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Istituto di Medicina Interna, Endocrinologia e Malattie del Metabolismo, Ospedale Garibaldi, Università di Catania (L.F., R.B., D.S., R.V.), 95123 Catania; Unità Operativa e di Ricerca di Endocrinologia, Istitito Scientifico Ospedale Casa Sollievo Sofferenza (R.D.P., V.T.), 71013 San Giovanni Rotondo (Foggia); and Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza" and Istituto Scientifico Mendel CSS (A.P.), 00100 Roma, Italy
Address all correspondence and requests for reprints to: Lucia Frittitta, M.D., Endocrinologia, Ospedale Garibaldi, Piazza Santa Maria di Gesù, 95123 Catania, Italy. E-mail: segmeint{at}mbox.unict.it
Abstract
PC-1 is a membrane glycoprotein that impairs insulin receptor
function. Its K121Q polymorphism is a genetic determinant of insulin
resistance. We investigated whether the PC-1 gene modulates insulin
sensitivity independently of weight status (i.e. both in
nonobese and obese individuals). Nondiabetic subjects [164 males, 267
females; age, 37 ± 0.6 yr, mean ±
SEM; body mass index (BMI), 32.7 ± 0.5
kg/m2], who were subdivided into 220 nonobese
(BMI 
29.9) and 211 obese, were studied. Although subjects were
nondiabetic by selection criteria, plasma insulin concentrations during
oral glucose tolerance test were higher (P < 0.05)
in Q allele-carrying subjects (K121Q or Q121Q genotypes),
compared with K121K individuals, in both the nonobese and obese groups.
Insulin sensitivity, measured by euglycemic clamp in a representative
subgroup of 131 of 431 randomly selected subjects, progressively
decreased (P < 0.001) from nonobese K121K [n
= 61; glucose disposal (M) = 34.9 ± 1.1 µmol/kg/min] to
nonobese Q (n = 21; M = 29.9 ± 2.0), obese K121K
(n = 31, M = 18.5 ± 1.2), and obese Q (n = 18,
M = 15.5 ± 1.2) carriers. The K121Q polymorphism was
correlated with insulin sensitivity independently
(P < 0.05) of BMI, gender, age, and waist
circumference. In conclusion, the Q121 PC-1 variant and obesity have
independent and additive effects in causing insulin resistance.
INSULIN RESISTANCE PLAYS a major role in the pathogenesis of type 2 diabetes mellitus and cardiovascular diseases (1, 2, 3). It represents a typical complex trait with environmental and genetic backgrounds both contributing to its pathogenesis (2, 3). The genetic background of insulin resistance is not completely understood, mainly because it is polygenic, i.e. it is determined by a variety of genes simultaneously involved, with each one having only a small effect (3). Unraveling the genetics of insulin resistance is further complicated by the frequent requirement of an interaction between environment and gene(s) for the phenotype to be expressed (3, 4).
A gene that may cause insulin resistance is PC-1, coding for a class II glycoprotein that negatively modulates insulin receptor function (5, 6, 7, 8, 9, 10, 11). In addition, the 6q22-q23 chromosomal region, which harbors the PC-1 gene, has been linked to insulin resistance (12). We have recently described a PC-1 gene polymorphism in which lysine (K) is replaced by glutamine (Q) at codon 121 (13). The Q121 variant associates with whole-body insulin resistance in most (13, 14), but not all (15), populations studied and impairs insulin signaling as indicated by functional studies in transfected cells (16). These data indicate that the PC-1 gene Q121 variant plays a role in human insulin resistance.
Whether PC-1 K121Q polymorphism interacts with obesity in causing insulin resistance is unknown. We have investigated this issue by studying a large cohort of nondiabetic Caucasians from Sicily with a wide range of body mass index (BMI).
Subjects and Methods
Experimental subjects
Four hundred thirty-one unrelated (164 males and 267 females;
age, 37 ± 1 yr; range, 13–72 yr; BMI, 32.7 ± 0.5
kg/m2; range, 18–59.3) nondiabetic (by American
Diabetes Association criteria) Caucasians from Sicily were
studied. Two hundred twenty subjects were nonobese (BMI 29.9
kg/m2), and 211 were obese. Overweight and obese
subjects (BMI 
25 kg/m2) were recruited from the
Outpatient Metabolic Clinic of our institution. Normal-weight subjects
were recruited from the staff of our hospital. Informed consent was
obtained from all participants in the study, which was approved by the
local ethics committees. All subjects were on a weight-maintaining
diet, and none were on medications known to interfere with measured
variables.
Metabolic studies
In all subjects, lipid profile and arterial blood pressure were measured. Serum cholesterol and triglycerides were evaluated by enzymatic methods (ILTest Cholesterol and ILTest Triglycerides, Instrumentation Laboratory, Milan, Italy). High-density lipoprotein fraction was separated by use of Mg2+ and dextran sulfate method (CHOL-HDL reagent, Sclavo Diagnostics, Siena, Italy).
The 75-g oral glucose tolerance test (OGTT) was performed after an overnight fast, and blood samples were collected at 0, 60, and 120 min. Plasma glucose was measured by the glucose oxidase method on a Beckman Glucose Analyzer 2 (Beckman Coulter, Inc., Fullerton, CA); plasma insulin was measured by microparticle enzyme immunoassay (Abbott IMx Insulin Assay, Abbott Laboratories, Abbott Park, IL).
Euglycemic hyperinsulinemic clamp was performed as previously described (13) in a subgroup of 131 randomly selected subjects representative of the entire cohort (60 males and 71 females; age, 36.4 ± 1 yr; range, 15–64 yr; BMI, 29.9 ± 1 kg/m2; range, 18.5–54).
Polymorphism screening
Exon 4 K121Q polymorphism was screened as previously described (13). Briefly, exon 4 amplimers were obtained using oligonucleotides Fw (5'-ctgtgttcactttggacatgttg-3') and Rw (5'-gacgttggaagataccaggttg-3') as primers. PCR products were digested using AvaII restriction enzyme and run on 12% native polyacrylamide gel for 2 h at 500 V. Then, gels were stained by silver nitrate. Three hundred ten subjects carried the K121K genotype (KK), 115 carried the K121Q genotype, and 6 carried the Q121Q genotype. Heterozygous or homozygous subjects for the 121Q polymorphism were considered together as a single group and indicated as Q. Both alleles were in Hardy-Weinberg equilibrium with the Q121 allele, having a frequency of 15.1%.
Statistical analysis
Values are given as mean ± SEM. Mean values from two groups were compared by t test or Mann-Whitney U test, as appropriate. Mean values from more than two groups were compared by one-way ANOVA test followed by Fisher’s projected least significant difference post hoc test of significance between groups. When subjects were grouped only for the PC-1 genotype, both in nonobese and obese subjects, glucose and insulin profiles during OGTT were compared by two-way ANOVA. When subjects were grouped for both the PC-1 genotype and the weight status (obese or nonobese) and gender, age, and waist circumference were considered as covariates, two-way ANOVA with repeated measures and Bonferroni correction for post hoc tests were applied to analyze glucose and insulin profiles during OGTT. When subjects were grouped for both PC-1 genotype and weight status with the above mentioned covariates and dependent variables were either glucose or insulin areas under the curves (AUCs) during OGTT or M value during clamp (i.e. no repeated measures), analysis of covariance and Bonferroni correction for post hoc tests were applied. Multiple regression analysis was performed to analyze the relationship between PC-1 genotype, BMI, gender, age, and waist circumference and either glucose or insulin AUC at OGTT or M value at clamp.
Results
Clinical features and fasting measurements
Clinical features of the subjects that were studied, subdivided
according to the PC-1 genotype and the absence or presence of obesity,
are shown in Table 1
. No difference in
the Q allele frequency was observed between nonobese and obese subjects
(14.8 and 15.4%, respectively). Compared with nonobese individuals,
obese subjects showed several abnormalities related to insulin
resistance, including higher concentrations of fasting glucose,
insulin, and triglycerides and higher values of waist circumference and
mean blood pressure (Table 1). In the Q carrier subgroup, gender, age,
and BMI were similar to the K121K carriers, and no significant
difference was observed in the parameters measured (both in obese and
nonobese subjects). Only serum cholesterol was significantly
(P < 0.05) higher in Q than in K121K nonobese
carriers. Therefore, under unstimulated conditions, the Q121 variant
did not affect the metabolic parameters in either the normal or the
obese subjects.
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A negative effect of the Q121 variant appeared under glucose
stimulation: plasma insulin profiles during OGTT were significantly
higher in Q carriers compared with K121K individuals, both among
nonobese (P < 0.05) and obese (P <
0.01) subjects (Fig. 1A). When all
subjects were grouped for both weight status
(i.e. presence or absence of obesity) and PC-1
genotype, with gender, age, and waist circumference as covariates,
plasma insulin concentrations during OGTT were significantly different
between obese vs. nonobese (F = 32.9; P
< 0.0001; data not shown) and Q vs. K121K (F = 9.8;
P = 0.002) individuals (Fig. 1C). No interaction
between PC-1 genotype and weight status was observed (F = 0.6;
P = 0.4).
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). This was not the case in obese individuals (Fig. 1B). When all subjects were grouped for both weight status and PC-1
genotype, with gender, age, and waist circumference as covariates,
plasma glucose concentrations were significantly higher (F = 13.3;
P < 0.0001) in obese in respect to nonobese subjects
(data not shown). In contrast, no difference was observed in Q compared
with K121K individuals (F = 2.4; P = 0.1) (Fig. 1D). No interaction between PC-1 genotype and weight status was
observed (F = 0.5; P = 0.5). Very similar results
were obtained when glucose and insulin AUC, instead of glucose
and insulin profiles, during OGTT were considered (data not shown). Insulin sensitivity measured by euglycemic hyperinsulinemic clamp
Whole-body insulin sensitivity (i.e. insulin-mediated
glucose disposal) was measured by euglycemic hyperinsulinemic clamp in
a subgroup of randomly selected individuals (n = 131; 82 nonobese
and 49 obese), representative of the entire cohort as far as gender,
age, and BMI are concerned (see Subjects and Methods). Also,
Q allele frequency in this subgroup was very similar to that of the
entire cohort (16.4 and 15.1%, respectively). M value of glucose
disposal (micromoles per kilogram per minute) progressively decreased
(P < 0.001) from nonobese K121K (34.9 ± 1.1;
n = 61) to nonobese Q (29.9 ± 2.0; n = 21), obese K121K
(18.5 ± 1.2; n = 31), and obese Q (15.5 ± 1.2; n
= 18) carriers (Fig. 2A). When all 131
subjects were grouped for both the weight status and the PC-1 genotype,
with gender, age, and waist circumference as covariates, the PC-1
genotype (F = 5.9; P = 0.02) (Fig. 2B), but not
weight status (F = 0.77; P = 0.4; data not shown),
was significantly associated with insulin resistance. No interaction
was observed between PC-1 genotype and weight status (F = 0.8;
P = 0.4). The lack of association between weight status
and insulin resistance is very likely due to the correction for waist
circumference, the latter being itself strongly associated with insulin
resistance (F = 22.5; P < 0.0001). This is also
suggested by multiple regression analysis (see below).
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Data were analyzed then by multiple regression analysis with the PC-1 genotype, BMI, gender, age, and waist circumference as independent variables and glucose or insulin AUC or M value as separate dependent variables. In detail, waist circumference and age (P = 0.0002 and P < 0.0001, respectively; coefficients, 3.9 and 3.5, respectively; R = 0.66, adjusted R2 = 0.44) were independently correlated with glucose AUC; the PC-1 genotype and waist circumference (P = 0.003 and P = 0.02, respectively; coefficients, 10,126.1 and 522.0, respectively; R = 0.57, adjusted R2 = 0.32) were independently correlated with insulin AUC; the PC-1 genotype and waist circumference (P = 0.02 and P = 0.048, respectively; coefficients, -3.5 and -0.3, respectively; R = -0.77, adjusted R2 = -0.58) were independently correlated with M value. Of the 58% variance of insulin sensitivity at euglycemic clamp explained by all of the above mentioned variables, PC-1 genotype alone accounted for 5.7%.
Discussion
We recently described a frequently occurring PC-1 amino acid variant in which K is replaced by Q at codon 121 (13). The Q121 PC-1 variant associates with insulin resistance in several (13, 14), but not all (15), populations that were studied. Intrafamily studies have confirmed this association (14), thus making unlikely false positive results due to population stratification (17, 18). Finally, when cDNAs of the two PC-1 variants were transfected in cultured cells, the Q121 PC-1 variant turned out to be a stronger inhibitor of insulin signaling than the wild-type PC-1 (16). Together, these data indicate that the Q121 PC-1 variant plays a role in causing insulin resistance.
We report here that, as indicated by hyperinsulinemia during OGTT and lower insulin-mediated glucose disposal at euglycemic clamp, both nonobese and obese individuals carrying the Q121 PC-1 variant are insulin resistant, compared with weight-matched K121K subjects. In addition, the Q121 PC-1 variant and waist circumference have independent and additive effects in causing insulin resistance, together accounting for nearly 60% of insulin sensitivity variability. The PC-1 genotype alone accounted for approximately 6% of insulin sensitivity variability, an effect for which the magnitude is quite compatible with a model of a complex trait recognizing a polygenic control.
Our findings are in concert with the leading hypothesis that insulin resistance recognizes multiple etiopathogenetic factors, including environmental determinants and a variety of genes that may be involved, each one having a small effect (i.e. polygenic background) (2). Recently, the interaction between obesity and amino acid variants of IRS proteins has been reported, with obesity being necessary for the genetic variant to cause insulin resistance (19, 20). In these studies, therefore, obesity played a permissive role for the gene negative effect to be manifest, indicating that the gene polymorphism per se was not sufficient to cause insulin resistance (19, 20). This is at variance with our present data with PC-1; the Q121 PC-1 variant is sufficient, independent of BMI and abdominal obesity, to cause insulin resistance. Our findings are also compatible with the possibility that obesity (21) and the Q121 PC-1 variant (16) impair insulin signaling and action through different molecular mechanisms. In fact, obesity is believed to cause insulin resistance through the overproduction of metabolites, cytokines, and hormones that interfere with insulin action at different postreceptor levels (21). At variance, the Q121 PC-1 variant shows, compared with the K121 PC-1 variant, a stronger direct interaction with the insulin receptor, which eventually ends up in a higher inhibition of its tyrosine-kinase activity and insulin action (16). The two mechanisms act, therefore, at different levels of the insulin signaling network and may well be independent and additive in causing insulin resistance.
In conclusion, whatever their BMI and fat distribution is, subjects carrying the Q121 PC-1 variant are insulin-resistant in respect to subjects with similar weight and with K121K genotype. Abdominal obesity and the Q121 PC-1 variant have additive and independent effects in further deteriorating insulin sensitivity.
Acknowledgments
Footnotes
This study was supported by grants from Ministero della Sanità (Ricerche Correnti 1999 and 2000 e Finalizzate 1999 and 2000) (to V.T.) and Cofinanziamento 2000 from Ministero Università Ricerca Scientifica e Tecnologica (to L.F.). L.F. is a recipient of a postdoctoral fellowship of the University of Catania.
Abbreviations: AUC, Area under the curve; BMI, body mass index; K, lysine; M, glucose disposal; OGTT, oral glucose tolerance test; Q, glutamine.
Received April 5, 2001.
Accepted August 31, 2001.
References
gene expression, is associated with a reduction of both whole body
insulin sensitivity and insulin receptor tyrosine-kinase activity. Diabetologia 40:282–289[CrossRef][Medline]
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