A Kinetic Analysis of Plasma Insulin Disappearance Glucose-Induced Insulin Delivery Rate in Diabetic Nondiabetic Acromegalics F. Maneschi, (with the technical

A. Pilo, and R. Navalesi

assistance

Derangements of insulin metabolism may have a role in the pathophysiology of diabetes in acromegaly. To investigate this point, we employed a technique that allows the measurement of the degradation rate, the distribution volumes of the hormone, and the posthepatic insulin delivery rate both in the fasting state and after intravenous (i.v.1 glucose. ‘*61-insulin was injected i.v. as a single bolus and the plasma disappearance curve of the immunoprecipitable radioactivity was sampled for 120 min. Immediately afterwards, an i.v. glucose tolerance test (GTT) was performed and both glucose and IRI levels were measured from samples taken for 90 min. By noncompartmental analysis of the plasma ‘261insulin curves were computed: (1) the initial distribution volume of insulin (IDV); (2) metabolic clearance rate (MCI?); (3) basal posthepatic delivery rate (IDR): and (4) total plasma equivalent distribution volume (TDV). By deconvolution analysis of glucose-induced immunoreactive insulin (IRI) curve and plasma disappearance curve of tracer insulin, the posthepatic secretion curve of the hormone was reconstructed. Nine acromegalic patients were studied; 4 had a normal glucose tolerance (NA). and 5 were diabetic (DA). Seven normals (N) were taken as a control group. In the acromegalics the IDV was enlarged compared to normals (2.6 + 0.2 versus 1.8 + 0.1 liter/sq m; p cc 0.01) and MCR was also greater (564 + 24 versus 473 + 24 ml/min x sq m; p < 0.05). In the DA the TDV was larger than in N (17.1 + 3.4 versus 8.4 + 0.7 liters/sq m; p cc0.05). In both NA (4.8 + 0.7 mU/min x sq m) and DA (8.6 f 1.9) the IDR was greater (p < 0.01) than N (2.3 r 0.9). Glucose-stimulated posthepatic insulin secretion showed, both in N and acromegalics, a first rapid burst secretion (Seer), which had its peak at 2-3 min and lasted for 6-10 min. and a more sustained second phase, Seer (6-90). which returned to the basal levels by the end of 90 min. In NA, Seer (O-6) was greater than in N (500 t 93 versus 232 + 39 mU/sq m: p < 0.05) and DA (174 + 39; p c 0.01). Seer (6-90) was significantly greater in both NA (1707 + 205) and DA (1752 + 391) than N (615 + 167 mU/sq m). When glucose-induced insulin secretion was expressed as a multiple of the basal insulin delivery the first phase of insulin secretion was superimposable in N and NA (17.7 + 2.7 versus 17.8 f 3.9) while it was reduced in DA (3.5 + 0.8; p < 0.01). The second phase was not significantly different in the 3 groups. All acromegalic patients showed: (1) an enlarged IDV, (2) an increased MCR, and (3) a higher secretion rate, both in the fasting state and after i.v. glucose. These alterations of insulin kinetics and secretion seem therefore to be Metabolism, Vol.28, No. 10 (October),1979

and and

of P. Cecchetti

and A. Masoni)

caused by acromegaly itself. On the other hand, an enlarged TDV, a marked decrease of the early phase of insulin secretion, and an additional factor of insulin resistance independent from GH were found in DA only, therefore they are more related to diabetes than to acromegaly.

I

N ACROMEGALY, higher than normal insulin concentrations have been reported both in the fasting state and after glucose.‘-3 These studies have assumed that changes in insulin concentrations directly reflect changes of insulin secretion. It is clear, however, that plasma levels of any hormone depend not only on secretion but also on degradation and distribution. It is therefore possible that in acromegaly, derangements of one or more of these factors could account for the variations of insulin concentrations; moreover, different alterations might occur in diabetic acromegalics and nondiabetic acromegalics. To evaluate possible derangements of insulin metabolism in acromegaly, we employed a technique that allowed the measurement of the degradation rate, the distribution volumes of the hormone and the posthepatic insulin secretion, both in the fasting conditions and after i.v. glucose. We studied four acromegalic patients with normal tolerance to carbohydrates (NA), as judged from a standard oral glucose tolerance test, and five diabetic acromegalics (DA). Seven normal subjects were taken as a control group. Our results enabled a distinction to be made between the alterations of insulin kinetics and secretion characteristic of acromegaly and those attributable to diabetes associated to acromegaly.

From the C.N.R. Clinical Physiology Laboratory and II Medical Clinic, University of Piss. Via Savi 8, 56100 Pisa, Italy and the Department of Medicine, Royal Postgraduate Medical School, London, England. Address reprint requests to Dr. A. Pilo, Clinical Physiology Laboratory. Via Savi 8. Pisa. Italy. 01979 by Grune & Stratton. Inc. 0026-0495/79/2810-0005$02.00/0

1011

1012

MANE’%+,

PILO. AND NAVALESI

Table 1. Clinical Data of Acromegalic Patients

Patient

sex

w

POd3ral

Basal

of

Duration

Age

Acromegaly

Index’

PWdlOUS

Treatment

fvr)

Fastmg

GH Levelt

Plasma

Glucoselmg/dl)

(ng/mli

1

F

55

164

11

None

2

M

50

126

22

X-ray (1958)

3

F

35

103

18

4

F

49

125

5

F

61

160

6

M

57

120

17

None

9.4

75

7

F

50

120

10

None

12.5

96

8

F

61

124

5

None

3.5

105

9

F

51

94

9

None

16.9

128

52 3

126 8

15 2

15.8 4.1

101

Mean SE

31.7

87

3.5

75

None

39.8

141

17

None

13.6

80

25

NOna

11.1

121

8

*Percent of their desirable body weight, according to the middle of the weight range for subjects of large frame from the 1959 Metropolitan Life Insurance Co. Tables. tMean of 3 consecutive plasma levels taken 30 and 10 min before and rmmedrately after oral glucoseadministratlon.

MATERIALS AND

METHODS

Patients Nine acromegalic patients were studied. Acromegaly was diagnosed on clinical grounds and confirmed by elevated glucose nonsuppressible growth hormone (GH) levels. Table 1 summarizes the pertinent clinical data for the acromegalic patients. Duration of acromegaly was assessed by changes of facial features from photographs. Patients I and 8 were mildly hypertensive, patient 6 had an arterial blood pressure of 2201120 mm Hg, and patient 2 had nontoxic goiter. The T, value of patient 7 was at the higher level of the normal range at the time of the study, and 6 mo later she developed thyrotoxicosis due to toxic goiter; TSH levels were normal on both occasions. Patient 3 was amenorrheic; patient 8 showed absence of circadian rhythm of cortisol. Other endocrine and metabolic disorders were excluded in all patients; none had signs of optic chiasmal compression. Family history of diabetes mellitus was negative in all patients. A standard oral glucose tolerance test (100 g of glucose) was performed in all patients. Figure 1 shows the respective glucose values in individual patients during these tests. Standard criteria were used to detect patients with abnormal carbohydrate tolerance.’ The control group consisted of 7 normal subjects, all within 10% of their ideal body weight; their age ranged from 32-57 yr. They had all negative family histories of diabetes mellitus, and were in good health as determined from clinical histories and routine laboratory tests. Neither the acromegalics nor the controls were taking any drugs at the time of the study. All patients ate the average Italian diet (43748% carbohydrates, 12%-l 7% proteins, and 35%40% fat). Fully informed consent was obtained in all cases.

Experimental

Protocol

All studies were performed in the morning. The patients were fasted overnight and their thyroid uptake was previously blocked by saturated potassium iodide. During the study the patients were sitting, and they rested for about 30 min before the beginning of it. Between 8:00 a.m. and 9:00

a.m., an indwelling plastic catheter was placed in the antecubital vein, then IO&150 FCi of ‘*‘I-insulin were injected i.v. as a single bolus in the other arm. Blood samples, taken at 2, 3, 4, 5, 7. 9, 12, 15, 20. 25, 30. and 40 min. and thereafter every IO min up to 2 hr. were collected into plastic tubes containing EDTA and sodium fluoride. Plasma was immediately separated by centrifugation at 4OC and the samples were immediately processed for the determination of immu‘2s1-insulin. Approximately 15 min after the noprecipitable end of the tracer study, 0.33 g/kg body weight of glucose (as a 40% aqueous solution) was injected rapidly i.v. Blood was collected every min up to I5 min and then at 17, 20. 25. 30, 40, 50, 60, 75, and 90 min into plastic tubes containing the aforementioned anticoagulant. Plasma, separated as above,

I

0

00

120

l&J

MINUTES

fig. 1. Plasma glucose levels of the nondiabetic [solid line) and diabetic acromegalics (dashed line) during the oral glucose tolerance test. Patient number for each curve is reported on the right.

1013

KINETICS OF INSULIN IN ACROMEGALY

was stocked at - 20°C for the determination endogenous insulin.

of glucose and

IDV MCR

Tracer Monocomponent crystalline pig insulin was labeled with ‘?- using lactoperoxidase and H202 as oxidizing agent; the labeling procedure has been reported in detail elsewhere.’

Analytical Determinations “‘I-insulin was determined by a Immunoprecipitable double antibody technique,6 usually within 2 hr from the end of the tracer study. Triplicate 0.2 ml samples of plasma were incubated with 0.1 ml of guinea pig anti-insulin serum (final dilution l/500) for 18 hr at 4OC. Then, 0.1 ml of rabbit serum anti guinea pig y-globulins (final dilution l/12) was added, and the mixture was incubated for 24 hr at 4OC. Phosphate buffer, pH 7.4, was used for dilutions. After centrifugation at 3000 x g for 45 min, the supernatant was discharged and the precipitated activity was counted in a well-type -r-counter. Two additional aliquots of plasma were incubated with 0.1 ml of nonimmune guinea pig serum for I8 hr at 4OC, then 0.1 ml of rabbit serum (anti guinea pig) y-globulins was added. The precipitate was separated as previously described. The radioactivity of this last precipitate was considered nonspecific, and it was subtracted from the immunoprecipitable ‘*‘I-insulin activity. Plasma concentration of endogenous insulin (IRI) was measured with “‘l-insulin as labeled antigen (prepared by the same procedure described for ‘ZSI-insulin). Bound/free insulin was separated by dextran-coated charcoal and by centrifugation at 3000 x g. Inter- and intra-assay variations were measured by 2 reference pools and were 14% and 7%. respectively. Endogenous GH was measured by a double antibody technique.’ Inter- and intra-assay variations were determined by 2 reference pools and were 10% and 6% for the high pool (7 ng/ml), and 8% and 4% for the low pool (I ng/ml). The samples with GH concentrations higher than the high pool were properly diluted to improve the precision of the assay. Plasma glucose was measured by the glucose-oxidase technique.’ DATA

ANALYSIS

The insulin kinetic parameters were evaluated from the plasma disappearance curve P(t) of immunoprecipitable ‘2SI-insulin by noncompartmental analysis. The assumptions and limitations of this approach were extensively reported elsewhere.’ The model employed is a minimal assumption one; it consists of a central compartment or initial distribution space (IDV) that is similar to the plasma pool and reversibly exchanges with the extravascular hormone pool, on which no definite compartmental structure is assumed. It is also assumed that all insulin input and degradation occur in the central compartment and no degradation occurs in the periphery. The following formulae were used,

k

ER RR ki k,

i TDV IDR

(initial distribution volume, liters/sq m) = dose/P(O) (1) (metabolic clearance rate, ml/min x sq m) = dose//,” P(t) dt (2) (fractional rate of exit from IDV or initial slope of the disappearance curve, min-‘) = [dP(t)/dt],_,/P(O) (3) (rate of exit from IDV, ml/min - sq m) =k-IDV (4) (reentry rate or reversible loss from IDV, ml/min . sq m) = ER-MCR (5) (fractional irreversible loss rate from IDV, min-‘) = MCR/IDV (6) (fractional reversible loss rate from IDV, min-‘I= RR/IDV (7) (mean residence time, min) = JO” tP(t)dt/j,” P(t) dt (8) (total plasma equivalent distribution volume, liters/sq m) = MCR . i (9) (basal posthepatic insulin delivery rate, mU/min - sq m) = MCR . IRI (10)

where dose indicates the immunoprecipitable fraction of the injected dose and IRI &U/ml) the basal plasma concentration of immunoreactive endogenous insulin. The integrals in equations (2) and (8) were computed according to the relations: l=P(t)

dt = J”‘P(t)

dt + P(120)/k

and l=tP(t)

dt = 112’+

P(t)dt

+ P(120)(1 + k . 120)/k’ The integrals from 0 to 120 min were computed by trapezoidal integration of the experimental points, while the contribution of the “tails” was estimated as usual by exponential extrapolation, “k” being the slope of the last 40-60 min of the plasma curve. Zero intercept P(0) and initial slope were obtained from monoexponential extrapolation of the first 5-min points. The glucose-stimulated insulin delivery function, D(t), is related to plasma insulin concentration I(t) (mu/ml) according to the following equation: T(t) = @t)*P(t)/dose

(11)

where P(t)/dose represents the unitary impulse response of the insulin system, and * indicates the convolution product. As the curves T(t) and

MANESCHI, PILO, AND NAVALESI

Experimental disapppearance curves of immuFig. 2. noprecipitable ‘“l-insulin (as mean k SE) measured in 7 normals (area within the full line). in 4 nondiabetic acromegalics (closed circles). and in 5 diabetic acromegalics (open circles).

p(t) were experimentally determined, o(t) could be computed minute by minute solving the integral equation (11) by a numerical deconvolution algorithm, which has been reported in detail elsewhere.” The first secretory peak was considered to be finished when insulin secretion function reached its first minimum value. This minimum was generally well determined, as it was preceded and followed for at least 3 min by decreasing and increasing secretion values, respectively. The average duration of this first phase was 6 min (range 5-7 min) in the 7 normals, 8 min

(range 6-10) in the 4 NA, and 8 min (range 6-10) in the 5 DA. The amount of insulin secreted during the whole duration of the i.v. GTT was obtained as the integral of the delivery function D(t) on the interval O-90 min. The integral of secretion on the interval C&6 min has also been calculated to estimate the insulin output of the acute phase in normals. In the computation of o(t), we considered as zero time the moment when half the glucose load was injected; the whole injection was usually performed within 1 min. To compare the first phase of insutin secretion, the O-6 min cumulative output was also computed in NA and DA, even if in these patients the duration of the first peak was slightly greater; for this reason the acute first-phase insulin response of the acromegalics was slightly underestimated. Plasma glucose fractional disapppearance rate (KG) was computed by linear regression analysis of the natural logarithms of plasma glucose concentration over the lo-60 min interval. Glucose area under the curve (GA) was computed by trapezoidal integration of the incremental values over the fasting level. RESULTS

Parameters Obtained by the Plasma Disappearance Curve of ‘Z51-lnsulin Figure 2 shows the mean plasma disappearance curves of immunoprecipitable ‘251-insulin in the 7 normal subjects, in nondiabetic acromegalits (NA) and in diabetic acromegalics (DA). The kinetic parameters referring to normals, NA and DA are reported in Tables 2, 3, and 4. As many of these parameters did not differ in NA

Table 2. ‘2?-lnsulin Kinetic Parameters of Normal Subjects Patient 1

IDV Iliter/sq ml

2.3

k (% min-‘) ER (ml/min

- sq m)

k, (% min-‘) MCR (ml/min k, (%

.

sq m)

min-‘1

RR (ml/min . sq m) T (min) TDV (literhq ml

2

1.7

3

1.3

4

1.6

5

1.9

6

1.9

7

1.6

Mean SE

1.8 + 0.1

33.0

43.0

43.0

35.0

40.0

36.0

36.0

38.0

763.0

739.0

571.0

542.0

763.0

680.0

574.0

662.0

24.0

30.0

33.0

26.0

28.0

25.0

24.0

27.0

551.0

511.0

448.0

407.0

544.0

460.0

390.0

473.0

9.0

13.0

9.0

9.0

228.0

124.0

135.0

16.8

20.1

12.6

15.0

9.3

10.3

5.6

6.1

212.0

11.0 219.0

12.0

11.0

+ 2.0 ? 37.0 + 3.0 t 24.0

11.0 * 2.0

220.0

184.0

16.1

22.0

21.5

17.8 f 1.3

8.7

10.1

8.4

8.4 + 0.7

189.0

i

16.0

IDV. initial distribution volume; k, fractional rate of exit from IDV; ER, exit rate from IDV; ki, fractional rate of irreversible loss from IDV: MCR, metabolic clearance rate; k,, fractional rate of reversible loss from IDV; RR, rata of reentry into IDV; ?, mean residence time; TDV. total plasma equivalent distribution volume.

1015

KINETICS OF INSULIN IN ACROMEGALY

Table 3. ‘*%lnsulin

Kinetic Parameters

of Nondiabetic

Acromegalics

Patient

k, (% min-‘) MCR (ml/min

.

597.0

786.0

sq ml

Mean SE 2.4 k 0.3.

3.0

31.0

37.0

35.0

803.0

1110.0

824.0

28.0

27.0

22.0

24.0

25.0

461.0

558.0

714.0

580.0

8.0

9.0

137.0

202.0

+ 55.0

245.0

13.9

19.4

k 52.0

14.3 * 2.0

5.6

6.6

7.8

13.8

8.5 k 1.8

23.3

4.8

7.9

9.4

11.4 * 4.1

TDV (liter/sq m) tGH (w/ml)

397.0

245.0

14.2

9.6

7 (min)

+ 3.0

10.0 * 2.0

13.0

8.0

+ 2.0 t 106.0

586.0

k, (% min-‘1

* sq m)

6

2.5

35.0

37.0

- sq m)

ER (ml/min

RR (ml/min

1.8

2.1

k 1% rnin-‘)

4

2

1

IDV (liter/sq m)

Ses Table 2 for explanation of abbreviations.

lp

< 0.05 in comparison with normals.

tMean GH concentration, measured from each 20-min sample during the 120 min of the disappearance curve of ‘%nsulin.

35 * 3 in DA and 38 f 2 in normals. The MCR values were similar in both groups of acromegalits (580 + 52 ml/min - sqminNAand551 f 20 in DA); when data from all these patients were pooled and compared with normals, a significant difference was found (564 + 24 versus 473 k 24, p -c 0.05). The fractional rate of irreversible loss (ki) was not significantly different in the 3 groups of subjects (27 k 3% min-’ in normals, 25 -c 3 in NA, and 23 + 4 in DA). The reversible loss rate or reentry rate (RR) of the tracer from its IDV was greater in acromegalics than in normals (3 17 f 46 versus 189 + 16 ml/min.m*, p -c 0.05), being greater in DA (374 ~tr63) than in NA (245 + 55). The fractional rate of reversible loss, k,, was significantly greater (p -c 0.05) in DA (14 + 3% min-‘) than both NA (10 + 2) and normals (11 + 2).

and DA, the mean f SE obtained from all acromegalic patients is reported in Table 5 also. Fast initial distribution volume of insulin (IDV) was similar in both groups of acromegalits, and when they were taken all together, it was significantly (p < 0.01) greater than normal (2.6 * 0.2 versus 1.8 * 0.1 liters/sq m). The rate of exit (ER) of tracer insulin from its IDV was greatest in DA (925 k 70 ml/min - sq m) and this value was significantly (p < 0.05) higher than normal (662 + 37), but not than DA (824 + 106); when the mean value from all acromegalics (876 + 60) was compared to normals a significant difference could be found. No significant difference was found in the hormone’s fractional rate of exit (k) from its IDV between the normals and acromegalics, either as a single group or all together. The respective values were 35 -t 2% min-’ in NA,

Table 4. “61-lnsulin Kinetic Parameters

of Diabetic Acromegalics

Patlent 3 IDV (liter/sq m)

2.8

k (% min-‘1 ER (ml/min

.

sq m)

k, (% min-‘1 MCR (ml/min

.

sq m)

k, (% min-‘1 RR (ml/min

§GH hg/ml)

m)

2.5

7

1.7

6

9

2.9

3.8

Mean SE

2.7 + 0.3.

38.0

36.0

40.0

37.0

24.0

35.0

1060.0

889.0

690.0

1079.0

905.0

925.0

22.0

22.0

29.0

612.0

550.0

501.0

16.0

- sq m)

i (min) TDV lter/sq

5

13.0

11.0

17.0 516.0 19.0

24.0 575.0 15.0

23.0

f 3.0 5 JO.Ot f 4.0

55 1 .o f 20.0’ 14.0 2 3.07

448.0

340.0

189.0

563.0

330.0

374.0

k 63.0’

33.5

23.0

14.3

52.0

32.0

31.0

+ 6.3*

20.5

12.6

7.1

26.9

18.3

17.1

+ 3.4*

13.7

11.1

9.6

2.4

9.6

9.3 + 1.9

See Table 2 for explanation of abbreviations.

lp

i 0.05 in comparison with normals.

tp < 0.01 in comparison with normals. $p c 0.05

in comparison with nondiabetic acromegalics.

$Mean GH concentration, measured from each 20-min sample during the 120 min of the disappearance curve of ‘Z61-insulin.

1016

MANESCHI,

Table 5. ‘2’l-lnsulin Kinetic Parameters Acromegalic

of all

Parameters of Insulin Secretion and Glucose Utilization

Patients Mean SE 2.6 ? 0.2f

IDV (liters/sq m) k (% min-‘) ER (ml/min

35.0

.

k, (% min-‘)

+ 60.0’

24.0

MCR (ml/min

.

+ 4.0

564.0

sq m)

k, (% mine’1 RR (ml/min

t 2.0

876.0

sq m)

+ 24.0*

12.0 + 4.0

* sq m)

317.0

f 46.0’

T (min)

23.5

TDV (liters/sq m)

13.3 + 2.5

$GH (ng/ml)

10.2 + 1.9

2 4.5

See Table 2 for explanation of abbreviations.

lp

< 0.05 in comparison with normals.

tp i 0.01 in comparison with normals. *Mean

GH

concentration,

measured

from

each

20-min

sample during the 120 min of the plasma disappearance curve of ‘251-insulin.

Mean residence time (t) and total plasmaequivalent distribution volume (TDV) were found to be significantly higher (p < 0.05) in DA than in normals (31.0 f 6.3 versus 17.8 _e 1.3 min, and 17.1 + 3.4 versus 8.4 + 017 Iiters/sq m). Conversely, NA showed values oft and TDV in the normal range, though not significantly different from those of DA, thus, when data from all acromegalics were compared to normals, no significant difference for i and TDV could be demonstrated. Table 6. Parameters

PLO, AND NAVALESI

Tables 6, 7, and 8 report the values of these parameters measured in normal subjects, NA, and DA. Compared to normals, both groups of acromegalic patients in the fasting state showed a significantly higher (p < 0.01) basal posthepatic insulin delivery rate (IDR), which was greater (p < 0.01) in DA than in NA (8.6 f 1.9 versus 4.8 + 0.7 mU/min - sq m). This resulted from higher IRI levels and from the greater MCR of the hormone in NA and DA. Figure 3 shows the pattern of glucose-stimulated posthepatic insulin secretion rate in the normal subjects. It can be seen that insulin secretion was characterized by a first burst occurring immediately after glucose administration, with a peak at 2-3 min and terminating after 6-7 min. This was followed by a second, more sustained, phase which returned to the basal levels by the end of the experiment. Figures 4 and 5 report the mean secretion curves of NA and DA. The insulin secretion curve of NA was higher than in the controls, although the normal pattern was maintained as both the acute and the late phases were similarly increased. In DA, while the total secretion was greater than that of normals, the acute phase, although present, was relatively reduced.

of Insulin Secretion

of Normal Subjects

Patient 1

FPG (mg/dl)

60.0

IRI (&Vml)

4.4

IDR (mU/min . sq m)

2.4

2

79.0

4.8 2.5

3

4

80.0

72.0

3.3 1.5

4.3 1.7

5

70.0

6.7 3.7

6

7

88.0

64.0

7.2 3.3

3.2 1.3

Mean SE

73.0

* 4.0

4.8 f 0.6 2.3 f 0.9

SecrlO-6) (mU/sq m)

276.0

346.0

229.0

102.0

367.0

127.0

177.0

232.0

t 39.0

Secr(6-90)

(mU/sq m)

351.0

714.0

633.0

278.0

1473.0

714.0

140.0

615.0

t 167.0

SecrtO-90)

(mU/sq m)

627.0

862.0

380.0

1840.0

841.0

317.0

847.0 f 194.0

First peak % of total A glucose (mg/dl) K, (% mine’) GA (g/d1

. min)

1060.0

31.0

30.0

27.0

20.0

20.0

187.0

132.0

158.0

235.0

193.0

1.67

2.33

1.49

4.0

1.6

1.45

15.0 232.0

1.60

1.41

56.0

28.0

219.0

194.0

1.34

+ 5.0 * 15.0

1.61 + 0.13

5.2

6.7

4.4

6.3

6.1

S,

19.2

23.0

25.5

10.0

16.5

6.3

22.7

17.7

2 2.7

9,

1.7

3.3

4.9

1.9

4.6

2.6

1.3

2.9

+ 0.5

s-

2.9

4.7

6.4

2.5

5.5

2.8

2.7

3.9 t 0.6

4.9 t 0.7

FPG. fasting plasma glucose; IRI, fasting plasma insulin concentration: IDR, basal posthepatic insulin delivery rate; Secr(O-6). of insulin secreted within the first 6 min; Secrt6-90).

amount

amount of insulin secreted from 6 to 90 min after glucose administration: % first

peak of total, amount of insulin secreted during the first 6 min as a percentage of the total insulin output: A glucose, difference between the peak glucose levels and the FPG; GA, glucose area above the fasting level; S, SecrlO-G)/IDR

x 6, amount of insulin secreted within the

first 6 min as a multiple of that delivered at basal rate during an equal time interval; S,, Secr(G-SO)/lDR 90.

x 84: S,, Secr(O-SO)/lDR

x

1017

KINETICS OF INSULIN IN ACROMEGALY

Table 7. Parameters

of Insulin Secretion

of Nondiabetic

Acromegalics

Patient 2

4

6

64.0

77.0

93.0

80.0

9.8

8.4

8.4

6.8

1 FPG (mg/dl) IRI (rU/ml)

- sq ml

IDR (mU/min

5.8

Secr(O-6) (mU/sq m)

4.7

4.0

Mean SE 78.0

f 6.0

8.3 f 0.6.

4.8

4.8 + 0.7’

285.0

529.0

454.0

733.0

500.0

f 93.0t

Secr(6-901

(mU/sq m)

1285.0

1691.0

2266.0

1587.0

1707.0

f 285.0*

Secr(O-90)

(mU/sq m)

1570.0

2220.0

2720.0

2320.0

2208.0

r 238.0”

18.0

24.0

17.0

32.0

23.0

147.0

234.0

157.0

85.0

156.0

0.9

0.88

1.13

First peak % of total A glucose (mg/dl) K, (% min-‘1

.

1.01

1.7

k 31.0 + 0.2

3.4

4.5

6.2

9,

8.2

22.0

16.2

25.5

17.8

+ 3.9

9,

2.6

4.9

5.6

3.8

4.2

f 0.7

ST /3 GH (ng/mO$

3.0

6.2

6.4

5.4

5.2 f 0.8

4.7

8.3

8.4

GA (g/d1

sq m)

23.5

1.5

r 3.0

3.9 k 1.0

11.2

i 4.1

See Table 6 for explanation of abbreviations. ‘p < 0.01 in comparison with normals. tp < 0.05 in comparison with normals. to, Mean GH concentration, measured from each lo-min sample during the 90 min of the i.v. GlT.

Total insulin output during the 90-min interval after glucose was higher than the normal mean value (847 + 194 mU/sq m) both in NA (2208 + 238) and in DA (1926 k 429). In normal subjects, the amount of insulin secreted during the first 6 min, i.e., during the acute phase, was 232 + 39 mU/sq m, and it accounted for 28 k 5% of the total insulin output. Seer (O-6) was significantly greater in NA (500 * 93, Table 8. Parameters

p < 0.05) in whom it was 23 f 3% of the Seer (CL90), whereas in DA it was smaller (174 + 39) than in controls and represented a significantly lower percentage of the total output (9 -e 1%). The secretion from 6-90 min was significantly greater both in NA and DA than in normals (1707 + 285 mU/sq m, and 1752 f 291 versus 615 + 167). GH levels were not significantly different in

of Insulin Secretion

of Diabetic Acromegalics

Patient

FPG (mg/dl) IRI (pU/ml) IDR (mU/min SecrIO-6)

.

sq m)

(mU/sq m)

7

3

5

168.0

128.0

14.9

18.6

9.1

8

9

98.0

104.0

112.0

10.8

18.9

14.5

10.2

5.4

271.0

91.0

167.0

253.0

Mean SE

8.3

9.8

89.0

122.0

* 12.0

15.5 + 1.5*t 8.6 ? 1.9.t 174.0

& 39.0t

Secr(6-90)

(mU/sq m)

2959.0

1159.0

1463.0

2337.0

841 .O

1752.0

2 391.0$

Secr(O-90)

(mU/sq ml

3230.0

1250.0

1630.0

2590.0

930.0

1926.0

+ 429.0$

8.0

7.0

10.0

10.0

10.0

138.0

89.0

108.0

145.0

221.0

0.59

0.52

1.01

0.82

1.15

GA (g/d1 . sq m)

6.0

4.0

2.7

5.3

5.0

4.6 f 0.6

S,

5.0

1.5

5.2

4.3

1.8

3.5 2 0.8.t

S*

3.9

1.4

3.2

2.8

1.2

2.5 ? 0.5

3.9

1.4

3.4

2.9

1.2

17.0

10.8

16.0

2.1

10.1

First peak % of total A glucose (mg/dl) K, (% min-‘1

?Gl-l (ng/ml)II

See Table 6 for explanation of abbreviations.

lp

< 0.01 in comparison with normals.

tp < 0.01 in comparison with nondiabetic acromegalics. $p < 0.05 in comparison with normals. $J < 0.05 in comparison with nondiabetic acromegalics. II@, Mean GH Concentration, measured from each IO-min sample during the 90 min of the i.v. GIT.

9.1 + 1.o*t 140.0 0.82

+ 23.0 + 0.1

l

2.6 2 0.55 11.2 * 2.7

1018

MANESCHI. PILO, AND NAVALESI

Fig. 3. Mean insulin secretion rate (lower curve). plasma glucose levels (middle curve), and plasma insulin levels (upper curve) in the 7 normal subjects during the i.v. GTT. The vertical bars indicate 1 SE.

the two groups of acromegalic patients (11.2 ? 4.1 in NA and 11.2 + 2.7 ng/ml in DA). The slight increase of GH levels found in DA after glucose administration was the result of a paradoxical rise of hormone concentrations in patients 3 and 7 after i.v. glucose. A linear positive correlation (r = 0.764. p < 0.05) was found in the 7 normal subjects between the basal insulin output (IDR) and the total amount of hormone secreted after glucose. This is in keeping with the results of Bagdade, Bierman, and Porte,” who found a linear positive correlation between the basal IRI levels and the insulin area after glucose administration in subjects with normal glucose tolerance. Therefore, in order to compare the incremental effects of glucose on insulin secretion above basal, Seer (O-6), Seer (6-90), and Seer (O-90) of the 3 groups of patients were expressed as multiples of their respective IDRs. When insulin secretion was expressed in this way, no difference was found in the acute phase (S,) between normals and NA (17.7 + 2.7 versus 17.8 t 3.9), while this was significantly reduced in DA (3.5 f 0.8, p < 0.01) in comparison with both NA and normals. There was no significant difference in the secretion from 6 to 90 min (S,) in the 3 groups, although NA had the highest and DA the lowest value. NA had a greater total secretion (S,) than

Fig. 4. Mean insulin secretion rate (lower curve). plasma glucose levels (middle curve). plasma insulin levels (upper curve), and Gli concentration (dashed line) in the 4 nondiabetic acromegalics during the iv. GTT. The vertical bars indicate 1 SE.

normals and DA, but it only reached statistical significance (p < 0.05) when compared with DA. It was not significantly different between normals and DA. The A glucose was greater in normals (194 ~fr 15 mg/dl) than in NA (156 + 3 1) and DA (140 +- 23); this was probably due to an enlarged glucose distribution volume which occurs in acromegalics, together with the fixed dose of glucose (0.33 g/kg body weight) which was infused in all groups. It is probably for the same reason that glucose areas (GA) were not different in the 3 groups. However, the glucose disappearance rate (KG) was greater in normals (1.61 + 0.13% mini’), than in acromegalics (1.13 t 0.2 in NA and 0.82 -t 0.1 in DA). DISCUSSION

The advantages and limitations of insulin kinetic studies with tracer hormone and deconvolution analysis of plasma concentration curves

KINETICS OF INSULIN IN ACROMEGALY

Fig. 5. Mean insulin secretion rate (lower curve), plasma glucose levels (middle curve), plasma insulin levels (upper curve). and GH concentration (dashed line) in the 5 diabetic ecromegalics. The vertical bars indicate 1 SE.

to determine insulin secretion in-humans have been reported in detail elsewhere.‘*” The fast initial distribution volume of insulin (IDV) can be considered to approximate the plasma volume (PV), although the IDV is slightly greater, due to some degradation of the hormone occurring before the complete mixing of the tracer hormone in the plasma.’ According to this, the enlarged IDV found in our acromegalic patients (61 ml/kg), in comparison with the normal value (48 ml/kg), may reflect an expansion of the plasma volume. Actually, Strauch et al.,” who measured the plasma volume in a large group of acromegalics using ‘251-albumin, found that PV was significantly greater than in normals (48 versus 43 ml/kg). Assuming that all the other factors influencing the extravascular diffusion of the hormone from IDV were constant, an enlargment of the PV would result in a greater rate of exit (ER) of insulin from IDV and of its two components: reversible (RR) and irreversible (MCR) exit rates. Alternatively, if factors other than a greater IDV were responsible for the higher RR and MCR of acromegalics, the ratios RR/IDV = k, and MCR/IDV = ki should be greater than normal. Therefore, the greater than normal k, of DA suggests an increase of vascular permeability, indeed, as much as IDV reflects PV, k, gives an estimate of vascular permeabili-

1019

ty. This is in keeping with the findings of increased vascular permeability in diabetesI and with our previous report of increased RR in nonketotic diabetes mellitus.’ The fact that k, of NA was similar to the normal value, indicates that their greater RRs were entirely due to the larger IDVs. The ki was not significantly different either in acromegalics or in normals, thus showing that the greater degradation of insulin (MCR) was accounted for by the larger IDV. In fact, a higher ER implies that a greater amount of hormone is made available to the extravascular catabolic sites. These sites responsible for the greater catabolism of insulin are thought to be the liver and the kidneys, indeed, in normal humans they account for most of insulin degradation’4.‘S and increases of both liver parenchyma1 massI and renal blood flowI have been reported in acromegaly. Futhermore, insulin clearance from peripheral tissues in acromegalits, measured in the forearms, was not different from normals,‘* and a direct effect of GH on insulin could be excluded, because it has been demonstrated in dogs” and in humans*’ that GH treatment does not modify the disappearance rate of insulin in vivo, and in vitro it reduces insulin degradation by rat renal and liver tissue.*’ From the pathophysiologic point of view, a 20% increase of MCR, if the secretion rate was unchanged, would lower the circulating levels of insulin, thus indirectly impairing glucose utilization. In acromegaly, therefore, together with the well recognized hyperinsulinemit effect of GH, the increased degradation of the hormone constitutes another factor why pancreatic secretion should be increased to achieve a normal glucose metabolism. Together with our previous finding of increased TDV in nonketotic diabetes mellitus,9 the larger total plasma equivalent distribution volume of insulin (TDV) found in DA, as compared with normals, points to a derangement of insulin distribution that seems to be related to diabetes mellitus. Nonetheless, the pathophysiologic meaning of larger TDV remains uncertain, as it may result from an enlargement of the physical distribution space, from accumulation of insulin in the extravascular space, or from reduced degradation at sites slowly exchanging with plasma, such as fat, skin, and muscle. In previous studies*,’ comparisons of insulin

1020

secretion between acromegalics and normals were based on IRI levels only. As the MCR of the hormone is actually 20% greater in acromegalics than in normals, the differences of insulin secretion were relatively underestimated. In our study, the determination of the “‘Iinsulin plasma disappearance curve has allowed us to measure the effective posthepatic insulin output in fasting conditions (IDR) and, by deconvolution analysis of the IRI curve after glucose, the reconstruction of the pattern of the glucose stimulated posthepatic insulin output. In the fasting state, NA had a twofold increase of IDR over normal values, with an increase of IRI levels of nearly the same extent. Their fasting plasma glucose (FPG). similar to that of normals (79 f 6 versus 73 k 4 mg/dl), shows that the greater insulin output was able to compensate both for the antiinsulin effect of GH and for the increased degradation of the hormone. In DA, on the contrary, the mean FPG was abnormally high (122 * 12 mg/dl) despite their even higher IDR and IRI values. This is in keeping with the results of Luft, Cerasi, and Hamberger,3 who also found higher basal IRI levels in diabetic than in nondiabetic acromegalits. Since GH levels and MCR values were similar in DA and NA, the higher FPG of DA implies another factor of insulin resistance that could not be overcome by the basal insulin output, although this was higher than in NA. A deficit of insulin receptors might be invoked to explain this additional factor of insulin resistance. However, none of I1 acromegalics with abnormal insulin receptors had diabetesz2 and in another 7 acromegalics, including 4 diabetics, no reduction of insulin receptors could be demonstrated.23 The additional factor of insulin resistance of DA is therefore due to some alterations of intracellular pathways of glucose metabolism independent from GH action. The temporal pattern of glucose stimulated posthepatic insulin secretion, which we have reconstructed in normals, was characterized by an acute phase occurring within the first 6-7 min, in agreement with the results of experiments performed in vitro with the isolated perfused pancreas,24 and in vivo with different to some of these techniques. 2s-27 According authors, the first rapid phase of insulin secretion plays an important role in the utilization of a glucose load; indeed, after i.v. glucose adminis-

MANESCHI,

PILO, AND NAVALESI

tration, a positive correlation was found between the disappearance rate of glucose and the amount of insulin released during the acute phase, and this phase was significantly reduced in diabetic patients, who showed a markedly reduced KG.” This hypothesis has been challenged by others, who could not find a correlation between the first phase of insulin secretion and the glucose disappearance rate after i.v. glucose.29 In our normal subjects, the correlation between the KG and the amount of insulin released downstream the liver in the acute phase was r = 0.681, with only a borderline significance (p < 0.1). However, when the glucose utilization was measured by the area under the glucose curve, a significant negative correlation was found with the acute phase, r = -0.863, p -c 0.05. Furthermore, no correlation existed between glucose utilization (expressed as KG or as area under the glucose curve) and the amount of insulin released from 6690 min or with the total insulin output. Therefore our data support the hypothesis that in normal subjects the first phase of insulin secretion is of great importance for the utilization of an intravenous glucose load. In NA the amount of insulin secreted during the acute and the late phase was greater than in normals. This is in keeping with the insulinogenic effect of GH3’ and with those reports of higher than normal IRI levels in nondiabetic acromegalics after glucose administration.3 The pattern of insulin secretion in NA was similar, however, to that of normals; moreover. when insulin secretion was expressed as a multiple of the IDR, the output of the acute phase in NA could be superimposed to that of normal subjects, and the difference in the late phase was cut off. This shows that the mechanisms by which glucose evokes insulin response in NA are, on the whole, similar to those of normal subjects. The comparison between acromegalics and normals on the utilization of the glucose load was made on the basis of KG only. In fact, the areas under the glucose curve were not comparable. In the fasting state NA achieved glucose levels similar to those of normals by their higher IDR, but after glucose they showed a lower KG (1.13 versus 1.6 1% min-‘) despite their massive insulin output. The difference of KG was only of borderline significance (p -c O.l), still, a reduction of glucose utilization after i.v. glucose was

1021

KINETICS OF INSULIN IN ACROMEGALY

reported by others.2*3 The interaction between GH and insulin seems to be different in the fasting state and after glucose load. This could be explained by a different sensitivity of the liver versus peripheral tissues to insulin and/or GH. In fact, these hormones have an opposite effect on both the hepatic output of glucose, which, in the postabsorptive state, is the only source of glucose,3’ and on the uptake of glucose by the tissues, which becomes the major determinant of glucose levels after an i.v. load.3’ In NA the slower KG suggests that the decrease of glucose uptake induced by GH3* cannot be completely corrected by insulin hypersecretion, while their normal FPG shows that the tendency to an increased hepatic glucose output induced by GH3* can be corrected by increased insulin secretion. Recently, however, it has been shown that in nondiabetic acromegalics, in vitro binding of insulin to its cell-surface receptors is inversely correlated to insulin concentrations and it becomes abnormal at insulin levels comparable to those obtained in vivo after glucose.** A similar phenomenon could provide another explanation for the reduced utilization of glucose observed in our NA after i.v. glucose. The time course of the glucose-induced insulin secretion in DA showed that the acute phase, although present, was blunted in comparison to that of normals and NA. On the contrary, no significant difference was found in the amount of hormone secreted after the initial burst and in

the total output. This is in keeping with previous reports of a deficient insulin response after i.v. glucose in diabetic acromegalics,3 and it resembles what has been found in patients with diabetes mellitus.2**33It seems therefore, that a selective deficit of the first phase of insulin secretion characterizes the pancreatic response to i.v. glucose in diabetes mellitus, both of the idiopathic type and in that associated with acromegaly. This is even more evident when insulin secretion was expressed as a multiple of its basal output. When expressed in this way, the difference in the first phase between DA and both NA and normals was amplified, while no significant difference was found in the insulin secretion of the late phase. In conclusion, in this study a distinction has been made between the features of insulin kinetics and secretion typical of acromegaly and those due to diabetes associated with acromegaly. Characteristic of acromegaly are: 1) a larger initial distribution volume of insulin, probably due to an expanded plasma volume; 2) an increased MCR of the hormone; and 3) a higher posthepatic delivery rate, both in the fasting state and after glucose. The distinctive features of diabetes associated with acromegaly are: 1) a larger total distribution volume of insulin; 2) an additional factor of insulin resistance independent from the effect of GH; and 3) a selective deficit of the acute phase of insulin release after i.v. glucose.

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6. Hales CN, Randle PJ: Immunoassay of insulin with insulin-antibody precipitate. Biochem J 88:137-146, 1963 7. Pennisi F: A fast procedure for radioimmunoassay of hGH. J Nucl Biol Med 12:137-138, 1968 8. Robin M, Saifer A: Determination of glucose in biologic fluids with an automated enzymatic procedure. Clin Chem 11:84&845,1965 9. Navalesi R, Pilo A, Ferrannini E: Kinetic analysis of plasma insulin disappearance in nonketotic diabetic patients and in normal subjects. J. Clin Invest 61:197-208, 1978 10. Pilo A, Ferrannini E, Navalesi R: Measurement of glucose-induced insulin delivery rate in man by deconvolution analysis. Am J Physiol233:E50&ES08, 1977 11. Bagdade JD, Bierman EL, Porte D Jr: The significance of basal insulin levels in the evaluation of insulin response to glucose in diabetic and nondiabetic subjects. J Clin Invest 46:1549-1557, 1967 12. Strauch G, Lego F, Therain F, et al: Reversible plasma and red cell volume increase in acromegaly. Acta Endccrinol (Kbh) 85:465478, 1977

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13. Parving HH, Munkgaard Rasmussen S: Transcapillary escape rate of albumin and plasma volume in short- and long-term juvenile diabetics. Stand J Clin Lab Invest 32:8 I 87. 1973 14. Madison LL: Roleof insulin in the hepatic handling of glucose. Arch Intern Med 123:28&292, 1969 IS. Rubenstein AH, Spitz I: Role of the kidney in insulin metabolism and excretion. Diabetes 17: 16 l-169, I968 16. Preisig R, Morris TQ, Shaver SC, et al: Volumetric, hemodynamic and excretory characteristics of the liver in acromegaly. J Clin Invest 45:1379-1387, 1966 17. lkkos D, Ljunggren J. Luft R: Glomerular filtration rates and renal plasma flow in acromegaly. Acta Endocrinol (Kbh) 21:226-236, 1956 18. Butterfield JH, Garratt CJ, Whichelow MJ: Periphera1 hormone action: studies on the clearance and the effect of (“‘I) lodo-insulin in the peripheral tissues of normal, acromegalic and diabetic subjects. Clin Sci 24:331-341, 1963 19. Pierluissi J, Norwich JK, Green GR, et al: Insulin kinetics in metasomatotropic diabetes. Metabolism 27:6170, 1978 20. Adamson U: Insulin-like and diabetogenic actions of human growth hormone (thesis). Stockholm, 1975 21. Mahler RJ, and Szabo 0: Early insulin sinergistic activity of growth hormone. Diabetes 18:550-555, 1969 22. Muggeo M, Bar RS, Roth J, et al: The insulin resistance of acromegaly: Evidence for two alterations in the insulin receptor on circulating monocytes. J Clin Endocrinol Metab 48:17-25, 1979 23. Archer JA, Gorden P, Gavin JR, et al: Insulin receptors in human circulating lymphocytes, applications to the study of insulin resistance in man. J Clin Endocrinol Metab 36~627-633, 1973 24. Curry DL, Bennett LL, Grodsky GM: Dynamics of

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insulin secretion in the perfused rat pancreas. Endocrinology 83572-584, 1968 25. Turner RC, Grayburn JA, Newman GB, et al: Measurement of insulin delivery rate in man. J Clin Endocrino1 Metab 33:279-286, 197 1 26. Cerasi E, Luft R: The plasma insulin response to glucose infusion in healthy subjects and in diabetes mellitus. Acta Endocrinol (Kbh) 55:278-304, 1967 27. Porte D Jr. Pupo AA: Insulin response to glucose: evidence for a two pool system in man. J Clin Invest 48:2309-23 19, 1969 28. Brunzell JD, Robertson RP, Lerner RL, et al: Relationship between fasting plasma glucose levels and insulin secretion during intravenous glucose tolerance tests. J Clin Endocrinol Metab 42:222-229, 1976 29. Reaven GM, Olefsky JM: Relationship between insulin response during the intravenous glucose tolerance test, rate of fractional glucose removal and the degree of insulin resistance in normal adults. Diabetes 23:45&459, 1974 30. Malaisse WJ. Malaisse-Lagae F, King S, et al: Effect of Growth Hormone on insulin secretion. Am J Physiol 215:423-428, 1968 3 I Felig P, Wahren J: Influence of endogenous insulin secretion on splanchnic glucose and aminoacid metabolism in man. J Clin Invest 50:17022171 I, 1971 32. Altszuler N, Steele R, Rathgeb 1. et al: Influence of growth hormone on glucose metabolism and plasma insulin levels in the dog. In Pecile A, Muller EE (eds): Growth Hormone, Proceedings of the First International Symposium, Excerpta Medica, International Congress Series No. 158.1973, ~~309-318 33. Lerner RL, Porte D Jr: Acute and steady-state insulin responses to glucose in nonobese diabetic subjects. J Clin Invest 51:1624-1631, 1972