IT IS WELL recognized that in subjects with noninsulin

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1 X/98/$03.00/0 Vol. 83, No. 2 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1998 by The Endocrine Society Disproportionately Elevated Proinsulin Levels Reflect the Degree of Impaired B Cell Secretory Capacity in Patients with Noninsulin-Dependent Diabetes Mellitus* MICHAEL E. RØDER, DANIEL PORTE, JR., ROBERT S. SCHWARTZ, AND STEVEN E. KAHN Divisions of Metabolism, Endocrinology, and Nutrition and Gerontology and Geriatric Medicine (R.S.S.), Department of Medicine, Veterans Affairs Puget Sound Health Care System, Harborview Medical Center (R.S.S.), and University of Washington, Seattle, Washington ABSTRACT An increased proportion of fasting proinsulin (PI) relative to immunoreactive insulin (IRI; increased PI/IRI) occurs in noninsulindependent diabetes mellitus (NIDDM). To determine whether the magnitude of the increase in PI/IRI is an indicator of the degree of reduced B cell secretory capacity, we measured fasting plasma glucose, PI, IRI, and PI/IRI and related them to maximal B cell secretory capacity (AIRmax) in 9 subjects with NIDDM [age, 61 3 yr; body mass index (BMI), kg/m 2 ; duration of NIDDM, yr; mean SEM] and in 10 healthy subjects matched for age and BMI (age, 61 6 yr; BMI, kg/m 2 ). AIRmax was quantified as the incremental insulin response to iv arginine at maximal glycemic potentiation (plasma glucose 25 mmol/l). Mean fasting plasma glucose was mmol/l (range, 7.5 Received February 21, Revision received October 8, Accepted October 17, Address all correspondence and requests for reprints to: Michael E. Røder, M.D., Rigshospitalet, Department of Nephrology and Endocrinology P, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. address: mroder@login.dknet.dk. * This work was supported by the Department of Veterans Affairs; the American Diabetes Association; the Danish Medical Research Council; NIH Grants DK-12829, DK-17047, and AG-08673; and the Bernhard and Marie Klein Foundation mmol/l) in NIDDM subjects and mmol/l in the controls. Fasting PI was higher in NIDDM ( ) than in controls ( pmol/l; P 0.01), but IRI levels were similar ( vs pmol/l; P NS). The PI/IRI ratio was significantly elevated in NIDDM compared to control subjects ( % vs %; P 0.01). After elevation of the glucose level to mmol/l (NIDDM) and mmol/l (controls), AIRmax was quantified as pmol/l in NIDDM and pmol/l in controls, (P 0.001). The PI/IRI ratio correlated inversely with AIRmax in the NIDDM patients (r 0.76; P 0.01). We conclude that the magnitude of the elevation in fasting PI/IRI is related to the reduction in AIRmax. Thus, the fasting PI/IRI ratio appears to be a marker of the degree of reduced AIRmax in NIDDM. (J Clin Endocrinol Metab 83: , 1998) IT IS WELL recognized that in subjects with noninsulin dependent diabetes mellitus (NIDDM), basal proinsulin immunoreactivity (PI) contributes approximately 2 3 times more to insulin immunoreactivity (IRI) than in normal healthy subjects (1 3). Thus, although fasting IRI has been found to be normal or even elevated in NIDDM (4), PI has been found to be disproportionately elevated relative to the IRI concentration. This alteration is not likely to be due to increased peripheral clearance of insulin, because the PI/IRI ratio is also elevated after acute B cell stimulation in subjects with NIDDM (5, 6). Further, when using C peptide as an index of B cell peptide release, the PI/C peptide ratio has been found to be elevated in NIDDM (7). The changes in B cell function in NIDDM have been systematically evaluated. All NIDDM subjects have absent or nearly absent acute insulin responses to glucose (AIRglucose) (8), a measure that is independent of glycemia in NIDDM. On the other hand, the acute insulin response to arginine, which is glucose modulated, is reduced throughout the glycemic range in NIDDM compared to that in weight- and age-matched normal subjects (9). The maximal potentiating effect of glucose on the acute insulin response to arginine (AIRmax) is a measure of the maximal insulin secretory capacity, which is reduced in NIDDM (9). In both Pima Indians and Japanese-Americans with NIDDM (2, 10), the fasting PI/IRI ratio was correlated with the degree of hyperglycemia. In contrast, an increased PI/IRI ratio was observed in a streptozocin model of subclinical diabetes (11) and in prediabetic, normoglycemic siblings of subjects with insulin-dependent diabetes who have impaired first phase insulin responses to glucose (12). Because B cell function, peripheral insulin sensitivity, and hepatic glucose production are all potentially of importance in determining the degree of hyperglycemia in NIDDM (4), the relationship between PI/IRI and hyperglycemia could potentially be explained by an association of the PI/IRI ratio with any of these three factors. As mentioned, the PI/IRI ratio is altered in NIDDM, but no data are available addressing whether this measure reflects the degree of reduced B cell secretory function. In this study we sought to determine whether the relationship between fasting glycemia and PI/IRI is due to an association with impaired B cell secretory capacity. Therefore, we measured the fasting PI and IRI levels and AIRmax in NIDDM subjects and in normal subjects to assess whether a relationship exists between this quantitative measure of B cell function and the PI/IRI ratio. 604

2 PROINSULIN AND B CELL FUNCTION IN NIDDM 605 Subjects and Methods Subjects Nine subjects with a history of NIDDM and 10 healthy subjects with no first degree relatives with diabetes mellitus and not receiving any medication participated in the study. Portions of the data from the control subjects have been published previously (13). The study was approved by the human subjects review committee at the University of Washington, and all participants gave informed consent. The known duration of NIDDM in the subjects was yr, and 7 subjects were being treated with sulfonylureas (6 with glyburide and 1 with glipizide). None of the subjects had previously been treated with insulin. In the subjects receiving hypoglycemic medication, this was discontinued at least 96 h (4 days) before the study. Neither the NIDDM subjects nor the normal subjects had any clinical evidence of kidney, hepatic, or cardiac disease. Study procedure All subjects were studied in the morning after a 10-h overnight fast. An iv line was established in each forearm: one for glucose infusion and arginine injection and one for blood sampling. The forearm used for blood sampling was wrapped in a heating pad to arterialize the samples. Thirty minutes after placement of the iv lines, three basal samples ( 15, 5, and 1 min) were obtained for PI and IRI measurements. At time zero, a variable rate 10% dextrose infusion was commenced to raise and clamp the plasma glucose level above 25 mmol/l over a period of 45 min. Necessary adjustments to the glucose infusion rate were made based on frequent bedside glucose measurements (Beckman, Palo Alto, CA). A glucose level greater than 25 mmol/l has been demonstrated to allow for the determination of maximal glycemic potentiation of the insulin response to the nonglucose secretagogue arginine (9). Prestimulus samples for IRI were drawn 40 and 45 min after commencement of the dextrose infusion. Then, 5 g arginine were injected iv over 30 s, and additional blood samples were drawn 2, 3, 4, and 5 min after the injection was completed. Assays and calculations PI was assayed by a previously described RIA that measures both intact proinsulin as well as proinsulin conversion intermediates [des(31,32)-, split(32 33)-, des(64,65)-, and split(65 66)-proinsulin) with 100% efficiency (14). The detection limit of this assay is 2 pmol/l, and it has intra- and interassay coefficients of variation of 10% and 14%, respectively. IRI was also measured by RIA (15). The antibody used in this IRI assay also cross-reacts 100% with proinsulin and its conversion intermediates (14). The detection limit of this assay is 10 pmol/l, with intra- and interassay coefficients of variation of 5% and 8%, respectively. The incremental IRI response to arginine at AIRmax was calculated as the mean of the samples drawn at 2, 3, 4, and 5 min minus the average of the two prestimulus values. Statistical analysis All data are expressed as the mean sem. Differences between the groups were tested by Student s two-tailed unpaired t test and by Mann- Whitney rank test. There were no differences in significance using either the parametric or the nonparametric test. The results presented are those obtained using the parametric tests. Potential relationships were analyzed using linear regression. Potential nonlinear relationships (16) were not analyzed due to the limited size of the material. Log transformation of the data did not change the results. P 0.05 was considered statistically significant. Results The clinical characteristics of the individual NIDDM subjects and normal subjects are listed in Table 1. Gender distribution was similar in the two groups, with all women except one in the NIDDM group being postmenopausal. The mean age of the NIDDM group was 61 3 yr, and the body mass index (BMI) was kg/m 2 with the normal TABLE 1. Clinical characteristics of patients and controls Subject no. Sex Age (yr) BMI (kg/m 2 ) DM duration (yr) DM treatment Patients 1 F Glyburide 2 F Diet 3 F Glipizide 4 M Glyburide 5 M Glyburide 6 F Diet 7 M Glyburide 8 M Glyburide 9 F Glyburide Controls 1 M M M F M M M F F F M/F, male/female. subjects being well matched for these two parameters (age, 61 6 yr; BMI, kg/m 2 ). In the subjects with NIDDM, the mean fasting plasma glucose concentration was (range, ) mmol/l, PI was (range, 14 60) pmol/l, and IRI was (range, ) pmol/l. Thus, the fasting PI/IRI ratio was % (range, 19 51%). The normal subjects had significantly lower mean fasting glucose [ (range, ) mmol/l], fasting PI [ (range, ) pmol/l], and fasting PI/IRI [ % (range, 10 17%; all P 0.01]. The fasting IRI concentration in the normal subjects was similar to that in the individuals with NIDDM [ (range, ) pmol/l; P NS]. During the glucose clamp, the blood glucose level was raised to mmol/l in the subjects with NIDDM and to mmol/l in the normal subjects. At this glucose level, AIRmax was quantified as (range, ) pmol/l in the NIDDM group and was more than 3-fold higher in the normal subjects [ (range, ) pmol/l; P 0.001]. When examining the NIDDM subjects separately, the fasting PI/IRI ratio correlated inversely with AIRmax using linear regression (r 0.76; P 0.01). This linear fit is shown in Fig. 1. Thus, in the NIDDM subjects, 58% of the variance in PI/IRI is explained by B cell secretory capacity. Analyzing the normal subjects separately showed no significant correlation between PI/IRI and AIRmax. In the NIDDM subjects there tended to be an inverse correlation between the fasting plasma glucose level and AIRmax (r 0.58; P 0.07). This curve fit is illustrated in Fig. 2. There was no relationship when only the normal subjects were examined. As described previously (2, 10), a positive relationship between fasting PI/IRI and fasting plasma glucose was demonstrated, but only when all 19 subjects were analyzed together (r 0.79; P ). However, when the control subjects were excluded from this analysis, the relationship failed to reach

3 606 RØDER ET AL. JCE&M 1998 Vol 83 No 2 FIG. 1. The relationship between the fasting PI/IRI ratio and AIRmax in the nine NIDDM subjects. The fitted curve represents a linear regression fit between the two variables (r 0.76; P 0.01). FIG. 2. The relationship fasting plasma glucose and AIRmax in the nine NIDDM subjects. The fitted curve represents a linear regression fit between the two variables (r 0.58; P 0.07). significance (r 0.33; P NS). There tended also to be an inverse relationship in the NIDDM subjects (r 0.48; P 0.16) between the fasting PI level and AIRmax, although it was not as strong as that between the PI/IRI ratio and AIRmax. As expected, a positive linear relationship was found in the normal subjects between fasting IRI and AIRmax (r 0.69; P 0.05), as both are dependent on insulin sensitivity (10, 16). However, this was not the case when only the NIDDM subjects were examined (r 0.23; P NS). Discussion It is well recognized that an elevated fasting PI/IRI ratio is a feature of NIDDM. This abnormality is present in a number of different populations with NIDDM (2, 3, 5 7, 10, 17). Absolute PI and IRI levels have been shown to be elevated under conditions of increased B cell secretory demand, without an increase in the PI/IRI ratio (10, 11, 18, 19). Thus, it has been suggested that in NIDDM, the PI/IRI ratio may reflect B cell dysfunction (20, 21), but this hypothesis has never been directly tested. This study represents the first demonstration that the fasting PI/IRI ratio and the degree of reduced B cell secretory function in NIDDM are related. In this study B cell function was quantified as the AIRmax, a measure known to be markedly reduced in subjects with NIDDM (9). This measure was inversely related to the fasting PI/IRI ratio, suggesting that in NIDDM, the fasting PI/IRI ratio provides a measure of the degree of reduced B cell secretory capacity. We have previously demonstrated a relationship between fasting glycemia and the PI/IRI ratio in two large cohorts of NIDDM subjects (2, 10), and the present findings provide an explanation for those observations. It appears that the degree of elevation in the PI/IRI ratio and thus the level of fasting hyperglycemia are related to the severity of B cell insufficiency to secrete insulin. This is supported by calculating AIRmax by true insulin values (subtracting poststimulus proinsulin values) in the NIDDM subjects from the present study. We found a relationship between true AIRmax and fasting PI/IRI (r 0.74; P 0.02) in the same order as previously calculated. The significant relationship between fasting plasma glucose and AIRmax that we observed suggests that the degree of impairment in B cell function is, in fact, a determinant of the degree of hyperglycemia, as previously suggested (22). Whether a progressive decrease in B cell function over time in NIDDM (or in pre-niddm) is associated with a progressive increase in PI/IRI cannot be determined from this crosssectional study. However, several studies from our laboratory using the same assays for PI and IRI (2, 6, 10) as well as work from other laboratories (5, 17) have consistently found a PI/IRI of 25 40% in different NIDDM populations. In our recent study of Japanese-American men, we found a modestly elevated fasting PI/IRI at the time of entry into the study in those subjects who later developed NIDDM (23). Similar findings have been reported in two other populations (24, 25). Since the PI/IRI ratio was less than 20% at baseline in those individuals with normal or impaired glucose tolerance who subsequently progressed to NIDDM (23), a progressive increase in PI/IRI probably takes place as B cell function declines and glycemia increases. Seven of the patients in this study were treated with sulfonylureas, which were withdrawn at least 4 days (96 h) before the study. This is sufficient time to clear these medications from the circulation (at least eight half-lives), but a biological effect from recent sulfonylurea treatment on B cell function cannot be definitively excluded. However, data from our group (26) and others (27) suggest that sulfonylurea treatment in NIDDM does not affect the PI/IRI ratio. In normal subjects it has been shown that both fasting IRI and AIRmax are regulated by the degree of insulin sensitivity (16). We indirectly confirmed this finding in the present study, in that fasting IRI levels correlated positively with AIRmax in our normal subjects. In NIDDM subjects there was no significant relationship between fasting IRI and AIRmax, as AIRmax in these subjects is determined by variations in both insulin sensitivity and the degree of B cell secretory insufficiency. As noted earlier, AIRmax is a more useful measure of B cell secretory function in hyperglycemic states than the acute insulin response to glucose, since the latter is completely lost in NIDDM when the fasting glucose level exceeds 6 7 mmol/l (28). Whether the reduced B cell function in NIDDM and the associated increase in fasting PI/IRI is a result of a primary B cell abnormality or whether it represents a toxic effect of the elevated glucose levels (29 31) is not entirely clear. Although it has been hypothesized that glucose toxicity could increase the secretion of PI relative to that of IRI, short term elevation of plasma glucose levels in healthy subjects have

4 PROINSULIN AND B CELL FUNCTION IN NIDDM 607 failed to provoke preferential secretion of PI when normal subjects were clamped at 11 mmol/l for 3 h (32) or at 8 mmol/l for 8 h (33). However, chronic hyperglycemia may affect proinsulin to insulin conversion differently (29). Further support for the concept that an elevated PI/IRI is the result of B cell dysfunction rather than hyperglycemia per se comes from the finding of an elevated PI/IRI in other conditions with normal fasting glucose values (34, 35). An elevated PI/IRI has also been reported in siblings of IDDM subjects who at the time had normal fasting glucose levels and reduced first phase insulin responses, but subsequently developed IDDM (12). In these individuals, the PI/IRI ratio also correlated inversely with B cell function. Furthermore, we observed an elevated PI/IRI ratio in healthy human subjects given glucocorticoids or GH (36) and in primates with fasting normoglycemia with experimental B cell dysfunction induced by streptozotocin (11). A primary impairment in proinsulin processing or increased secretory demand on the B cells could lead to an increased PI/IRI ratio in NIDDM (21). Although the contribution of increased secretory demand cannot be fully determined, it does appear that impaired B cell function may be essential, because an increase in B cell demand from obesity-associated insulin resistance with normal B cell function (2, 18, 37), experimental insulin resistance produced by nicotinic acid (38), or even severe insulin resistance associated with congenital muscle fiber myopathy (19) is not associated with an increase in PI/IRI. On the contrary, a tendency toward a decrease in PI/IRI was observed in obesity (2, 10, 37) and in experimental insulin resistance (38). Also, in the face of abnormal B cell function, not all data support the idea that increasing demand increases PI/IRI. Thus, the addition of nicotinic acid to streptozotocin-treated monkeys tended to decrease the PI/IRI ratio (11). Finally, results obtained after surgical reductions in B cell mass have been somewhat disparate (8, 39). Recently, we reported that hemipancreatectomy in relatives of IDDM subjects was associated with an increased PI/IRI ratio (39), but a similar reduction in islet cell mass in dogs was not associated with the same finding (8). It should be added that the group of subjects studied by Seaquist et al. (39) had several characteristics different from normal subjects. A few of them were actually hyperglycemic at the time of examination and many had a diabetes-prone human leukocyte antigen haplotype (DR3 and/or DR4). 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