© 1998 American Heart Association, Inc.
Original Contributions |
Plasma Leptin Levels
Interaction of Obesity With a Common Variant of Insulin Receptor Substrate-1
Presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[suppl]:I-34).
From the Departments of Internal Medicine (F.K.) and Surgery (E.H.), Krankenhaus Hallein, and the Department of Laboratory Medicine (C.W., D.B., W.P.), Landeskrankenanstalten, Salzburg, Austria.
Correspondence to Franz Krempler, MD, Department of Internal Medicine, Krankenhaus Hallein, Bürgermeisterstrasse 34, A-5400 Hallein, Austria. E-mail w.patsch{at}lkasbg.gv.at
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Abstract |
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Abstract—Obesity is associated with insulin resistance and other major cardiovascular risk factors. A common amino acid polymorphism at codon 972 of the insulin receptor substrate-1 (IRS-1) has been shown to interact with obesity in the expression of insulin resistance. The plasma concentration of the adipocyte-specific hormone leptin is increased in obesity and is correlated with adipose tissue mass. Because in vitro studies demonstrated inhibitory effects of leptin on insulin signaling, leptin may be involved in obesity-associated insulin resistance. To gain insight into the relationship between insulin and leptin in obesity, we studied plasma leptin levels and several cardiovascular risk factors, as well as their modification by the IRS-1 codon 972 genotype, in 156 obese individuals and 131 lean control subjects. In both groups, 10% of the subjects were heterozygous for the IRS-1 codon 972 variant. Obese individuals harboring the IRS-1 variant displayed significantly lower plasma concentrations of leptin than obese subjects without the polymorphism (means, 26.7 versus 37.8 ng/mL, P<0.0293). In a subgroup of obese patients, leptin mRNA abundance was measured in the adipose tissue and was significantly lower in carriers of the IRS-1 variant than in subjects with the wild-type variant (P<0.0291). Our data suggest that insulin signaling influences plasma leptin concentrations at the mRNA expression level and argue against leptin as a major causative factor of insulin resistance.
Key Words: obesity • leptin • insulin resistance • insulin receptor substrate-1
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Introduction |
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Obesity is associated with insulin resistance and hyperinsulinemia. Both conditions increase the risk for coronary heart disease.1 2 Hyperinsulinemia seems to predict ischemic heart disease independent of other risk factors.3 The mechanism(s) underlying the association between insulin resistance and obesity is not completely understood. Leptin, the product of the ob gene, is secreted specifically by adipocytes, binds to hypothalamic receptors encoded by the db gene, and conveys the size of the adipose tissue mass to centers controlling appetite.4 5 6 Administration of recombinant leptin to ob/ob mice resulted in decreased food intake, increased energy expenditure, and improved insulin resistance before affecting body weight.7 8 9 Because the reduction in glucose and insulin exhibited a more rapid time course than the changes in body weight, the inhibitory effect of leptin on neuropeptide Y gene expression and interactions with peripheral cells have been implicated in the improvement of insulin sensitivity.10 Moreover, leptin increased glucose turnover in lean wild-type mice.11 Insulin administration, on the other hand, was shown to increase leptin expression in rodents.12 In the common forms of human obesity, the relation of leptin to insulin resistance is less clear, because both leptin and insulin levels are often increased.13 Associations of plasma levels of leptin and insulin have been reported in subjects with or without insulin resistance.14 Three-hour euglycemic hyperinsulinemic clamp studies failed to increase leptin mRNA abundance in human adipose tissues,15 while infusion of insulin during a period of 8.5 hours induced an increase in plasma leptin levels.14 Moreover, hyperinsulinemia during a period of 72 hours increased serum leptin concentrations in a dose-dependent manner.16 Thus, prolonged hyperinsulinemia seems to enhance leptin expression in humans also. However, leptin itself has been suspected to be a causative factor of insulin resistance.17 Studies in cultured human and rat hepatoma cell lines have shown that leptin attenuates several insulin signals.18
A common sequence polymorphism in the insulin receptor substrate-1 (IRS-1) gene that predicts the replacement of glycine by arginine at codon 972 has been shown to be more frequent in patients with non–insulin-dependent diabetes mellitus than in control subjects and may therefore contribute to insulin resistance.19 Interestingly, this IRS-1 polymorphism appeared to interact with obesity, because it potentiated insulin resistance in young obese subjects.20 To determine the role of insulin signaling on leptin levels and to ascertain potential influences of leptin on the interaction of obesity with the IRS-1 variant, we studied both control and obese subjects exhibiting sequence variations at the IRS-1 gene locus. Part of this study was presented at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997.21
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Methods |
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Study Population
This study included 287 unrelated white subjects; 156 subjects were obese patients referred consecutively for weight reduction surgery, and 112 of these obese subjects fulfilled the criteria for bariatric surgery22 and underwent a gastric banding procedure. Adipose tissue samples were collected from comparable anatomic sites of the omentum (referred to as intraperitoneal) and the abdominal wall (referred to as extraperitoneal). One hundred thirty-one subjects were lean, healthy individuals. Among obese subjects, 6 females used oral contraceptive drugs and 27 subjects used medications, including thyroid hormones (4 subjects), calcium channel blockers or angiotensin-converting enzyme inhibitors (9 subjects), hypoglycemic drugs (1 subject), allopurinol (4 subjects), H2-receptor antagonists or antacids (3 subjects), benzodiazepines or tricyclic antidepressants (7 subjects), and theophylline and selective ß2-adrenergic agonists by inhalation (2 subjects). Study subjects gave their written informed consent and the study protocol was approved by the institutional review board for human studies.
Body mass index (BMI, kilograms per meter squared) was calculated from measurements of weight and height. Sitting blood pressure was measured after a 5-minute rest. Smoking status was assessed with interviews. Subjects were classified as diabetic if they were using hypoglycemic medications or had fasting-plasma glucose concentrations >140 mg/dL.
Biochemical Measurements
After patients fasted overnight, venous blood was collected at 8
AM into tubes containing EDTA. Fasting-plasma glucose was
measured using a hexokinase/glucose-6-phosphatase dehydrogenase method.
Plasma insulin was measured using immunoassay (MEIA, Abbott
Laboratories). Plasma cholesterol and
triglyceride were measured with enzymatic procedures using
a Hitachi 717 analyzer (Boehringer Mannheim
Diagnostics) and the respective enzymatic kits (catalog
Nos. 1489437 and 1058550, Boehringer Mannheim
Diagnostics). HDL cholesterol was determined in
supernatants after precipitation of plasma with phosphotungstic
acid/MgCl2, and LDL cholesterol was
calculated according to the formula of Friedewald et
al.23 Levels of apoA-I and apoB were determined
using nephelometric procedures (Array 360, Beckman). Plasma leptin
levels were measured with a Leptin RIA kit (Linco Inc) using an
antibody raised against highly purified human leptin and recombinant
human leptin as a tracer and standard, respectively. The interassay and
intra-assay coefficients of variation were 8% and 7%,
respectively.
Isolation of RNA From Adipose Tissues and Northern Blot
Analysis
Total RNA was isolated from 2 g of adipose tissue according
to the method of Chomczynski and Sacchi.24 The
integrity of RNA was ascertained by the electrophoretic pattern of rRNA
in formaldehyde gels.25 Relative abundance of
leptin mRNA was determined using Northern blot analysis as
outlined by Thomas.26 Total adipose tissue
RNA (30 µg) was denatured with 1 mol/L glyoxal and 50% DMSO and
separated by electrophoresis in 1.2% agarose. The RNA was transferred
to S&S Nytran membranes (Schleicher & Schüll) by capillary
blotting and hybridized to full-length 32P-leptin
cDNA labeled by the random priming method using
[32P]dCTP (3000 Ci/mmol, Amersham). The
relative abundance of leptin mRNA was determined from the intensities
of bands, which were quantified on the autoradiographs by densitometry
using the Molecular Analyst software (Bio-Rad). The relative abundance
of leptin mRNA was corrected for by the relative abundance of GAPDH
(GenBank accession No. M33197). After the leptin probe was stripped
according to the manufacturer's recommendations, membranes were
hybridized with a 553 bp–containing human
32P-GAPDH cDNA probe. The GAPDH cDNA probe was
obtained with reverse transcription and polymerase chain reaction (PCR)
amplification of adipose tissue RNA using 5'-CACCACCATGGAGAAGGCTGG-3'
(+306, +326) and 5'-GAAGTCAGAGGAGACCACCTG-3' (+858, +838) as upstream
and downstream primers, respectively. The numbers in parentheses
designate the 5' and 3' ends in the cDNA relative to the translation
start site. Primers were synthesized using a Beckman Oligo 1000 DNA
Synthesizer (Beckman Instruments Inc). Leptin and GAPDH mRNA signals on
different membranes were normalized by using overlapping RNA samples in
Northern blots. To obtain a measure of individual leptin mRNA
expression, the average value was calculated from the
intraperitoneal and extraperitoneal measurements
and referred to as "total leptin expression."
DNA Preparation and IRS-1 Typing
DNA was isolated from white blood cells using the QIAamp Tissue
Kit (Qiagen Inc). For typing of the IRS-1 polymorphism at amino
acid residue 972, the PCR conditions and primers described by Almind et
al19 were used. After digestion of PCR
products with 10 U of BstN1 (New England Biolabs) for 3
hours at 60°C, DNA fragments were separated in 3% agarose gels. For
quality control purposes, 
10% of samples were reanalyzed
and showed complete agreement with their original assignment.
Statistical Analyses
Allele frequencies of the 972 polymorphism were
estimated by gene counting. One-way ANOVA was used to test the
distributions of the continuous variables among strata. The
variation in plasma leptin levels attributable to the IRS-1
polymorphism was calculated using the
R2 from the ANOVA. Kruskal-Wallis test was
used, or a transformation was made on the original variable, if the
equal variance and normality assumptions of the 1-way ANOVA were
rejected. To compare categorical variables, a contingency
2 test was used. A factorial ANOVA was used to
determine the interaction between obesity and IRS-1 polymorphism on
leptin and other continuous variables adjusted for sex.
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Results |
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This study examined 156 obese patients and 131 controls. Table 1
shows sex, age, BMI, blood pressure,
and prevalence of diabetes in our study subjects. About 4 times more
women than men were referred for weight reduction surgery. Nonobese
controls and obese patients were similar in average and median age (39
and 36 years in controls versus 36 and 35 years in obese patients,
respectively). Both average and median BMI was much higher in obese
patients than in controls (43.7 and 42.4 kg/m2
versus 23.3 and 23.1 kg/m2). Systolic and
diastolic blood pressure readings were significantly higher
in obese patients than in controls. Overt diabetes was present in 8
obese subjects compared with 1 subject in the nonobese group
(P=0.0401). The proportion of cigarette smokers was similar
in lean and overweight individuals (P=0.7101).
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The profiles of cardiovascular risk factors were markedly different between obese and nonobese individuals. Average values for glucose (91.4 versus 70.7 mg/dL), insulin (10.9 versus 6.7 µU/mL), triglyceride (342 versus 121 mg/dL), and apoB (99.5 versus 86.2 mg/dL) were significantly higher in obese individuals than in lean individuals (all P<0.001). Average values for HDL cholesterol (34.0 versus 53.3 mg/dL) and apoA-I (117.7 versus 141.8 mg/dL) were significantly lower in obese subjects than in controls (both P<0.001). Exclusion of diabetic subjects did not alter these differences. No difference in plasma cholesterol was observed between obese patients and controls (197.6 versus 195.6 mg/dL). Average and median levels of leptin were significantly higher in cases than in controls (36.8 versus 8.6 ng/mL and 33.2 versus 7.1 ng/mL, P<0.001).
In the entire study group, 29 subjects (10.1%) were heterozygous for
the glycine to arginine substitution at codon 972 of IRS-1 (Table 2), and the frequency of this
polymorphism was similar in obese and nonobese individuals. Neither
in the obese group nor in the control group was a subject homozygous
for the IRS-1 variant identified. Among all clinical and biochemical
data studied, only plasma leptin levels in obese subjects exhibited a
significant difference by IRS-1 genotype. ANOVA showed that
obese carriers of the IRS-1 variant had significantly lower average
plasma leptin levels than obese wild-type subjects (26.7 versus 37.8
ng/mL, P<0.029) (Table 2). Adjustment for sex and BMI
slightly strengthened the significance (P<0.023). Median
values were also significantly different (23.5 versus 34.3 ng/mL,
P<0.018) (Kruskal-Wallis). The IRS-1 codon 972
polymorphism accounted for 3.05% of the variation of serum leptin
levels in our obese subjects. The average leptin values in lean
subjects were 9.4 versus 8.6 ng/mL (P<0.636) and median
values were 8.8 versus 7.0 ng/mL (P<0.436). Moreover, a
significant interaction between the IRS-1 codon 972 variant and obesity
status was observed with respect to leptin levels
(P<0.035). Exclusion of diabetic subjects and subjects
using oral contraceptives or any kind of medication did not
substantially alter the association of IRS-1 genotype with
leptin levels. Among obese subjects, the average glucose and insulin
levels tended to be higher in IRS-1 variant carriers than in IRS-1
wild-type subjects (Table 2). The only nonobese patient with diabetes
was a carrier of the IRS-1 variant.
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In a subgroup of obese patients, relative leptin mRNA abundance in
adipose tissue was determined (Table 3).
Patients in the studied subgroup exhibited similar differences in
plasma leptin levels by IRS-1 genotype as patients in the
entire study group (40.8 versus 28.5 ng/mL, P<0.028). In
carriers of the IRS-1 variant, total leptin expression was
significantly lower than in wild-type patients
(P<0.029).
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Discussion |
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Circulating leptin levels are correlated with the mass of adipose tissue. Factors other than body weight may also play a role in the regulation of leptin. Several studies indicate that leptin expression is stimulated by insulin,14 16 whereas other studies suggest that high leptin concentrations may contribute to insulin resistance.17 18 In the present study, plasma leptin concentrations were significantly lower in obese carriers of the IRS-1 variant than in obese wild-type carriers, indicating that the insulin-signaling pathway is part of the regulatory system of leptin. Our results are therefore consistent with studies that reported stimulation of leptin expression and an increase of serum leptin by insulin.14 16 27
In obese carriers of the IRS-1 variant, fasting glucose and insulin
levels were higher than in wild-type subjects (Table 2
), albeit these
differences were not statistically significant. Similar results were
reported by Clausen et al,20 who also found
higher glucose and insulin levels in their obese IRS-1 codon 972
variant subjects. These investigators used additional tests to
demonstrate a difference in insulin sensitivity between IRS-1 wild-type
and variant subjects. It is therefore reasonable to assume that the
higher insulin and glucose levels in our obese subjects harboring the
IRS-1 codon 972 variant reflect diminished insulin sensitivity. Hence,
our observations do not support a major role of leptin in promoting
insulin resistance. Nevertheless, minor or transient effects of
leptin-reducing insulin sensitivity may have been concealed by more
dominant, leptin-independent influences of the IRS-1 codon 972
variant.
The frequency of the IRS-1 variant in our study population was comparable to that of other studies17 and was similar in lean and obese subjects. We can therefore conclude that, at least in our study population, the IRS-1 codon 972 polymorphism itself is not a causative factor for obesity. However, interactions of subclinical genetic defects may influence the course and expression of multifactorial diseases. Injection of MSG in neonatal mice resulted in a more severe syndrome of obesity in IRS-1–deficient animals than in controls.28 Moreover, heterozygosity for both insulin receptor and IRS-1 null alleles increased the incidence of overt diabetes in transgenic mice several-fold.29 It is thus possible that interactions of the IRS-1 codon 972 polymorphism with other predisposing factors are involved in the pathogenesis of obesity.
Arteriovenous balance studies have shown that leptin synthesis is the
principal determinant of leptin plasma levels,30
and studies in rodents have demonstrated that insulin increases the
abundance of leptin mRNA in adipose tissue.12
Evidence to suggest that the insulin-signaling pathway is part of the
regulatory system of leptin in humans comes from leptin mRNA expression
data in a subgroup of our obese patients. In carriers of the IRS-1
variant, leptin expression levels were significantly lower than in
wild-type carriers (Table 3).
Experiments in myeloid progenitor cells have provided biochemical
evidence that the IRS-1 codon 972 variant impairs insulin-stimulated
signaling along the phosphatidylinositol 3-kinase pathway but also
tends to reduce the association of IRS-1 with
Grb2.31 Whether these pathways are also involved
in the regulation of leptin expression is not known. IRS-1 is also used
by other upstream activators such as insulin-like growth
factor-1 or interleukin-4.32 Moreover, treatment
of cultured murine adipocytes with tumor necrosis factor-
(TNF-)
converts IRS-1 into an inhibitor of the insulin receptor
tyrosine kinase activity, thus attenuating insulin receptor
signaling.33 In human obesity, the expression of
TNF- is correlated with the extent of obesity and
hyperinsulinemia.34 An
interaction of the functionally defective IRS-1 codon 972 variant with
TNF- or other mediators that are overexpressed in obesity might
contribute to our finding that leptin levels in plasma are reduced only
in obese carriers, but not in lean carriers, of the IRS-1 variant.
In our obese patients, the IRS-1 codon 972 polymorphism was associated with 30% lower plasma leptin levels and accounted for 3% of the variation in leptin concentration. Comuzzie et al35 conducted a genome-wide scan and multipoint linkage analysis in pedigrees of Mexican Americans and showed that chromosomal region 2p21 accounted for 47% of the variation of serum leptin levels. Compared with this quantitative trait locus, possibly harboring several sequence substitutions in 1 or more genes, the effect of the single amino acid substitution at residue 972 of IRS-1 seems relatively small. However, in vivo studies indicate that the effect of insulin on leptin plasma concentrations is moderate. Thus, most functional mutations of the insulin-signaling components cannot be expected to have a major impact on the variation in plasma leptin levels in the population, if their frequency is as low as that of the IRS-1 polymorphism. Nevertheless, we cannot rule out the possibility that the lower leptin levels in obese carriers of the IRS-1 variant resulted from linkage disequilibrium with a functional mutation in a different gene.
Although leptin is very effective in lowering food intake and body weight in ob/ob mice, animal models with diet-induced obesity exhibit, like many obese human subjects, increased plasma leptin concentrations.36 Higher doses of leptin are required in these animal models in comparison with ob/ob mice to achieve weight reduction.9 In a recent report, low plasma concentrations of leptin predicted weight gain in Pima Indians.37 The identification of obese subjects with inappropriately low leptin levels, as reported here in carriers of the IRS-1 codon 972 polymorphism, may therefore identify a subset of patients who may be candidates for therapeutic regimens aimed at reducing body weight by increasing leptin levels.
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Acknowledgments |
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This study was supported by a grant of the Medizinische Forschungsgesellschaft Salzburg (to F.K.), by the Jubilaeumsfondsprojekt Nr. 5175 of the Oesterreichische Nationalbank, and by a grant of Dr Karl Thomae GmbH, Biberach, Germany (to W.P.).
Received December 23, 1997; accepted April 20, 1998.
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