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Original Studies |
Istituto di Medicina Interna, Endocrinologia e Malattie del Metabolismo, Ospedale Garibaldi, Università di Catania (D.S., G.P., R.V., L.F.), 95123 Catania; Clinica Chirurgica, Ospedale Vitt. Emanuele, Università di Catania (A.I.), 95123 Catania; and Divisione ed Unità di Ricerca di Endocrinologia, Istituto Scientifico Casa Sollievo della Sofferenza, San Giovanni Rotondo (V.T.), 71013 Foggia, Italy
Address correspondence and requests for reprints to: Lucia Frittitta, M.D., Endocrinologia, Ospedale Garibaldi, P.zza S. M. Gesù, 95123 Catania, Italy. E-mail segmeint{at}mbox.unict.it
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Abstract |
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 Top Abstract IntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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IR content was significantly reduced in insulin-resistant subjects both nonobese and obese, compared with insulin-sensitive subjects (2.32 ± 0.26, 2.36 ± 0.18, and 3.45 ± 0.42 ng/mg protein, respectively, P = 0.002). In contrast, IGF-I-R content was similar in the three groups. Muscle HR content was not different in insulin-sensitive vs. insulin-resistant subjects (both nonobese and obese) (4.90 ± 0.46, 4.69 ± 0.29, and 4.91 ± 0.25 ng/mg protein, respectively, P = not significant). These studies indicate that, in insulin-resistant subjects without diabetes or severe obesity, muscle IR content but not IGF-I-R or HR content is reduced. They do not suggest, therefore, a primary (genetic) role of increased HR as a cause of IR decrease and insulin resistance.
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Introduction |
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TopAbstractIntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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ß-heterodimer and one type I
insulin-like growth factor receptor (IGF-I-R) ß-heterodimer, are
present in tissues and cells coexpressing both IR and IGF-I-R
(1, 2, 3). These HRs behave like IGF-I-R rather than IRs
because they bind to and are activated by IGF-I with an affinity
similar to that of typical IGF-I-R, whereas they bind insulin with a
much lower affinity (3, 4). Although the mechanisms regulating HR formation are not known, random assembly of heterogeneous dimers, made available during IR and IGF-I-R syntheses, is believed to be the most likely evidence. If this is the case, factors affecting availability of either IR or IGF-I-R heterodimers will influence the cell/tissue abundance of HRs (5).
Clinical interest for HRs has been raised by recent observations that
the relative abundance of HRs is increased in skeletal muscle tissue of
patients with type 2 diabetes with respect to control subjects
(6). In these studies, HRs were measured as the
125I-IGF-I fraction that was first bound to a
solid phase anti-IGF-I-R antibody and then immunoadsorbed with
an anti-IR antibody. Data were expressed as the fraction of total
labeled IGF-I binding (to both typical IGF-I-Rs and to HR). An
increased formation of HRs was hypothesized to contribute to the
insulin resistance by drawing away ß-heterodimer to the formation
of IR homodimers and consequently reducing the cell IR content, cell
insulin binding, and responsivity (6, 7). Chronic
hyperinsulinemia has been hypothesized as a possible cause of increased
HR formation in type 2 diabetic patients (7). This
hypothesis was supported by the observation that patients with
hyperinsulinemia due to insulinoma had a relative abundance of HR
higher than normal subjects (7). In insulinoma patients,
however, hyperinsulinemia does not reflect that observed in type 2
diabetic patients both because insulin plasma levels are usually more
elevated and because hyperinsulinemia caused by insulinoma is a primary
phenomenon and not the consequence of insulin resistance as it is in
diabetic patients. The reasons and the role of increased HRs in insulin
resistance, therefore, remain unclear.
To investigate this problem, we first set up direct measurements of IR, IGF-I-R, and HR by specific enzyme-linked immunosorbent assays (ELISAs) and then measured HR content in muscle tissue of healthy nonobese, nondiabetic subjects either insulin sensitive or insulin resistant. In these insulin-resistant individuals, insulin resistance precedes more relevant metabolic abnormalities like obesity and/or diabetes and is believed to be mostly genetically determined (8, 9). We measured IR, IGF-I-R, and HR also in muscle tissue of moderately obese, nondiabetic patients, a model in which acquired insulin resistance may be considered a predominant feature, in addition to the genetic component. The results indicate that: 1) the HR content in human muscle tissue is higher than expected by the random assembly model; 2) HR content is not increased in muscle tissue of insulin-resistant subjects who are not severely obese or diabetic; and 3) the moderate chronic hyperinsulinemia typical of insulin resistance does not cause HR increase. Therefore, HRs do not seem to contribute to insulin resistance in these subjects.
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Materials, Subjects, and Methods |
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TopAbstractIntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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Triton X-100, bacitracin, phenylmethyl sulfonyl fluoride (PMSF),
and BSA were purchased from Sigma (St. Louis, MO). The
following monoclonal antibodies to the IR were used: MA-20, which
reacts with an -subunit epitope close to the insulin binding site
(10) and does not recognize HR; CT-1, which reacts with
ß-subunit at residues 1344–1355 (11); and 83–7, which
reacts with an epitope at the -subunit (12) and
recognizes both IR and HR. Monoclonal antibodies to the IGF-R were:
IR3, which reacts with the -subunit at residues 223–274 and
recognizes typical IGF-1-R (13); and 17–69, which reacts
with the -subunit residues 440–586 and recognizes both the IGF-1-R
and HR (14, 15). Antibody MA-20 was kindly provided by Dr.
I. D. Goldfine (University of California–San Francisco, San
Francisco, CA). Antibodies CT-1, 83-7, and 17-69 were kindly
provided by Dr. Siddle (University of Cambridge, Cambridge, UK).
Monoclonal antibody IR3 was purchased from Calbiochem
Oncogene Research Products (Cambridge, MA). For immunoblotting, the
following antibodies were used: an anti-IR ß-subunit polyclonal
antibody (Transduction Laboratories, Lexington, KY) and a chicken
polyclonal antibody against the -subunit of IGF-I-R (UBI, Lake
Placid, NY). Plasma insulin and IGF-I levels were measured (by
immunoradiometric assay) with commercially available kits (for insulin:
IMx system insulin, Abbott Laboratories, Daimabot, Tokyo,
Japan; for IGF-I: Diagnostics Systems Laboratories, Inc.,
Webster, TX).
Subjects
Fourteen moderately obese [body mass index (BMI), 27.5–35.2 kg/m2] patients and 21 nonobese (BMI, <27) subjects undergoing elective abdominal surgery were studied after giving informed consent, according to the Declaration of Helsinki. All subjects were not diabetic as evaluated by oral glucose tolerance test, according to WHO criteria. In all of them "homeostasis model assessment" (HOMA), an index of insulin sensitivity, was calculated according to the formula: insulin/(22.5e-ln glucose) (16). The 21 nonobese subjects were subdivided according to HOMA values into 11 insulin-sensitive (having HOMA <1.4, the median value) and 10 insulin-resistant (HOMA, >1.4) subjects. Moreover, in 8 obese patients and in 13 nonobese individuals who gave informed consent, insulin-mediated whole-body glucose utilization was also determined by the euglycemic hyperinsulinemic clamp technique after an overnight fast. Insulin-stimulated glucose disposal was evaluated with an insulin infusion at a constant rate (1.0 mU/kg-1·min-1) and a variable glucose infusion to maintain plasma glucose within 10% of the baseline value, as described previously (17).
All subjects were fed a weight-maintaining diet (50% carbohydrate, 30% lipid, and 20% protein) for the 8 days preceding the study.
Tissue specimens
Human muscle tissue specimens were obtained from the human
rectus abdominus skeletal muscle during elective abdominal surgery from
all subjects studied, 3–5 days after insulin sensitivity studies.
After adipose tissue was dissected and blood was removed, muscle
specimens were immediately frozen in liquid nitrogen. Soluble extracts
were prepared from frozen muscle tissue as described previously
(18). Briefly, muscle tissue (
150 mg) was pulverized
under liquid nitrogen and then homogenized in a 2-mL buffer [50 mmol/L
HEPES, 150 mmol/L NaCl, and 2 mmol/L PMSF (pH 7.6)] at 4 C using a
polytron homogenizer for 10 sec at medium speed. Triton X-100 was added
to a final concentration of 1%, and the homogenates were solubilized
for 60 min at 4 C. The material was then centrifuged at 100,000 x
g for 60 min at 4 C, and the supernatants were used for the IR,
IGF-I-R, and HR content measurement. The protein content was measured
by the Bradford method (19).
IR, IGF-I-R, and HR measurements
By ELISA—IR ELISA. IR muscle content was measured by a
specific ELISA as described previously (20). Briefly,
wells in Maxisorb plates (Nunc, Roskilde, Denmark) were precoated
(overnight incubation at 4 C) with anti-IR antibody MA-20 (2 µg/mL).
After washing to remove unbound antibody, wells were blocked with 1%
BSA and then 100–200 µg protein extracts were added to each well,
and IR was allowed to bind overnight at 4 C. After extensive washing,
biotinylated anti-IR antibody -CT-1 was added [0.3 µg/mL in 50
mM HEPES (pH 7.4), 150 mM NaCl, 0.05% Tween
20, 1 mM PMSF, 1 mg/mL bacitracin, and 1% BSA). After
2 h at 22 C, the peroxidase-streptavidin was added, and 30 min
later wells were washed again, and then 100 µL biotinyl-tyramide
solution were added. After a 15-min incubation at 22 C, wells were
washed with TBST (20 mM Tris, 150 mM
NaCl, and 0.05% Tween 20) and streptavidin-horseradish
peroxidase solution was added (30 min at 22 C). After further extensive
washing, the peroxidase activity was determined colorimetrically by
adding 3.3'.5.5'-tetramethylbenzidine at a concentration of 0.4 g/L in
an organic base, and the absorbance was measured at 451 nm.
The IR standard was purified from NIH-3T3 cells transfected with human IR complementary DNA, as described previously (20). No appreciable interference was caused by adding IGF-I-R or IR/IGF-I-R to the IGF-I-R standard curve (20).
IGF-I-R ELISA. IGF-1-R was measured by immunocapturing
receptors with anti-IGF-I-R IR-3 monoclonal antibody (2 µg/mL) and
revealed by biotinylated anti-IGF-I-R 17-69 monoclonal antibody
(20). The IGF-I-R standard was obtained from Chinese
hamster ovary cells transfected with human IGF-I-R complementary DNA,
as described previously (21). No appreciable interference
was caused by adding IR or HR to the IGF-I-R standard curve
(20). About 200–400 µg protein extracts were added to
each well, and the assay was performed as described above.
Assay sensitivity was linear from 0.125–1.0 ng/well, intra- and interassay coefficients of variation were less than 10% (20).
HR ELISA. HRs were measured by immunocapturing receptors
with anti-IR 83-7 monoclonal antibody (2 µg/mL) and using
biotinylated anti-IGF-I-R 17-69 monoclonal antibody (0.3 µg/mL) as a
revealing second antibody (20). The HR standard was
obtained from human placenta, as described previously (3).
About 100–300 µg protein extracts were added to each well, and the
assay was performed as described above. A typical standard curve for HR
hybrid ELISA using a purified HR preparation is shown in Fig. 1
. The minimal detectable amount of
hybrids was 0.125 ng/100 µL (1.25 ng/mL). The assay was linear from
0.125–1.0 ng/well. No appreciable interference was caused by adding 1
ng IR or IGF-1-R to the HR standard curve (20). Intra- and
interassay coefficients of variation were less than 10%. Multiple
dilution of solubilized muscle tissue produced a dose-response curve
parallel to that obtained with the purified HR standard (Fig. 1).
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By Western blot. To validate the ELISA methods, IR, IGF-I-R,
and HR content were measured by Western blot analysis in muscle tissues
from six subjects showing a wide range of both IR and HR content as
measured by ELISA. Muscle extracts (1.5 mg protein) were
immunoprecipitated (2 h, 4 C) with anti-IR antibody MA-20 (4.0
µg/sample) or anti-IGF-I-R antibody -IR3 (4.0 µg/sample) for
IGF-I-R or anti-IGF-I-R receptor antibody 17-69 (4 µg/sample) for HR.
Samples were then precipitated with rabbit antimouse Ig-G prebound to
protein A Sepharose and subjected to SDS-PAGE in a 7.5% polyacrylamide
gel under reducing conditions. Proteins were transferred (3 h at 4 C)
to nitrocellulose membranes that were first blocked with 10% BSA, then
washed with TBST buffer (10 mM Tris, 150 mM
NaCl, and 0.5% Tween 20), and incubated with an anti IR
ß-subunit polyclonal antibody (1 µg/mL) for IR and HR or with a
polyclonal antibody against the -subunit of the IGF-I-R for IGF-I-R.
After 16 h at 4 C, membranes were incubated for 1 h with a
rabbit antimouse serum conjugated with horseradish peroxidase, and the
reaction was developed according to an enhanced chemiluminescence
detection system. The specific signaling was revealed by
autoradiography.
Predicted value of IR/IGF-I-R and statistical analysis
If we assume that HRs are formed by random assembly of
ß-heterodimers of IR and IGF-I-R, before they are assembled
with homologous ß-heterodimers to form typical IR and IGF-I-R, the
predicted HR formation can be calculated on the basis of the
cell/tissue IR and IGF-I-R content. If the total concentrations of IR
and IGF-I-R half receptors are I and G, respectively, and these half
receptors combine randomly, then the predicted relative concentrations
of HRs would be I2:G2:2IG
(22). Thus, on the basis of random assembly, the expected
HR content of each specimen can be calculated as equal to
2.
IR.IGF-I-R (22).
Unpaired Student’s t test and ANOVA were used to compare mean values. Linear regression analysis was used to calculate numerical correlation between variables.
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Results |
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TopAbstractIntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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The clinical characteristics of the 35 subjects studied are shown
in Table 1. According to HOMA values, the
21 nonobese subjects were divided as insulin sensitive and insulin
resistant. Age, gender, and fasting plasma glucose (FPG) were neither
different in these two groups nor in comparison with the 14 obese
patient group. BMI was similar in the two groups of nonobese subjects.
Obese subjects had a significantly higher BMI (P <
0.001) and were insulin resistant by HOMA (P < 0.01)
with respect to the 11 nonobese insulin-sensitive subjects. This
difference in insulin sensitivity was confirmed by the euglicemic
hyperinsulinemic clamp in randomly selected individuals from each
group: the M value was significantly (P < 0.01) lower
in nonobese insulin-resistant (by HOMA) individuals (n = 5;
M = 4.4 ± 0.2) and obese patients (n = 7; M =
4.1 ± 0.7) compared with the eight insulin-sensitive subjects
(M = 6.8 ± 0.6). FPG was significantly higher
(P < 0.01) in obese patients compared with both
insulin-sensitive and insulin-resistant nonobese subjects. Fasting
plasma insulin concentrations were significantly higher in
insulin-resistant subjects (both nonobese and obese) compared with
insulin-sensitive subjects (P < 0.004). The average
plasma IGF-I concentration was similar in the three groups (Table 1).
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Skeletal muscle IR content was significantly reduced in insulin-resistant subjects (both nonobese and obese) with respect to insulin-sensitive subjects (2.32 ± 0.26, 2.36 ± 0.18, and 3.45 ± 0.42 ng/mg protein, respectively; P = 0.002). In contrast, skeletal muscle IGF-1-R content was similar in the three groups (0.66 ± 0.06, 0.71 ± 0.09, and 0.82 ± 0.08 ng/mg of protein, respectively; P = not significant). When all subjects were considered together, muscle IR content was inversely correlated with fasting plasma insulin levels (r = -0.37; P = 0.03) and muscle IGF-I-R content was inversely correlated with plasma IGF-I levels (r = -0.45; P < 0.05).
HR content in skeletal muscle tissue
Skeletal muscle HR content ranged from 2.7–8.0 ng/mg
protein and was not different in insulin-resistant subjects (both
nonobese and obese), when compared with insulin-sensitive nonobese
subjects (4.69 ± 0.29, 4.91 ± 0.25, and 4.90 ± 0.46
ng/mg protein, respectively; P = not significant)
(Table 2).
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). Skeletal muscle HR content was significantly correlated with IGF-I-R content (r = 0.45, P < 0.01) while the correlation with IR content did not reach the significance level (r = 0.31, P = 0.06). No correlation was observed between either plasma insulin (r = 0.18, P = 0.3) or plasma IGF-I concentrations and HR content (r = 0.10, P = 0.6).
Western blot studies
To validate the ELISA immunoassay, quantitation of IR, IGF-I-R,
and HR was assessed also by Western blot. A single band of 97 kDa
corresponding to the ß subunits of the IR and the HR and a single
band of 130 kDa corresponding to the subunits of IGF-I-R were
revealed (Fig. 2). The intensity of the
bands was quantitated by scanning densitometry and was significantly
correlated to results obtained in the same six subjects with the ELISA
measurements of IR (r = 0.94; P < 0.02), HR
(r = 0.8; P < 0.05), and IGF-I-R (r = 0.92;
P < 0.001).
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Discussion |
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TopAbstractIntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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No difference in muscle HR content was observed in the three groups of subjects, despite the observation that both lean insulin-resistant individuals and obese patients were clearly hyperinsulinemic and had a significantly reduced IR content in muscle, a likely consequence of a down-regulation effect (26, 27) as indicated by the significant inverse correlation between plasma insulin levels and muscle IR content.
In lean or moderately obese insulin resistant subjects, therefore,
insulin resistance and hyperinsulinemia are associated with (and
probably cause) reduced muscle IR content but are not associated with
changes of either IGF-I plasma levels or IGF-I-R content in skeletal
muscle. In these subjects, the muscle HR content is not altered,
arguing against the possibility of a primary role of increased HR
formation (and IR ß heterodimer sequestration) as a cause of
insulin resistance. This conclusion is also supported by the
consideration that a 30% decrease of muscle IR content is unlikely to
cause insulin resistance because of the presence of spare receptors: a
more marked IR reduction may be necessary to become a limiting step for
insulin action (28); more so when hyperinsulinemia is
present. Therefore, although the HR/IR ratio is slightly increased in
subjects with insulin resistance, the present findings do not support a
clinical relevance of this condition. Finally, our data also argue
against the possibility that the moderate hyperinsulinemia (2- to
3-fold increase) secondary to insulin resistance may per se
cause an increased HR formation in human muscle tissue.
Differences between the present data and previous data may be explained both by differences in the technique used to measure HR in the type of patients investigated. The ELISA assays that we have used provide a direct measurement of muscle receptor protein content and very well reflect measurements obtained by Western blot studies. The coprecipitation method, instead, provides HR measurement data as the percentage of bound 125I-IGF that is immunoprecipitated by an anti-IR antibody. By calculating total IGF-I binding by competition/inhibition studies, it is then possible to extrapolate the HR aliquot. Of course the two methods rely on different assumptions and do not necessarily provide similar results.
Relevant differences between the present study and previous
studies also regard the subjects studied. Severely obese (average BMI,
>40) and diabetic patients were investigated in previous studies. In
those patients, complex metabolic and hormone abnormalities are present
and may contribute to abnormalities in muscle HR content. For instance,
an important decrease of IGF-I plasma levels and the consequent
up-regulation of IGF-I-R muscle content was observed in those patients.
Increased IGF-I-R synthesis may provide overabundant was ß
heterodimers that become available for HR formation.
Additional information obtained in the present study concerns the HR
formation in of IR and IGF-I-R ß heterodimers. In human muscle
tissues this may well be the case because the measured value of HR
content was directly correlated with the value that can be predicted on
the basis of IR and IGF-I-R content in each muscle specimen. Measured
values, however, were always higher than predicted values, suggesting
that in muscle, at variance with other tissues (20, 22, 23), some mechanisms may provide a more efficient assembly of
available IR and IGF-I-R ß heterodimers into HR rather than into
typical IRs or IGF-I-Rs. Alternatively, in muscle tissue, HR
degradation and recycling may be slower than that of IR and IGF-I-R,
resulting in an overall HR content higher than expected. The biological
relevance of this observation, if any, remains to be understood.
Received November 22, 1999.
Revised May 8, 2000.
Accepted July 19, 2000.
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References |
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TopAbstractIntroductionMaterials, Subjects, and MethodsResultsDiscussionReferences |
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