Chronic
Hyperinsulinemia
Decreases
Owen P. McGuinness,
Insulin
Sharon R. Myers,
Action
But Not Insulin
Sensitivity
Doss Neal, and Alan D. Cherrington
Hyperinsulinemia and insulin resistance are commonly seen in obese and non-insulin-dependent diabetes mellitus (NIDDM) patients, suggesting a causal link exists between hyperinsulinemia and insulin resistance. In a previous study, we demonstrated that chronic (28 days) intraportal hyperinsulinemia (50% increase in basal insulin levels) resulted in a decrease in insulin action as assessed by a one-step euglycemic hyperinsulinemic clamp. Since only one dose of insulin was used during the clamp, it was not possible to determine if the decrease in insulin action was due to a change in insulin sensitivity and/or maximal insulin responsiveness. In the present study, insulin resistance was induced as before, but insulin action was assessed in overnight fasted conscious dogs using a four-step euglycemic hyperinsulinemic clamp (1,2, 10, and 15 mU/kg/min). Insulin responsiveness was assessed before the induction of chronic hyperinsulinemia (day 0). and after 28 days of hyperinsulinemia (day 28). Transhepatic glucose balance and whole-body glucose utilization were measured to allow assessment of both the hepatic and peripheral effects of insulin. Chronic hyperinsulinemia increased basal insulin levels from 13 + 2 to 21 r 4 pU/mL. After 4 weeks of chronic hyperinsulinemia, maximal insulin-stimulated glucose utilization was decreased 23% + 4% (P < .05) and insulin sensitivity (ED,) was not significantly altered. During the four-step clamp, the liver was a major site of glucose utilization. The liver was responsible for 13% of the total glucose disposal rate on day0 (2.9 mg/kg/min) at the highest insulin infusion rate (15 mU/kg/min:2.000 @/mL). On day28, insulin suppression of endogenous glucose production and stimulation of hepatic glucose uptake was similar (3.3 mg/kg/min) to that seen on day 0, suggesting that the decrease in maximal insulin responsiveness did not involve the liver. In conclusion, insulin resistance produced by a mild hyperinsulinemia is due to a suppression of maximal extrahepatic insulin-stimulated glucose uptake, suggesting -- that a postreceptor defect is present in extrahepatic tissues. 0 1990 by W. 6. Saunders Company.
I
NSULIN RESISTANCE is commonly seen in situations in which hyperinsulinemia is also present (ie, non-insulindependent diabetes mellitus [NIDDM], obesity, and pregnancy).’ Previous work by ourselves and others have demonstrated that induction of mild hyperinsulinemia can lead to an insulin resistant state.2-4 However, it remains unclear as to the site of the resistance (ie, liver, muscle, fat) or the mechanism by which resistance is brought about (receptor and/or postreceptor defect). The type of resistance (receptor and/or postreceptor defect) that develops from chronic hyperinsulinemia is variable and to some extent depends on the characteristics of the 2-4 Exposure to pharmacologic insulin levhyperinsulinemia. els for brief (-4 hours) or more prolonged periods (14 days) decreased insulin receptor number and caused a postreceptor defect in rat adipocytes.5’6 Mild hyperinsulinemia for a brief duration (20 to 40 hours) brought about a postreceptor defect without an accompanying change in receptor number in man.3,4 A much longer duration of exposure may have been required to result in receptor down-regulation. Since in our previous study the duration of hyperinsulinemia was considerably longer (28 days) than in the human studies (20 to 40 hours) and the resistance was somewhat greater (34% v 20%) the cause of the resistance may differ from that seen in man. From our previous work’ we were unable to localize the site or the mechanism of the resistance, because only a single-step euglycemic hyperinsulinemic clamp was used to assess insulin action. The goal of the present study was to determine if the decrease in insulin action seen after 28 days of hyperinsulinemia was due to a decrease in insulin sensitivity and/or a decrease in maximal insulin responsiveness of the liver and/or the periphery. Using a four-step euglycemic hyperinsulinemic clamp, the doseresponse relationship between insulin concentration and insulin-stimulated glucose uptake by the liver and by the Metabolism, Vol39,
No 9 (September), 1990: pp 931-937
periphery before and after chronic exposure to hyperinsulinemia for 28 days was assessed. METHODS
Animal Preparation Experiments were performed in five conscious mongrel dogs (18 to 26 kg), receiving a diet consisting of Kal-Kan meat (Vernon, CA)
and Wayne Dog Chow (Purina Mills, St Louis, MO) once daily. The composition of the diet was 52% carbohydrate, 31% protein, 11% fat, and 6% fiber based on dry weight. Two weeks before the first experiment, a laparotomy was performed under general anesthesia (sodium pentobarbital; 30 mg/kg). Silastic catheters were placed into a mesenteric and a splenic vein for the chronic infusion of insulin. Blood sampling catheters were inserted into the portal vein and the left hepatic vein. In addition, a catheter was inserted into the femoral artery following a cut-down in the left inguinal area. The catheters were then filled with saline containing heparin (200 IU/mL). The infusion catheters were exteriorized, and brought through a subcutaneous tunnel to the back between the clavicles, where they were placed in a subcutaneous pocket. The portal and hepatic blood sampling catheters were exteriorized through the incision used to perform the laparotomy and placed in a subcutaneous pocket. The arterial blood sampling catheter was placed under the skin in the inguinal region. Catheters were removed from their respective pockets under local anesthesia on the day of the first study. After the completion of the first study (day 0) the sampling catheters were placed under the skin and the wound was closed. The dogs received ampicillin (1 g/d for 2 days) to minimize the possibility of From the Department of Molecuiar Physiology and Biophysics, Vanderbilt University, Nashville, TN. Supported by a grant from the Juvenile Diabetes Association and by the Hormone and Animal core labs of the Diabetes Research and Training Center (DK20593-12). Address reprint requests to Owen P. MeGuinness. PhD. 702 Light Hall. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232. m 1990 by W.B. Saunders Company. OOZO-0495/90/3909-0009$03.00/0 931
932
infection. The infusion catheters were left exteriorized for the chronic infusion of insulin. All animals studied had (1) a good appetite (consuming all of the daily ration); (2) normal stools; (3) a hematocrit above 38%; and (4) a leukocyte count below 18,000 rL on days 0 and 28.
Experimental Design On day 0, after an overnight fast, a four-step, 400-minute euglycemic hyperinsulinemic clamp was performed (see below) to assess insulin responsiveness. At the end of the experiment the dog was placed in a jacket (Alice King Chatham, Los Angeles, CA), containing a pocket into which was placed a portable infusion pump. On day 1 a chronic intraportal infusion of insulin (425 NI_J/kg/min) was begun using a portable Millhill infuser (Harvard Apparatus, South Natick, MA; 60 rL/h). Porcine insulin, the structure of which is identical to canine insulin,’ was dissolved in saline containing heparin (30 U/mL) and dog plasma (3%). The solution was then filtered (0.2 pm) under sterile conditions before infusion. A fresh solution was prepared on each of the 28 infusion days to prevent aggregation of the insulin.” On day 28, insulin responsiveness was determined as on day 0.
Euglycemic Hyperinsulinemic Protocol On Days 0 and 28 Angiocaths (Deseret Med, Sandy, Utah; 1g-gauge) were inserted percutaneously into both cephalic veins and a saphenous vein. A primed (50 &i) constant infusion of 3-‘H glucose (0.4 rCi/min) and an infusion of indocyanine green (0.1 mg/m’/min) were begun through the right cephalic vein and continued throughout the entire experiment. The experiment consisted of three periods, an equilibration period (- 120 to -40 minutes), a control period (-40 to 0 minutes), and an experimental period (0 to 400 minutes). When studies were performed on day 28, the chronic intraportal insulin infusion was continued until the end of the control period (0 minutes) at which time it was discontinued. After the control period in both studies, a four-step euglycemic hyperinsulinemic clamp was begun. Insulin was infused through the left cephalic vein in a stepwise manner (1, 2, IO, and 15 mu/kg/ min), allowing 100 minutes for each step. A variable glucose infusion was begun through the saphenous vein; the arterial glucose concentration was measured every 5 minutes and the glucose infusion rate was adjusted to maintain euglycemia. On day 28 the glucose concentration was clamped at the level seen on day 0. Arterial blood samples were taken at -40, - 20, and 0 minutes, as well as at 30,60, 70, 80, 90, and 100 minutes during each loo-minute step in the experimental period. Portal and hepatic vein blood samples were taken at -40, -20, and 0 minutes during the control period, and at 80.90, and 100 minutes during each lOO-minute step.
Tracer Methods and Calculations The rates of total glucose production (R,) and utilization (Rd) were calculated according to the method of Wall et al’ as simplified by DeBodo et al.‘” Endogenous glucose production was calculated as the difference between R, and the exogenous glucose infusion rate. The tracer method has been shown to systematically underestimate glucose flux during a euglycemic clamp, resulting in negative endogenous glucose production.’ The cause of the error has not been elucidated; however, a combination of modelling errors and tracer contamination have been postmated to explain the error.i’-‘3 In the present study we did not obtain negative numbers; however, this does not indicate that the calculated endogenous glucose production during the clamp is correct. During the last 30 minutes of each step the coefficient of variation of the specific activity was less than 5%. Net hepatic glucose balance (NHGB) was calculated using the formula (H - [0.28A + 0.72P]) x0.73 xHBF, where H, A, and P
McGUlNNESS
ET AL
are the plasma glucose concentrations in the hepatic vein, femoral artery, and portal vein, respectively; 0.28 and 0.72 represent the percent contribution of the hepatic artery and portal vein, respectively, to total hepatic blood flow”‘; and 0.73 is an experimentally derived factor which converts plasma glucose values to blood glucose values in the dog”; and HBF is total hepatic blood flow. Extrahepatic glucose utilization was calculated as the difference between the exogenous glucose infusion rate and net hepatic glucose uptake. We did not have hepatic balance data for two of the animals because the hepatic vein sampling catheter did not work on day 28.
Processing of Blood Samples Blood samples were drawn into heparinized syringes and transferred to chilled tubes containing Na,EDTA (15 mg). Plasma glucose was assayed immediately using a Beckman glucose analyzer. Plasma treated with 500 KIU of Trasylol (FBA Pharmaceuticals, New York, NY) was assayed for immunoreactive glucagon using 30-k antiserum of Unger16; (coefficient of variation [CV], 8%). Immunoreactive insulin” was assayed using a sephadex-bound antibody technique (Pharmacia Diagnostics, Piscataway, NJ; CV. 11%). Polyethylene glycol (PEG) pretreatment of plasma was not required since dogs do not develop antibodies to insulin following insulin treatment.‘” Plasma cortisol was assayed with Clinical Assays Gamma Cost radioimmunoassay kit (Travenil-Genetech Diagnostics, Cambridge, MA; CV, 6%). Indocyanine green dye was measured spectrophotometrically (805 nm) to estimate total hepatic blood flow.19
Porcine insulin was purchased from Eli Lilly and Co (Indianapolis, IN). Glucagon 30-k antiserum was obtained from the University of Texas Southwestern Medical School (Dallas, TX). Purified glucagon and “51-glucagon were obtained from Novo Research Institute (Copenhagen, Denmark). Cortisol assay kits were obtained from Upjohn Diagnostics (Kalamazoo, MN) 3-JH-glucose was purchased from New England Nuclear Research Products (Wilmington, DE).
Statistics Statistical analysis was performed paired Student’s t test.
using one-way
ANOVA
and
RESULTS
During tion on day
the control
period,
28 was significantly
the average lower
than
glucose
concentra-
on day 0 (94 + 7
and 115 f 3 mg/dL; P < .OS; Fig 1). Glucose turnover on day 28 and 0 were similar (3.07 + 0.35 and 3.37 t 0.62 mg/kg/min, respectively). The exogenous intraportal insulin infusion produced a mild hyperinsulinemia on day 28,21 f 4 wU/mL, as compared with 13 + 2 rU/mL on day 0, (P < .05; Table 1). The average glucagon concentrations on days 28 and 0 were not significantly different (146 i 34 and 98 + 16 pg/mL, respectively; Table 2). The average cortisol concentrations (Fg/DL) on days 28 and 0 were 3.4 f 0.4 and 2.8 f 0.2, respectively. The average body weight was similar on days 28 and 0 (21.6 2 1.4 and 21.1 k 1.5 kg, respectively). The average glucose concentrations in both groups are shown in Fig 1. The exogenous glucose infusion rates on days 0 and 28 during the last 30 minutes of each step of the euglycemic hyperinsulinemic clamp were 6.0 of-0.8, 12.1 k 1.7, 19.5 f 2.0,and20.3 + 1.9ondayO,and6.8 _tO.8,9.6 _t
HYPERINSULINEMIA
AND INSULIN SENSlTIVlTY
933
Table 2. Plasma Glucagon Concentrations
During a Four-Step
Euglycemic Clamp Before (Day 0) and After a 28-Day Hyperinaulinemia
”
01
I
!X
INSULININFUSIONRATE
-40
d
Day 0
Day 28
Basal
98 + 16
146 * 34
1
69i
2
58 * 8
86 * 12
10
54*
14
58 f 8
15
56 ? 14
57 & 9
11
92 * 15
NOTE. Data expressed as mean f SEM (n = 5).
IdO Time
PlasmaGlucsgon(pg/mL)
ExogenousInsulin WJ/kg/min)
h\\\\V
(mu/kg/mh)
,,
Exposure to
(Dey 28)
360
260
40b
(minutes)
Arterial glucose concentration in dogs before (day 0) Fig 1. and after a 28-day exposure to hyperinaulinemia (426 N/kg/min: day 28) during a four-step euglycemic hyperinaulinemic clamp are presented. Data are expressed as means + SEM.
1.1, 16.5 + 1.8, and 16.4 + 1.2 mg/kg/min onday 28 for the insulin infusion rates of 1, 2, 10, and 15 mU/kg/min, respectively (Fig 2). The average insulin levels for those insulin infusion rates on days 0 and 28 are presented in Table 1. The arterial glucagon concentration decreased progressively throughout the study on days 0 and 28 (Table 2). The average R, during the last 30 minutes of each step was 7.4 T 0.9, 14.0 * 1.620.9 i 2.0, and 23.0 + 2.0 mg/kg/min on day 0, and 8.1 + 1.3, 11.0 r 1.3, 17.3 f 1.8, and 17.8 * 1.3 mg/kg/min on day 28. The relationship between the insulin concentration and the average rate of glucose utilization is presented in Fig 3. Following chronic insulin administration, the maximal rate of glucose utilization was decreased significantly (23% * 4%; P < .05). However, when the data are plotted as a percent of the maximal rate of glucose utilization, no significant change in the ED,, was detected. Net hepatic glucose balance (NHGB) was sensitive to the Table 1. Plasma Insulin Concentrations
actions of insulin (Table 3). NHGB was 2.38 + 0.66 and 2.29 + 0.71 mg/kg/min during the control period on days 0 and 28 and the liver switched to net consumption at very high insulin levels (- 2.89 + 0.96 and -3.30 + 0.66 mg/kg/min on days 0 and 28). The relationship between insulin concentration and suppression of hepatic glucose production, or stimulation of hepatic glucose uptake, was not altered by chronic insulin infusion. To further localize the site of the whole-body insulin resistance, extrahepatic glucose utilization was calculated. The insulin resistance was localized to the periphery (utilization decreased 23% + 3%; Fig 4). In addition, the calculated ED,, was unchanged by the exogenous insulin infusion (Fig 4). DISCUSSION
These data suggest that prolonged intraportal hyperinsulinemia (425 lU/kg/min) leads to insulin resistance charac25-
20 -ti
o-o Day 0 ~0~28 m---e mean f sem
IX
INSULIN INFUSION RATE (mu/kg/min)
During a Four-Step
Euglycemic Clamp Before (Day 0) and After (Day 28) Exposure to Hyperinaulinemia EXOgeIlOU~ Insulin ImU/ka/minl Basal
for 28 Days
Day 0 uJlrnL)
Day 26 ul/mL)
13 & 2
21 *4*
1
58 * 3
2
120 f 18
72 f 14 154 f 26
10
1,134
* 102
1.072
* 47
15
2,044
+ 293
1,996
+ 191
NOTE. lP -c -05 as compared with day 0. Data expressed as mean f SEM (n = 5).
0
100
200 Xme (minutia)
300
400
Exogenous glucose infusion rates in dogs before (day 0) Fig 2. and after a 28-day exposure to hyperinaulinemia (426 AU/kg/min: day 28) during a four-step euglyoemic hyperinaulinemic clamp are presented. Data are expressed aa means f SEM.
McGUlNNESS
934
A Day 0
O--O
Day 26
l
.’ “*
B .f..
G-2 N 3
iE - .l-x ‘Z $
tiz
Day26
l
75. DayQ
IOO50-
0-0
n/
::
/:
was unchanged. However, suppression of hepatic glucose production and pancreatic glucagon secretion and stimulation of hepatic glucose uptake by insulin were not altered. To assess the magnitude of the decrease of insulin action in extrahepatic tissues, the rate of extrahepatic glucose utilization was calculated. Chronic hyperinsulinemia decreased (23%) maxima1 insulin-stimulated extrahepatic glucose utilization without altering insulin sensitivity. Following chronic hyperinsulinemia, maximal extrahepatic glucose disposal was decreased (23%) and insulin sensitivity remained unaltered. These results are similar to those described by Rizza et al and Marangou et all.“ in man. Hyperinsulinemia for 40 and 20 hours, respectively, decreased maximal insulin stimulated glucose utilization (12% and 20%) without altering insulin binding in freshly isolated adipocytes. However, they did not differentiate between the contribution the liver and the periphery play in the development of the resistance. Others have found both decreases in insulin sensitivity and responsiveness following hyperinsulinemia,’ but they have relied on changes in insulin action in adipocytes to reflect changes in whole-body action. Since
:
8s 2
:
. “0
ET AL
25-
Z
/A
0, 10
100
INSULIN
25
A Day0
t
O-----O
1000
CONCENTRATION W/ml)
Fig 3. (A) Average tracer-determined glucose utilixstion rates during the last 30 minutes of each step of a four-step euglycsmic hyperinsulinemic clamp bafore (day 0) and after e 28-day exposure to hyperinaulinemie (425 @J/kg/min: day 28) are presented. Asterisks indicste that a significant diierence exists on days 0 compared with day 28. Date are expressad es means f SEM. (8) The tracer-determined glucose utilization retea are expressed ea a percent of the maximal glucose utilization rate during the lest 30 minutes of each step of a four-step euglycemic hyperinaulinemic clamp before (day 0) and after a 28-dey exposure to hyperinaulinsmia (426 /.dl/kg/min: day28). Data are expressed as means + SEM (n = 51.
-lb
160
lob0
100
1000
B Day0
o---o
Day 26
*
*
terized by a decrease in maximal insulin responsiveness, which appears to be localized in muscle and/or fat rather than in the liver. Following exposure to hyperinsulinemia for 28 days, whole-body maximal insulin responsiveness was decreased by 23% f 4% and whole body insulin sensitivity Table 3. Net Hepatic Glucose 8alance During a Four-Step Euglycemic Clamp Before (Day 0) and After a 28-Day Exposure to Hyperinaulinemie (Dey 28) EXogelWJS Insulin (mU/ks/min! Basal 1
Day0 fma/ke/min) 2.38 f 0.66
Day 28 hne/ke/min) 2.29 zt 0.71
-0.10
* 0.29
- 1.04 * 0.55
2
-0.55
f 0.26
-0.88
10
-1.83
+ 0.13
-2.26
f 0.53 * 0.70
15
-2.89
+ 0.96
-3.30
f 0.66
NOTE. Negative balance indicates net uptake of glucose. Data srs expressed ss mean + 6EM h = 3).
01 10
INSULIN CONCENTRATION W/m0 Fig 4. (A) Average extrahepatic glucose utilization retea are presented during the lest 30 minutes of each step of a four-step euglycemic hyperinaulinemic clemp before iday 01 end sfter a 28-dey exposure to hyperinaulinemis 1426 AU/kg/min; dsy 28). Dete are expressed es mesna f 8EM (n = 31. (81 Extrahepstic glucoas utilixstlon rate is expresssd as a percent of the meximal glucose utilixetion rate during the lsat 30 minutes of esch step of a four-•teg euglyoemic hyperinaulinemic clamp before ldey 0) and after a 28 day exposure to hyperinaulinemia (426 fiU/kg/min; day 28). Dete ere expressed as mssna + 8EM In = 3).
HYPERINSULINEMIA
AND INSULIN SENSlTlVll-Y
adipocytes do not represent a major site of glucose removal and their response to hyperinsulinemia may not always parallel that of muscle,*’ extrapolating these results to data obtained in vivo is tenuous. The present study clarifies the major role extrahepatic tissues play in disposing of glucose during a euglycemic hyperinsulinemic clamp (disposing 96% of the exogenous glucose infusion during the first two insulin infusion rates) and in the decreased insulin action seen following chronic hyperinsulinemia. In our previous work,* chronic intraportal hyperinsulinemia decreased insulin action by 35% k 8%, which is a greater decrease than that observed in the present study (21% f 4%) at a comparable insulin infusion rate (2 mU/kg/min) during the euglycemic clamp. The magnitude of the chronic hyperinsulinemia was similar in both studies, from 13 to 21 &J/mL in the present study and from 15 to 23 pU/mL in our previous study. There are several possible explanations for this small difference in insulin resistance. First, it may be due to the different basal insulin action on day 0 in the two studies. Glucose utilization was approximately 33% lower on day 0 in the present study than in our previous study during the euglycemic hyperinsulinemic clamp. This in turn is partially explained by the fact that the 2 mU/kg/min step in the present study was 100 minutes in length rather than 180 minutes as in our previous study. Insulin action has been shown to continuously increase over time during a euglycemic hyperinsulinemic clamp.** In our experience during a euglycemic hyperinsulinemic clamp (2 mU/kg/min), insulin action was 8% lower after 100 minutes of hyperinsulinemia than after 180 minutes. The remaining factor, which may account for the somewhat smaller effect of chronic hyperinsulinemia, is dog to dog variation. A saline control group was not included in this study. However, based on our previously reported results, insulinstimulated glucose uptake during a euglycemic hyperinsulinemic clamp (2 mU/kg/min) was not altered after 28 days of saline infusion.2 Therefore, changes in insulin action in the present study cannot be explained by time-dependent decreases in insulin action. Insulin responsiveness of muscle is underestimated by the euglycemic hyperinsulinemic clamp because, although arterial euglycemia was maintained during the clamp, extracellular glucose concentration decreased as the rate of glucose utilization by muscle increased. However, the extracellular glucose concentration influences the rate of glucose uptake. Accurately estimating the extracellular glucose concentration during the clamp is difficult. The arteriovenous glucose concentration difference across the forelimb in man at insulin levels comparable to that seen at the highest insulin infusion rate used in the present study (> 1,000 gU/mL) was approximately 60 mg/dL,23 when the arterial glycemia was clamped at 90 mg/dL. If we assume that the muscle venous glucose concentration reflects the extracellular glucose concentration and we normalize the glucose disposal rate to the arterial glycemia, maximal insulin responsiveness will be increased. The effect on ED,, is difficult to predict, since the impact of glycemia on glucose uptake is dependent on the prevailing insulin and glucose levels. On day 28 in the present study, glucose disposal was 23% lower than on day 0. Consequently,
935
the venous glucose concentration will be higher at comparable arterial glycemia, and therefore the correction in maximal glucose responsiveness will be less. If the data from the present study are corrected, the maximal rate of glucose disposal is approximately 28%, rather than 23%, lower on day 28 than on day 0. The liver was a significant site of glucose utilization at the two highest doses of insulin used. During the euglycemic hyperinsulinemic clamp the liver switched from being a net producer to a net consumer of glucose. As the insulin levels increased to 58 rU/mL on day 0 during the first step of the clamp, hepatic glucose production was completely suppressed (-0.10 c 0.29 mg/kg/min). During the second step of the clamp (insulin concentration = 120 rU/ml) the liver switched to consumption (-0.55 + 0.26 mg/kg/min). In man, an insulin infusion rate (1 mU/kg/min), producing similar insulin levels (101 + 5 pU/mL), suppressed splanchnit glucose production and also converted the splanchnic bed to net consumption (0.42 k 0.09 mg/kg/min).24 In the present study, as insulin levels increased to approximately 1,000 &l/mL, hepatic glucose uptake increased to approximately 2 mg/kg/min, which is considerably larger than has been seen in man at comparable insulin levels both at euglycemia (0.68 + 0.13 mg/kg/min) and at hyperglycemia (1.07 + 0.25 mg/kg/min), even when the difference between splanchnic and hepatic glucose production is taken into account.24 At the highest dose of insulin used (15 mU/kg/min), the liver took up approximately 3.0 mg/kg/min or about 13% of total glucose disposal on day 0 and about 17% on day 28. Therefore, pharmacologic hyperinsulinemia in the presence of euglycemia can convert the liver to a significant consumer of glucose. However, in the presence of physiologic levels of insulin, other factors (ie, hyperglycemia and route of glucose delivery) I*.*5in addition to insulin regulate hepatic glucose uptake. Surprisingly, the uptake of glucose by the liver, as opposed to the periphery, did not completely saturate with an insulin infusion rate of 15 mU/kg/min. Therefore, insulin sensitivity of the peripheral tissues may not be less than that of the liver with regard to glucose uptake. However, it is possible that the rate of stimulation of hepatic glucose uptake by insulin may be slower than its stimulation of peripheral glucose uptake. Therefore, the further increase in hepatic glucose uptake may simply reflect the sluggishness of the process. The experimental design did not allow us to directly assess hepatic insulin sensitivity, because we did not prevent the expected decrease in glucagon during the clamp.26 Recently, it has been shown that the liver is sensitive to subtle decreases in glucagon in the presence of hyperinsulinemia,*’ leading one to overestimate hepatic insulin sensitivity. As the insulin levels were increased to 58 and 72 pU/mL on days 0 and 28, respectively, hepatic glucose production was completely suppressed and, in fact, the hepatic glucose balance was slightly negative on day 28. Based on these results alone, one might conclude that the liver was more sensitive to insulin on day 28. However, previous work indicates that it is the change in glucagon level, and possibly the absolute glucagon level, which determines the hepatic response.*’ The greater
McGUlNNESS
936
decrease in glucagon in concert with the slightly higher insulin levels on day 28 probably explains the apparent increase in suppression of hepatic glucose production on day 28 at the first step of the clamp. On days 0 and 28, plasma glucagon levels during the last two steps of the clamp did not change, allowing us to more clearly assess hepatic insulin action. Insulin stimulation of hepatic glucose uptake was not altered by chronic hyperinsulinemia. These results are similar to those obtained by Rizza et al,’ in which chronic hyperinsulinemia did not alter insulin suppression of hepatic glucose production. However, as was discussed, the use of the tracer method to measure endogenous glucose production during a euglycemic hyperinsulinemic clamp is associated with some undefined problems.28,29 In addition, since glucagon data were not reported, those results are difficult to interpret. The mechanism for the development of the peripheral resistance is unknown. One possibility is that throughout the 28 days the animal experienced hypoglycemic episodes resulting in a change in counterregulatory hormones which, when administered in pharmacologic quantities, are known to alter insulin action.30.3’ Based on our previously reported data* in which a 24-hour profile of glucose and counterregulatory hormones was obtained during the intraportal infusion
ET AL
of insulin, no significant hypoglycemia or change in the counterregulatory hormones was detected. However, we cannot rule out that a subtle increase in counterregulatory hormone production contributed to the insulin resistance produced in this model. The results of the present study and previous studies in man indicate that the majority of the defect seen in a hyperinsulinemic state is at the postreceptor leve1.2’4Whether it involves the glucose transport system or a subsequent step remains unclear. Garvey et a13’ demonstrated that chronic (24-hour) exposure of adipocytes to severe hyperinsulinemia (2,400 $J/mL) decreased both insulin binding and the coupling between insulin binding and insulin-stimulated glucose transport. Since insulin binding was not measured in the present study, we can only speculate that a receptor defect is not present, although this is supported by the lack of a change in insulin sensitivity. In conclusion, these studies indicate that a mild hyperinsulinemia produced by a chronic intraportal infusion of insulin leads to a decrease in insulin action. This decrease in insulin action was due to a decrease in maximal extrahepatic insulin responsiveness, without a concomitant change in peripheral or hepatic insulin sensitivity, suggesting that a postreceptor defect is present.
REFERENCES 1. McGuinness OP, Steiner KE, Abumrad NN, et al: Insulin action in vivo, in Alberti KGMM, Krall LP (eds): Diabetes Annual, vol 3. Amsterdam, The Netherlands, Elsevier Science, 1987, pp 398-432. 2. McGuinness OP, Friedman A, Cherrington AD: intraportal hyperinsulinemia decreases insulin stimulated uptake in the dog. Metabolism (in press)
Chronic glucose
3. Rizza RA, Mandarin0 LJ, Genest J, et al: Production of insulin resistance by hyperinsulinemia in man. Diabetologia 28:70-75, 1985
4. Marangou AG. Weber KM, Baston RC, et al: Metabolic consequences of prolonged hyperinsulinemia in humans, evidence for induction of insulin insensitivity. Diabetes 35: 1383- 1389, 1986 5. Kobayashi M, Olefsky J: Effect of experimental hyperinsulinemia on insulin binding and glucose transport in isolated rat adipcytes. Am J Physiol235:E53-E62, 1978 6. Gavin J, Roth J, Neville D, et al: Insulin dependent regulation of insulin receptor concentration, A rapid demonstration in cell culture. Proc Natl Acad Sci USA 71:84-88, 1974 7. Smith LF: Species variation in the amino insulin. Am J Med 40:662-666, 1966 8. Loughheed WD, Woulfe-Flanagan Insulin aggregation in artifical delivery 1-9, 1980
acid sequence
of
H, Clement JR, et al: systems. Diabetologia 19:
9. Wall JS, Steele R, DeBodo RC, et al: Effect of insulin on utilization of glucose and production of circulating glucose. Am J Physiol 189:43-50, 1957 10. DeBodo RC, Steele R, Altzuler regulation of carbohydrate metabolism: Ret Prog Horm Res 19:445-88, 1963
NR, et al: The hormonal studies with “C-glucose.
11. Finegood DT, Bergman RN, Vranic M: Modelling error and apparent isotope discrimination confound estimation of endogenous glucose production during euglycemic glucose clamps. Diabetes 37:1025-1034, 1988 12. McMahon MM, Schwenk WF, Haymond MW, et al: Underestimation of glucose turnover measured with [6-‘HI- and [6,6-*HZ]But not (6-‘4C] glucose during hyperinsulinemia in humans. Diabetes 38:97- 107, 1989
13. Yki-Jarvinen H, Consoli A, Nurjahan N, et al: Mechanism for the underestimation of isotopically determined glucose disposal. Diabetes 38:744-51, 1989 14. Greenway CV, Stark RD: Hepatic vascular bed. Physiol Rev 51:23-65, 1971 15. Adkins-Marshall BA, Myers SR, Hendrick GK, et al: Importance of the route of glucose delivery to hepatic glucose balance in the conscious dog. J Clin Invest 79:557-565, 1987 16. Agular-Parada E, Eisentraut AM, Unger RH: Pancreatic glucagon secretion in normal and diabetic subjects. Am J Med Sci 257:415-419, 1968 17. Wide L, Porath J: Radioimmunoassay of sephadex-coupled antibodies. Biochem 260,1966
of proteins with the use Biophys Acta 130:257-
18. Vranic M, Kawamori S, Pek N, et al: The essentiality of insulin and the role of glucagon in regulating glucose utilization and production during strenuous exercise in dogs. J Clin Invest 57:245255. 1976 19. Leevy CM, Mendenhall CL, Lesko W, et al: Estimation of hepatic blood flow with indocyanine green. J Clin Invest 41:1169-79, 1962 20. James DE, Jenkins AB, Kraegen EW: Heterogeneity of insulin action in individual muscles in vivo: Euglycemic clamp studies in rats. Am J Physiol248:E567-E574, 1985 21. Wardzala LJ, Hiesham M, Pofcher E, et al: Regulation of glucose utilization in adipose cells and muscle after long term experimental hyperinsulinemia in rats. J Clin Invest 76:460-469, 1985 22. Doberne L, Greenfield MS, Schultz B, et al: Enhanced glucose utilization during prolonged glucose clamp studies. Diabetes 30:829-835, 1981 23. Yki-Jarvinen H, Young AA, Lamkin C, et al: Kinetics of glucose disposal in whole body and across the forearm in man. J Clin Invest 79:1713-1719, 1987 24. Defronzo RA, Ferrannini E, Hendler R, et al: Regulation of splanchnic and peripheral glucose uptake by insulin and hyperglycemia in man. Diabetes 32:35-45, 1983
HYPERINSULINEMIA
AND INSULIN SENSITIVITY
25. Cherrington AD, Stevenson RW, Steiner KE, et al: Insulin, glucagon and glucose as regulators of hepatic glucose uptake and production in vivo. Diabetes Metab Rev 3:307-318, 1987 26. Samols E, Weir GC, Bonner-Weir S: Intraislet insulinglucagon-somatostatin relationships, in Lefebvre P (ed): Glucagon II. Handbook of Experimental Pharmacology, vol 66. Berlin, FRG, Springer Verlag, 1983, pp 133-173 27. Myers SR, Adkins-Marshall B, Williams PE, et al: Subtle changes in glucagon during the euglycemic clamp can markedly alter the apparant sensitivity of the liver to insulin. Diabetologia 31:525a, 1988 (abstr) 28. Bergman RN, Finegood DT, Ader M: Assessment of insulin sensitivity in vivo. Endocrine Rev 6:45-89, 1985 29. Finegood DT, Bergman RN, Vranic M: Modelling error and
937
apparent isotope discrimination confound estimation of endogenous glucose production during euglycemic glucose clamps. Diabetes 37:1025-1034, 1988 30. Scheidegger K, Robbins DC, Daforth E: Effects of chronic beta receptor stimulation on glucose metabolism. Diabetes 33:11441149,1984 3 I. Rizza RA, Mandarin0 LJ, Gerich JE: Cortisol-induced insulin resistance in man: Impaired suppression of glucose production and stimulation of glucose utilization due to a post receptor defect on insulin action. J Clin Endocrinol Metab 54:131-138, 1982 32. Garvey WT. Olefsky JM, Marshall S: Insulin receptor down regulation is linked to an insulin-induced post receptor defect in the glucose transport system in rat adipocytes. J Clin Invest 76:22-30, 1985
Hyperinsulinemia
Decreases
Owen P. McGuinness,
Insulin
Sharon R. Myers,
Action
But Not Insulin
Sensitivity
Doss Neal, and Alan D. Cherrington
Hyperinsulinemia and insulin resistance are commonly seen in obese and non-insulin-dependent diabetes mellitus (NIDDM) patients, suggesting a causal link exists between hyperinsulinemia and insulin resistance. In a previous study, we demonstrated that chronic (28 days) intraportal hyperinsulinemia (50% increase in basal insulin levels) resulted in a decrease in insulin action as assessed by a one-step euglycemic hyperinsulinemic clamp. Since only one dose of insulin was used during the clamp, it was not possible to determine if the decrease in insulin action was due to a change in insulin sensitivity and/or maximal insulin responsiveness. In the present study, insulin resistance was induced as before, but insulin action was assessed in overnight fasted conscious dogs using a four-step euglycemic hyperinsulinemic clamp (1,2, 10, and 15 mU/kg/min). Insulin responsiveness was assessed before the induction of chronic hyperinsulinemia (day 0). and after 28 days of hyperinsulinemia (day 28). Transhepatic glucose balance and whole-body glucose utilization were measured to allow assessment of both the hepatic and peripheral effects of insulin. Chronic hyperinsulinemia increased basal insulin levels from 13 + 2 to 21 r 4 pU/mL. After 4 weeks of chronic hyperinsulinemia, maximal insulin-stimulated glucose utilization was decreased 23% + 4% (P < .05) and insulin sensitivity (ED,) was not significantly altered. During the four-step clamp, the liver was a major site of glucose utilization. The liver was responsible for 13% of the total glucose disposal rate on day0 (2.9 mg/kg/min) at the highest insulin infusion rate (15 mU/kg/min:2.000 @/mL). On day28, insulin suppression of endogenous glucose production and stimulation of hepatic glucose uptake was similar (3.3 mg/kg/min) to that seen on day 0, suggesting that the decrease in maximal insulin responsiveness did not involve the liver. In conclusion, insulin resistance produced by a mild hyperinsulinemia is due to a suppression of maximal extrahepatic insulin-stimulated glucose uptake, suggesting -- that a postreceptor defect is present in extrahepatic tissues. 0 1990 by W. 6. Saunders Company.
I
NSULIN RESISTANCE is commonly seen in situations in which hyperinsulinemia is also present (ie, non-insulindependent diabetes mellitus [NIDDM], obesity, and pregnancy).’ Previous work by ourselves and others have demonstrated that induction of mild hyperinsulinemia can lead to an insulin resistant state.2-4 However, it remains unclear as to the site of the resistance (ie, liver, muscle, fat) or the mechanism by which resistance is brought about (receptor and/or postreceptor defect). The type of resistance (receptor and/or postreceptor defect) that develops from chronic hyperinsulinemia is variable and to some extent depends on the characteristics of the 2-4 Exposure to pharmacologic insulin levhyperinsulinemia. els for brief (-4 hours) or more prolonged periods (14 days) decreased insulin receptor number and caused a postreceptor defect in rat adipocytes.5’6 Mild hyperinsulinemia for a brief duration (20 to 40 hours) brought about a postreceptor defect without an accompanying change in receptor number in man.3,4 A much longer duration of exposure may have been required to result in receptor down-regulation. Since in our previous study the duration of hyperinsulinemia was considerably longer (28 days) than in the human studies (20 to 40 hours) and the resistance was somewhat greater (34% v 20%) the cause of the resistance may differ from that seen in man. From our previous work’ we were unable to localize the site or the mechanism of the resistance, because only a single-step euglycemic hyperinsulinemic clamp was used to assess insulin action. The goal of the present study was to determine if the decrease in insulin action seen after 28 days of hyperinsulinemia was due to a decrease in insulin sensitivity and/or a decrease in maximal insulin responsiveness of the liver and/or the periphery. Using a four-step euglycemic hyperinsulinemic clamp, the doseresponse relationship between insulin concentration and insulin-stimulated glucose uptake by the liver and by the Metabolism, Vol39,
No 9 (September), 1990: pp 931-937
periphery before and after chronic exposure to hyperinsulinemia for 28 days was assessed. METHODS
Animal Preparation Experiments were performed in five conscious mongrel dogs (18 to 26 kg), receiving a diet consisting of Kal-Kan meat (Vernon, CA)
and Wayne Dog Chow (Purina Mills, St Louis, MO) once daily. The composition of the diet was 52% carbohydrate, 31% protein, 11% fat, and 6% fiber based on dry weight. Two weeks before the first experiment, a laparotomy was performed under general anesthesia (sodium pentobarbital; 30 mg/kg). Silastic catheters were placed into a mesenteric and a splenic vein for the chronic infusion of insulin. Blood sampling catheters were inserted into the portal vein and the left hepatic vein. In addition, a catheter was inserted into the femoral artery following a cut-down in the left inguinal area. The catheters were then filled with saline containing heparin (200 IU/mL). The infusion catheters were exteriorized, and brought through a subcutaneous tunnel to the back between the clavicles, where they were placed in a subcutaneous pocket. The portal and hepatic blood sampling catheters were exteriorized through the incision used to perform the laparotomy and placed in a subcutaneous pocket. The arterial blood sampling catheter was placed under the skin in the inguinal region. Catheters were removed from their respective pockets under local anesthesia on the day of the first study. After the completion of the first study (day 0) the sampling catheters were placed under the skin and the wound was closed. The dogs received ampicillin (1 g/d for 2 days) to minimize the possibility of From the Department of Molecuiar Physiology and Biophysics, Vanderbilt University, Nashville, TN. Supported by a grant from the Juvenile Diabetes Association and by the Hormone and Animal core labs of the Diabetes Research and Training Center (DK20593-12). Address reprint requests to Owen P. MeGuinness. PhD. 702 Light Hall. Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232. m 1990 by W.B. Saunders Company. OOZO-0495/90/3909-0009$03.00/0 931
932
infection. The infusion catheters were left exteriorized for the chronic infusion of insulin. All animals studied had (1) a good appetite (consuming all of the daily ration); (2) normal stools; (3) a hematocrit above 38%; and (4) a leukocyte count below 18,000 rL on days 0 and 28.
Experimental Design On day 0, after an overnight fast, a four-step, 400-minute euglycemic hyperinsulinemic clamp was performed (see below) to assess insulin responsiveness. At the end of the experiment the dog was placed in a jacket (Alice King Chatham, Los Angeles, CA), containing a pocket into which was placed a portable infusion pump. On day 1 a chronic intraportal infusion of insulin (425 NI_J/kg/min) was begun using a portable Millhill infuser (Harvard Apparatus, South Natick, MA; 60 rL/h). Porcine insulin, the structure of which is identical to canine insulin,’ was dissolved in saline containing heparin (30 U/mL) and dog plasma (3%). The solution was then filtered (0.2 pm) under sterile conditions before infusion. A fresh solution was prepared on each of the 28 infusion days to prevent aggregation of the insulin.” On day 28, insulin responsiveness was determined as on day 0.
Euglycemic Hyperinsulinemic Protocol On Days 0 and 28 Angiocaths (Deseret Med, Sandy, Utah; 1g-gauge) were inserted percutaneously into both cephalic veins and a saphenous vein. A primed (50 &i) constant infusion of 3-‘H glucose (0.4 rCi/min) and an infusion of indocyanine green (0.1 mg/m’/min) were begun through the right cephalic vein and continued throughout the entire experiment. The experiment consisted of three periods, an equilibration period (- 120 to -40 minutes), a control period (-40 to 0 minutes), and an experimental period (0 to 400 minutes). When studies were performed on day 28, the chronic intraportal insulin infusion was continued until the end of the control period (0 minutes) at which time it was discontinued. After the control period in both studies, a four-step euglycemic hyperinsulinemic clamp was begun. Insulin was infused through the left cephalic vein in a stepwise manner (1, 2, IO, and 15 mu/kg/ min), allowing 100 minutes for each step. A variable glucose infusion was begun through the saphenous vein; the arterial glucose concentration was measured every 5 minutes and the glucose infusion rate was adjusted to maintain euglycemia. On day 28 the glucose concentration was clamped at the level seen on day 0. Arterial blood samples were taken at -40, - 20, and 0 minutes, as well as at 30,60, 70, 80, 90, and 100 minutes during each loo-minute step in the experimental period. Portal and hepatic vein blood samples were taken at -40, -20, and 0 minutes during the control period, and at 80.90, and 100 minutes during each lOO-minute step.
Tracer Methods and Calculations The rates of total glucose production (R,) and utilization (Rd) were calculated according to the method of Wall et al’ as simplified by DeBodo et al.‘” Endogenous glucose production was calculated as the difference between R, and the exogenous glucose infusion rate. The tracer method has been shown to systematically underestimate glucose flux during a euglycemic clamp, resulting in negative endogenous glucose production.’ The cause of the error has not been elucidated; however, a combination of modelling errors and tracer contamination have been postmated to explain the error.i’-‘3 In the present study we did not obtain negative numbers; however, this does not indicate that the calculated endogenous glucose production during the clamp is correct. During the last 30 minutes of each step the coefficient of variation of the specific activity was less than 5%. Net hepatic glucose balance (NHGB) was calculated using the formula (H - [0.28A + 0.72P]) x0.73 xHBF, where H, A, and P
McGUlNNESS
ET AL
are the plasma glucose concentrations in the hepatic vein, femoral artery, and portal vein, respectively; 0.28 and 0.72 represent the percent contribution of the hepatic artery and portal vein, respectively, to total hepatic blood flow”‘; and 0.73 is an experimentally derived factor which converts plasma glucose values to blood glucose values in the dog”; and HBF is total hepatic blood flow. Extrahepatic glucose utilization was calculated as the difference between the exogenous glucose infusion rate and net hepatic glucose uptake. We did not have hepatic balance data for two of the animals because the hepatic vein sampling catheter did not work on day 28.
Processing of Blood Samples Blood samples were drawn into heparinized syringes and transferred to chilled tubes containing Na,EDTA (15 mg). Plasma glucose was assayed immediately using a Beckman glucose analyzer. Plasma treated with 500 KIU of Trasylol (FBA Pharmaceuticals, New York, NY) was assayed for immunoreactive glucagon using 30-k antiserum of Unger16; (coefficient of variation [CV], 8%). Immunoreactive insulin” was assayed using a sephadex-bound antibody technique (Pharmacia Diagnostics, Piscataway, NJ; CV. 11%). Polyethylene glycol (PEG) pretreatment of plasma was not required since dogs do not develop antibodies to insulin following insulin treatment.‘” Plasma cortisol was assayed with Clinical Assays Gamma Cost radioimmunoassay kit (Travenil-Genetech Diagnostics, Cambridge, MA; CV, 6%). Indocyanine green dye was measured spectrophotometrically (805 nm) to estimate total hepatic blood flow.19
Porcine insulin was purchased from Eli Lilly and Co (Indianapolis, IN). Glucagon 30-k antiserum was obtained from the University of Texas Southwestern Medical School (Dallas, TX). Purified glucagon and “51-glucagon were obtained from Novo Research Institute (Copenhagen, Denmark). Cortisol assay kits were obtained from Upjohn Diagnostics (Kalamazoo, MN) 3-JH-glucose was purchased from New England Nuclear Research Products (Wilmington, DE).
Statistics Statistical analysis was performed paired Student’s t test.
using one-way
ANOVA
and
RESULTS
During tion on day
the control
period,
28 was significantly
the average lower
than
glucose
concentra-
on day 0 (94 + 7
and 115 f 3 mg/dL; P < .OS; Fig 1). Glucose turnover on day 28 and 0 were similar (3.07 + 0.35 and 3.37 t 0.62 mg/kg/min, respectively). The exogenous intraportal insulin infusion produced a mild hyperinsulinemia on day 28,21 f 4 wU/mL, as compared with 13 + 2 rU/mL on day 0, (P < .05; Table 1). The average glucagon concentrations on days 28 and 0 were not significantly different (146 i 34 and 98 + 16 pg/mL, respectively; Table 2). The average cortisol concentrations (Fg/DL) on days 28 and 0 were 3.4 f 0.4 and 2.8 f 0.2, respectively. The average body weight was similar on days 28 and 0 (21.6 2 1.4 and 21.1 k 1.5 kg, respectively). The average glucose concentrations in both groups are shown in Fig 1. The exogenous glucose infusion rates on days 0 and 28 during the last 30 minutes of each step of the euglycemic hyperinsulinemic clamp were 6.0 of-0.8, 12.1 k 1.7, 19.5 f 2.0,and20.3 + 1.9ondayO,and6.8 _tO.8,9.6 _t
HYPERINSULINEMIA
AND INSULIN SENSlTIVlTY
933
Table 2. Plasma Glucagon Concentrations
During a Four-Step
Euglycemic Clamp Before (Day 0) and After a 28-Day Hyperinaulinemia
”
01
I
!X
INSULININFUSIONRATE
-40
d
Day 0
Day 28
Basal
98 + 16
146 * 34
1
69i
2
58 * 8
86 * 12
10
54*
14
58 f 8
15
56 ? 14
57 & 9
11
92 * 15
NOTE. Data expressed as mean f SEM (n = 5).
IdO Time
PlasmaGlucsgon(pg/mL)
ExogenousInsulin WJ/kg/min)
h\\\\V
(mu/kg/mh)
,,
Exposure to
(Dey 28)
360
260
40b
(minutes)
Arterial glucose concentration in dogs before (day 0) Fig 1. and after a 28-day exposure to hyperinaulinemia (426 N/kg/min: day 28) during a four-step euglycemic hyperinaulinemic clamp are presented. Data are expressed as means + SEM.
1.1, 16.5 + 1.8, and 16.4 + 1.2 mg/kg/min onday 28 for the insulin infusion rates of 1, 2, 10, and 15 mU/kg/min, respectively (Fig 2). The average insulin levels for those insulin infusion rates on days 0 and 28 are presented in Table 1. The arterial glucagon concentration decreased progressively throughout the study on days 0 and 28 (Table 2). The average R, during the last 30 minutes of each step was 7.4 T 0.9, 14.0 * 1.620.9 i 2.0, and 23.0 + 2.0 mg/kg/min on day 0, and 8.1 + 1.3, 11.0 r 1.3, 17.3 f 1.8, and 17.8 * 1.3 mg/kg/min on day 28. The relationship between the insulin concentration and the average rate of glucose utilization is presented in Fig 3. Following chronic insulin administration, the maximal rate of glucose utilization was decreased significantly (23% * 4%; P < .05). However, when the data are plotted as a percent of the maximal rate of glucose utilization, no significant change in the ED,, was detected. Net hepatic glucose balance (NHGB) was sensitive to the Table 1. Plasma Insulin Concentrations
actions of insulin (Table 3). NHGB was 2.38 + 0.66 and 2.29 + 0.71 mg/kg/min during the control period on days 0 and 28 and the liver switched to net consumption at very high insulin levels (- 2.89 + 0.96 and -3.30 + 0.66 mg/kg/min on days 0 and 28). The relationship between insulin concentration and suppression of hepatic glucose production, or stimulation of hepatic glucose uptake, was not altered by chronic insulin infusion. To further localize the site of the whole-body insulin resistance, extrahepatic glucose utilization was calculated. The insulin resistance was localized to the periphery (utilization decreased 23% + 3%; Fig 4). In addition, the calculated ED,, was unchanged by the exogenous insulin infusion (Fig 4). DISCUSSION
These data suggest that prolonged intraportal hyperinsulinemia (425 lU/kg/min) leads to insulin resistance charac25-
20 -ti
o-o Day 0 ~0~28 m---e mean f sem
IX
INSULIN INFUSION RATE (mu/kg/min)
During a Four-Step
Euglycemic Clamp Before (Day 0) and After (Day 28) Exposure to Hyperinaulinemia EXOgeIlOU~ Insulin ImU/ka/minl Basal
for 28 Days
Day 0 uJlrnL)
Day 26 ul/mL)
13 & 2
21 *4*
1
58 * 3
2
120 f 18
72 f 14 154 f 26
10
1,134
* 102
1.072
* 47
15
2,044
+ 293
1,996
+ 191
NOTE. lP -c -05 as compared with day 0. Data expressed as mean f SEM (n = 5).
0
100
200 Xme (minutia)
300
400
Exogenous glucose infusion rates in dogs before (day 0) Fig 2. and after a 28-day exposure to hyperinaulinemia (426 AU/kg/min: day 28) during a four-step euglyoemic hyperinaulinemic clamp are presented. Data are expressed aa means f SEM.
McGUlNNESS
934
A Day 0
O--O
Day 26
l
.’ “*
B .f..
G-2 N 3
iE - .l-x ‘Z $
tiz
Day26
l
75. DayQ
IOO50-
0-0
n/
::
/:
was unchanged. However, suppression of hepatic glucose production and pancreatic glucagon secretion and stimulation of hepatic glucose uptake by insulin were not altered. To assess the magnitude of the decrease of insulin action in extrahepatic tissues, the rate of extrahepatic glucose utilization was calculated. Chronic hyperinsulinemia decreased (23%) maxima1 insulin-stimulated extrahepatic glucose utilization without altering insulin sensitivity. Following chronic hyperinsulinemia, maximal extrahepatic glucose disposal was decreased (23%) and insulin sensitivity remained unaltered. These results are similar to those described by Rizza et al and Marangou et all.“ in man. Hyperinsulinemia for 40 and 20 hours, respectively, decreased maximal insulin stimulated glucose utilization (12% and 20%) without altering insulin binding in freshly isolated adipocytes. However, they did not differentiate between the contribution the liver and the periphery play in the development of the resistance. Others have found both decreases in insulin sensitivity and responsiveness following hyperinsulinemia,’ but they have relied on changes in insulin action in adipocytes to reflect changes in whole-body action. Since
:
8s 2
:
. “0
ET AL
25-
Z
/A
0, 10
100
INSULIN
25
A Day0
t
O-----O
1000
CONCENTRATION W/ml)
Fig 3. (A) Average tracer-determined glucose utilixstion rates during the last 30 minutes of each step of a four-step euglycsmic hyperinsulinemic clamp bafore (day 0) and after e 28-day exposure to hyperinaulinemie (425 @J/kg/min: day 28) are presented. Asterisks indicste that a significant diierence exists on days 0 compared with day 28. Date are expressad es means f SEM. (8) The tracer-determined glucose utilization retea are expressed ea a percent of the maximal glucose utilization rate during the lest 30 minutes of each step of a four-step euglycemic hyperinaulinemic clamp before (day 0) and after a 28-dey exposure to hyperinaulinsmia (426 /.dl/kg/min: day28). Data are expressed as means + SEM (n = 51.
-lb
160
lob0
100
1000
B Day0
o---o
Day 26
*
*
terized by a decrease in maximal insulin responsiveness, which appears to be localized in muscle and/or fat rather than in the liver. Following exposure to hyperinsulinemia for 28 days, whole-body maximal insulin responsiveness was decreased by 23% f 4% and whole body insulin sensitivity Table 3. Net Hepatic Glucose 8alance During a Four-Step Euglycemic Clamp Before (Day 0) and After a 28-Day Exposure to Hyperinaulinemie (Dey 28) EXogelWJS Insulin (mU/ks/min! Basal 1
Day0 fma/ke/min) 2.38 f 0.66
Day 28 hne/ke/min) 2.29 zt 0.71
-0.10
* 0.29
- 1.04 * 0.55
2
-0.55
f 0.26
-0.88
10
-1.83
+ 0.13
-2.26
f 0.53 * 0.70
15
-2.89
+ 0.96
-3.30
f 0.66
NOTE. Negative balance indicates net uptake of glucose. Data srs expressed ss mean + 6EM h = 3).
01 10
INSULIN CONCENTRATION W/m0 Fig 4. (A) Average extrahepatic glucose utilization retea are presented during the lest 30 minutes of each step of a four-step euglycemic hyperinaulinemic clemp before iday 01 end sfter a 28-dey exposure to hyperinaulinemis 1426 AU/kg/min; dsy 28). Dete are expressed es mesna f 8EM (n = 31. (81 Extrahepstic glucoas utilixstlon rate is expresssd as a percent of the meximal glucose utilixetion rate during the lsat 30 minutes of esch step of a four-•teg euglyoemic hyperinaulinemic clamp before ldey 0) and after a 28 day exposure to hyperinaulinemia (426 fiU/kg/min; day 28). Dete ere expressed as mssna + 8EM In = 3).
HYPERINSULINEMIA
AND INSULIN SENSlTlVll-Y
adipocytes do not represent a major site of glucose removal and their response to hyperinsulinemia may not always parallel that of muscle,*’ extrapolating these results to data obtained in vivo is tenuous. The present study clarifies the major role extrahepatic tissues play in disposing of glucose during a euglycemic hyperinsulinemic clamp (disposing 96% of the exogenous glucose infusion during the first two insulin infusion rates) and in the decreased insulin action seen following chronic hyperinsulinemia. In our previous work,* chronic intraportal hyperinsulinemia decreased insulin action by 35% k 8%, which is a greater decrease than that observed in the present study (21% f 4%) at a comparable insulin infusion rate (2 mU/kg/min) during the euglycemic clamp. The magnitude of the chronic hyperinsulinemia was similar in both studies, from 13 to 21 &J/mL in the present study and from 15 to 23 pU/mL in our previous study. There are several possible explanations for this small difference in insulin resistance. First, it may be due to the different basal insulin action on day 0 in the two studies. Glucose utilization was approximately 33% lower on day 0 in the present study than in our previous study during the euglycemic hyperinsulinemic clamp. This in turn is partially explained by the fact that the 2 mU/kg/min step in the present study was 100 minutes in length rather than 180 minutes as in our previous study. Insulin action has been shown to continuously increase over time during a euglycemic hyperinsulinemic clamp.** In our experience during a euglycemic hyperinsulinemic clamp (2 mU/kg/min), insulin action was 8% lower after 100 minutes of hyperinsulinemia than after 180 minutes. The remaining factor, which may account for the somewhat smaller effect of chronic hyperinsulinemia, is dog to dog variation. A saline control group was not included in this study. However, based on our previously reported results, insulinstimulated glucose uptake during a euglycemic hyperinsulinemic clamp (2 mU/kg/min) was not altered after 28 days of saline infusion.2 Therefore, changes in insulin action in the present study cannot be explained by time-dependent decreases in insulin action. Insulin responsiveness of muscle is underestimated by the euglycemic hyperinsulinemic clamp because, although arterial euglycemia was maintained during the clamp, extracellular glucose concentration decreased as the rate of glucose utilization by muscle increased. However, the extracellular glucose concentration influences the rate of glucose uptake. Accurately estimating the extracellular glucose concentration during the clamp is difficult. The arteriovenous glucose concentration difference across the forelimb in man at insulin levels comparable to that seen at the highest insulin infusion rate used in the present study (> 1,000 gU/mL) was approximately 60 mg/dL,23 when the arterial glycemia was clamped at 90 mg/dL. If we assume that the muscle venous glucose concentration reflects the extracellular glucose concentration and we normalize the glucose disposal rate to the arterial glycemia, maximal insulin responsiveness will be increased. The effect on ED,, is difficult to predict, since the impact of glycemia on glucose uptake is dependent on the prevailing insulin and glucose levels. On day 28 in the present study, glucose disposal was 23% lower than on day 0. Consequently,
935
the venous glucose concentration will be higher at comparable arterial glycemia, and therefore the correction in maximal glucose responsiveness will be less. If the data from the present study are corrected, the maximal rate of glucose disposal is approximately 28%, rather than 23%, lower on day 28 than on day 0. The liver was a significant site of glucose utilization at the two highest doses of insulin used. During the euglycemic hyperinsulinemic clamp the liver switched from being a net producer to a net consumer of glucose. As the insulin levels increased to 58 rU/mL on day 0 during the first step of the clamp, hepatic glucose production was completely suppressed (-0.10 c 0.29 mg/kg/min). During the second step of the clamp (insulin concentration = 120 rU/ml) the liver switched to consumption (-0.55 + 0.26 mg/kg/min). In man, an insulin infusion rate (1 mU/kg/min), producing similar insulin levels (101 + 5 pU/mL), suppressed splanchnit glucose production and also converted the splanchnic bed to net consumption (0.42 k 0.09 mg/kg/min).24 In the present study, as insulin levels increased to approximately 1,000 &l/mL, hepatic glucose uptake increased to approximately 2 mg/kg/min, which is considerably larger than has been seen in man at comparable insulin levels both at euglycemia (0.68 + 0.13 mg/kg/min) and at hyperglycemia (1.07 + 0.25 mg/kg/min), even when the difference between splanchnic and hepatic glucose production is taken into account.24 At the highest dose of insulin used (15 mU/kg/min), the liver took up approximately 3.0 mg/kg/min or about 13% of total glucose disposal on day 0 and about 17% on day 28. Therefore, pharmacologic hyperinsulinemia in the presence of euglycemia can convert the liver to a significant consumer of glucose. However, in the presence of physiologic levels of insulin, other factors (ie, hyperglycemia and route of glucose delivery) I*.*5in addition to insulin regulate hepatic glucose uptake. Surprisingly, the uptake of glucose by the liver, as opposed to the periphery, did not completely saturate with an insulin infusion rate of 15 mU/kg/min. Therefore, insulin sensitivity of the peripheral tissues may not be less than that of the liver with regard to glucose uptake. However, it is possible that the rate of stimulation of hepatic glucose uptake by insulin may be slower than its stimulation of peripheral glucose uptake. Therefore, the further increase in hepatic glucose uptake may simply reflect the sluggishness of the process. The experimental design did not allow us to directly assess hepatic insulin sensitivity, because we did not prevent the expected decrease in glucagon during the clamp.26 Recently, it has been shown that the liver is sensitive to subtle decreases in glucagon in the presence of hyperinsulinemia,*’ leading one to overestimate hepatic insulin sensitivity. As the insulin levels were increased to 58 and 72 pU/mL on days 0 and 28, respectively, hepatic glucose production was completely suppressed and, in fact, the hepatic glucose balance was slightly negative on day 28. Based on these results alone, one might conclude that the liver was more sensitive to insulin on day 28. However, previous work indicates that it is the change in glucagon level, and possibly the absolute glucagon level, which determines the hepatic response.*’ The greater
McGUlNNESS
936
decrease in glucagon in concert with the slightly higher insulin levels on day 28 probably explains the apparent increase in suppression of hepatic glucose production on day 28 at the first step of the clamp. On days 0 and 28, plasma glucagon levels during the last two steps of the clamp did not change, allowing us to more clearly assess hepatic insulin action. Insulin stimulation of hepatic glucose uptake was not altered by chronic hyperinsulinemia. These results are similar to those obtained by Rizza et al,’ in which chronic hyperinsulinemia did not alter insulin suppression of hepatic glucose production. However, as was discussed, the use of the tracer method to measure endogenous glucose production during a euglycemic hyperinsulinemic clamp is associated with some undefined problems.28,29 In addition, since glucagon data were not reported, those results are difficult to interpret. The mechanism for the development of the peripheral resistance is unknown. One possibility is that throughout the 28 days the animal experienced hypoglycemic episodes resulting in a change in counterregulatory hormones which, when administered in pharmacologic quantities, are known to alter insulin action.30.3’ Based on our previously reported data* in which a 24-hour profile of glucose and counterregulatory hormones was obtained during the intraportal infusion
ET AL
of insulin, no significant hypoglycemia or change in the counterregulatory hormones was detected. However, we cannot rule out that a subtle increase in counterregulatory hormone production contributed to the insulin resistance produced in this model. The results of the present study and previous studies in man indicate that the majority of the defect seen in a hyperinsulinemic state is at the postreceptor leve1.2’4Whether it involves the glucose transport system or a subsequent step remains unclear. Garvey et a13’ demonstrated that chronic (24-hour) exposure of adipocytes to severe hyperinsulinemia (2,400 $J/mL) decreased both insulin binding and the coupling between insulin binding and insulin-stimulated glucose transport. Since insulin binding was not measured in the present study, we can only speculate that a receptor defect is not present, although this is supported by the lack of a change in insulin sensitivity. In conclusion, these studies indicate that a mild hyperinsulinemia produced by a chronic intraportal infusion of insulin leads to a decrease in insulin action. This decrease in insulin action was due to a decrease in maximal extrahepatic insulin responsiveness, without a concomitant change in peripheral or hepatic insulin sensitivity, suggesting that a postreceptor defect is present.
REFERENCES 1. McGuinness OP, Steiner KE, Abumrad NN, et al: Insulin action in vivo, in Alberti KGMM, Krall LP (eds): Diabetes Annual, vol 3. Amsterdam, The Netherlands, Elsevier Science, 1987, pp 398-432. 2. McGuinness OP, Friedman A, Cherrington AD: intraportal hyperinsulinemia decreases insulin stimulated uptake in the dog. Metabolism (in press)
Chronic glucose
3. Rizza RA, Mandarin0 LJ, Genest J, et al: Production of insulin resistance by hyperinsulinemia in man. Diabetologia 28:70-75, 1985
4. Marangou AG. Weber KM, Baston RC, et al: Metabolic consequences of prolonged hyperinsulinemia in humans, evidence for induction of insulin insensitivity. Diabetes 35: 1383- 1389, 1986 5. Kobayashi M, Olefsky J: Effect of experimental hyperinsulinemia on insulin binding and glucose transport in isolated rat adipcytes. Am J Physiol235:E53-E62, 1978 6. Gavin J, Roth J, Neville D, et al: Insulin dependent regulation of insulin receptor concentration, A rapid demonstration in cell culture. Proc Natl Acad Sci USA 71:84-88, 1974 7. Smith LF: Species variation in the amino insulin. Am J Med 40:662-666, 1966 8. Loughheed WD, Woulfe-Flanagan Insulin aggregation in artifical delivery 1-9, 1980
acid sequence
of
H, Clement JR, et al: systems. Diabetologia 19:
9. Wall JS, Steele R, DeBodo RC, et al: Effect of insulin on utilization of glucose and production of circulating glucose. Am J Physiol 189:43-50, 1957 10. DeBodo RC, Steele R, Altzuler regulation of carbohydrate metabolism: Ret Prog Horm Res 19:445-88, 1963
NR, et al: The hormonal studies with “C-glucose.
11. Finegood DT, Bergman RN, Vranic M: Modelling error and apparent isotope discrimination confound estimation of endogenous glucose production during euglycemic glucose clamps. Diabetes 37:1025-1034, 1988 12. McMahon MM, Schwenk WF, Haymond MW, et al: Underestimation of glucose turnover measured with [6-‘HI- and [6,6-*HZ]But not (6-‘4C] glucose during hyperinsulinemia in humans. Diabetes 38:97- 107, 1989
13. Yki-Jarvinen H, Consoli A, Nurjahan N, et al: Mechanism for the underestimation of isotopically determined glucose disposal. Diabetes 38:744-51, 1989 14. Greenway CV, Stark RD: Hepatic vascular bed. Physiol Rev 51:23-65, 1971 15. Adkins-Marshall BA, Myers SR, Hendrick GK, et al: Importance of the route of glucose delivery to hepatic glucose balance in the conscious dog. J Clin Invest 79:557-565, 1987 16. Agular-Parada E, Eisentraut AM, Unger RH: Pancreatic glucagon secretion in normal and diabetic subjects. Am J Med Sci 257:415-419, 1968 17. Wide L, Porath J: Radioimmunoassay of sephadex-coupled antibodies. Biochem 260,1966
of proteins with the use Biophys Acta 130:257-
18. Vranic M, Kawamori S, Pek N, et al: The essentiality of insulin and the role of glucagon in regulating glucose utilization and production during strenuous exercise in dogs. J Clin Invest 57:245255. 1976 19. Leevy CM, Mendenhall CL, Lesko W, et al: Estimation of hepatic blood flow with indocyanine green. J Clin Invest 41:1169-79, 1962 20. James DE, Jenkins AB, Kraegen EW: Heterogeneity of insulin action in individual muscles in vivo: Euglycemic clamp studies in rats. Am J Physiol248:E567-E574, 1985 21. Wardzala LJ, Hiesham M, Pofcher E, et al: Regulation of glucose utilization in adipose cells and muscle after long term experimental hyperinsulinemia in rats. J Clin Invest 76:460-469, 1985 22. Doberne L, Greenfield MS, Schultz B, et al: Enhanced glucose utilization during prolonged glucose clamp studies. Diabetes 30:829-835, 1981 23. Yki-Jarvinen H, Young AA, Lamkin C, et al: Kinetics of glucose disposal in whole body and across the forearm in man. J Clin Invest 79:1713-1719, 1987 24. Defronzo RA, Ferrannini E, Hendler R, et al: Regulation of splanchnic and peripheral glucose uptake by insulin and hyperglycemia in man. Diabetes 32:35-45, 1983
HYPERINSULINEMIA
AND INSULIN SENSITIVITY
25. Cherrington AD, Stevenson RW, Steiner KE, et al: Insulin, glucagon and glucose as regulators of hepatic glucose uptake and production in vivo. Diabetes Metab Rev 3:307-318, 1987 26. Samols E, Weir GC, Bonner-Weir S: Intraislet insulinglucagon-somatostatin relationships, in Lefebvre P (ed): Glucagon II. Handbook of Experimental Pharmacology, vol 66. Berlin, FRG, Springer Verlag, 1983, pp 133-173 27. Myers SR, Adkins-Marshall B, Williams PE, et al: Subtle changes in glucagon during the euglycemic clamp can markedly alter the apparant sensitivity of the liver to insulin. Diabetologia 31:525a, 1988 (abstr) 28. Bergman RN, Finegood DT, Ader M: Assessment of insulin sensitivity in vivo. Endocrine Rev 6:45-89, 1985 29. Finegood DT, Bergman RN, Vranic M: Modelling error and
937
apparent isotope discrimination confound estimation of endogenous glucose production during euglycemic glucose clamps. Diabetes 37:1025-1034, 1988 30. Scheidegger K, Robbins DC, Daforth E: Effects of chronic beta receptor stimulation on glucose metabolism. Diabetes 33:11441149,1984 3 I. Rizza RA, Mandarin0 LJ, Gerich JE: Cortisol-induced insulin resistance in man: Impaired suppression of glucose production and stimulation of glucose utilization due to a post receptor defect on insulin action. J Clin Endocrinol Metab 54:131-138, 1982 32. Garvey WT. Olefsky JM, Marshall S: Insulin receptor down regulation is linked to an insulin-induced post receptor defect in the glucose transport system in rat adipocytes. J Clin Invest 76:22-30, 1985
