Effect of growth hyperinsulinemia syndrome hormone treatment on associated with Turner Sonia Caprio, MD, Susan D. Boulware, MD, Martin Press, MD, Robert S. Sherwin, MD, Karen Rubin, ME), Thomas O. Carpenter, MD, Gerd Plewe, MD, and William V. Tamborlane, MD From the Departments of Pediatrics and Internal Medicine and the Clinical Research Centers, Yale University School of Medicine, New Haven, Connecticut, and the University of Connect- icut Health Center, Farmington To determine whether the insulin resistance in patients with Turner syndrome, which may be exaggerated by treatment with human growth hormone, leads to excessive insulin secretion, we applied the hyperglycemic glucose-clamp technique to produce a standard hyperglycemic stimulus (6.9 mmol/L, or 125 mg/dl, greater than fasting plasma glucose level for 120 minutes) in seven pa- tients with Turner syndrome and in seven healthy children. These studies were repeated in the patients after 6 to 12 months of therapy with growth hormone. Fasting plasma levels of insulin were comparable in control subjects and patients before therapy but increased significantly in the patients after 6 to 12 months of treatment with growth hormone. Despite identical glucose increments in the two groups during the glucose-clamp procedure, both first- and second- phase insulin responses were significantly greater in the patients than in the control subjects. Moreover, the hyperlnsulinemic responses to glucose were markedly exaggerated in the patients after their treatment with growth hormone, reaching values (first phase 474 • 100 pmol and second phase 826 +_ 100 pmol; p <0,02 vs pretreatment values) that were almost threefold greater than those in control subjects (p <0.001). Nevertheless, the rate of insulin-stimulated glucose metabolism during the last 60 minutes of the clamp procedure was similar in all three groups of studies. Glycosylated hemoglobin, total cholesterol level, and blood pressure remained normal in patients after therapy with growth hormone. We conclude that glucose-stimulated insulin response is increased in patients with Turner syndrome and that these alterations are further exaggerated by treatment with growth hormone. These hyperinsulinemic responses appear to compensate for reductions in insulin sensitivity. (J PEDIATR 1992;120:238-43) Recent studies using the euglycemic insulin-clamp tech- nique in nondiabetic girls with Turner syndrome indicate Supported by grants RR00125, RR06022, and DK20495 from the National Institutes of Health and by a "Career Development Award" (S.C.) and a fellowshipgrant (S.D.B.) from the Juvenile Diabetes Foundation International. Submitted for publication May 24, 1991; accepted Aug. 26, 1991. Reprint requests: Sonia Caprio, MD, Department of Pediatrics, Divisionof Endocrinology,333 Cedar St., Yale University School of Medicine, New Haven, CT 06510. 9/20/33349 that insulin resistance is an early metabolic defect that pri- marily involves nonoxidative pathways of intracellular glu- cose metabolism. 1,2 Insulin resistance in adults without Turner syndrome leads to compensatory hyperinsulinemia that in turn may contribute to the development of hyper- tension, dyslipoproteinemia, and type II diabetes. 3, 4 Such considerations may be important in the treatment of girls with Turner syndrome because adult women with this dis- order are at increased risk for the development of essential hypertension and type II diabetes. 5 Moreover, increasing numbers of young patients are being treated with growth- 238
Effect of growth hormone treatment on hyperinsulinemia associated with Turner syndrome
Feb 22, 2023
To determine whether the insulin resistance in patients with Turner syndrome,
which may be exaggerated by treatment with human growth hormone, leads to
excessive insulin secretion, we applied the hyperglycemic glucose-clamp
technique to produce a standard hyperglycemic stimulus (6.9 mmol/L, or 125
mg/dl, greater than fasting plasma glucose level for 120 minutes) in seven patients with Turner syndrome and in seven healthy children
Welcome message from author
These studies were repeated in the patients after 6 to 12 months of therapy with growth hormone. Fasting plasma levels of insulin were comparable in control subjects and patients before therapy but increased significantly in the patients after 6 to 12 months of treatment with growth hormone.
Transcript
PII: S0022-3476(05)80434-2hormone treatment on associated with Turner
Sonia Capr io , MD, Susan D. Boulware, MD, Mart in Press, MD, Robert S. Sherwin, MD, Karen Rubin, ME), Thomas O. Carpen te r , MD, Gerd Plewe, MD, and William V. Tambor lane , MD
From the Departments of Pediatrics and Internal Medicine and the Clinical Research Centers, Yale University School of Medicine, New Haven, Connecticut, and the University of Connect- icut Health Center, Farmington
To determine whether the insulin resistance in patients with Turner syndrome, which may be exaggera ted by treatment with human growth hormone, leads to excessive insulin secretion, we app l ied the hyperg lycemic g lucose-c lamp technique to produce a standard hyperg lycemic stimulus (6.9 mmol/L, or 125 mg/dl , greater than fasting p lasma glucose level for 120 minutes) in seven pa- tients with Turner syndrome and in seven healthy children. These studies were repeated in the patients after 6 to 12 months of therapy with growth hormone. Fasting plasma levels of insulin were comparab le in control subjects and patients before therapy but increased signif icantly in the patients after 6 to 12 months of treatment with growth hormone. Despite ident ical g lucose increments in the two groups during the g lucose-c lamp procedure, both first- and second- phase insulin responses were signif icantly greater in the patients than in the control subjects. Moreover, the hyperlnsul inemic responses to glucose were markedly exaggera ted in the patients after their treatment with growth hormone, reaching values (first phase 474 • 100 pmol and second phase 826 +_ 100 pmol; p <0,02 vs pretreatment values) that were almost threefold greater than those in control subjects (p <0.001). Nevertheless, the rate of insulin-stimulated glucose metabol ism during the last 60 minutes of the c lamp procedure was similar in all three groups of studies. Glycosylated hemoglobin, total cholesterol level, and b lood pressure remained normal in patients after therapy with growth hormone. We conc lude that glucose-st imulated insulin response is increased in patients with Turner syndrome and that these alterations are further exaggera ted by treatment with growth hormone. These hyperinsul inemic responses appear to compensate for reductions in insulin sensitivity. (J PEDIATR 1992;120:238-43)
Recent studies using the euglycemic insulin-clamp tech- nique in nondiabetic girls with Turner syndrome indicate
Supported by grants RR00125, RR06022, and DK20495 from the National Institutes of Health and by a "Career Development Award" (S.C.) and a fellowship grant (S.D.B.) from the Juvenile Diabetes Foundation International. Submitted for publication May 24, 1991; accepted Aug. 26, 1991. Reprint requests: Sonia Caprio, MD, Department of Pediatrics, Division of Endocrinology, 333 Cedar St., Yale University School of Medicine, New Haven, CT 06510. 9/20/33349
that insulin resistance is an early metabolic defect that pri- marily involves nonoxidative pathways of intracellular glu- cose metabolism. 1,2 Insulin resistance in adults without Turner syndrome leads to compensatory hyperinsulinemia that in turn may contribute to the development of hyper- tension, dyslipoproteinemia, and type II diabetes. 3, 4 Such considerations may be important in the treatment of girls with Turner syndrome because adult women with this dis- order are at increased risk for the development of essential hypertension and type II diabetes. 5 Moreover, increasing numbers of young patients are being treated with growth-
238
Volume 120 Hyperinsulinemia and Turner syndrome 2 3 9 Number 2, Part 1
Table I. Clinical characteristics of subjects
Patient Age Tanner Height Weight BSA BMI No, (yr) stage* (cm) (kg) (m 2) (kg/m 2) HbA4 (%) Karyotype
1 12 I 124 33 1.06 21.8 5.5 45X 2 13 II-III 132 40 1.25 23.2 5.5 45X 3 12 I 134 40 1.21 22.3 5.3 45X 4 1 l I 120 26 0.92 17.8 5.8 45X/XX 5 10 I 123 24 0.92 16.1 5.7 45X/46XY 6 15 II-III 138 57 1.40 30.2 5.0 45X/46XY 7 9 I 110 20 0.78 16.5 5.5 45X
Mean _+ SEM 11.7 • 0.7 126 _+ 3.6 34.3 • 4.7 1.07 + 0.08 21.1 • 1.9 5.47 _+_+ 0.09 Control subjects 10.8 • 0.8 144 • 0.05 36 + 5 1.18 • 0.10 17.3 _ 0.74 - -
(mean • SEM)
Age range for the control group is 8 to 14 years. BSA, Body surface area; BMI, body mass index; HbA1, hemoglobin A~. *Pubic hair development.
promoting agents that may decrease responsiveness to in- sulin. 6
Plasma insulin levels after oral glucose ingestion in
patients with Turner syndrome have been reported to be
high, normal, or low. 7, 8 These contrasting results may be
related to the inclusion in the studies of patients who varied
widely in age, degree of carbohydrate intolerance, and type
of hormonal replacement. In addition, factors other than
changes in insulin sensitivity can influence the plasma insu-
lin response to orally administered glucose, including
differences in the rate of absorption and the size of the glu-
cose load. loss of early (first-phase) insulin release. 9 and al-
tered hepatic clearance of insulin. 1~
We undertook our study to define more precisely the ef-
fects of both Turner syndrome and its t reatment with bio-
synthetic human growth hormone on pancreatic 6-cell
function. We used the glucose-clamp technique 11 to provide
a standardized, square-wave, hyperglycemic stimulus for
comparison of early and late plasma insulin responses, with
simultaneous measurements of C-peptide to assess alter-
ation in hepati 9 clearance of insulin. The patients were
studied before and after 6 to 12 months of t reatment with
human growth hormone.
Subjects. Seven children with Turner syndrome (9 to 15
years of age) were studied (Table I); five had Tanner stage
I pubic hair development and two had Tanner stage II to II1
pubic hair development. All patients were in good general
health, had normal thyroid activity, fasting plasma glucose
levels, and hemoglobin A1 levels (Table II), and had stim-
ulated growth hormone values > 10 ng/dl . None had been
treated with estrogen, androgen, or other hormone therapy,
or had any breast development. Responses in the patients
were compared with those in seven healthy children matched
for age and as closely as possible for body surface area CTa-
Table II. Effect of t reatment with human growth
hormone on biochemical and clinical responses
Before After treatment treatment
Fasting plasma insulin 52 + 6 115 • 21" (pmol/L)
HbAi (%) 5.47 _~ 0.09 5.7 z .19 Total cholesterol (mg/dl) 238 _ 9 219 + 21 Growth velocity (cm/yr) 3.6 _~ 0.46 9 _~ 0.7* Systolic blood pressure 114 __ 7 112 _~ 6
(mm Hg) Diastolic blood pressure 61 _~ 5 65 z 4
(ram Hg)
HbAi, Hemoglobin AI. *p <0.001
ble I). Four control subjects had Tanner stage I pubic hair
and breast development and three had Tanner stage II to IV
PUbi c hair and breast development. All subjects and their
Parents gave written informed consent before taking part in
the study; the protocol was approved by the human inves-
tigation committee of the Yale University School of Med-
icine. Subjects were studied in the Yale Children's Clinical
Research Center on the mormng after an overnight fast ( 10
to 12 hours). Two intravenous catheters were inserted be-
fore the clamp studies: one was placed in an antecubital vein
for administration of test substances and the other was
placed into a vein of the dorsal part of the hand of the con-
tralateral arm for blood sampling. The hand used for blood
sampling was kept in a heated box (60 ~ to 65 ~ C) to "ar-
terialize" venous blood samples. After a rest period of 30 to
60 minutes baseline fasting blood samples were obtained for
the measurement of glucose, insulin, C-peptide, hemoglobin
A1, and total cholesterol levels. The subjects lay supine in
2 4 0 Caprio et al. The Journal of Pediatrics February 1992
Fig. I. Changes in plasma levels of glucose, insulin, and C-pep- tide during glucose-clamp procedure in patients with Turner syn- drome before (pre-GH) and after (post-GH) treatment with growth hormone and in control subjects (shaded areas). Data are mean + SEM for patients and control subjects.
a quiet room with a constant ambient temperature of 21 ~
to 24 ~ C. Hyperglycemic glucose-clamp study. Measurements of
height, weight, and blood pressure, and assessment of stage of sexual development, were obtained when the subjects
were admitted to the Yale Children's Clinical Research Center. lnsulin responses were assessed with the use of the hyperglycemic glucose-clamp technique on the next morn- ing. With this procedure plasma level of glucose was raised sharply, by 6.9 mmol/L (125 mg/dl), to greater than fast- ing values by infusing, in a decreasing logarithmic manner, a priming dose of glucose to achieve rapidly the desired hy- perglycemic plateau. The plasma glucose level (measured at 5-minute intervals) was kept at this hyperglycemic plateau for 120 minutes by appropriate adjustments of a variable- rate infusion of 20% dextrose solution. Blood samples were also obtained at 2, 4, 6, 8, and I0 minutes, and every I0 minutes thereafter for 120 minutes for measurement of plasma levels of insulin and C-peptide. Urine was collected at the beginning and at the end of the procedure for deter- mination of glucose content.
To determine changes in insulin secretion, six patients had a second hyperglycemic glucose-clamp study per- formed after 6 to 12 months of therapy with growth hormone. Growth hormone (Protopin) at a dose of 0.125 mg/kg was administered three times per week. All patients received their usual dose of growth hormone on the evening before each of these studies.
Analyses. Plasma and urine levels of glucose were mea- sured by the glucose-oxidase method by means of a Beck- man glucose analyzer (Beckman Instruments Inc., Fuller- ton, Calif.). Plasma levels of insulin and C-peptide were measured by a double-antibody radioimmunoassay proce- dure j2, 13 Total hemoglobin A1 was measured chromato-
graphically with a microcolumn (Isolab, Inc., Akron, Ohio). Calculations. Plasma insulin response is biphasic during
the hyperglycemic glucose clamp. The first-phase (0 to l 0 minutes) and second-phase (10 to 120 minutes) responses were calculated as the mean hormone concentration during the respective periods. The rate of insulin-stimulated glu- cose metabolism (M) during the hyperglycemic glucose clamp was calculated at 20-minute intervals according to the following equation: M = INF - UC - SC, where INF is the glucose infusion rate, UC is the correction for urinary loss of glucose, and SC is the correction for changes in the glucose space. 1l The average rate of glucose metabolism during the last 60 minutes of each clamp study was used for comparison between the study groups. In addition, for each study the rate of glucose metabolism was divided by the mean plasma insulin level during the last 60 minutes of the clamp to obtain the ratio M/I , an index of sensitivity to in- sulin) 4
All data are expressed as mean _+ SEM. Statistical analyses were performed by means of analysis of variance and two-tailed Student unpaired t tests. Differences within the patient group before and after treatment were compared
Volume 120 Hyperinsulinemia and Turner syndrome 2 4 1 Number 2, Part 1
by paired t tests. Differences were considered statistically significant at p <0.05.
R E S U L T S
As shown in Fig. 1 and Table II, fasting plasma concen- trations of glucose were similar in the patients with Turner syndrome before and after treatment and in control sub- jects. In addition, during the clamp procedure the plasma concentration of glucose was raised to the same hypergly- cemic plateau during allithree groups of studies (Fig. 1).
Fasting plasma levels of insulin and C-peptide were com- parable in control subjects and patients before therapy, but both increased significantly in the patients after 6 to 12 months of therapy with growth hormone (p <0.05; Table II, Fig. 1). The identical hyperglycemic stimulus achieved during the clamp study produced similar time courses and biphasic patterns of insulin responses in patients with Turner syndrome and in control subjects (Fig. 1); however, the magnitude of the insulin response was significantly greater in the patients. On the other hand, C-peptide responses in the patients before treatment were not differ- ent from those in the control subjects (Fig. 1). As shown in Fig. 2, even before therapy with growth hormone first- and second-phase insulin responses were significantly higher in the patients than in control subjects (first phase, 309 +_ 37 vs 180 _+ 36 pmol/L, p <0.05; second phase, 481 _+ 72 vs 266 _+ 57 pmol/L, p <0.02). Moreover, the hyperinsuline- mic responses to glucose were markedly exaggerated in the patients after treatment with growth hormone, reaching values (first phase, 474 _+ 100 pmol/L; second phase, 826 _+ 100 pmol/L; p <0.02 vs pretreatrrient values) that were almost threefold greater than those in control subjects (p <0.001). Therapy with growth hormone also increased first- and second-phase C-peptide responses (p <0.05 for pretreatment and control values; Fig. 2).
The insulin-stimulated glucose metabolism during the last 60 minutes of the clamp procedure was similar in the patients (315 _+ 37 mg/m 2 body surface area.rain) and control subjects (266 _+ 31 mg/m 2, rain; p not significant), even though mean plasma levels of insulin during that pe- riod were twofold greater in the patients. As a result, the M/ [ ratio (see Methods section) was markedly lower in the patients than in control subjects (0.58 _+ 0.07 vs 0.99 _+ 0.14; p <0.02). After treatment with growth hormone the M / I ratio was further reduced by almost 50% in the patients (0.29 _+ 0.02; p <0.01 vs control and pretreatment values).
The clinical and biochemical responses to 6 to 12 months of treatment with growth hormone are summarized in Ta- ble II. Therapy with growth hormone resulted in a sharp increase in growth velocity (p <0.01), but no statistical changes in blood pressure or in cholesterol and hemoglobin
1000
400 E
200 13_
Phase I
Controls Turner
Phase II
Controls Turner
Phase II
Fig. 2, Mean +_ SEM first-phase (0 to 10 minutes) and second- phase (10 to 120 minutes) plasma insulin and C-peptide responses during glucose-clamp procedure in control subjects (open bars) and patients with Turner syndrome before (hatched bars) and after (shaded bars) therapy with growth hormone. *p <0.05; **p <0.01.
A1 levels were noted. Results of studies of liver function were within the normal range before and after treatment (data not shown).
D I S C U S S I O N
In our study we used the hyperglycemic glucose-clamp procedure to estimate glucose-stimulated insulin responses because this method is free of many of the confounding factors that limit interpretation of plasma responses to in- sulin during oral glucose-tolerance testing. Most important, use of this method provided the opportunity to apply an identical hyperglycemic stimulus to the patients before and after treatment with growth hormone and to a group of healthy, age-matched control children.
Before receiving any hormonal replacement therapy, our young patients with Turner syndrome had normal fasting plasma glucose, plasma insulin, and glycosylated hemoglo- bin levels. Nevertheless, plasma insulin responses during the hyperglycemic glucose-clamp procedures were nearly two- fold greater in the patients than in control subjects. These exaggerated insulin responses could not be attributed to differences in muscle mass or relative adiposity, or to delayed or defective early release of insulin as seen near the onset of type I and type II diabetes. On the other hand, el-
2 4 2 Caprio et al. The Journal of Pediatrics February 1992
evated concentrations of circulating insulin in the patients effectively compensated for the syndrome-induced impair- ments in insulin action; the rates of insulin-stimulated glu- cose metabolism during the last 60 minutes of the hyper- glycemic plateau were similar in patients and control sub- jects.
Although plasma insulin responses to glucose Were increased in the patients before treatment, first- and second-phase C-peptide responses were not significantly different from those in normal subjectS. Insulin is extracted by th e liver but C-peptide is nottS; reduced hepatic clear- ance of insulin may have contributed to the hyperinsuline- mic responses. These data must be interpretedwith Caution, however, because of the small number of patients in our study. On the other hand, both c-pept~de and insulin responses Were markedly increased by therapy with growth hormone, indicating that administration of exogenous growth hormone sharply stimulated B-cell secretion Of insulin.
The most striking alterations wer e observed after 6 to 12 months of treatment with exogenous growth hormone. Al- though fasting glucose and glycosylated hemoglobin levels were unchanged, fasting insulin levels rose, glucose-stimu- lated insulin responses were increased three to four times, and insulin sensitivity, as estimated by the M / I ratio, Was reduced to 25% of normal values. Such marked chaiiges in insulin secretion and action were unexpected in View of the more modest and variable changes in insulin levels after treatment of Turne r syndrome with growth hormone re- cently reported by Wilson et al. 16 Although similar doses and dosage schedules of growth hormone were employed, the two studies are not readily comparable because Wilson et al. used oral glucose-tolerance testing to evaluate insulin resp0nses. 17 Moreover, that study lacked concurrent com- parison With a grouP of healthy children and did not correct fo r the interval between the last injection of growth hormone and administration of the glucose-tolerance test.
The diabetogenic potential of growth hormone is of par- ticular concern when this agent is used to treat conditions such a s Turner syndrome, which is Characterized by in- creased risks of impaired glucose tolerance and diabetes. In addition, increasing evidence suggests that insulin resis- tance and hyperinsulinemia per se may contribute to the development of hypertension, dyslipoproteinemia, and ath- erosclerosis, as well as of type II diabetes. 17-2~ Blood pres- sure; total cholesterol level, and glycosylated hemoglobin level did not change in our small set of patients. However, further studies in much larger groups of subjects treated and observed for more prolonged periods are needed to evaluate the potential risks of growth hormone therapy for patients with Turner syndrome. Furthermore, the complications of
therapy with growth hormone might have been greater if an older population of patients had been studied. Of course, growth hormone-induced increases in plasma insulin level may also have beneficial effects in the growing child; increases in circulating insulin may contribute to the increased growth rates observed in our patients by amplify- ing insulin's effect on other insulin-sensitive substrates, such as amino acids. 21, z2
We are grateful to Angela Notar Francesco and the staff of the Children's Clinical Research Center for the excellent care given to our patients during these studies. We are indebted to the staff of the Core Laboratory of the Clinical Research Center for technical assistance.
R E F E R E N C E S
1. Caprio S, Boulware S, Diamond M, et al. Insulin resistance: an early metabolic effect of Turner syndrome. J Clin Endo- crinol Metab 1991;72:832-6.
2. Stoppoloni G, Prisco F, Alfano C, Iafusco D, Marrozzo G, Gaolisso G. Characteristics of insulin resistance in Turner syndrome. Diabetes Metab 1990;16:267-71.
3. DeFronzo RA, Ferrannini E. Insulin resistance a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dis- lipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991;14:173-94.
4. Reaven GM. Role of insulin resistance in human disease. Di- abetes 1988;37:1595-1607.
5. Lippe BM. Primary ovarian failure. In: Kaplan SA, ed. Clin- ical pediatric endocrinology. Philadelphia: WB Saunders, 1990:325-66.
6. Bratusch-Marrain PR, Smith D, DeFronzo RA. The effect of growth hormone on glucose metabolism and insulin secretion in man. J Clin End0crino[ Metab 1982;55:973-81.
7. Karp M, Sniz A, Dozon M, et al. Glucose tolerance tests and insulin response in juveniie patients with gonadal dysgenesis. Mod Probl…
Sonia Capr io , MD, Susan D. Boulware, MD, Mart in Press, MD, Robert S. Sherwin, MD, Karen Rubin, ME), Thomas O. Carpen te r , MD, Gerd Plewe, MD, and William V. Tambor lane , MD
From the Departments of Pediatrics and Internal Medicine and the Clinical Research Centers, Yale University School of Medicine, New Haven, Connecticut, and the University of Connect- icut Health Center, Farmington
To determine whether the insulin resistance in patients with Turner syndrome, which may be exaggera ted by treatment with human growth hormone, leads to excessive insulin secretion, we app l ied the hyperg lycemic g lucose-c lamp technique to produce a standard hyperg lycemic stimulus (6.9 mmol/L, or 125 mg/dl , greater than fasting p lasma glucose level for 120 minutes) in seven pa- tients with Turner syndrome and in seven healthy children. These studies were repeated in the patients after 6 to 12 months of therapy with growth hormone. Fasting plasma levels of insulin were comparab le in control subjects and patients before therapy but increased signif icantly in the patients after 6 to 12 months of treatment with growth hormone. Despite ident ical g lucose increments in the two groups during the g lucose-c lamp procedure, both first- and second- phase insulin responses were signif icantly greater in the patients than in the control subjects. Moreover, the hyperlnsul inemic responses to glucose were markedly exaggera ted in the patients after their treatment with growth hormone, reaching values (first phase 474 • 100 pmol and second phase 826 +_ 100 pmol; p <0,02 vs pretreatment values) that were almost threefold greater than those in control subjects (p <0.001). Nevertheless, the rate of insulin-stimulated glucose metabol ism during the last 60 minutes of the c lamp procedure was similar in all three groups of studies. Glycosylated hemoglobin, total cholesterol level, and b lood pressure remained normal in patients after therapy with growth hormone. We conc lude that glucose-st imulated insulin response is increased in patients with Turner syndrome and that these alterations are further exaggera ted by treatment with growth hormone. These hyperinsul inemic responses appear to compensate for reductions in insulin sensitivity. (J PEDIATR 1992;120:238-43)
Recent studies using the euglycemic insulin-clamp tech- nique in nondiabetic girls with Turner syndrome indicate
Supported by grants RR00125, RR06022, and DK20495 from the National Institutes of Health and by a "Career Development Award" (S.C.) and a fellowship grant (S.D.B.) from the Juvenile Diabetes Foundation International. Submitted for publication May 24, 1991; accepted Aug. 26, 1991. Reprint requests: Sonia Caprio, MD, Department of Pediatrics, Division of Endocrinology, 333 Cedar St., Yale University School of Medicine, New Haven, CT 06510. 9/20/33349
that insulin resistance is an early metabolic defect that pri- marily involves nonoxidative pathways of intracellular glu- cose metabolism. 1,2 Insulin resistance in adults without Turner syndrome leads to compensatory hyperinsulinemia that in turn may contribute to the development of hyper- tension, dyslipoproteinemia, and type II diabetes. 3, 4 Such considerations may be important in the treatment of girls with Turner syndrome because adult women with this dis- order are at increased risk for the development of essential hypertension and type II diabetes. 5 Moreover, increasing numbers of young patients are being treated with growth-
238
Volume 120 Hyperinsulinemia and Turner syndrome 2 3 9 Number 2, Part 1
Table I. Clinical characteristics of subjects
Patient Age Tanner Height Weight BSA BMI No, (yr) stage* (cm) (kg) (m 2) (kg/m 2) HbA4 (%) Karyotype
1 12 I 124 33 1.06 21.8 5.5 45X 2 13 II-III 132 40 1.25 23.2 5.5 45X 3 12 I 134 40 1.21 22.3 5.3 45X 4 1 l I 120 26 0.92 17.8 5.8 45X/XX 5 10 I 123 24 0.92 16.1 5.7 45X/46XY 6 15 II-III 138 57 1.40 30.2 5.0 45X/46XY 7 9 I 110 20 0.78 16.5 5.5 45X
Mean _+ SEM 11.7 • 0.7 126 _+ 3.6 34.3 • 4.7 1.07 + 0.08 21.1 • 1.9 5.47 _+_+ 0.09 Control subjects 10.8 • 0.8 144 • 0.05 36 + 5 1.18 • 0.10 17.3 _ 0.74 - -
(mean • SEM)
Age range for the control group is 8 to 14 years. BSA, Body surface area; BMI, body mass index; HbA1, hemoglobin A~. *Pubic hair development.
promoting agents that may decrease responsiveness to in- sulin. 6
Plasma insulin levels after oral glucose ingestion in
patients with Turner syndrome have been reported to be
high, normal, or low. 7, 8 These contrasting results may be
related to the inclusion in the studies of patients who varied
widely in age, degree of carbohydrate intolerance, and type
of hormonal replacement. In addition, factors other than
changes in insulin sensitivity can influence the plasma insu-
lin response to orally administered glucose, including
differences in the rate of absorption and the size of the glu-
cose load. loss of early (first-phase) insulin release. 9 and al-
tered hepatic clearance of insulin. 1~
We undertook our study to define more precisely the ef-
fects of both Turner syndrome and its t reatment with bio-
synthetic human growth hormone on pancreatic 6-cell
function. We used the glucose-clamp technique 11 to provide
a standardized, square-wave, hyperglycemic stimulus for
comparison of early and late plasma insulin responses, with
simultaneous measurements of C-peptide to assess alter-
ation in hepati 9 clearance of insulin. The patients were
studied before and after 6 to 12 months of t reatment with
human growth hormone.
Subjects. Seven children with Turner syndrome (9 to 15
years of age) were studied (Table I); five had Tanner stage
I pubic hair development and two had Tanner stage II to II1
pubic hair development. All patients were in good general
health, had normal thyroid activity, fasting plasma glucose
levels, and hemoglobin A1 levels (Table II), and had stim-
ulated growth hormone values > 10 ng/dl . None had been
treated with estrogen, androgen, or other hormone therapy,
or had any breast development. Responses in the patients
were compared with those in seven healthy children matched
for age and as closely as possible for body surface area CTa-
Table II. Effect of t reatment with human growth
hormone on biochemical and clinical responses
Before After treatment treatment
Fasting plasma insulin 52 + 6 115 • 21" (pmol/L)
HbAi (%) 5.47 _~ 0.09 5.7 z .19 Total cholesterol (mg/dl) 238 _ 9 219 + 21 Growth velocity (cm/yr) 3.6 _~ 0.46 9 _~ 0.7* Systolic blood pressure 114 __ 7 112 _~ 6
(mm Hg) Diastolic blood pressure 61 _~ 5 65 z 4
(ram Hg)
HbAi, Hemoglobin AI. *p <0.001
ble I). Four control subjects had Tanner stage I pubic hair
and breast development and three had Tanner stage II to IV
PUbi c hair and breast development. All subjects and their
Parents gave written informed consent before taking part in
the study; the protocol was approved by the human inves-
tigation committee of the Yale University School of Med-
icine. Subjects were studied in the Yale Children's Clinical
Research Center on the mormng after an overnight fast ( 10
to 12 hours). Two intravenous catheters were inserted be-
fore the clamp studies: one was placed in an antecubital vein
for administration of test substances and the other was
placed into a vein of the dorsal part of the hand of the con-
tralateral arm for blood sampling. The hand used for blood
sampling was kept in a heated box (60 ~ to 65 ~ C) to "ar-
terialize" venous blood samples. After a rest period of 30 to
60 minutes baseline fasting blood samples were obtained for
the measurement of glucose, insulin, C-peptide, hemoglobin
A1, and total cholesterol levels. The subjects lay supine in
2 4 0 Caprio et al. The Journal of Pediatrics February 1992
Fig. I. Changes in plasma levels of glucose, insulin, and C-pep- tide during glucose-clamp procedure in patients with Turner syn- drome before (pre-GH) and after (post-GH) treatment with growth hormone and in control subjects (shaded areas). Data are mean + SEM for patients and control subjects.
a quiet room with a constant ambient temperature of 21 ~
to 24 ~ C. Hyperglycemic glucose-clamp study. Measurements of
height, weight, and blood pressure, and assessment of stage of sexual development, were obtained when the subjects
were admitted to the Yale Children's Clinical Research Center. lnsulin responses were assessed with the use of the hyperglycemic glucose-clamp technique on the next morn- ing. With this procedure plasma level of glucose was raised sharply, by 6.9 mmol/L (125 mg/dl), to greater than fast- ing values by infusing, in a decreasing logarithmic manner, a priming dose of glucose to achieve rapidly the desired hy- perglycemic plateau. The plasma glucose level (measured at 5-minute intervals) was kept at this hyperglycemic plateau for 120 minutes by appropriate adjustments of a variable- rate infusion of 20% dextrose solution. Blood samples were also obtained at 2, 4, 6, 8, and I0 minutes, and every I0 minutes thereafter for 120 minutes for measurement of plasma levels of insulin and C-peptide. Urine was collected at the beginning and at the end of the procedure for deter- mination of glucose content.
To determine changes in insulin secretion, six patients had a second hyperglycemic glucose-clamp study per- formed after 6 to 12 months of therapy with growth hormone. Growth hormone (Protopin) at a dose of 0.125 mg/kg was administered three times per week. All patients received their usual dose of growth hormone on the evening before each of these studies.
Analyses. Plasma and urine levels of glucose were mea- sured by the glucose-oxidase method by means of a Beck- man glucose analyzer (Beckman Instruments Inc., Fuller- ton, Calif.). Plasma levels of insulin and C-peptide were measured by a double-antibody radioimmunoassay proce- dure j2, 13 Total hemoglobin A1 was measured chromato-
graphically with a microcolumn (Isolab, Inc., Akron, Ohio). Calculations. Plasma insulin response is biphasic during
the hyperglycemic glucose clamp. The first-phase (0 to l 0 minutes) and second-phase (10 to 120 minutes) responses were calculated as the mean hormone concentration during the respective periods. The rate of insulin-stimulated glu- cose metabolism (M) during the hyperglycemic glucose clamp was calculated at 20-minute intervals according to the following equation: M = INF - UC - SC, where INF is the glucose infusion rate, UC is the correction for urinary loss of glucose, and SC is the correction for changes in the glucose space. 1l The average rate of glucose metabolism during the last 60 minutes of each clamp study was used for comparison between the study groups. In addition, for each study the rate of glucose metabolism was divided by the mean plasma insulin level during the last 60 minutes of the clamp to obtain the ratio M/I , an index of sensitivity to in- sulin) 4
All data are expressed as mean _+ SEM. Statistical analyses were performed by means of analysis of variance and two-tailed Student unpaired t tests. Differences within the patient group before and after treatment were compared
Volume 120 Hyperinsulinemia and Turner syndrome 2 4 1 Number 2, Part 1
by paired t tests. Differences were considered statistically significant at p <0.05.
R E S U L T S
As shown in Fig. 1 and Table II, fasting plasma concen- trations of glucose were similar in the patients with Turner syndrome before and after treatment and in control sub- jects. In addition, during the clamp procedure the plasma concentration of glucose was raised to the same hypergly- cemic plateau during allithree groups of studies (Fig. 1).
Fasting plasma levels of insulin and C-peptide were com- parable in control subjects and patients before therapy, but both increased significantly in the patients after 6 to 12 months of therapy with growth hormone (p <0.05; Table II, Fig. 1). The identical hyperglycemic stimulus achieved during the clamp study produced similar time courses and biphasic patterns of insulin responses in patients with Turner syndrome and in control subjects (Fig. 1); however, the magnitude of the insulin response was significantly greater in the patients. On the other hand, C-peptide responses in the patients before treatment were not differ- ent from those in the control subjects (Fig. 1). As shown in Fig. 2, even before therapy with growth hormone first- and second-phase insulin responses were significantly higher in the patients than in control subjects (first phase, 309 +_ 37 vs 180 _+ 36 pmol/L, p <0.05; second phase, 481 _+ 72 vs 266 _+ 57 pmol/L, p <0.02). Moreover, the hyperinsuline- mic responses to glucose were markedly exaggerated in the patients after treatment with growth hormone, reaching values (first phase, 474 _+ 100 pmol/L; second phase, 826 _+ 100 pmol/L; p <0.02 vs pretreatrrient values) that were almost threefold greater than those in control subjects (p <0.001). Therapy with growth hormone also increased first- and second-phase C-peptide responses (p <0.05 for pretreatment and control values; Fig. 2).
The insulin-stimulated glucose metabolism during the last 60 minutes of the clamp procedure was similar in the patients (315 _+ 37 mg/m 2 body surface area.rain) and control subjects (266 _+ 31 mg/m 2, rain; p not significant), even though mean plasma levels of insulin during that pe- riod were twofold greater in the patients. As a result, the M/ [ ratio (see Methods section) was markedly lower in the patients than in control subjects (0.58 _+ 0.07 vs 0.99 _+ 0.14; p <0.02). After treatment with growth hormone the M / I ratio was further reduced by almost 50% in the patients (0.29 _+ 0.02; p <0.01 vs control and pretreatment values).
The clinical and biochemical responses to 6 to 12 months of treatment with growth hormone are summarized in Ta- ble II. Therapy with growth hormone resulted in a sharp increase in growth velocity (p <0.01), but no statistical changes in blood pressure or in cholesterol and hemoglobin
1000
400 E
200 13_
Phase I
Controls Turner
Phase II
Controls Turner
Phase II
Fig. 2, Mean +_ SEM first-phase (0 to 10 minutes) and second- phase (10 to 120 minutes) plasma insulin and C-peptide responses during glucose-clamp procedure in control subjects (open bars) and patients with Turner syndrome before (hatched bars) and after (shaded bars) therapy with growth hormone. *p <0.05; **p <0.01.
A1 levels were noted. Results of studies of liver function were within the normal range before and after treatment (data not shown).
D I S C U S S I O N
In our study we used the hyperglycemic glucose-clamp procedure to estimate glucose-stimulated insulin responses because this method is free of many of the confounding factors that limit interpretation of plasma responses to in- sulin during oral glucose-tolerance testing. Most important, use of this method provided the opportunity to apply an identical hyperglycemic stimulus to the patients before and after treatment with growth hormone and to a group of healthy, age-matched control children.
Before receiving any hormonal replacement therapy, our young patients with Turner syndrome had normal fasting plasma glucose, plasma insulin, and glycosylated hemoglo- bin levels. Nevertheless, plasma insulin responses during the hyperglycemic glucose-clamp procedures were nearly two- fold greater in the patients than in control subjects. These exaggerated insulin responses could not be attributed to differences in muscle mass or relative adiposity, or to delayed or defective early release of insulin as seen near the onset of type I and type II diabetes. On the other hand, el-
2 4 2 Caprio et al. The Journal of Pediatrics February 1992
evated concentrations of circulating insulin in the patients effectively compensated for the syndrome-induced impair- ments in insulin action; the rates of insulin-stimulated glu- cose metabolism during the last 60 minutes of the hyper- glycemic plateau were similar in patients and control sub- jects.
Although plasma insulin responses to glucose Were increased in the patients before treatment, first- and second-phase C-peptide responses were not significantly different from those in normal subjectS. Insulin is extracted by th e liver but C-peptide is nottS; reduced hepatic clear- ance of insulin may have contributed to the hyperinsuline- mic responses. These data must be interpretedwith Caution, however, because of the small number of patients in our study. On the other hand, both c-pept~de and insulin responses Were markedly increased by therapy with growth hormone, indicating that administration of exogenous growth hormone sharply stimulated B-cell secretion Of insulin.
The most striking alterations wer e observed after 6 to 12 months of treatment with exogenous growth hormone. Al- though fasting glucose and glycosylated hemoglobin levels were unchanged, fasting insulin levels rose, glucose-stimu- lated insulin responses were increased three to four times, and insulin sensitivity, as estimated by the M / I ratio, Was reduced to 25% of normal values. Such marked chaiiges in insulin secretion and action were unexpected in View of the more modest and variable changes in insulin levels after treatment of Turne r syndrome with growth hormone re- cently reported by Wilson et al. 16 Although similar doses and dosage schedules of growth hormone were employed, the two studies are not readily comparable because Wilson et al. used oral glucose-tolerance testing to evaluate insulin resp0nses. 17 Moreover, that study lacked concurrent com- parison With a grouP of healthy children and did not correct fo r the interval between the last injection of growth hormone and administration of the glucose-tolerance test.
The diabetogenic potential of growth hormone is of par- ticular concern when this agent is used to treat conditions such a s Turner syndrome, which is Characterized by in- creased risks of impaired glucose tolerance and diabetes. In addition, increasing evidence suggests that insulin resis- tance and hyperinsulinemia per se may contribute to the development of hypertension, dyslipoproteinemia, and ath- erosclerosis, as well as of type II diabetes. 17-2~ Blood pres- sure; total cholesterol level, and glycosylated hemoglobin level did not change in our small set of patients. However, further studies in much larger groups of subjects treated and observed for more prolonged periods are needed to evaluate the potential risks of growth hormone therapy for patients with Turner syndrome. Furthermore, the complications of
therapy with growth hormone might have been greater if an older population of patients had been studied. Of course, growth hormone-induced increases in plasma insulin level may also have beneficial effects in the growing child; increases in circulating insulin may contribute to the increased growth rates observed in our patients by amplify- ing insulin's effect on other insulin-sensitive substrates, such as amino acids. 21, z2
We are grateful to Angela Notar Francesco and the staff of the Children's Clinical Research Center for the excellent care given to our patients during these studies. We are indebted to the staff of the Core Laboratory of the Clinical Research Center for technical assistance.
R E F E R E N C E S
1. Caprio S, Boulware S, Diamond M, et al. Insulin resistance: an early metabolic effect of Turner syndrome. J Clin Endo- crinol Metab 1991;72:832-6.
2. Stoppoloni G, Prisco F, Alfano C, Iafusco D, Marrozzo G, Gaolisso G. Characteristics of insulin resistance in Turner syndrome. Diabetes Metab 1990;16:267-71.
3. DeFronzo RA, Ferrannini E. Insulin resistance a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dis- lipidemia, and atherosclerotic cardiovascular disease. Diabetes Care 1991;14:173-94.
4. Reaven GM. Role of insulin resistance in human disease. Di- abetes 1988;37:1595-1607.
5. Lippe BM. Primary ovarian failure. In: Kaplan SA, ed. Clin- ical pediatric endocrinology. Philadelphia: WB Saunders, 1990:325-66.
6. Bratusch-Marrain PR, Smith D, DeFronzo RA. The effect of growth hormone on glucose metabolism and insulin secretion in man. J Clin End0crino[ Metab 1982;55:973-81.
7. Karp M, Sniz A, Dozon M, et al. Glucose tolerance tests and insulin response in juveniie patients with gonadal dysgenesis. Mod Probl…
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