Decreased hepatic insulin extraction in subjects with mild glucose intolerance

Decreased

Hepatic Insulin Extraction in Subjects Mild Glucose Intolerance Enzo Bonora. lvana Zavaroni,

with

Carlo Coscelli, and Ugo Butturini

The fact that hyperinsulinemia occurs in simple obesity and mild glucose intolerance has been well established. Altered hepatic insulin extraction may influence the levels of circulating hormone. The simultaneous measurement of insulin and C-peptide concentrations in peripheral blood enables an in vivo estimation of hepatic insulin removal. To evaluate hepatic insulin extraction, insulin and C-peptide responses to oral glucose were studied in 176 obese and nonobese subjects with normal, impaired. or diabetic glucose tolerance. Insulin levels as well as insulin incremental areas in glucose intolerant subjects were significantly higher than in weight-matched controls. The levels of C-peptide as well as C-peptide incremental areas were only slightly enhanced in subjects with impaired glucose tolerance, whereas they were reduced in subjects with diabetic tolerance. The molar ratios of C-peptide to insulin, both in the fasting state and after ingestion. of glucose, as well as the relationship between the incremental areas of the two peptides were used as measures of hepatic insulin extraction. They were significantly reduced in glucose intolerant subjects and, to a lesser extent, in nondiabetic obese subjects. These results indicate that peripheral hyperinsulinemia in subjects with simple obesity or impaired glucose tolerance is a result of both pancreatic hypersecretion and diminished hepatic insulin extraction. In subjects with a more severe degree of glucose intolerance, decreased hepatic insulin removal is the primary cause of hyperinsulinemia.

H

YPERINSULINEMIA is a well-recognized feature of simple obesity,‘,’ and mild glucose intolerance.3*4 Generally the explanation of its cause has been increased insulin secretion, even though the possibility of an alteration in hepatic degradation, kinetics, or metabolic clearance of the hormone has been raised for a long time. The studies undertaken so far have shown different and sometimes opposite results,5m24 but it has been largely accepted that hepatic degradation, kinetics, and the rate of metabolic clearance of insulin do not drastically differ between nondiabetic obese or mildly glucose-intolerant subjects and normal persons. Nevertheless, insulin removal by perfused liver from obese-hyperglycemic (ob/ob) mice and from spontaneously obese rats has been shown to be decreased.25,26 These two observations, along with the large extraction of insulin by the liver’*‘“*’ emphasize again that hyperinsulinemia in subjects with simple obesity or glucose intolerance may be secondary to an abnormal hepatic insulin metabolism. The most valid method to estimate the exact extent of hepatic esxtraction of insulin is the simultaneous measurement of the hormone concentrations in portal and hepatic veins and in the hepatic artery, along with the estimation of blood flow in these three vessels. Nevertheless, this technique is extremely invasive and not completely safe. Moreover, it cannot be used in

From the Istituto di Clinica Medica Generale e Terapia Medica. Parma University Medical School, Parma, Italy. Accepted for publication December 8, 1982. Supported by grant No. 80.00396.04 from the C.N.R. Address reprint requests to Dr. Enzo Bonora. Istituto di Clinica Medica Generale e Terapia Medica. Via Gramsci, 14. 43100 Parma, Italy. 0 I983 by Grune & Stratton, Inc. 0026-0495/83/3205-0004$01.00/0 430

metabolic studies involving a large number of subjects. The cleavage of proinsulin in the pancreatic betacell yields insulin and C-peptide, which are secreted into the portal blood in equimolar amounts*’ and consequently traverse the hepatic bed. In the course of this, about half the amount of insulin delivered to the hepatic bed is extracted by the liver.5s’4m2’ Unlike insulin, C-peptide is removed only to a minor extent by the liver,28-32 whereas most C-peptide is degraded by the kidney.29.33 Also the half-life of the two peptides is different, the half-life of C-peptide being longer than that of insulin..3’a33 Because of the aforementioned reasons, plasma C-peptide levels in peripheral blood seem to provide a more reliable picture of actual beta-cell secretion.3”36 Moreover, simultaneous measurement of insulin and C-peptide in peripheral blood has provided a noninvasive method for assessing hepatic insulin extraction.37 Two recent studies performed by this method in obese humans reached different conclusions. In 1979 Savage et a13*suggested that over the range of insulin concentrations normally observed in human subjects alterations in hepatic insulin extraction do not contribute to hyperinsulinemia. On the contrary, Faber et al39 more recently demonstrated that a decreased fractional hepatic extraction of insulin is a prominent feature of obesity and contributes to the hyperinsulinemia of obese subjects. These two reports referred to a limited number of nondiabetic obese subjects. Using in part the same methodology of Savage and Faber and their coworkers, we examined a large population of both obese and nonobese subjects whose glucose tolerance was normal, impaired, or diabetic. The aim of our investigation was to determine whether a diminished hepatic insulin Metabolism, Vol. 32, No. 5 (May), 1983

INSULIN

REMOVAL

IN

GLUCOSE

439

INTOLERANCE

extraction may contribute to the hyperinsulinemia observed in obesity and mild glucose intolerance. SUBJECTS AND METHODS In this study, I76 subjects (93 females and 83 males) with fasting plasma glucose concentrations lower than 140 mg/lOOml (7.8 mmoie/liter) were examined. They had a mean age of 34.5 + 3.2 years (range: 14-55). Their percentage of ideal body weight (% IBW) ranged from 78% to 251% Ideal body weight was estimated according to the method of Lorentz et aLa Subjects were considered obese when their weight exceeded their IBW by 25% and nonobese when their weight did not exceed their IBW by 10%. Subjects with hepatic or renal disorders were excluded from the study. All subjects received a diet containing more than 250 gm carbohydrate daily for J minimum of three days before the study. After a I2-hour overnight fast, each subject received an oral glucose load (100 gm). Blood samples were obtained from an antecubital vein through an indwelltng catheter kept patent by a slow saline infusion before and 30, 60. i 20, and I80 minutes after the glucose ingestion. Blood was drawn tnt,) chilled plastic tubes containing EDTA and aprotinin, centriI’uged at 4 C. and the plasma stored in a frozen state until analysis. Plasma glucose was measured by a gluco-oxidase method and insulin dnti C-peptide by radioimmunoassays, as described by Hales and Kandle4’ and Kaneko et aL4’ respectively. Hepatic insulin extraction was assessed in three ways: (I) from the molar ratio between C-peptide and insulin in the fasting state; (2) from the difference between the mean of all the molar ratios of C-peptide to insulin after glucose ingestion and the fasting Cpeptide/insulin molar ratio; and (3) from the difference between the incremental areas of C-peptide and insulin divided by the incremental irea of C-peptide.‘Y C;lucose tolerance was estimated according to the 1979 National Diabetes Data Group criteria.J’ Subjects with glucose levels between I40 and 200 mg/lOO ml at 120 minutes and higher than 200 mg/lOO ml at 30 or 60 minutes after the glucose ingestion were considered to hav? an impaired glucose tolerance. Subjects with plasma glucose concentrations higher than 200 mg/ 100 ml both at 120 and at 30 or 60 minutes after the glucose load were considered to have a diabetic glutose tolerance. In the figures subjects with normal body weight and glucose tolerance are indicated as NN, subjects with normal bodv weight and impaired glucose tolerance as NIGT. and subjects with normal body weight and diabetic tolerance as ND. Analogously, subjects with obesity and normal, impaired, or diabetic glucose tolerance are indicated as ON, OIGT. and OD. respectively. 1r1order to minimize changes in the distribution spaces of glucose, insulin, and C-peptide as a result of differences in body weight, weight-matched groups of subjects were taken into consideration in our :,tudy. T?e analysis of variance (ANOVA) and the Student t-test for unp;.ired and paired data were used for statistical analysis of the resu’ta. A I data are presented as mean + SEM. Table 1. Characteristics

RESULTS

On the basis of their percentage of ideal body weight, 72 subjects of the study were classified as nonobese and 104 as obese. According to the glucose concentrations shown during oral glucose tolerance testing (OGTT), they were scored as having a normal (n = 84), impaired (n = 49), or diabetic (n = 43) glucose tolerance. Therefore. the study population was divided into three classes of nonobese subjects with normal, impaired, or diabetic tolerance, and three classes of obese individuals with normal, impaired, or diabetic glucose tolerance. Table 1 summarizes the characteristics of these six groups with regard to age and body weight. The glucose, insulin, and C-peptide responses to oral glucose are depicted in Figure I. A clear-cut difference in plasma glucose concentrations was observed, as expected, in the three groups, considering subjects either nonobese or obese. Subjects with impaired and, to a lesser extent, diabetic glucose tolerance after oral glucose exhibited insulin levels significantly higher than those of weight-matched individuals with normal glucose tolerance at many time points of the test. In contrast, C-peptide responses to glucose in weightmatched groups with normal or impaired glucose tolerance were quite close, whereas in subjects with a diabetic tolerance, mainly in the presence of obesity, the C-peptide levels were lower than in weightmatched normotolerant individuals. These results were confirmed by the evaluation of insulin and C-peptide incremental areas after glucose. Subjects with both impaired (P < 0.001) and diabetic (P < 0.05) glucose tolerance exhibited insulin incremental areas significantly higher than weight-matched normotolerant individuals. In contrast, the C-peptide incremental areas were only slightly higher than in controls in subjects with impaired glucose tolerance (P = ns.). and lower than in controls in individuals with a more severe glucose intolerance (P = ns. among nonobese subjects; P -c0.005 among obese subjects) (Figs. 2 and 3). Fasting molar ratios of C-peptide to insulin tended to decrease with the impairment of glucose tolerance toward diabetes both within the nonobese and the of the Study Population

Number

Age (years)

Percentage of Ideal Body Weight

Body

GllX0se

of

Group

Weight

Tolerance

Pat,allts

1

Normal

Normal

30

35.8

+ 2.3

15-55

2

Normal

Impaired

23

40.3

k 2.1

3

Normal

Diabetic

19

42.0

+ 2.6

4

Obese

Normal

54

38.4

k

1.6

Mean f SEM

Range

Mean 2 SEM

Range

99.3

+

1.5

87-109

21-54

99.0

i

1.5

86-108

23-55

100.2

?

1.8

78-109

14-55

166.7

+ 4.9

125-251

5

Obese

Impaired

26

42.5

f

2.1

15-55

164.7

k 7.0

132-229

6

Obese

Diabetic

24

44.7

t

1.6

28-55

162.4

+ 7.3

127-232

BONORA ET AL

440

PG -Vi .

OBESES

NORMALWEIGHTS

12. .

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IRI

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1.0.

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0.6.

0.6. .

.

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obese class (P < 0.05 in impaired glucose tolerant versus nondiabetic obese subjects; P -e0.005in diabetic tolerant versus nondiabetic obese individuals; Fig. 4). After the glucose load there was a significant increase of the molar ratio of C-peptide to insulin in both nonobese and obese subjects with normal glucose tolerance (P -c0.001). In groups with glucose intolerance, the increase in the ratio was not as great in nonobese subjects and was completely absent in obese individuals (Table 2). This phenomenon was more evident in subjects with mild glucose intolerance compared with subjects with a more severe glucose intolerance (Fig. 5).

l&J

min

Fig. 1. Plasma glucose (PG), insulin (VW, and C-peptide (CPR) levels during oral glucose tolerance testing. * = P < 0.05 in normotolerant versus impaired glucose tolerant subjects. * = P -c 0.05 in normotolerant versus diabetic glucose tolerant subjects.

The ratios between the difference of incremental areas of C-peptide and insulin and the incremental area of C-peptide in the six groups are reported in Fig. 6. There were significant differences between the glucose intolerant patients and weight-matched controls. Moreover, it is very interesting that the values obtained in individuals with impaired glucose tolerance were lower than those observed in subjects with diabetic tolerance. DISCUSSION

AND

CONCLUSIONS

In healthy humans, in the last phase of an OGTT the molar ratio of C-peptide to insulin tends to become

441

INSULIN REMOVAL IN GLUCOSE INTOLERANCE

3.9

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troversial, as other investigators have reached different results and conclusions. Waldhausl et a14’ failed to observe any variation in insulin removal by the liver during OGTT, and the same conclusion was suggested by Savage et al.38 Conversely, a marked reduction in the percentage of insulin that is extracted by the liver after glucose was reported by Kaplan and Madison,48 Honey and Prince,49 and Trandberg and Thorell,” but in their studies the route of glucose administration was different. In our study, the molar ratios of C-peptide to insulin increased after oral glucose in both nonobese and obese subjects with a normal glucose tolerance. Within the limitations of the methodology, this may suggest increased hepatic insulin extraction after oral glucose. In contrast, in glucose intolerant groups the Cpeptidelinsulin ratios did not change after glucose ingestion; in these subjects the increase of hepatic insulin removal after glucose was inhibited. Moreover, the molar ratios of C-peptide to insulin after glucose as well as the relationship between the incremental areas

i . . l

553.

..

.

:

5.0.

l

0 IGT

.

.

o”o

Fig. 2. Plasma insulin incremental areas after glucose load (nmole x l-’ x 160 min-‘I. ANOVA: F = 30.16, P < 0.01 in nonobese and F = 20.42. P-c 0.01 in obese subjects. * = P < 0.001 in normotolerant versus impaired glucose tolerant subjects. -b = P <: 0.05 in normotolerent versus diabetic glucose tolerant subjects.

higher than in the fasting state, since C-peptide levels remain elevated after those of insulin have returned toward basal levels.35.36.39This occurrence is generally accepted to be a consequence of the lower metabolic clearance rate29 as well as the longer half-life3’,33 of C-peptide in comparison with insulin. The phenomenon, however, may be also due to an increased hepatic extraction of insulin after oral glucose. There has been controversy in the literature concerning the effect of glucose on hepatic extraction of insulin. In a recent report, Jaspan et al44 demonstrated that following glucose ingestion concomitant with increased insulin delivery to the liver there is a striking increase in hepatic insulin extraction. This report is in accordance with previous studies by Erwald et al,” Kaden et al,16 Waddell and Sussman,45 Field et al,46 and also seems to agree with the results obtained by Faber et al39 in their nonobese subjects. The problem, however, is still con-

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Fig. 3. Plasma C-Peptide incremental areas after oral glucose (nmole x I-’ x 160 min-‘). ANOVA: F = 4.23, P < 0.01 within nonobese and F = 446, P < 0.01 within obese subjects. * = P i 0.005 in normotolerant versus diabetic tolerant obese subjects.

442

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of the two peptides were significantly lower in glucose intolerant individuals than in weight-matched controls. These results support the hypothesis that subjects with impaired or diabetic glucose tolerance had decreased fractional hepatic extraction of insulin after oral glucose. Furthermore, among obese subjects impaired insulin removal by the liver seems to be present also in the fasting state, since the fasting molar ratios of C-peptide to insulin of obese glucose-intolerant individuals were significantly lower than those of weight-matched controls.

Fig. 4. Molar ratios of C-peptide to insulin in fasting state. ANOVA: F = 0.85, P = n.s. within nonobese and F = 8.47, P < 0.01 in obese subjects. * = P < 0.05 in normotolerant versus impaired glucose tolerant obese subjects: b = P < 0.005 in normotolerant versus diabetic glucose tolerant obese subjects.

Subjects with simple obesity, as a group, did not show significant differences with respect to nonobese healthy individuals in C-peptide/insulin molar ratios after glucose or in the relationship between C-peptide and insulin incremental areas. However, referring to the slower metabolic clearance of C-peptide compared with insulinz9 and to the well established beta-cell hypersecretion in simple obesity,‘,’ higher molar ratios of C-peptide to insulin after a glucose load and higher differences between C-peptide and insulin incremental areas would have been expected in this group com-

Table 2. Change in the Molar Ratio of C-Peptide to Insulin During Oral Glucose Tolerance Testing B

Group

M

D

P

NonobeseSubjects Normaltolerance

3.20

k 0.32

5.05

*

+ 1.87

k 0.33

Impaired

3.15

+ 0.36

3.46

‘- 0.55

+0.31

k 0.57

N.S.

2.62

+ 0.23

3.35

t

+0.73

+- 0.22

N.S.

0.001

Diabetic Obese

tolerance

tolerance

Impaired Diabetic

tolerance

l P < 0.05 -c 0.005

0.28

0.001

tolerance

ratio;

M

=

mean

of all the

3.89

k 0.31

5.45

+ 0.54

+ 1.62

+ 0.43

2.89

+ 0.28’

2.74

+ 0.26

-0.15

+ 0.20

N.S.

2.34

k 0.20t

2.41

+ 0.19

f0.06

f

N.S.

ratios

following

data.

tP

0.50

Subjects

Normal

B = fasting

tolerance

versus versus

normotolerant normotolerant

obese obese

subjects. subjects.

glucose

ingestion;

D = difference

between

M and

0.19

B; P =

significance

of r-test

for paired

443

INSULIN REMOVAL IN GLUCOSE INTOLERANCE

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. . .

7.

b

5.

l

:

. 4. t 3. :

.

:

l

ON

oh

-3.

. -4.

.

NN

N

;*GT I&

CtD

Fig. 5. Differences between the mean of all molar ratios of C-peptide to insulin after glucose loading and the fasting ratio of C-peptide to insulin in each individual. ANOVA: F = 3.64, P < 0.05 in nonobese and F = 6.26, P < 0.01 in obese subjects. * = P-c 0.05 in normotolerant versus impaired glucose tolerant subjects; * = P < 0.05 in normotolerant versus diabetic glucose tolerant subjects.

pared with the group of healthy subjects. Thus, even in simple obesity, a reduced hepatic insulin removal may be present as also suggested by Faber et a1.39Nevertheless, the hyperinsulinemia found in nondiabetic obese subjects to a greater extent is the consequence of true pancreatic hypersecretion, since they also showed an elevated C-peptide response to oral glucose. The results we have obtained in this study are also compatible with an hypothesis of an altered metabolic clearance rate of C-peptide in obese and glucose intolerant subjects. C-Peptide metabolism has not been investigated in these categories of individuals. Most C-peptide is extracted by the kidneys,29*33 and the glomerular filtration rate in obese subjects is elevated as in insulin-dependent diabetics.” In the latter, however, the metabolic clearance rate of C-peptide is the same as that in normal subjects.33 The renal extraction of C-peptide correlates linearly with the levels of the

peptide in the renal artery,29 and the saturation is achieved only for extremely high C-peptide concentrations not obtainable in vivo.52 Kuzuya et ai53 observed high C-peptide concentrations in peripheral blood as well as low C-peptide urinary extraction rates in patients with renal failure with a positive and significant correlation between plasma C-peptide and serum creatinine. No subject investigated in this study had renal diseases or a reduction in glomerular filtration rate, however. The finding by Kilhl et a13” and by Canivet et a13’ that the liver does extract C-peptide to a small extent suggests that changes in hepatic removal of the peptide may contribute to variations in its circulating levels. There should be no metabolic need for the liver to bind or remove C-peptide from the circulations4 but there might be changes in the amount of C-peptide nonspecifically trapped on its passage through the liver. This phenomenon should account for the high C-peptide levels observed in cirrhosis of the 1iver.j' At any rate, no subject in this study had hepatic disorders. Last, as the metaboiic clearance rate of C-peptide did not vary at different peptide concentrations in a constant-infusion study,33 a significant concentration dependency of C-peptide metabolic clearance rate seems improbable. For all these reasons, it is unlikely that C-peptide mebolism is altered in subject with obesity or glucose intolerance or both. On the other hand, an impaired C-peptide metabolism seems improbable also, as in this case in glucose intolerant subjects the ratios and relationships of C-peptide to insulin would be higher. Indeed, a concomitant decrease in the uptake of both peptides by the liver would partially annihilate that reduction in the ratios and relationship of C-peptide to insulin caused by the impaired removal of insulin alone. If we assume that the metabolism of C-peptide is not altered, the high levels of circulating insulin in peripheral blood should be a consequence of a decreased hepatic or extrahepatic metabolic clearance of the hormone. Since hepatic insulin catabolism accounts for the major fraction of the overall metabolic clearance of insulin, the differences we found between normal, obese, and mildly diabetic subjects should primarily reflect changes in hepatic insulin extraction. Under normal physiologic conditions, it has been estimated that about one-half of the extrapancreatic insulin is actually bound to hormone receptors in liver, muscle, adipose tissue, and other tissues.“j Most of the insulin degradation has been demonstrated to follow hormone-receptor binding.5ti1 Obesity and mild glucose intolerance are well known insulin-resistant states in which a reduced binding of insulin to its own receptors is present.2d According to the relationship between insulin degradation and the status of insulin

444

BONORA ET AL

.

1 .o-

.

4:

0.8.

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0.6.

0.7.

0.6.

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ob

Fig. 6. Difference between C-peptide and insulin incremental areas divided by C-Peptide area in each individual. ANOVA: F = 10.00, P < 0.01 in nonobese and F = 6.32, P < 0.01 in obese subjects. * = P < 0.001 in normotolerant versus impaired glucose tolerant subjects; Q = P -c 0.006 in normotolerant versus diabetic glucose tolerant obese subjects.

receptors, a receptor-dependent reduction of hepatic insulin degradation might be suggested.62 This may contribute to the hyperinsulinemia in obese and mildly glucose intolerant subjects. This would agree with the recent report by Flier et a1,63who demonstrated an association between marked target-cell resistance to insulin and impaired in vivo insulin clearance, suggesting an important role for receptor-mediated pathways in insulin clearance. On the other hand, data from Jaspan et a144are consistent with the postulate that hepatic insulin extraction may be related to the extent of insulin action. This may explain the increased hepatic removal of insulin after a glucose load in healthy humans coincident with increased hepatic insulin action,44 as well as the reduction of insulin

extraction by the liver that occurs in glucose intolerant, insulin-resistant subjects. In conclusion, subjects with mild glucose intolerance and, to a lesser extent, individuals with simple obesity seem to have a decreased hepatic insulin extraction. This impairment of insulin removal from the blood may be caused by a reduced function of hepatic insulin receptors. In subjects with simple obesity or impaired glucose tolerance in which a pancreatic hypersecretion is actually present, the phenomenon contributes only in part to the peripheral hyperinsulinemia. In individuals with a more pronounced glucose intolerance, decreased hepatic insulin extraction is the primary and sole cause of the high concentrations of circulating insulin after a glucose load.

-0.6.

.

.

*

NN

NIGT

l

ND

ON

OIGT

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by liver.

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