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Fluctuations in postabsorptive blood glucose in relation to insulin release
Fluctuations in Postabsorptive Blood Glucose In Relation to Insulin Release ByGEORGE E.
ANDERSON,YUSUF KOLOGLUANDCONSTANTINPAPADOPOULOS
In the dog multiple glucose determinations in rapid succession (at 15-second intervals) on hepatic-venous blood were correlated with assays of immunoassayable insulin concentrations in pancreaticvenous blood. Within a period of 9% minutes, the following observations were made: (1) Pulsatile rises in hepatic-venous glucose are followed within approximately 15 seconds by corresponding increases in the insulin content of the effluent of the pancreas. (2) As the insulin concentration in the pancreatic veins increases, glucose level in the hepatic veins decreases. (3) With decrease in the glucose release, there is a simultaneous decrease in the insulin content of hepatic-venous blood. (4) A return of hepatic-venous glucose concentration is associated with a coincidental return of its insulin concentration. Two opposite modalities of insulin
control are suggested: in the first, pulses of increased hepatic-venous glucose output seem to promote the release of insulin into the pancreatic veins; in the second, an increasing insulin concentration in the pancreatic veins seems to be associated not only with precipitous reduction in hepatic-venous glucose but also with inhibition by the liver of insulin output into the periphery. In all probability, both processes represent a feedback regulatory mechanism whereby during postabsorption the liver controls the production of glucose as well as the availability to the tissues of insulin in accordance with peripheral needs. As originally shown by Madison, th!s control is mediated by the liver through variations in its release of hepatic-venous glucose. (Metabolism 16: No. 7, July, 586-596, 1967)
I
N DOG and in man the fasting blood glucose concentration, both arterial and venous, changes rapidly from moment to moment throughout the entire postabsorptive period. v The resulting wave-like fluctuations can be traced to their origin in hepatic-venous blood. Under normal conditions, the peripheral oscillations vary between 1 and 36 mg./lOO ml. (over the standard error incidental to the glucose method). Determinations of arterial, peripheralvenous, and hepatic-venous glucose levels at 30-second intervals indicate that these changes are totally irregular and unpredictable but hover about a relatively unchanging (or slowly changing) mean. In 1952 Bondy catheterized the hepatic veins of 11 human subjects and could find “no cuase for these sudden ‘spikes’ of glucose release [from the liver] . . . rapid unpredictable fluctuations occur in splanchnic balance . . . these variations are so large as to interfere seriously with attempts to estiFrom the Department of Medicine, The Brooklyn-Cumberland Medical Center, an afjiliate of the State University of New York, Downstate Medical Center. This study was supported by a grant from the John A. Hartford Foundation, Inc. Received for publication December 19, 1966. GEORGE E. ANDERSON, M.D.: Clinical Professor of Medicine, State University of New York, Downstate Medical Center; Senior Physician, Brooklyn-Cumberland Medical Center. YUSUF KOLOCLU, M.D.: Clinical Instructor of Medicine, State University of New York, Downstate Medical Center. CONSTANTIN PAPADOPOULOS, M.D.: Clinical Instructor of Surgery, State University of New York, Downstate Medical Center; Vascular Surgeon to the Brooklyn-Cumberlund Medical Center. 586
BLOOD
GLUCOSE
IN INSULIN
RELEASE
587
mate the mean balance over any protracted period of time.“3 Since in that study blood samples were drawn simultaneously from the hepatic vein and peripheral artery with no correction for the circulation time between these sites, peripheral arterial concentrations not infrequently were found to be higher than those at the point of origin in the hepatic veins. By careful timing of the blood circulation between the hepatic vein and a femoral artery of dogs, it was subsequently shown in our laboratory that the larger hepatic venous fluctuations are paralleled grossly by similar oscillations in the femoral artery1 at lower glucose levels. This was established by laparotomizing dogs, cannulating in each an hepatic vein and femoral artery, and timing the circulation of injected fluorescein between the two sites with the aid of a Wood lamp. Blood samples were then drawn at 30-second intervals from both vessels, separating the sampling of hepatic venous blood from that of the femoral artery by the number of seconds found to be the circulation time between these two sites. Glucose was subsequently determined (in duplicate) on each sample. The complete irregularity in configuration, rate, rhythm, and size of the hepatic-venous glucose emanations made possible the identification of similar clusters of fluctuations in the arterial blood at lower levels. A gross parallelism was consistently found to exist between the glucose curves from both sources, the femoral arterial blood, as would be anticipated, demonstrating a few seconds later a lower but parallel level of glucose concentration (Fig. 1). By a similar technic these glucose fluctuations were followed from the arterial to the venous side of the circulation in extremities. The original hepatic-venous fluctuations travel accordingly from their source in the liver and are projected into the larger blood vessels of the body. Fig. 2 illustrates examples of their presence in the peripheral veins of several normal subjects, both human and canine. When 10 or more sequential glucose determinations are made at 6-second intervals from a single peripheral vein and these are plotted in relation to time, it is found that there usually results a two-phase fluctuation, the completed up-plus-down strokes of which take approximately one minute. Since the rise and fall in glucose level in this brief period of time was the dominant finding in what otherwise appeared to be a completely irregular phenomenon, the present investigation was undertaken to study the relationship, if any, during postabsorption between glucose fluctuations appearing in the hepatic veins and the concentration of insulin in the venous effluent of the pancreas. Briefly, within approximately 15 seconds after the appearance of each major elevation of glucose in the hepatic veins, there occurred a sharp release of insulin into the pancreatic veins. It is suggested that during postabsorption these momentary accessions in hepatic glucose output, reflected in the arterial system. serve to trigger the release of insulin at a time when there is little or no absorption of nutrient from the intestinal tract. An effective feedback mechanism might thus be established, limiting the output of glucose by the liver as well as regulating the retention of insulin by that organ4-” and, secondarily, controlling peripheral insulin levels.
588
ANDERSON,KOLOCLUANDPAPADOPOULOS
: ,r i:
FEMORAL ARTEllA
Fig. l.-Gross parallelism between fasting hepatic-venous and femoral glucose fluctuations in a normal dog. (To accentuate the parallelism glucagon ministered by splenic artery at the point of time indicated.)
arterial was ad-
METHODS Glucose determinations were made by the Autoanalyzer* (modified Hoffman’*’ method). Except for steady monitoring by the Autoanalyzer, all determinations were made on plasma. Analyses were made of errors inherent in the method of glucose determination. In samples numbering 40, the Standard Deviation and the 95 per cent Confidence Limits established at three levels were respectively: 85 mg./lOO ml., 1.67 and 84.5 to 85.5; 200 mg./lOO ml., 4.17 and 198.7 to 201.3; 300 mg./lOO ml., 3.65 and 298.8 to 301.2. Fourteen hours after the last ingestion of food of a liberal mixed diet, an hepatic vein in each of 6 large full-grown normal mongrel dogs was cannulated under pentobarbital anaesthesia and closed oxygen insufflation for respiration. The transthoracic route was used. All dogs were heparinized as well as heavily ergotaminized (dihydroergotamine 0.5 mg/K-I.V.) to blot out adventitious adrenal medullary influence on blood glucose levels. A relatively large pancreatic vein was isolated and also cannulated. At 15-second intervals blood was withdrawn from the hepatic vein and at 15- to 30-second intervals, from the pancreatic vein. The total period of blood sampling in all 6 dogs ranged from 6 to 9 minutes. The pancreatic-venous specimens in one of these dogs ( 18 within a period of 6 minutes) were subjected to assay for their concentration of insulin. Dr. Sheldon J. Bleiche? of the Jewish Hospital of Brooklyn and Dr. Harry N. Antoniades” of the Protein Foundation Laboratories of Boston and the School of Public Health of Harvard University very kindly ‘Technicon
Company,
Chauncey,
New York.
BLOOD
GLUCOSE
IN INSULIN
589
RELEASE
Fig. 2.-Examples of postabsorptive venous glucose fluctuations on 30-second samnling: (a) in normal human subjects, (b) in normal zontal line in each graph represents mean blood glucose. Note: in the lower the mean blood glucose level, the greater the tendency to of oscillations.
in man and dog dogs. The horinormal subjects, wider amplitude
agreed to assay for insulin independently and blindly in their respective laboratories aliquots of all of the pancreatic venous specimens. These assays were carried out by the and by the coated charcoal method of e-antibody method of Morgan and Lazarow” Bleicher and his group? The readings from both sources were correlated with our own simultaneous findings of hepatic-venous glucose concentration during this brief period. Dr. Antoniades assayed for their insulin concentration not only the pancreatic-venous specimens, but, in addition, all of the hepatic-venous samples from the dog, specimens A, B, C, D, etc., in Fig. 3 and Table 1. By the use of a fluorescein-Wood-lamp technic, in which fluorescein was injected into one vessel and its arrival at and exit from another vessel was demonstrated by Wood lamp, the circulation time between the hepatic vein and the arterial supply to the pancreas was measured. In a similar manner, the circulation time between the hepatic vein and the venous effluent of the pancreas was determined. &WLTS
It will be noted that, although the absolute values for insulin concentration in the two laboratories differ (Fig. 3), the curue.s of increasing or decreasing insulin concentration were found grossly to parallel each other and to show
590
ANDERSON,
KOLOGLU
AND PAPADOPOULOS
among the specimens significant variation from moment to moment. This variation is far beyond that which might be anticipated to occur as intrinsic standard error peculiar to the method of assay. The rapid changes in insulin concentration appear to have occurred in relation to similarly sharp variation in hepatic-venous glucose concentration preceding each insulin reading. The insulin changes seem to ensue puri passe with the rises and falls in glucose concentration, there being a lag of 15 seconds. The insulin values in pancreaticblood increase within approximately 15 seconds after each major rise in hepaticvenous glucose takes place. This is documented in Fig. 3 and Table 1, in which a preliminary run of five 1Ssecond hepatic-venous glucose readings is shown to precede the taking of concurrent samples from the pancreatic vein. The hepatic-venous glucose values are labelled in sequence: A, B, C? etc., the pancreatic-venous insulin values, from 1 to 18. The 15-second time scale of Fig. 3 serves to indicate the time lag between the respective individual spikes and falls in hepatic-venous glucose and the corresponding rises and falls in concentration of pancreatic-venous insulin. The correlation is indicated by E, F, I, K, M, etc. The rises and falls in hepatic-venous glucose 1 1 1 i ’ 12567 are rapidly reflected in similar crests and troughs of insulin concentration in the pancreatic veins. This repetitive process, occurring every few seconds, revealed itself to be an ordered sequence linking the two factors. One should be mindful of the fact that in a totally irregular mechanism, as is the emanation of the glucose oscillations from the liver,3 there are bound to occur discrepancies in timing relationships. These are obvious in Fig. 3 but are not sufficiently great over the brief period of 6 or 7 minutes of blood sampling to compromise the principles aimed at in these procedures. Similarly, it may be anticipated that the theoretical changes in blood circulation rate through the liver during the few minutes while the sampling was in process would be negligible. Regarding the sharp momentary oscillations, it seems clear that the individual spikes in hepatic-venous glucose are associated with similarly sharp releases of insulin (Fig. 3). In all 6 dogs as the postabsorptive state progressed, the amplitude of interspersed individual oscillations (e.g., M, Y in Fig. 3) increased in spite of a steadily declining mean blood glucose (from M through EE). At a time when the mean hepatic venous glucose dipped into a noticeably sharper decline (from R thru EE) there occurred a progressive but sharp rise in the pancreatic-venous concentration of insulin (Fig. 3) -over 400 per cent rise in one laboratory (from 60 to 250 PU within 4 minutes), 375 per cent in the other laboratory (from 200 to 750 PU ) . Coincidental with this rise in pancreatic-venous insulin there was pari passu a fall in the concentration of insulin in blood after it passed through the liver, i.e., in the hepatic veins (Table 1). Hepatic-venous insulin declined to a low of 4 pU/ml. at a time when pancreatic-venous insulin hovered about 250 ,U/ml. and hepatic-venous glucose had dropped to a nadir of 22 mg./lOO ml. These findings would suggest a second hepatic regulatory mechanism controlling insulin-glucose homeostasis.
BLOOD
GLUCOSE
IN INSULIN
591
RELEASE
;yEPAW
VENOU : ’ .G PUCOSE:
Fig. X-In dog, postabsorptive hepatic-venous glucose oscillations (designated as A,B,C, etc.) correlated with insulin release into the pancreatic veins (specimens 1-18). Correlation is recorded over a total period of 6% minutes (designated, e.g. E, F, M, 1 1 I etc.). 127
592
ANDERSON, Table
Pancreatic-Venous Insulin (pU/ml.)
Hepatic-Venous GlUCOSC ml.)
c- 48 D-134 E-128 F- 66 G- 78 H- 84 I-90
l + + +
J- 94 + K- 80 L- 92 + M-124 +
l-90 2-80 3-100 4100 S-100 6-60 7-60
N- 60 O-60 + P-121
8-70
Q- 62 +
9-80
82
S- 46 T- 72 U- 80 v-52 w- 50 x- 30 Y-118
Simultaneous Concentration of Hepatic-Venous Insulin tau/ml.) 12
A-130 B- 98
R-
AND PAPADOPOULOS
I.-Hepatic-Venous Glucose Concentrations at 15Second Inter&s with Pancreatic-Venous and Hepatic-Venous Insulin Concentrations*
Correlated
(mg./lOO
KOLOGLU
+ + *
IO-80 11-100 12-100
* +
13-140 14120 15-170
z-64 AA- 48 BB- 22
* + e
CCDDEEFF-
56 58 30 32
+
GGHHII-
44 52 56
X-120 17-250 18-250
20 20 15 12 50 20 12 12 50 12 12 10 6 12 10 25 20 15 10 50 10 10 10 4 4 4 4 4 4 4 4 12 10 40
JJ- 22 KK-130 MM- 88 *There is a lag of approximately 15 seconds between the glucose reading and that of pancreatic-venous insulin, e.g., E-128 (mg.JlOO ml.) + l-90 (aU/ml.; F-66 (mg./lOO ml.) *
Z-80 ( jJJ/ml.)
, etc. DISCUSSION
It would appear that increases in insulin concentration in the pancreatic veins are preceded by pulsatile rises in hepatic-venous glucose during fasting, ( 1) since the relationship between the two factors is so consistent, and (2) since the circulation time between the hepatic vein and the pancreas approxi-
BL~~DGLUCOSEININSULINRELEASE
593
mately accounts for the time-lag. The circulation time between the hepatic veins and the arterial supply to the pancreas was found to range from 4 to 6 seconds; that between the hepatic veins and pancreatic veins, from 6 to 8 seconds. Accordingly, the pancreatic tissue was usually exposed for approximately 2 to 4 seconds to the specific segment of blood (with its brief rise in glucose) as it traversed the capillary bed of that organ. The release of insulin from the pancreas in response to glucose is therefore presumably immediate upon exposure, rather than time-consuming or after a prolonged latent period. Two opposite modalities of insulin control are presented: the first, pulses of increased hepatic-venous glucose, seem to initiate the release of insulin into the pancreatic veins; the second, an increasing insulin concentration in the pancreatic veins, seems to be associated not only with precipitous reduction in the overall output of glucose into the hepatic veins but also with inhibition by the liver of the release of insulin into the periphery. In all probability, both processes represent a feedback regulatory mechanism whereby during postabsorption the production of insulin as well as its availability to the tissues is controlled by the liver through variations in its release of insulin and glucose into the hepatic veins. These findings support those of Madison: “Insulin administered in a manner which minimized the counter-regulatory responses to a falling blood glucose concentration resulted in a prompt decline in hepatic glucose output. . . . ” 4 In our experiments “the counter-regulatory” factor was “minimized” by ergotaminization. The coincidental marked falls in hepatic-venous glucose, and the sharp rises in pancreatic-venous insulin probably indicate deprivation in the animal of adrenal medullary influence as this is effected by ergotaminization “minimizing the counter-regulatory responses.” In lieu of insulin’s being “administered” by portal vein, the concentration of the hormone in this venous system spontaneously increased approximately 400 per cent within a period of four minutes. This phase of insulin increase in all probability lasted samples was only 1 or 2 minutes after the last of the 18 pancreatic-venous withdrawn for insulin assay, since within 1% minutes after this last sampling, hepatic-venous insulin had again climbed (Table 1) from a concentration of 4 /lU to 40 pU/ml. At the same time hepatic-venous glucose had risen within 2X minutes from its nadir of 22 mg./lOO ml. (BB in Table 1) to 130 mg./lOO ml. (LL). S imultaneously there was an increase in mean hepaticvenous glucose value. Apparently, the sharp momentary oscillations in hepaticvenous glucose tend to cluster about a broad or sinusoidal curve of changing mean glucose levels over several minutes (Fig. 3). The normal existence of rapid changes in blood glucose in dog and in man was originally reported by our laboratory in 1956.l Subsequently these brief rises and falls in glucose levels were found to be present without exception in the fasting peripheral venous blood of 250 human subjects when these individuals were tested at 30-second intervals in an effort to obtain more accurate fasting glucose levels for insulin sensitivity tests. The amplitude of the glucose excursions tends to increase as the postabsorp-
594
ANDZRSON,
KOLOGLU
AND PAPADOPOULOS
Fig. 4.- In a dog, simultaneous glucose readings from the same vein: (a) by Autoanalyzer, (b) by 30-second intermittent aspiration through an l&gauge needle. The former is the arithmetical mean of the multiple readings. The recorded curve of continuous monitoring by Autoanalyzer (a) was traced on tracing paper from right to left (the direction of the pen-writer), reversed and superimposed from left to right on the graph of plotted 30-second samplings.
tive period progresses and the mean blood glucose level declines (Fig. 2). The oscillations, however, immediately disappear in dogs when the liver is clamped off; 1 instead there occurs a slow, even, steady decline in blood glucose until the animal’s decease. This fact seems to define their origin to the liver. Widespread single observations of blood glucose, i.e., specimens aspirated at 20-, lo- or even 5-minute intervals, are not sufficiently sensitive to pick up the minute critical changes occurring in such a dynamically rapid-moving process as is blood glucose regulation. The process is far better represented by a composite of many of these pictures in rapid succession yielding thereby a concept of activity in slow motion. [The entire action in the present demonstration (Fig. 3) took only 9W minutes.] Determinations at 15- or 30-second intervals bring out fine points of sequence commonly missed by more static conventional methods of investigation. This principle would seem to hold also in respect to the frequency with which determinations of insulin concentration are made. Rapid momentary glucose oscillations are not visualized when peripheral venous blood is continuously monitored by the Autoanalyzer.8,11 However, when an l&gauge needle is inserted into the same vein as the indwelling cannula of the analyzer (about one-half inch away from its opening), simultaneous sampling through both channels is possible; the Autoanalyzer registers a continuous, smooth, uninterrupted, nonfluctuant reading (“a” in Fig. 4), whereas simultaneous staccato 30-second withdrawals show rises and falls typical of those found in all dogs and human subjects when an intermittent aspiration technic is used (‘b”). It may be that blood which has traveled the length of the analyzer’s cannula and tubing at the slow rate of 0.33 mI./min. (approximately 20 ml./hr.) has become homogenized in this tubing before it
BLOOD
GLUCOSE
IN INSULIN
595
RELEASE
reaches its entrance into the Autoanalyzer. The reading from the latter was found to represent the arithmetical mean of all the simultaneous 30-second determinations. Proof of this is suggested by the observation that the level established by the Autoanalyzer is identical with that in blood which has slowly and simultaneously been aspirated by needle and syringe from the same vein over a Sminute period. Lambert and Hoet I2 recently noted (in their studies on diurnal insulin levels in peripheral blood) that not infrequently some of the highest insulin values are found at 3:00 or 4:00 A.M., many hours after the last ingestion of food. Our findings suggest a partial answer to this enigma. Postabsorptively, the release of insulin secondary to brief episodes of spontaneous glycogenolysis rather than (as is more usually observed) to the ingestion of food could account for this phenomenon. Spurts of release of glucose appearing in the arteries would be anticipated to increase in tempo and intensity as the postabsorptive period progresses, since the magnitude of the hepatic-venous glucose oscillations increases as time elapses. These would be expected to cause corresponding releases of insulin into the pancreatic effluent. These studies inevitably lead to a pertinent question: What originally causes the sharp releases of hepatic-venous glucose? Preliminary studies in this laboratory have suggested that measurably increased “glucagon-like” activity (GLA) in pancreatic-venous blood precedes the individual spurts of hepatic-venous glucose output. This fact suggests that the rises in hepaticvenous glucose are preceded by, and may even be produced by, similarly timed releases of GLA or of glucagon from the pancreas or small-bowel wall. This triggering of glucose output by the liver would constitute at least one basic mechanism whereby glucagon or related substances might indirectly “stimulate” 13*14the release of insulin or enhance its activity. ACKNOWLEDGMENT The authors are most grateful to Dr. Harry N. Antoniades of the Protein Foundation Laboratory, Boston, Mass., and Dr. Sheldon J. Bleicher of the Jewish Hospital, Brooklyn, N. Y., who kindly carried out the necessary insulin assay procedures. We acknowledge with thanks the technical advice of Dr. Martin G. Goldner and our associates, Dr. Eugenie The M. Fribourg, Stanley S. Bergen, Jr., Robert W. Hillman, and Seymour Werthamer. technical services of James H. Wilds, MS., Agnes C. Dann, R.N., and Fernando Figoralo, D.V.M., is also appreciated.
REFERENCES 1.
2.
3.
Anderson, G. E., Hillman, R. W., Van Elk, I. F. A., and Perfetto, A. J.: Postabsorptive undulations and oscillations in blood glucose. Amer. J. Clin. Nutr. 4:673, 1956. Hansen, K. M.: Oscillations in the blood sugar of fasting normal persons. Acta Med. Stand. Suppl. 4:27, 1923. Bondy, P. K.: Spontaneous fluctua-
4.
5.
tions in glucose content of hepatic venous blood in resting normal human beings. J. Clin. Invest. 31:231, 1952. Madison, L. L., Mebane, D., Lecocq, F., and Combes, B.: Physiological significance of the secretion of endogenous insulin into the portal circulation. Diabetes 12:8, 1963. Madison, L. L., Combes, B., Adams,
ANDERSON,
596
6.
7.
8.
9.
R.: Insulin’s control of the role of the liver in the disposition of a glucose load in diabetic and non-diabetic dogs. J. Clin. Invest. 39:1009, 1960. Combes, B., Adams, R. H., Strickland, W., and Madison, L. L.: The physiological significance of the secretion of endogenous insulin into the portal circulation. IV Hepatic uptake of glucose during glucose infusion in non-diabetic dogs. J. Clin. Invest. 40:1706, 1961. Hoffman, W. S.: A rapid photoelectric method for determination of glucose in blood and urine. J. Biol. Chem., 120: 51, 1937. Weller, C., Lindner, M., Macaulay, A., Ferrari, A., and Kessler, G.: Continuous in viva determination of blood glucose in human subjects. Ann. N. Y. Acad. SC. 87:658, 1960. Herbert, V., Lau, K. S., Gottlieb, C. W., and Bleicher, S. J.: Coated charcoal
10.
11.
12.
13.
14.
KOLOGLU
AND PAPADOPOULOS
immunoassay of insulin. J. Clin. Endocrin. 25: 1375, 1965. Morgan, C. R., and Lazarow, A.: Immunoassay of insulin: two antibody system. Diabetes 12: 115, 1963. Burns, T. W., Bregant, R., Van Peenan, H. J., and Hood, T. E.: Observations on blood glucose concentration of human subjects during continuous sampling. Diabetes 14: 186, 1965. Lambert, A. E., and Hoet, J. J.: Diurnal pattern of plasma insulin concentration in the human. Diabetologia 2: 69, 1966. Samols, E., Marri, G., Marks, V.: Promotion of insulin secretion by glucagon. Lancet 2:415, 1965. Samols, E.: Effects of Glucagon: Insulin Release in Vivo. Presented at the Research Symposium on Glucagon, American Diabetes Ass., San Francisco, Calif., October 8, 1966 (unpublished).
ANDERSON,YUSUF KOLOGLUANDCONSTANTINPAPADOPOULOS
In the dog multiple glucose determinations in rapid succession (at 15-second intervals) on hepatic-venous blood were correlated with assays of immunoassayable insulin concentrations in pancreaticvenous blood. Within a period of 9% minutes, the following observations were made: (1) Pulsatile rises in hepatic-venous glucose are followed within approximately 15 seconds by corresponding increases in the insulin content of the effluent of the pancreas. (2) As the insulin concentration in the pancreatic veins increases, glucose level in the hepatic veins decreases. (3) With decrease in the glucose release, there is a simultaneous decrease in the insulin content of hepatic-venous blood. (4) A return of hepatic-venous glucose concentration is associated with a coincidental return of its insulin concentration. Two opposite modalities of insulin
control are suggested: in the first, pulses of increased hepatic-venous glucose output seem to promote the release of insulin into the pancreatic veins; in the second, an increasing insulin concentration in the pancreatic veins seems to be associated not only with precipitous reduction in hepatic-venous glucose but also with inhibition by the liver of insulin output into the periphery. In all probability, both processes represent a feedback regulatory mechanism whereby during postabsorption the liver controls the production of glucose as well as the availability to the tissues of insulin in accordance with peripheral needs. As originally shown by Madison, th!s control is mediated by the liver through variations in its release of hepatic-venous glucose. (Metabolism 16: No. 7, July, 586-596, 1967)
I
N DOG and in man the fasting blood glucose concentration, both arterial and venous, changes rapidly from moment to moment throughout the entire postabsorptive period. v The resulting wave-like fluctuations can be traced to their origin in hepatic-venous blood. Under normal conditions, the peripheral oscillations vary between 1 and 36 mg./lOO ml. (over the standard error incidental to the glucose method). Determinations of arterial, peripheralvenous, and hepatic-venous glucose levels at 30-second intervals indicate that these changes are totally irregular and unpredictable but hover about a relatively unchanging (or slowly changing) mean. In 1952 Bondy catheterized the hepatic veins of 11 human subjects and could find “no cuase for these sudden ‘spikes’ of glucose release [from the liver] . . . rapid unpredictable fluctuations occur in splanchnic balance . . . these variations are so large as to interfere seriously with attempts to estiFrom the Department of Medicine, The Brooklyn-Cumberland Medical Center, an afjiliate of the State University of New York, Downstate Medical Center. This study was supported by a grant from the John A. Hartford Foundation, Inc. Received for publication December 19, 1966. GEORGE E. ANDERSON, M.D.: Clinical Professor of Medicine, State University of New York, Downstate Medical Center; Senior Physician, Brooklyn-Cumberland Medical Center. YUSUF KOLOCLU, M.D.: Clinical Instructor of Medicine, State University of New York, Downstate Medical Center. CONSTANTIN PAPADOPOULOS, M.D.: Clinical Instructor of Surgery, State University of New York, Downstate Medical Center; Vascular Surgeon to the Brooklyn-Cumberlund Medical Center. 586
BLOOD
GLUCOSE
IN INSULIN
RELEASE
587
mate the mean balance over any protracted period of time.“3 Since in that study blood samples were drawn simultaneously from the hepatic vein and peripheral artery with no correction for the circulation time between these sites, peripheral arterial concentrations not infrequently were found to be higher than those at the point of origin in the hepatic veins. By careful timing of the blood circulation between the hepatic vein and a femoral artery of dogs, it was subsequently shown in our laboratory that the larger hepatic venous fluctuations are paralleled grossly by similar oscillations in the femoral artery1 at lower glucose levels. This was established by laparotomizing dogs, cannulating in each an hepatic vein and femoral artery, and timing the circulation of injected fluorescein between the two sites with the aid of a Wood lamp. Blood samples were then drawn at 30-second intervals from both vessels, separating the sampling of hepatic venous blood from that of the femoral artery by the number of seconds found to be the circulation time between these two sites. Glucose was subsequently determined (in duplicate) on each sample. The complete irregularity in configuration, rate, rhythm, and size of the hepatic-venous glucose emanations made possible the identification of similar clusters of fluctuations in the arterial blood at lower levels. A gross parallelism was consistently found to exist between the glucose curves from both sources, the femoral arterial blood, as would be anticipated, demonstrating a few seconds later a lower but parallel level of glucose concentration (Fig. 1). By a similar technic these glucose fluctuations were followed from the arterial to the venous side of the circulation in extremities. The original hepatic-venous fluctuations travel accordingly from their source in the liver and are projected into the larger blood vessels of the body. Fig. 2 illustrates examples of their presence in the peripheral veins of several normal subjects, both human and canine. When 10 or more sequential glucose determinations are made at 6-second intervals from a single peripheral vein and these are plotted in relation to time, it is found that there usually results a two-phase fluctuation, the completed up-plus-down strokes of which take approximately one minute. Since the rise and fall in glucose level in this brief period of time was the dominant finding in what otherwise appeared to be a completely irregular phenomenon, the present investigation was undertaken to study the relationship, if any, during postabsorption between glucose fluctuations appearing in the hepatic veins and the concentration of insulin in the venous effluent of the pancreas. Briefly, within approximately 15 seconds after the appearance of each major elevation of glucose in the hepatic veins, there occurred a sharp release of insulin into the pancreatic veins. It is suggested that during postabsorption these momentary accessions in hepatic glucose output, reflected in the arterial system. serve to trigger the release of insulin at a time when there is little or no absorption of nutrient from the intestinal tract. An effective feedback mechanism might thus be established, limiting the output of glucose by the liver as well as regulating the retention of insulin by that organ4-” and, secondarily, controlling peripheral insulin levels.
588
ANDERSON,KOLOCLUANDPAPADOPOULOS
: ,r i:
FEMORAL ARTEllA
Fig. l.-Gross parallelism between fasting hepatic-venous and femoral glucose fluctuations in a normal dog. (To accentuate the parallelism glucagon ministered by splenic artery at the point of time indicated.)
arterial was ad-
METHODS Glucose determinations were made by the Autoanalyzer* (modified Hoffman’*’ method). Except for steady monitoring by the Autoanalyzer, all determinations were made on plasma. Analyses were made of errors inherent in the method of glucose determination. In samples numbering 40, the Standard Deviation and the 95 per cent Confidence Limits established at three levels were respectively: 85 mg./lOO ml., 1.67 and 84.5 to 85.5; 200 mg./lOO ml., 4.17 and 198.7 to 201.3; 300 mg./lOO ml., 3.65 and 298.8 to 301.2. Fourteen hours after the last ingestion of food of a liberal mixed diet, an hepatic vein in each of 6 large full-grown normal mongrel dogs was cannulated under pentobarbital anaesthesia and closed oxygen insufflation for respiration. The transthoracic route was used. All dogs were heparinized as well as heavily ergotaminized (dihydroergotamine 0.5 mg/K-I.V.) to blot out adventitious adrenal medullary influence on blood glucose levels. A relatively large pancreatic vein was isolated and also cannulated. At 15-second intervals blood was withdrawn from the hepatic vein and at 15- to 30-second intervals, from the pancreatic vein. The total period of blood sampling in all 6 dogs ranged from 6 to 9 minutes. The pancreatic-venous specimens in one of these dogs ( 18 within a period of 6 minutes) were subjected to assay for their concentration of insulin. Dr. Sheldon J. Bleiche? of the Jewish Hospital of Brooklyn and Dr. Harry N. Antoniades” of the Protein Foundation Laboratories of Boston and the School of Public Health of Harvard University very kindly ‘Technicon
Company,
Chauncey,
New York.
BLOOD
GLUCOSE
IN INSULIN
589
RELEASE
Fig. 2.-Examples of postabsorptive venous glucose fluctuations on 30-second samnling: (a) in normal human subjects, (b) in normal zontal line in each graph represents mean blood glucose. Note: in the lower the mean blood glucose level, the greater the tendency to of oscillations.
in man and dog dogs. The horinormal subjects, wider amplitude
agreed to assay for insulin independently and blindly in their respective laboratories aliquots of all of the pancreatic venous specimens. These assays were carried out by the and by the coated charcoal method of e-antibody method of Morgan and Lazarow” Bleicher and his group? The readings from both sources were correlated with our own simultaneous findings of hepatic-venous glucose concentration during this brief period. Dr. Antoniades assayed for their insulin concentration not only the pancreatic-venous specimens, but, in addition, all of the hepatic-venous samples from the dog, specimens A, B, C, D, etc., in Fig. 3 and Table 1. By the use of a fluorescein-Wood-lamp technic, in which fluorescein was injected into one vessel and its arrival at and exit from another vessel was demonstrated by Wood lamp, the circulation time between the hepatic vein and the arterial supply to the pancreas was measured. In a similar manner, the circulation time between the hepatic vein and the venous effluent of the pancreas was determined. &WLTS
It will be noted that, although the absolute values for insulin concentration in the two laboratories differ (Fig. 3), the curue.s of increasing or decreasing insulin concentration were found grossly to parallel each other and to show
590
ANDERSON,
KOLOGLU
AND PAPADOPOULOS
among the specimens significant variation from moment to moment. This variation is far beyond that which might be anticipated to occur as intrinsic standard error peculiar to the method of assay. The rapid changes in insulin concentration appear to have occurred in relation to similarly sharp variation in hepatic-venous glucose concentration preceding each insulin reading. The insulin changes seem to ensue puri passe with the rises and falls in glucose concentration, there being a lag of 15 seconds. The insulin values in pancreaticblood increase within approximately 15 seconds after each major rise in hepaticvenous glucose takes place. This is documented in Fig. 3 and Table 1, in which a preliminary run of five 1Ssecond hepatic-venous glucose readings is shown to precede the taking of concurrent samples from the pancreatic vein. The hepatic-venous glucose values are labelled in sequence: A, B, C? etc., the pancreatic-venous insulin values, from 1 to 18. The 15-second time scale of Fig. 3 serves to indicate the time lag between the respective individual spikes and falls in hepatic-venous glucose and the corresponding rises and falls in concentration of pancreatic-venous insulin. The correlation is indicated by E, F, I, K, M, etc. The rises and falls in hepatic-venous glucose 1 1 1 i ’ 12567 are rapidly reflected in similar crests and troughs of insulin concentration in the pancreatic veins. This repetitive process, occurring every few seconds, revealed itself to be an ordered sequence linking the two factors. One should be mindful of the fact that in a totally irregular mechanism, as is the emanation of the glucose oscillations from the liver,3 there are bound to occur discrepancies in timing relationships. These are obvious in Fig. 3 but are not sufficiently great over the brief period of 6 or 7 minutes of blood sampling to compromise the principles aimed at in these procedures. Similarly, it may be anticipated that the theoretical changes in blood circulation rate through the liver during the few minutes while the sampling was in process would be negligible. Regarding the sharp momentary oscillations, it seems clear that the individual spikes in hepatic-venous glucose are associated with similarly sharp releases of insulin (Fig. 3). In all 6 dogs as the postabsorptive state progressed, the amplitude of interspersed individual oscillations (e.g., M, Y in Fig. 3) increased in spite of a steadily declining mean blood glucose (from M through EE). At a time when the mean hepatic venous glucose dipped into a noticeably sharper decline (from R thru EE) there occurred a progressive but sharp rise in the pancreatic-venous concentration of insulin (Fig. 3) -over 400 per cent rise in one laboratory (from 60 to 250 PU within 4 minutes), 375 per cent in the other laboratory (from 200 to 750 PU ) . Coincidental with this rise in pancreatic-venous insulin there was pari passu a fall in the concentration of insulin in blood after it passed through the liver, i.e., in the hepatic veins (Table 1). Hepatic-venous insulin declined to a low of 4 pU/ml. at a time when pancreatic-venous insulin hovered about 250 ,U/ml. and hepatic-venous glucose had dropped to a nadir of 22 mg./lOO ml. These findings would suggest a second hepatic regulatory mechanism controlling insulin-glucose homeostasis.
BLOOD
GLUCOSE
IN INSULIN
591
RELEASE
;yEPAW
VENOU : ’ .G PUCOSE:
Fig. X-In dog, postabsorptive hepatic-venous glucose oscillations (designated as A,B,C, etc.) correlated with insulin release into the pancreatic veins (specimens 1-18). Correlation is recorded over a total period of 6% minutes (designated, e.g. E, F, M, 1 1 I etc.). 127
592
ANDERSON, Table
Pancreatic-Venous Insulin (pU/ml.)
Hepatic-Venous GlUCOSC ml.)
c- 48 D-134 E-128 F- 66 G- 78 H- 84 I-90
l + + +
J- 94 + K- 80 L- 92 + M-124 +
l-90 2-80 3-100 4100 S-100 6-60 7-60
N- 60 O-60 + P-121
8-70
Q- 62 +
9-80
82
S- 46 T- 72 U- 80 v-52 w- 50 x- 30 Y-118
Simultaneous Concentration of Hepatic-Venous Insulin tau/ml.) 12
A-130 B- 98
R-
AND PAPADOPOULOS
I.-Hepatic-Venous Glucose Concentrations at 15Second Inter&s with Pancreatic-Venous and Hepatic-Venous Insulin Concentrations*
Correlated
(mg./lOO
KOLOGLU
+ + *
IO-80 11-100 12-100
* +
13-140 14120 15-170
z-64 AA- 48 BB- 22
* + e
CCDDEEFF-
56 58 30 32
+
GGHHII-
44 52 56
X-120 17-250 18-250
20 20 15 12 50 20 12 12 50 12 12 10 6 12 10 25 20 15 10 50 10 10 10 4 4 4 4 4 4 4 4 12 10 40
JJ- 22 KK-130 MM- 88 *There is a lag of approximately 15 seconds between the glucose reading and that of pancreatic-venous insulin, e.g., E-128 (mg.JlOO ml.) + l-90 (aU/ml.; F-66 (mg./lOO ml.) *
Z-80 ( jJJ/ml.)
, etc. DISCUSSION
It would appear that increases in insulin concentration in the pancreatic veins are preceded by pulsatile rises in hepatic-venous glucose during fasting, ( 1) since the relationship between the two factors is so consistent, and (2) since the circulation time between the hepatic vein and the pancreas approxi-
BL~~DGLUCOSEININSULINRELEASE
593
mately accounts for the time-lag. The circulation time between the hepatic veins and the arterial supply to the pancreas was found to range from 4 to 6 seconds; that between the hepatic veins and pancreatic veins, from 6 to 8 seconds. Accordingly, the pancreatic tissue was usually exposed for approximately 2 to 4 seconds to the specific segment of blood (with its brief rise in glucose) as it traversed the capillary bed of that organ. The release of insulin from the pancreas in response to glucose is therefore presumably immediate upon exposure, rather than time-consuming or after a prolonged latent period. Two opposite modalities of insulin control are presented: the first, pulses of increased hepatic-venous glucose, seem to initiate the release of insulin into the pancreatic veins; the second, an increasing insulin concentration in the pancreatic veins, seems to be associated not only with precipitous reduction in the overall output of glucose into the hepatic veins but also with inhibition by the liver of the release of insulin into the periphery. In all probability, both processes represent a feedback regulatory mechanism whereby during postabsorption the production of insulin as well as its availability to the tissues is controlled by the liver through variations in its release of insulin and glucose into the hepatic veins. These findings support those of Madison: “Insulin administered in a manner which minimized the counter-regulatory responses to a falling blood glucose concentration resulted in a prompt decline in hepatic glucose output. . . . ” 4 In our experiments “the counter-regulatory” factor was “minimized” by ergotaminization. The coincidental marked falls in hepatic-venous glucose, and the sharp rises in pancreatic-venous insulin probably indicate deprivation in the animal of adrenal medullary influence as this is effected by ergotaminization “minimizing the counter-regulatory responses.” In lieu of insulin’s being “administered” by portal vein, the concentration of the hormone in this venous system spontaneously increased approximately 400 per cent within a period of four minutes. This phase of insulin increase in all probability lasted samples was only 1 or 2 minutes after the last of the 18 pancreatic-venous withdrawn for insulin assay, since within 1% minutes after this last sampling, hepatic-venous insulin had again climbed (Table 1) from a concentration of 4 /lU to 40 pU/ml. At the same time hepatic-venous glucose had risen within 2X minutes from its nadir of 22 mg./lOO ml. (BB in Table 1) to 130 mg./lOO ml. (LL). S imultaneously there was an increase in mean hepaticvenous glucose value. Apparently, the sharp momentary oscillations in hepaticvenous glucose tend to cluster about a broad or sinusoidal curve of changing mean glucose levels over several minutes (Fig. 3). The normal existence of rapid changes in blood glucose in dog and in man was originally reported by our laboratory in 1956.l Subsequently these brief rises and falls in glucose levels were found to be present without exception in the fasting peripheral venous blood of 250 human subjects when these individuals were tested at 30-second intervals in an effort to obtain more accurate fasting glucose levels for insulin sensitivity tests. The amplitude of the glucose excursions tends to increase as the postabsorp-
594
ANDZRSON,
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AND PAPADOPOULOS
Fig. 4.- In a dog, simultaneous glucose readings from the same vein: (a) by Autoanalyzer, (b) by 30-second intermittent aspiration through an l&gauge needle. The former is the arithmetical mean of the multiple readings. The recorded curve of continuous monitoring by Autoanalyzer (a) was traced on tracing paper from right to left (the direction of the pen-writer), reversed and superimposed from left to right on the graph of plotted 30-second samplings.
tive period progresses and the mean blood glucose level declines (Fig. 2). The oscillations, however, immediately disappear in dogs when the liver is clamped off; 1 instead there occurs a slow, even, steady decline in blood glucose until the animal’s decease. This fact seems to define their origin to the liver. Widespread single observations of blood glucose, i.e., specimens aspirated at 20-, lo- or even 5-minute intervals, are not sufficiently sensitive to pick up the minute critical changes occurring in such a dynamically rapid-moving process as is blood glucose regulation. The process is far better represented by a composite of many of these pictures in rapid succession yielding thereby a concept of activity in slow motion. [The entire action in the present demonstration (Fig. 3) took only 9W minutes.] Determinations at 15- or 30-second intervals bring out fine points of sequence commonly missed by more static conventional methods of investigation. This principle would seem to hold also in respect to the frequency with which determinations of insulin concentration are made. Rapid momentary glucose oscillations are not visualized when peripheral venous blood is continuously monitored by the Autoanalyzer.8,11 However, when an l&gauge needle is inserted into the same vein as the indwelling cannula of the analyzer (about one-half inch away from its opening), simultaneous sampling through both channels is possible; the Autoanalyzer registers a continuous, smooth, uninterrupted, nonfluctuant reading (“a” in Fig. 4), whereas simultaneous staccato 30-second withdrawals show rises and falls typical of those found in all dogs and human subjects when an intermittent aspiration technic is used (‘b”). It may be that blood which has traveled the length of the analyzer’s cannula and tubing at the slow rate of 0.33 mI./min. (approximately 20 ml./hr.) has become homogenized in this tubing before it
BLOOD
GLUCOSE
IN INSULIN
595
RELEASE
reaches its entrance into the Autoanalyzer. The reading from the latter was found to represent the arithmetical mean of all the simultaneous 30-second determinations. Proof of this is suggested by the observation that the level established by the Autoanalyzer is identical with that in blood which has slowly and simultaneously been aspirated by needle and syringe from the same vein over a Sminute period. Lambert and Hoet I2 recently noted (in their studies on diurnal insulin levels in peripheral blood) that not infrequently some of the highest insulin values are found at 3:00 or 4:00 A.M., many hours after the last ingestion of food. Our findings suggest a partial answer to this enigma. Postabsorptively, the release of insulin secondary to brief episodes of spontaneous glycogenolysis rather than (as is more usually observed) to the ingestion of food could account for this phenomenon. Spurts of release of glucose appearing in the arteries would be anticipated to increase in tempo and intensity as the postabsorptive period progresses, since the magnitude of the hepatic-venous glucose oscillations increases as time elapses. These would be expected to cause corresponding releases of insulin into the pancreatic effluent. These studies inevitably lead to a pertinent question: What originally causes the sharp releases of hepatic-venous glucose? Preliminary studies in this laboratory have suggested that measurably increased “glucagon-like” activity (GLA) in pancreatic-venous blood precedes the individual spurts of hepatic-venous glucose output. This fact suggests that the rises in hepaticvenous glucose are preceded by, and may even be produced by, similarly timed releases of GLA or of glucagon from the pancreas or small-bowel wall. This triggering of glucose output by the liver would constitute at least one basic mechanism whereby glucagon or related substances might indirectly “stimulate” 13*14the release of insulin or enhance its activity. ACKNOWLEDGMENT The authors are most grateful to Dr. Harry N. Antoniades of the Protein Foundation Laboratory, Boston, Mass., and Dr. Sheldon J. Bleicher of the Jewish Hospital, Brooklyn, N. Y., who kindly carried out the necessary insulin assay procedures. We acknowledge with thanks the technical advice of Dr. Martin G. Goldner and our associates, Dr. Eugenie The M. Fribourg, Stanley S. Bergen, Jr., Robert W. Hillman, and Seymour Werthamer. technical services of James H. Wilds, MS., Agnes C. Dann, R.N., and Fernando Figoralo, D.V.M., is also appreciated.
REFERENCES 1.
2.
3.
Anderson, G. E., Hillman, R. W., Van Elk, I. F. A., and Perfetto, A. J.: Postabsorptive undulations and oscillations in blood glucose. Amer. J. Clin. Nutr. 4:673, 1956. Hansen, K. M.: Oscillations in the blood sugar of fasting normal persons. Acta Med. Stand. Suppl. 4:27, 1923. Bondy, P. K.: Spontaneous fluctua-
4.
5.
tions in glucose content of hepatic venous blood in resting normal human beings. J. Clin. Invest. 31:231, 1952. Madison, L. L., Mebane, D., Lecocq, F., and Combes, B.: Physiological significance of the secretion of endogenous insulin into the portal circulation. Diabetes 12:8, 1963. Madison, L. L., Combes, B., Adams,
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R.: Insulin’s control of the role of the liver in the disposition of a glucose load in diabetic and non-diabetic dogs. J. Clin. Invest. 39:1009, 1960. Combes, B., Adams, R. H., Strickland, W., and Madison, L. L.: The physiological significance of the secretion of endogenous insulin into the portal circulation. IV Hepatic uptake of glucose during glucose infusion in non-diabetic dogs. J. Clin. Invest. 40:1706, 1961. Hoffman, W. S.: A rapid photoelectric method for determination of glucose in blood and urine. J. Biol. Chem., 120: 51, 1937. Weller, C., Lindner, M., Macaulay, A., Ferrari, A., and Kessler, G.: Continuous in viva determination of blood glucose in human subjects. Ann. N. Y. Acad. SC. 87:658, 1960. Herbert, V., Lau, K. S., Gottlieb, C. W., and Bleicher, S. J.: Coated charcoal
10.
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14.
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AND PAPADOPOULOS
immunoassay of insulin. J. Clin. Endocrin. 25: 1375, 1965. Morgan, C. R., and Lazarow, A.: Immunoassay of insulin: two antibody system. Diabetes 12: 115, 1963. Burns, T. W., Bregant, R., Van Peenan, H. J., and Hood, T. E.: Observations on blood glucose concentration of human subjects during continuous sampling. Diabetes 14: 186, 1965. Lambert, A. E., and Hoet, J. J.: Diurnal pattern of plasma insulin concentration in the human. Diabetologia 2: 69, 1966. Samols, E., Marri, G., Marks, V.: Promotion of insulin secretion by glucagon. Lancet 2:415, 1965. Samols, E.: Effects of Glucagon: Insulin Release in Vivo. Presented at the Research Symposium on Glucagon, American Diabetes Ass., San Francisco, Calif., October 8, 1966 (unpublished).