Serum Proinsulin in Children and Adolescents with Chemical Diabetes A. L. Rosenbloom, M.D.* J. 1. Starr, M.D.J D. Juhn, M.S.Jand A. H. Rubenstein, M.D.^ Gainesville and Chicago

SUMMARY Proinsulin levels (PLC) in serum were determined after gel filtration on specimens obtained during oral glucose tolerance testing (OGTT) in seven patients with repeated abnormality in OGTT and in a group of seven control subjects matched for age. Fasting and thirty-, sixty-, and 120-minute postglucose venous samples were analyzed for glucose (PG) and immunoreactive insulin (IRI) as well as PLC. PG and IRI mean concentrations were greater at all testing times in the patient population, but the mean IRI/PG was significantly higher in the patients only at fasting. PLC mean levels were higher in the patients but not to a level of significance. Percentage of total IRI attributable to PLC at each time point was identical between the two groups. The apparent diminished effectiveness of circulating insulin in chemical diabetes cannot be attributed to an abnormal proportion of proinsulin. DIABETES 24:753-57, August, 1975.

Chemical diabetes has been observed in children with symptoms suggestive of hypoglycemia, 1 ' 2 among siblings of children with overt (insulin-dependent) diabetes, 3 " 6 in children with a history of idiopathic hypoglycemia of infancy, 78 and in those with random glucosuria, a i ( ) stress hyperglycemia, 10 and obesity. 11 " 14 Even when the latter group is excluded, these children have demonstrated normal or elevated serum immunoassayable insulin levels in response to glucose ingestion. 1 2 ' 5 ' 6 ' 1 1 0 1 5 This study was designed to ascertain whether an increased proportion of the total circulating immunoreactive insulin (IRI) in these patients' sera might be proinsulin or its closely related two-chain intermediates (proinsulin-like components, PLC). 16 Because proinsulin, the single-chain precursor of insulin, and its intermediates are biologically less active From the Division of Genetics, Endocrinology and Metabolism, Department of Pediatrics, University of Florida College of Medicine, Gainesville 32610* and the Department of Medicine, University of Chicago, Chicago, Illinois 60637. t Address reprint requests to A. L. Rosenbloom, Department of Pediatrics, Box 739, University of Florida College of Medicine, Gainesville, Fla. 32610. Accepted for publication May 8, 1975. AUGUST, 1975

than insulin, 17 such a finding could explain the apparent ineffectiveness of high serum insulin levels in maintaining normal carbohydrate homeostasis. MATERIALS AND METHODS Subjects. Control subjects were seven healthy volunteers aged seventy-five to 187 months without a firstor second-degree relative with diabetes mellitus. Seven patients with chemical diabetes who had been followed in our clinic for more than three years were studied. They had had at least two oral glucose tolerance tests (OGTT) demonstrating abnormality by our criteria (see below). The clinical data are summarized in table 1. Two control subjects (MR, TE) and four patients (DH, KG, BZ, KC) had begun adolescence or had completed sexual development. None was greater than 7 per cent above ideal body weight for height according to the Iowa (U.S.A.) graphs. Three of the patients were detected during a study of siblings of children with insulin-dependent diabetes;' three were investigated because of symptoms of hypoglycemia1 and one because she had asymptomatic glucosuria. 10 Glucose tolerance testing. All tests were performed on the General Clinical Research Center of the Shands Teaching Hospital. The subjects consumed a normal diet prior to testing, which was confirmed by history to contain at least 50 per cent carbohydrate. Intercurrent or recent illness was a contraindication to testing. Following an overnight fast of ten hours, an indwelling needle was placed in an arm vein and all specimens withdrawn therefrom; the needle was kept open with an occasional flush from an attached syringe containing heparinized saline. Following the withdrawal of a fasting specimen, a cola-flavored carbohydrate solution (Glucola) was ingested; the dose was 1.75 gm. per kilogram body weight to a maximum of 75 gm. Further blood specimens were taken at one-half, one, two, three, and four hours. Laboratory methods. Glucose (PG) was measured in 753

SERUM PROINSULIN IN CHEMICAL DIABETES

TABLE 1 Clinical data of control and diabetic subjects

Patients MJ BH KB DH KG BZ KC Mean ± S.D.

Sex. F M M F F F F

Controls WA HR WW DR ER MR TE Mean ± S.D.

M M M F M F F

Chronologic age (mo.) 72 123 146 154 160 202 216 153.3 ± 48.2§

Height age (mo.) 70 100 117 180* 160 180* 180* 127 ± 46§

% IBWt 107 94 99 100 96 84 94 96.3 ± 7.0§

75 87 106 122 153 125 187 122 + 38.5§

70 98 104 114 142 168 180* 125 ± 40§

103 95 95 98 94 102 95 97.4 ± 3.7§

Follow-up Years NL 3 0 2 2 0 1 2

4.5 4 4.5 4.5 3 3 3.5

No. of OGTT* B 2 3 4 1 1 2 0

c3 3 6 2 6 1 3

*Female adult height reached at fifteen years (180 mo.). tIBW = ideal body weight. . tNL = normal, B = borderline, C = chemical diabetic. §Difference between means insignificant (p>0.20).

venous plasma by the ferricyanide method on the AutoAnalyzer (Technicon methodology N2A). Total serum IRI was measured by a modification 18 of the double-antibody method of Morgan and Lazarow.1'1 Sera were separated on Bio-Gel P-30 columns equilibrated in borate buffer, pH 8.4, and.the IRI-like activity in each fraction was determined by the insulin immunoassay. Two well-separated peaks corresponding to the PLC (first peak) and IRI (second peak) were found. By using standards of human proinsulin and insulin, the absolute amounts of each peptide in the original serum could be calculated by reading the PLC fraction against the proinsulin standard and those in

the IRI region from the insulin standard (table 2). 2 0 The sum of the PLC fractions measured against the insulin standard was expressed as a percentage of the total IRI concentration in all the fractions; this indicated the proportion of total serum insulin immunoreactivity due to cross-reaction with PLC (figure 1). OGTT criteria. The diagnosis of chemical diabetes was made on the basis of at least two abnormal glucose tolerance tests during the period of study using criteria developed in our laboratory; 21 these criteria are comparable to those devised by others for children 22 as well as adults. 23 We have previously

TABLE 2 Serum insulin and proinsulin levels (ng./ml.)* during oral glucose tolerance testing of controls and chemical-diabetes patients Insulin WA 0 0.28 30 60 1.52 120 0.76

HR 0.12 1.32 1.56 0.88

WW 0.16 1.60 0.48 0.76

Controls DR ER 0.20 0.16 1.04 1.40 2.80 0.76 0.56 0.84

0 0.22 30 60 0.79 120 0.57

0.14 0.50 0.65 0.69

0.06 0.36 0.35 0.60

0.08 0.55 1.20 0.93

0.22 0.53 0.46 0.30

MR 0.44 3.36 3.12 2.92

TE Mean 0.44 0.26 1.56 1.71 4.00 2.04 2.08 1.26

0.44 1.56 1.83 1.26

0.15 0.91 2.29 1.80

0.19 0.74 1.08 0.89

SD MJ 0.13 0.56 0.83 2.54 1.30 5.06 0.89 2.24 Proinsulin 0.13 0.30 0.44 0.87 0.73 1.64 0.51 1.57

BH 0.20 2.12 2.76 0.96

KB 0.32 4.64 4.28 3.12

KG 0.36 2.88 3.60 1.68

Patients DH BZ 0.85 0.64 4.97 3.04 5.16 2.57 1.60 2.20

KC Mean 0.56 0.50 5.64 3.69 7.48 4.42 4.72 2.36

SD 0.22 1.37 1.69 1.24

0.17 0.48 1.17 0.83

0.02 1.34 1.67 1.72

0.40 1.46 2.56 1.86

0.30 1.46 2.19 1.16

0.22 2.20 5.37 3.91

0.12 NS 0.55

Serum proinsulin in children and adolescents with chemical diabetes.

Proinsulin levels (PLC) in serum were determined after gel filtration on specimens obtained during oral glucose tolerance testing (OGTT) in seven pati...
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