt,O6 .50 zlz.07 .38 It.09 .42 zt.07 .40 AZ.09

4.58

5 10 20 30 40 50

85 &ll 116 All 139 zt14 165 *14 171 It15 182 zt19 182 zt18

477 ztlO7 455 AZ95 419 A57 443 A43 438 zt67 556 A75 460 +69

I41 *12 169 A15 185 It15 209 ItI5 213 Et20 232 A29 219

zk25

5

120 &ll 81

3~8 10 20 30

data

Values (see

49 zt5 30 zt4 25 +4

163 429 110 A19 69 AZ9 50 +7 47 Zt&

are means

&

99 Et11 73 +7 46

+6 33 &5 28 Et5 SE.

Four

t

4.15 3.99

infusion

15 *3 20 Et4 28 rt5 33 zk5 36 zt6 32 It4 52 +4

47 It8 59 It9 80 &II 95 It15 100 *13 103 &13 103 Al3

87 AZ12 63

zt8 51 zt9 36 *7 33 tt7 of the

400

r

3.89

-60

108 All 110 *12 103 It12 JO7 ztll 105 All 101 &12 103 *14

157 ItI0 166 +11 163 A11 163 ZtlO 157 zt8 153 zt8 148 It11

.66 zt.07 l 53 It.07 .48 zt.06 .44 zt.06 -47 It.07 .46 zt.08 .4F h-08

20.58

104 +11 107 ZtlO 301 Ztll 107 &12 103 *11 94 *11 91 rtl0

155 Et9 161 *9 159

38.01

158 zt13 166 A17

-51 &.U5 .45 It.05 .44 zt.04 .4-l h.04 .39 +.06 -37 &.08 -40 A.07

166 A13 170 &I3 174 &I3 165 *12 152 +13

.28 1t.C6 .20 A.08 . 15 rt.09 . 15 h-08 .16 AZ.09

10.01

17.74 17.68 16.76

d.z9 159 &10 363

34

94 It9 92 zt9 93 =t8 92 +10 83 It9 19 dogs

have

been

excluded

.40 -20 oL 1

-30 18.12 16.01 16.04

37.05 37.79 38.63 36.22 34.34 37.52

PostiT2flision 2

500

VEIN tn=lO) ARTERY (n=l5)

6.21

Second infusion 2

i

2 7 E

PORTAL FEMORAL

4.78 2,17 1.50 1.25

from

these

RESULTS).

risen to a plateau of 50 + 4 pU/ml. The values for Eh during infusion were compared with the mean control values by the paired f test. Significant elevations were found at 2 min following the commencement of infusion (0.66 zt .07, P < .OOl) and at 5 min (0.53 & .07, P < -025). During the remainder of the first period of insulin infusion, & was consistently above control, but the differences were not significant. The elevation of Eh at the Z-

0

I

I

30 60 MINUTES

90

I

I

120

FIG. 2. Effect qf insulin infusion on portal vein and arterial insulin concentrations and hepatic extraction of insulin. Values are means + SE for 15 dogs with exception of those for portal vein insulin concentrations. Latter are means & SE of values in 10 dogs that had portal vein insulin recovery in range 65-135 yO-

and 5-min points is felt to be due to failure to account fully for dead space within the infusion system, so that the anticipated concentration of insulin has not yet been achieved in the portal vein. This source of error does not apply to the remainder of the experiment when the infusion is running continuously. The 2- and 5-min values have, therefore, been omitted from the data in Fig. 2. During the second stage of infusion, 50.8 & 1.7 mU/min were infused, and once again a plateau of arterial insulin concentration was reached between 20 and 50 min at a level of 175 & 8 pU/ml. i!& did not differ significantly from control, the mean value between 20 and 50 min being 0.39 & .03. However, this value was significantly lower than the value of 0.46 b .03 f ound during the plateau of the first infusion period (t = 2.702, P < .Ol). D uring the 30 min following the cessation of insulin infusion, arterial insulin levels fell rapidly with an initial disappearance half-time over the first 10 min of 4 min. The log concentration disappearance, however, was not linear with respect to time, suggesting the presence of an additional slower component. & fell markedly to a nadir of 0.15 & .O8 between 10 and 20 min after the infusidn was stopped. The values were significantly below control at 5 min (P < .05) and at 10, 20, and 30 min (P < .025) following the cessation of the infusion, so that control values were not reestablished by the end of the experiment. Mesenteric insulin extraction. In 7 of the 15 dogs, control mesenteric vein insulin levels (I, in APPENDIX) were slightly above arterial, making the calculation of control En impossible. As mentioned previously, this presumably reflects contamination with pancreatic blood. During steady-

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1584

HARDING,

state infusion, this was not a since arterial insulin concentrations exceeded those of the mesenteric vein in all 15 dogs (Table 3). E, averaged 0.33 rt 0.02 for the first period and 0.43 =t 0.01 for the second. However, after the cessation of infusion the mesenteric vein insulin in eight dogs was 127 =t 37 % of the arterial value at 20 min postinfusion and 147 & 54 % at 30 min, indicating an average net efflux of insulin from the mesenteric circulation. This was not felt to reflect contamination with pancreatic venous blood, as these dogs did not exhibit mesenteric vein insulin concentrations greater than arterial during the control period. Sytamic insulin extraction. The APPENDIX describes a mathematical model of the kinetics of insulin secretion and its extraction by vascular beds. This model is depicted in Fig. 3. Equation 4 of this model yields E,, the mean coefficient of insulin extraction in the systemic circulation. This is derived from the hepatic artery and portal vein plasma flows, hepatic vein (I& and systemic arterial (Ia) insulin concentrations, and the systemic plasma flow (QJ All these parameters are measured in the present study with the exception of Qs, which is the ascending aorta flow minus hepatic and mesenteric flow. Dedichen and Schenk (4) measured the distribution of cardiac output using electromagnetic flow probes in ventilated, barbital-anesthetized mongrel dogs of weights similar to our dogs’. Their value for total hepatic blood flow of 2 1.2 ml/kg per min corresponds closely to ours of 23.7 ml/kg per min. Assuming the same distribution of cardiac output in the two groups of dogs, a mean value for Qs of 1,085 ml plasma per minute may be derived for the present study. It should be noted that Qs is a simple inverse determinant of E,, and its value, therefore, only determines the magnitude of E, rather than the relative magnitude of any changes. Using this value for Qs together with the mean values from the 15 dogs studied for the other parameters, we can calculate values for Es. During the control period, Es averaged 0.107 & 0.009; whereas between 20 and 50 min of the first infusion, it was 0.135 & 0.012. This latter was significantly (P < 0.05) different from the control value. During the second infusion period, E, was 0.059 =I= 0.004. Hejutic glucose output (HGO). During the control

ES

Direct measurement of the quantities of insulin entering and leaving the liver affords the most accurate estimate of hepatic removal of insulin from the circulation. Ideally, a representative sample should be obtained from the same segment of blood as it first enters the liver and then leaves it. To approach this, hepatic vein samples were obtained 30 s after portal vein samples. In two experiments, recovery as a single sharp peak in the hepatic vein within 60 s of 5 1 and 23 % of the 1251-labeled insulin injected suggested that this peak represents unaltered insulin not extracted by the liver. The wide variation in recovery (9-303 %) from the portal vein sampling catheter of insulin infused some 10 cm upstream emphasizes the difficulties involved in obtaining representative portal vein insulin samples necessary for the measurement of insulin balance across the liver, at least under the conditions of our experiments. The finding of an overall recovery of 110 % with an equal proportion of anomalously high or low values suggests random variations in lamination of blood flow relative to the position

vascular

3.

Model for insulin beds (see APPENDIX

secretion for legend).

I

I

I

30

60

90

I

120

MINUTES

. FIG.

FIG.

FIELD

DISCUSSION

I

;--.

AND

period, hepatic glucose output averaged 36 rtr 3 mg/min when the arterial plasma glucose was 106 =t 3 mg/ 100 ml. The changes in both these parameters during the insulin infusion are shown in Fig. 4. HGO fell to a nadir of 16 & 5 mg/min 20 min after the onset of insulin infusion. The values at 10, 20, and 30 min were significantly (P < .025) below control. After this time, however, HGO returned toward control values. The higher rate of insulin infusion was not associated with as marked a fall, and during the last 30 min of the infusion, HGO had returned to control values although arterial glucose levels continued to fall to a nadir of 60 & 4 mg/lOO ml at the end of the infusion. Following cessation of insulin infusion, HGO increased and was still rising at the end of the period of observation, when it was 67 & 12 mg/min.

0

,

BLOOM,

and

extraction

by

various

centration 15 dogs.

4. Effect of insulin infusion on and hepatic glucose output,

arterial Values

plasma glucose cow are means =k SE for

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HEPATIC

INSULIN

EXTRACTION

DURING

INSULIN

INFUSION

of the catheter tip in the transverse plane of the vessel. The literature contains previous examples of such lamination of flow in the portal vein. We have reported an experiment (7) in which, during glucose-stimulated insulin secretion, hepatic vein and arterial insulin levels rose before those in the portal vein. Erwald et al. (5) explained portal vein insulin levels inappropriate to the prevailing systemic levels in several of their subjects on the basis of lamination of portal vein flow. Kanazawa et al. (9) found that India ink injected into the portal vein via the superior pancreaticoduodenal vein was distributed in a selective and inconstant pattern among the various hepatic lobes. Furthermore, in four of six experiments in which hepatic blood samples were obtained from each lobe, there was evidence that pancreatic insulin secretion was distributed in the same pattern as the India ink. Our use of a catheter with multiple holes did not overcome the sampling errors produced by this phenomenon, and caution should be exercised in interpretation of studies based on portal vein insulin concentrations. Selective distribution of insulin to the different hepatic lobes will also result in differential insulin concentrations within the hepatic venous bed, as found by Kanazawa and his colleagues (9). Therefore, hepatic vein sampling is another possible source of error in these studies. The ocfinding of hepatic vein insulin casional inappropriate levels lower than systemic has been reported by others (4, 9, 15) and ourselves (7), when we catheterized blindly the hepatic vein from the inferior vena cava. However, in the present study, hepatic vein samples were obtained from the left common hepatic vein, which carries approximately 40 % of hepatic venous blood (20). Evidence that such samples adequately reflect mixed hepatic venous insulin concentration was obtained in three experiments in which the IVC was ligated immediately below the hepatic veins. Mean steady-state concentrations in the LCHV over a 70-min period were 83, 97, and 99 % of the corresponding samples taken from the vena cava at a point farthest away from the liver where most mixing is presumed to have occurred (Table 2). The greater variation in vena caval insulin levels taken opposite the entry of the hepatic veins (low IVC) suggests that this catheter was more liable to sample small streams from lobes being perfused with variable insulin concentrations. No obvious abnormality of catheter placement was found in the two dogs excluded from the study because of clearly erroneous hepatic vein results. In the remainder of the dogs, whereas there were occasional time points when the hepatic vein insulin concentration was less than arterial, there were no cases in which such an anomaly was consistent+ Therefore, calculation of Eh from the arterial, mesenteric vein, and left common hepatic vein plasma insulin concentrations, together with the known insulin infusion rate and hepatic artery and portal vein plasma flows, constitutes the most valid measurement during steady-state infusion. This does not apply to the measurements immediately following the start of the infusion, in which the increased extraction demonstrated appears to be artifactual. The control & of 0.42 & 0.02 corresponds closely to our previously reported value (7) and to those of other investigators (10, 12, 14, 19). Although the slight rise in

1585

El, during the first infusion period (Fig. 2) is not statistically significant, some increase in tissue extraction, either in the liver or elsewhere, appears to be necessary to account for the 20 min taken for arterial insulin levels to rise to a steady level consistent with the abrupt fourfold increase in insulin ?ecretion” produced by the infusion. Blackard and Nelson (2) found a sharp peak of portal vein insulin occurred at 1 min after glucose infusion in man whereas the peripheral levels peaked at 5 min. It seems unlikely that this lag is due to transit time, which should not be grossly different from our value of 30 s determined in the dog; it would be consistent with a transient increase in tissue extraction. Also, in our previous report (7), hepatic insulin clearance increased within 5 min of the onset of glucose-induced insulin secretion. The transitory increase in the proportion of insulin leaving the blood during passage through the liver may represent equilibration of a tissue insulin compartment or compartments within the liver to the higher plasma concentrations. This equilibration may in part be attributable to the binding of insulin to vascular endothelium, as has been postulated by Rasio and Conard (17). Increasing the insulin infusion rate to 50.8 mU/min did not significantly increase & (Fig. 2), and arterial insulin levels rose more abruptly. By 2 min the mean rise was value), whereas 2 40 pU/ml (897 0 a b ove the previous min after the onset of the first infusion, the mean rise was only 4 $-J/ml (24 %)* A s arterial insulin concentration rose to a plateau between 20 and 50 min, & tended to fall slightly, but not significantly. However, during this time, arterial arterial insulin levels were proportionately greater in relation to the infusion rate than during the first infusion period. During the control period, the ratio of arterial insulin to insulin secretion was 2.9; during the first infusion (20-50 min), 1.9; during the second infusion, 3.1. This ratio should remain constant provided there is no change in either blood flow or tissue extraction coefficients. As blood flows were unchanged (Table 3), the reduction of this ratio during the “equilibrium” portion of the first infusion period must be due to a persisting increase in either Eh or E,. Although the rise in & at this time achieved statistical significance, variations in J!& are more effective in modifying systemic insulin levels than are changes in E, because of the higher concentrations of insulin presented to the liver. Figure 5 plots the relationship between the total amount of insulin presented to the liver and the arterial insulin concentration, which should also remain constant if extractions do not change. Figure 5, which used all the individual time plots for each dog during the control and 20- to 50-min periods of each infusion, demonstrates that during the equilibrium period of the first infusion, the rise in arterial insulin levels was not commensurate with the increased amounts of insulin presented to the liver. The time course of this divergence from linearity is similar to that observed when intraduodenal glucose was administered to dogs (7). F’ivc minutes after glucose was given, the ratio of arterial plasma insulin to total insulin delivered to the liver decreased and remained below the control ratio for 60 min. After this time, the ratio was the same as during the control period. The return of this relationship to control values, and not below, during the infusion of 50.8 mU/min of insulin, achieving arterial levels averaging

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1586

HARDING,

7.0

6.0

50

100

ARTERIAL

I50

INSULIN

250

200

AU/

300

ml

FIG. 5. Relationship between arterial insulin concentration and amount of insulin reaching liver, Small blank circles (0) represent data obtained in all dogs during control period. Small filled circles represent individual values obtained from 20 to 50 min during first infusion, and small triangles, individual values from same period of second infusion. Large symbol, Q are mean values for each period. Line going through origin and control points indicates relationship between arterial insulin and insulin presented to liver if hepatic extraction remained constant.

175 I,cU/ml, and the associated return of both & and E, to control levels under these conditions indicate that extraction of insulin by the liver and other tissues is not a saturable process at least up to these levels, which are as high as those found physiologically. In the isolated perfused rat liver, hepatic insulin extraction exhibited saturation kinetics only at perfusate levels in excess of 2,500 pU/ml (18), well-above the highest portal vein levels achieved in either this study or in response to glucose in dogs (7). Waddell and Sussman (24) found no evidence of saturation of hepatic extraction at portal vein insulin levels of 500 pU/ml, and Turner et al. (23) and Silvers et al. (21) also concluded that the liver had a large capacity to remove insulin. In addition to hepatic extraction of insulin, the remainder of insulin clearance occurs in the systemic and mesenteric circuits (Fig. 3). The present data also permit calculation of plasma clearance in these compartments. In the first and second infusion periods, respectively, mesenteric clearances were 45.0 and 63.6 ml/min, whereas systemic clearances of the were 146.5 and 55.3 ml/ min. This large contribution prehepatic splanchnic circulation to insulin disposal has not previously been recognized. As stated earlier, mesenteric clearance during the control period cannot be quantitated because of the possible contamination of mesenteric vein blood with pancreatic venous blood. The sites and distribution of the systemic clearance are not discernible from the present data.

BLOOM,

AND

FIELD

The peripheral plasma insulin response to an abrupt increase in pancreatic insulin secretion will be modulated by a slight increase in hepatic insulin uptake and by a more pronounced increase in peripheral tissue uptake requiring approximately 1 h for equilibration. Such a mechanism was postulated in general terms by Kanazawa et al. (9) to account for their observation that peripheral plasma insulin curves were not a simple reflection of those found in the pancreaticoduodenal vein. Upon cessation of insulin infusion, a rapid decrease in percent uptake of insulin was observed in each vascular bed with, in some instances, a net efflux of insulin being demonstrated. This washing out of insulin may be due to displacement of insulin bound to capillaries as demonstrated by Rasio (16) and could affect the time course of the disappearance of insulin from the plasma. Variations in the rate of this washout from tissues with different rates of perfusion might contribute to the nonlinearity of the log concentration-disappearance curve of insulin which was observed in this study. The fall in hepatic glucose output from a control value of 36 =I= 3 mg/min to a nadir of 16 =t 5 mg/min at 20 min after the onset of insulin infusion is similar to that observed by Madison et al. (11) in dogs with portocaval shuns. However, in their studies, a progressive fall of HGO was observed during the infusion of 46 mU/min of insulin, so that after 70 min it had fallen to 7.4 mg/min compared with a control value of 43.3 mg/min. In the present study, control values had been regained by this time. The essential difference between their preparation and ours is the intact portal circulation in our dogs which ensures that the liver received high portal vein levels of glucagon, should this hormone be secreted in response to hypoglycemia. Studies currently in progress indicate that a significant rise in dog portal vein glucagon levels occurs in response to the hypoglycemia found during the first period of insulin infusion in the present study. Madison et al. (11) stressed the importance of avoiding profound hypoglycemia in the assessment of hepatic response to insulin. In the present HGO started to increase toward control study, however, values 30 min after the start of insulin infusion when the arterial plasma glucose had fallen to only 90 & 4 mg/ 100 ml from a mean control value of 106 & 3 mg/ 100 ml (Fig. 4). Despite this, arterial plasma glucose continued to fall to a nadir of 60 & 4 mg/lOO ml at the end of the infusion when HGO was back to control values. This suggests that increased peripheral glucose utilization is the major factor in maintaining insulin-induced hypoglycemia. In this connection, the persistent increase in E, found during insulin infusion may have physiological significance. When the insulin infusion was stopped a prompt rise in HGO occurred, suggesting that insulin still exerted an inhibitory effect on this function even though the value had returned to control level during the infusion. APPENDIX Figure 3 is a schematic representation of insulin kinetics under steady-state conditions. The body has been divided into hepatic (h), mesenteric (m), and remaining systemic (s) vascular beds, each of which removes insulin from the circulation with a coefficient of extraction, E, which is the percent extraction by that vascular bed expressed as a proportion of unity. The vessels connecting these beds each carry an insulin flux (F, mU/min) which is the product of

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HEPRTIC

INSULIN

EXTRACTION

DURING

INSULIN

INFUSION

plasma flow (Q, ml/min) and plasma insulin concentration (I, pU/ml) times 10- 3. Input of insulin into the system representing pancreatic insulin secretion plus-or-minus insulin infused into the portal vein is expressed as S (mU/min). The flow and fluxes and insulin concentrations are designated by the following subscripts (see Fig. 3) : a, ascending aorta; ha, hepatic artery; m, arterial supply entering mesenteric bed including the spleen; s, remaining systemic arterial flow; v, systemic venous return; hv, hepatic vein; p, portal vein, cv, pooled venous return. It is assumed that no extraction occurs across the cardiopulmonary circuit and the spleen is considered a representative part of the mesenteric-portal circuit. The following equations may then be derived.

1587

Since

the amount

I&ha

of insulin

+

QP

=

I&ha Iv

+

=

entering

Qs)

=

IhvQhv

+

QP

+

I&&,3

+

Qp

and leaving +

+

Ihv(Qha

QJ Q)

is the same

LQs

=

Qs,

the heart

-

+

hw(Q~m

+

Qp)

+

LQs

QJ

S

or

and F,

=

QP

x

I,

I,

extracted

by gut

= Q&

-

Q

e+

Substituting

Ip)

for IV in equation

E, = Qs I, -

&a

1, -

by the liver

=

QhaJa

Eh

=

+

QpIp + S -

hv(Qha;Qp)

Ihv(Qha

+

QJ

h.(Qha

+

Q,)

QPIa - Qs 1, + Qha&xv+ Qp hw Qs L

Ihv(Qhn + Qp) - L(QI~ + QJ Qs L

F2s =

extracted

-

3 gives

and

insulin

1)

( e+ s

in which I, is the insulin concentration in portal vein prior to entry of S from pancreas and is equivalent to the insulin concentration in the mesenteric vein. In Table 3 the portal vein insulin concentration includes insulin secreted by the pancreas or infused during the experiment and is not equivalent to I, in the following equations. insulin

= I,

FAS =

(%a

+

QJ Qs

(Ihv 1,

-

Ifi) (4)

and

insulin

extracted

Qha

Ia +

Qp

Ip +

s -

Qha

Ia +

Qp

systemically

= Q&

I,

-

+

s

(2)

I,)

and

(3) total

plasma

flow Q = Qha + Qp + Qs

We are grateful to Joan Martell and James Stout for invaluable technical assistance and to Barbara Sheehan, Mary Matthews, and Gail Mashiter for help in preparing the manuscript. We are especially indebted to Dr. Richard N. Bergman, who provided a simplified solution for the calculation of Es as presented in the APPENDIX. This work was supported by Public Health Service Grant AM12974 from the National Institutes of Health. Present address of P. E. Harding: Endocrine Unit, Royal Adelaide Hospital, Adelaide, Australia. Received

for

publication

8 November

1973.

REFERENCES G. E., Y. KOLOGU, AND C. PAPADOPOULAS. Fluctuations to insulin release. post-absorptive blood glucose in relation Metabolism 16: 586-596, 1967. BLACKARD, We G., AND N. C. NELSON. Portal and peripheral vein immunoreactive insulin concentrations before and after glucose infusions. Diabetes I9 : 302-306, 1970, Growth hormone-induced diaCAMPBELL, J., AND K. RASTOGI. betes and high levels of serum insulin in dogs. Diabetes 15 : 30-43, 1966. DEDICHEN, H., AND W. G. SCHENK, JR. Cardiac output and its regional distribution in the dog. J. Thoracic Cardiovasc. Surg. 61: 110-120, 1971. ERWALD, R., R, HED, A. NYGREN, S. RODJMARK, L. SUNDBLAD, AND K. L. WIECHEL. Insulin concentration in portal and peripheral venous blood after oral glucose in human pancreatitis. Acta Med, Stand. 194: 103-109, 1973. ISHIWATA, K., G. HETENYI, JR., AND M. VRANIC. Elect of D-glucose or D-ribose on/the turnover of glucose in pancreatectomized dogs maintained on a matched intraportal infusion of insulin. Diabetes 18 : 820-827, 1969. KADEN, M., P. E. HARDING, AND J. B. FIELD. Effect of intraduodenal glucose administration on hepatic extraction of insulin in the anesthetized dog. J. C&n. Invest. 52 : 2016-2028, 1973. KANAZAWA, Y., T, KUZWYA, AND T. IDE. Insulin output via the pancreatic vein and plasma insulin response to glucose in dogs. Am. J. Physiol. 2 15 : 620-626, 1968.

1. ANDERSON,

2.

3.

4.

5.

6.

7.

8.

Y., T. KUZUYA, T. IDE, AND K. KOSARA. Plasma insulin responses to glucose in femoral, hepatic and pancreatic veins in dogs. Am. J. Physioi. 211: 442-448, 1966. KAPLAN, N., AND L, L. MADISON. Effects of endogenous insulin secretion on the magnitude of hepatic binding of labelled insulin during a single transhepatic circulation in human subjects. C/in. Res. 7 : 248, 1959. MADISON, L. I,. , B. COMBES, R. ADAMS, AND W. ST~CICKLAND. The physiological significance of the secretion of endogenous insulin into the portal circulation. III. Evidence for a direct immediate effect of insulin on the balance of glucose across the liver. J. C/in. Invest. 39 : 507-522, 1960. MADISON, L. L., AND N. KAPLAN. The hepatic binding of I13r labelled insulin in human subjects during a single transhepatic circulation. J. Lab. C&n. Med. 52 : 927, 1958, MARSHALL, A., R. L. GINGERICH, AND P. H. WRIGHT. Hepatic metabolism of insulin in vitro. Clin. Res. 18 : 33, 1970. MORTIMORE, G. E,, F. TIETZE, AND D. STETTEN, JR. Metabolism perfused rat liver and hindof insulin-1131 : studies in isolated, limb preparations. Diabetes 8 : 307-3 14, 1959. RAPPAPORT, A. M., J. K, DAVIDSON, T, KAWAMURA, B. J. LIN, S. ZELIN, J. HENDERSON, AND R. E. HA~ST. Quantitative determination of insulin output following an intravenous glucose tolerance test in the dog. Can. J. Physiol. Pharmacol. 46 : 373-381,

9. KANAZAWA,

in

10.

Il.

12.

13. 14.

15.

1968.

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1588 16. CASIO,

displacement of insulin from blood capillaries. 19, 1969. RASIO, E., AND V. CONARD. Absorption and release of insulin by the vascular endothelium, Diabetologia 5: 53, 1969. RUBENSTEIN, A. H., L. A. POTTENGER, M. MAKO, G. S. GETZ, AND D. F. STEINER. The metabolism of insulin and proinsulin by the liver. J. Clin. Invest. 51 : 912-92 1, 1972. SAMOLS, E., AND J. A. RYDER. Studies on tissue uptake of insulin in man using a differential immunoassay for endogenous and exogenous insulin. S. Clin. Invest. 40 : 2092-Z 102, 1961. SHOEMAKER, W. C., W. F. WALKER, T. B. VAN ITALLIE, AND F. D. MOORE. A method for the simultaneous catheterization of major hepatic vessels in a chronic canine preparation. Am. J. Physiol. 196: 311-314, 1959. Diabetologia

17. 18.

19.

20.

HARDING, E. A. The

5 : 4 16-4

BLOOM,

AND

FIELD

21. SILVERS, A., J. FARQUHAR, R. LERNER, AND G. M. REAVEN. Evaluation of the dog as an experimental model for the study of insulin distribution and degradation in man. J. Lab. Clin. Med. 75: 175-184, 1970. 22. SONKSEN, P. H., J. R. MCCORMICK, R. H. EGDAHL, AND J. S. SOELDNER. Distribution and binding of insulin in the dog hindlimb. Am. J. Physiol. 221: 1672-1680, 1971. 23. TURNER, R. C.,J. A. GRAYBURN, G. B. NEWMAN, AND J. D. N. NABARRO. Measurement of insulin delivery rate in man. J. Clin. Endocrinol. 33 : 279-286, 1971. 24. WADDELL, W. R., AND K. E. SUSSMAN. Plasma insulin after diversion of portal and pancreatic venous blood to the vena cava, J. A#.

Physiol.

22:

808-812,

1967.

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