(Hypertension. 1996;27:1273-1276.)
© 1996 American Heart Association, Inc.
Articles |
Effect of Race and Hypertension on Plasma Amylin Concentrations
From the Department of Psychiatry, University of California, San Diego (J.E.D., R.N.), and Amylin Pharmaceuticals, La Jolla, Calif (O.K., J.K.).
Correspondence to Dr Joel E. Dimsdale, UCSD, La Jolla, CA 92093-0804.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Amylin is a recently discovered peptide hormone composed of 37 amino acids that is cosecreted with insulin by pancreatic ß cells. Amylin has been reported to be present in increased amounts in insulin-resistant subjects who are hyperinsulinemic. Because blacks and whites differ in the prevalence of both hypertension and diabetes, we examined amylin levels in 77 individuals; 42 were black (11 hypertensive and 31 normotensive) and 35 were white (10 hypertensive and 25 normotensive) individuals who were either healthy control subjects or hypertensive subjects not receiving antihypertensive medication. Plasma amylin concentrations were measured in two separate monoclonal antibody–based immunofluorescent sandwich-type assays. The F002-2 capture antibody binds amylin plus at least two additional amylin-like peptides, and the F024-4 capture antibody detectably binds only the amylin peptide. There was a significant race-by-diagnosis interaction for levels of amylin immunoreactivity during a 2-hour glucose tolerance test (P<.005 for F002-2 antibody and P<.05 for F024-4 antibody). Highest levels were found in black hypertensive subjects. The results appear to fit with previously observed differences in metabolic status between blacks and whites and with the association between hypertension and alterations in metabolic status.
Key Words: amylin • insulin • glucose • race • peptides
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Approximately 30 years ago, Welborn and coworkers1 described an association between hypertension and hyperinsulinemia. Subsequently, numerous investigators have reported insulin resistance in subjects with essential hypertension by demonstrating a decrease in insulin-mediated glucose disposal in these individuals.2 The relationship between insulin resistance and hypertension has also been reported to be affected by racial influences.3 To date, efforts to demonstrate a direct link between elevated insulin levels and elevations in blood pressure have failed to yield convincing evidence of a causal relationship,4 although acute, sustained intravenous insulin infusion has been shown to increase norepinephrine release5 and sympathetic firing.6 This has led to the suggestion that the link between hypertension and hyperinsulinemia may be indirect via genetic or commonly acquired factors. Still other researchers have proposed that other ß-cell secretory products, such as proinsulin, 32-33 split proinsulin, or amylin, might represent the link between insulin resistance, obesity, and hypertension.7 8
Amylin is a 37–amino acid peptide that has recently been identified and demonstrated to be cosecreted with insulin.9 10 In animal models, amylin has been shown to have multiple effects, including inhibition of insulin secretion, modification of insulin action in skeletal muscle, slowing of gastric emptying, and stimulation of plasma renin activity.11 12 13 Knowledge of circulating amylin levels in insulin-resistant subjects with hypertension could provide useful insights to follow up recent reports documenting elevated plasma amylin concentrations in a group of hypertensive subjects who were predominantly white (Reference 1414 and personal communication, G. Pacini, 1994) and other reports that black individuals have higher insulin levels during both fasting and carbohydrate challenge (eg, glucose tolerance test).3 Additional insights into this area would be welcomed in view of the increased incidence of both non–insulin-dependent diabetes mellitus and hypertension among blacks.15 16
We examined blood pressure, glucose, and amylin levels during both fasting and a 2-hour glucose tolerance test in 77 black and white nondiabetic subjects who were all either normotensive or unmedicated hypertensive. Plasma amylin concentrations were measured with newly validated monoclonal-based, two-site immunofluorescent assays, obviating the need for extraction of plasma samples before assay.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Subjects were recruited by word-of-mouth referral and in response to advertisements. All signed informed consent. All hypertensive subjects had been off antihypertensive medication for at least 4 weeks before participating in the study. No subject had a history of diabetes. The diagnosis of hypertension was based on repeated blood pressure measurements on two screening occasions. On each occasion, blood pressure was taken three times with an appropriately sized cuff after the subject had been seated for more than 5 minutes. Subjects whose average blood pressure on both occasions exceeded 140 mm Hg systolic or 90 mm Hg diastolic were considered hypertensive. Obesity was measured as body mass index (kilograms per meter squared).
Subjects were admitted to the University of California, San Diego,
Clinical Research Center and placed on an isocaloric diet providing 200
mEq sodium and 100 mEq potassium per day for 1 day before testing.
Table 1
shows the subject characteristics.
|
An indwelling catheter was placed in a forearm vein on the second evening, and after an overnight fast, the subjects completed a 2-hour glucose tolerance test the following morning. A 75-g glucose load was administered, and blood samples were drawn through the catheter just before subjects drank the carbohydrate load and 30, 60, 90, and 120 minutes later. Blood samples for glucose were collected in tubes containing sodium benzoate, insulin in untreated tubes, and amylin in tubes containing EDTA. Samples were stored on crushed ice and promptly centrifuged. Plasma was stored at -70°C until the assays could be performed. Glucose was measured with a glucose analyzer (Yellow Springs Instrument Co). Insulin was measured with a double-antibody radioimmunoassay by a modification of the method of Morgan and Lazarow.17
Amylin concentrations were measured with two separate monoclonal antibody–based, two-site immunofluorescent assays. These assays differed only in their capture antibodies. One assay uses monoclonal antibody F002-2, which has been shown to bind to both human amylin and some amylin-like peptides but does not react with rat amylin.18 The amylin-like peptides have been shown to consist of two distinct peaks on reversed-phase high-performance liquid chromatography that are distinct from the amylin peak and have higher molecular weights. The other assay uses monoclonal antibody F024-4, which does not bind these additional peaks of amylin immunoreactivity in human plasma and exhibits 100% cross-reactivity with rat amylin.19 Thus, this latter antibody is specific for the intact human amylin molecule.
The assays were performed by coating black, 96-well microtiter plates (Dynatech) with either F002-2 or F024-4 antibody by overnight incubation at 4°C with 50 µL antibody (20 µg/mL) in 50 mmol/L carbonate buffer, pH 9.5. The plates were then washed with Tris-buffered saline with 0.1% Tween 20 (wash buffer) and blocked with 1% nonfat dry milk powder in the same carbonate buffer. Samples were thawed on ice and centrifuged at 800g to remove precipitates. Aliquots (50 µL) of samples and standards were added to the plates in triplicate and incubated for 1 hour at room temperature. After washing with wash buffer, the detection antibody, F025-27, conjugated to alkaline phosphatase was added.19 Antibody-enzyme conjugation was performed with the maleimide alkaline phosphatase conjugation kit from Pierce Immunochemical Co. The antibody conjugate was incubated for 3 hours at room temperature, followed by washing with Tris-buffered saline. A fluorescent signal was generated by the addition of the substrate 4-methylumbelliferyl phosphate at 50 µg/mL in diethanolamine (1 mol/L)/MgCl (0.5 mmol/L), pH 9.8. After 40 minutes of substrate turnover, the plates were read with a Dynatech Microfluor plate reader and the data analyzed with 1224 Multicalc software (Wallac). Concentrations of amylin or amylin-like peptides were determined by comparison with an eight-point standard curve. The minimum detectable concentration in both assays was 2 pmol/L. The interassay coefficient of variation was less than 15%, and the intra-assay coefficient of variation was less than 10% for both assays.
Fasting levels of insulin, glucose, and amylin were contrasted across the four groups (blacks and whites, with and without hypertension) with ANOVA. Insulin, glucose, and amylin responses to glucose challenge were summarized as areas under the curve (AUC).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Subject characteristics are summarized in Table 1
. The groups were
similar in age and body mass index. Blacks and whites had comparable
blood pressures. Hypertensive subjects had higher diastolic
pressures than normotensive subjects (P<.016). All subjects
exhibited normal glucose tolerance after ingestion of a 75-g glucose
load according to the criteria of the National Diabetes Data Group, ie,
fasting plasma glucose less than 7.8 mmol/L and 2-hour
post–glucose load plasma glucose less than 7.8
mmol/L.20
Table 2 summarizes the ANOVAs on fasting samples by race
and diagnosis for glucose, insulin, and amylin (as measured by both the
F002-2 and F024-4 antibody assays). No racial differences or
race-by-hypertension interactions were noted in fasting glucose
concentrations, although the hypertensive subjects had slightly higher
glucose values than the normotensive subjects (P<.01).
Blacks had significantly higher fasting insulin concentrations
(P<.025). Although hypertension was not associated with
higher fasting insulin values, there was a trend for a
race-by-diagnosis interaction (ie, black hypertensive subjects
had higher concentrations, P=.09). A significant interaction
between race and hypertension was seen in circulating plasma
amylin-like peptide concentrations as measured by the F002-2 assay
(P=.005) such that black hypertensive subjects had higher
concentrations of these peptides. No differences were observed between
the groups for the amylin concentrations measured by the F024-4 assay
(P=.892). Thus, circulating concentrations of
amylin-like peptides were increased in this cohort of black
hypertensive subjects but not in the white hypertensive subjects.
|
Table 2 also summarizes the ANOVA analyses for the various area
under the curve (AUC) values during the 2-hour glucose tolerance test.
No significant differences in glucose or insulin AUC were noted. There
was a significant race-by-diagnosis interaction affecting the
amylin-like peptide concentrations with the F002-2 assay
(P=.005), such that black hypertensive subjects had higher
values. A similar interaction (P=.04) was observed for
amylin concentrations measured with the F024-4 antibody assay. Figs 1 and 2 show plasma amylin responses
throughout the glucose tolerance tests as measured by both the F002-2
and F024-4 assays.
|
), black normotensive
(
), white hypertensive (
), and black hypertensive (
)
subjects with an F002-2 capture antibody that detects amylin and at
least two additional larger molecular weight species.
|
We also examined the association between fasting glucose, insulin, and amylin concentrations. As expected, fasting glucose and insulin concentrations were significantly correlated (r=.471, P<.0001). The two amylin measurements were even more strongly correlated (r=.688, P<.0001). The F002-2 concentrations were significantly associated with the corresponding insulin concentrations (r=.577, P<.0001) and, to a lesser extent, with glucose (r=.237, P<.05). The F024-4 measurements were not associated with either fasting glucose or insulin.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The concentrations of plasma amylin-like immunoreactivity were highest in black hypertensive subjects, a race-by-diagnosis interaction, which may imply a genetic predisposition. Although the results of the F002-2 and F024-4 assays were strongly correlated (r=.69, P<.0001), the effect of hypertension was most pronounced in the F002-2 assay, which detects both human amylin and the larger molecular weight species contributing to amylin-like activity. This raises the possibility of altered processing of the amylin peptide by the ß cell in these subjects. This altered processing could represent either altered cleavage of a precursor molecule or alterations in the posttranslational modifications of the amylin molecule.
Differences in circulating plasma amylin concentrations in hypertensive individuals are of interest in view of the proposed linkage between ß-cell hypersecretion and hypertension.1 2 3 Previous reports have demonstrated elevated plasma insulin concentration in hypertensive whites,1 2 an observation not seen in the present study. However, recent studies have failed to confirm the proposed causal relationship between hyperinsulinemia and hypertension, increasing interest in the possibility that another ß-cell peptide may be involved, with insulin being an "innocent bystander."8 Although a previous report described a relationship between elevated plasma amylin concentrations and hypertension in a white population,14 no published data exist regarding the influence of race on circulating plasma amylin levels. However, a recently published abstract reported a trend for increased amylin levels in blacks and hypertensive subjects.21
The present data support the existence of a relationship between hypertension and elevated circulating levels of amylin-like immunoreactivity that appears to be accentuated in the subset of black hypertensive subjects. Given our relatively small sample size, additional studies are required to confirm these observations and explore the underlying mechanisms. Such studies need to examine whether the presence of increased circulating amounts of the larger molecular weight amylin-like peptides indicates an alteration in peptide processing by the ß cell and whether these peptides play a role in the pathogenesis of either hypertension or insulin resistance.
|
Acknowledgments |
|---|
This work was supported in part by grants HL-36005 and RR 00827 from the National Institutes of Health, Bethesda, Md. The authors thank Amylin Pharmaceuticals for running the amylin assays.
Received November 8, 1995; first decision December 18, 1995; accepted February 12, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Welborn TA, Brechenridge A, Rubinstein AH, Dollery CT, Fraser TR. Serum-insulin in essential hypertension and in peripheral vascular disease. Lancet. 1966;1:1336-1337. [Medline] [Order article via Infotrieve]
2. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico MA, Oleggini M, Pedrinelli R, Brandi L, Bevilacqua S. Insulin resistance in essential hypertension. N Engl J Med. 1987;317:350-357. [Abstract]
3. Saad MF, Lillioja S, Nyomba BL, Castillo C, Ferraro R, De Gregorio M, Ravussin E, Knowler WC, Bennett P, Howard B. Racial difference in the relation between blood pressure and insulin resistance. N Engl J Med. 1991;324:733-739. [Abstract]
4. Williams B. Insulin resistance: the shape of things to come. Lancet. 1994;344:521-524. [Medline] [Order article via Infotrieve]
5. Liang C-S, Doherty JU, Faillace R, Maekawa K, Arnold S, Gavras H, Hood BW Jr. Insulin infusion in conscious dogs: effects on systemic and coronary hemodynamics, regional blood flows, and plasma catecholamines. J Clin Invest. 1982;69:1321-1336.
6. Rowe JW, Young JB, Minaker KL, Stevens AL, Pallotta J, Landsberg L. Effect of insulin and glucose infusions on sympathetic nervous system activity in normal man. Diabetes. 1981;30:219-225. [Medline] [Order article via Infotrieve]
7. Young AA, Rink TJ, Vine W, Gedulin B. Amylin and syndrome X. Drug Dev Res. 1994;32:90-99.
8. Nagi DK, Hendra TJ, Ryle AJ, Cooper TM, Temple RC, Clark P, Schneider A, Hales C, Yudkin J. The relationship of concentrations of insulin, intact proinsulin and 32-33 split proinsulin with cardiovascular risk factors in type 2 (non-insulin-dependent) diabetic subjects. Diabetologia. 1990;33:532-537. [Medline] [Order article via Infotrieve]
9. Westermark P, Wernstedt C, O'Brien TD, Hayden DW, Johnson KH. Islet amyloid in type 2 human diabetes mellitus and adult diabetic cats contains a novel putative polypeptide hormone. Am J Pathol. 1987;127:414-417. [Abstract]
10.
Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Reid
KB. Purification and characterization of a peptide from
amyloid-rich pancreases of type 2 diabetic patients.
Proc Natl Acad Sci U S A. 1987;84:8628-8632.
11. Peiro E, Degano P, Silvestre RA, Marco J. Inhibition of insulin release by amylin is not mediated by changes in somatostatin output. Life Sci. 1991;49:761-765. [Medline] [Order article via Infotrieve]
12. Frontoni S, Choi SB, Banduch D, Rossetti L. In vivo insulin resistance induced by amylin primarily through inhibition of insulin-stimulated glycogen synthesis in skeletal muscle. Diabetes. 1991;40:568-573. [Abstract]
13. Wookey P, Berka J, Rumble J, Kelly D, Du H, Cooper M. Amylin binding sites in rat kidney and stimulation of plasma renin activity: characterization using antagonists. Diabetologia. 1994;37:A116. Abstract.
14. Kautaky-Willer A, Thomaseth K, Pacini G, Clodi M, Ludvik B, Streli C, Waldhausi W, Prager R. Role of islet amyloid polypeptide secretion in insulin-resistant humans. Diabetologia. 1994;37:188-194. [Medline] [Order article via Infotrieve]
15. Harris MI. Non-insulin dependent diabetes mellitus in black and white Americans. Diabetes Metab Rev. 1990;6:71-90. [Medline] [Order article via Infotrieve]
16. Hall WD, Saunders E, Shulman N, eds. Hypertension in Blacks: Epidemiology, Pathophysiology, and Treatment. Chicago, Ill: Year Book Medical Publishers Inc; 1985.
17. Morgan C, Lazarow A. Immunoassay of insulin: two antibody systems. Plasma levels of normal, subdiabetic and diabetic rats. Diabetes. 1963;12:115-126.
18. Rittenhouse J, Janes SM, Bierle JR, Park DM, Phelps JL, Koda JE. Isolation and characterization of human amylin. Protein Sci. 1994;3:66. Abstract.
19. Percy AJ, Trainor D, Rittenhouse J, Janes S, Koda JE. Sensitive two-site enzyme immunoassays for amylin and amylin-like peptides in human plasma. Diabetologia. 1994;37(suppl 1):A117. Abstract.
20. National Diabetes Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes. 1979;28:1039-1057. [Medline] [Order article via Infotrieve]
21. Kailasam M, Fineman M, Koda J, Parmer R, O'Connor D, Circulating amylin in human essential hypertension: racial differences and early elevations in subjects at genetic risk of hypertension. J Invest Med. 1995;43(suppl 2):266A. Abstract.
This article has been cited by other articles:
 |
|
 |
|
  K. Ku, K. Nakayama, Y. Saitoh, S. Nosaka, T. Kitano, T. Hanada, H. Nagami, K. Yamada, and K. Minami Long-Term Follow-up (8 to 17 Years) After Thromboexclusion Operation for Thoracic Aortic Aneurysms Ann. Thorac. Surg., August 1, 1997; 64(2): 399 - 403. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||