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Original Articles |
Divisions of Internal Medicine I (G.P., A.B., S.B., L.L.), Nuclear Medicine (P.S.), Diagnostic Radiology (P.S., A.D.M.), and Laboratory Medicine-Centro San Luigi (L. S.), Unit of Clinical Spectroscopy (G.P., P.S., A.D.M., L.L.), Università Vita e Salute San Raffaele, Istituto Scientifico H San Raffaele, 20132 Milan; and International Center for the Assessment of Nutritional Status, Università degli Studi di Milano (E.P., G.T., L.L.), 20100 Milan, Italy
Address all correspondence and requests for reprints to: Gianluca Perseghin, M.D., Division of Internal Medicine I, Laboratory of Amino Acids and Stable Isotopes/Unit of Clinical Spectroscopy, Via Olgettina 60, 20132 Milan, Italy. E-mail: perseghin.gianluca{at}hsr.it
Abstract
Plasma free fatty acids and intramyocellular triglycerides (IMCL) content modulate whole body insulin sensitivity in humans. To test whether the interactions between fatty acid metabolism and insulin action in nonobese humans are related to gender factors, we studied 15 young, normal weight, healthy men and 15 women matched for life habits and whole body insulin sensitivity, determined with the euglycemic-hyperinsulinemic clamp, by means of indirect calorimetry to assess substrate oxidation, localized 1H nuclear magnetic resonance spectroscopy of calf muscles to assess IMCL content, and dual energy x-ray absorption to assess body composition. In addition, to test whether perturbation of the feminine hormonal milieu modifies these interactions, we studied 15 matched females using oral steroidal contraception (OSC). Insulin sensitivity in women, notwithstanding increased body fatness, plasma free fatty acids, IMCL content, and circulating ß-hydroxybutyrate levels and reduced lipid oxidation, was similar to that in men. Women using OSC showed a 40% reduction of insulin sensitivity associated with increased plasma free fatty acids, ß-hydroxybutyrate, cholesterol, and triglycerides levels and a slight increment in IMCL content compared with women with intact hormonal cycles. In all groups the IMCL content was inversely related to insulin sensitivity. In conclusion, nonobese, healthy, young women are as insulin sensitive as men, notwithstanding the higher levels of postabsorptive circulating and tissue-stored fatty acids; OSC-induced insulin resistance is associated with abnormal fatty acid metabolism and loss of this gender-related feature.
THE INTERACTIONS between fatty acid metabolism and insulin sensitivity have been a matter of debate since 1963 when Randle introduced the concept of substrate competition (1). Several studies showed detrimental effects of increasing concentrations of plasma free fatty acids (FFA) on insulin sensitivity in healthy subjects (2, 3, 4) and an association between insulin resistance and increased plasma FFA levels in insulin-resistant states (5, 6, 7). It was hypothesized that defects in fatty acid metabolism may be crucial for the development of insulin resistance in the pathogenesis of type 2 diabetes (8). Fatty acids may induce insulin resistance because they impair insulin-stimulated glucose oxidation and muscle glycogen synthesis secondary to a defective insulin-dependent stimulation of muscle glucose transport/phosphorylation (9, 10), closely resembling the features of insulin resistance in type 2 diabetic patients (11, 12) and their nondiabetic relatives (13). This defect is due to an impairment of insulin’s ability to activate the insulin receptor substrate/phosphoinositol 3-kinase signaling pathway (14). Nuclear magnetic resonance (NMR) spectroscopy is performed in vivo in humans to noninvasively assess intracellular substrate concentrations (15), and the 1H spectrum of human muscle tissue contains two compartments of triglycerides: one represents the lipids within the fat cells, and the other the lipids located within the cytoplasm of muscle cells (16, 17). The assessment of the intramyocellular triglycerides (IMCL) content has been recently validated in vivo in human tissues (18), and this local parameter is considered to be a predictor of whole body insulin sensitivity; in fact, soleus muscle IMCL content was inversely associated with insulin sensitivity in normal humans (19), insulin-resistant offspring of type 2 diabetic patients (20, 21), and type 2 diabetic and obese patients (22).
The present study was performed in healthy subjects to assess which parameters of fatty acid metabolism affect insulin sensitivity, measuring 1) postabsorptive serum lipid profile and plasma FFA, 2) calf muscle (soleus and tibialis anterior) IMCL content using localized 1H NMR spectroscopy, 3) whole body fatty acid oxidation using indirect calorimetry, and 4) body fat content and distribution using dual energy x-ray absorption (DEXA), and to understand whether these relationships differently interact in nonobese men and women. In addition, to test whether perturbation of the feminine hormonal milieu was able to induce insulin resistance and whether this was associated with abnormal fatty acid metabolism we studied an additional group of healthy, young women using oral steroidal contraception (OSC), which is known to be characterized by reduced circulating endogenous estrogens. To avoid the confounding effect of other factors (age, diet, exercise, ethnicity, obesity, family history of diabetes, and metabolic diseases), we carefully selected young, healthy, nonobese, and nonexercising subjects who were not taking any medications (7). The results demonstrate for the first time in vivo in humans that women have whole body insulin sensitivity similar to men notwithstanding higher postabsorptive circulating and tissue-stored fatty acids and that OSC-induced insulin resistance is associated with derangement of fatty acid metabolism and loss of the gender-related feature.
Subjects and Methods
Subjects
Subjects participating in the study were recruited at the
Istituto Scientifico H San Raffaele. Criteria for the inclusion were 1)
no family history of diabetes, obesity, and hypertension traced through
their grandparents; 2) age (24–40 yr); 3) Caucasian race; 4) body
weight within 10% of the ideal body weight according to the 1983
Metropolitan Life Insurance tables; 5) sedentary life style; and 6) no
history of hypertension, endocrine/metabolic disease, or cigarette
smoking; habitual physical activity was assessed using a questionnaire
(23). Women were (women using OSC) or were not using OSC
(women with intact hormonal cycles) for at least 6 months. The clinical
and laboratory characteristics of the three groups are summarized in
Table 1
. All subjects were in good health
as assessed by medical history, physical examination, hematological,
and urinalysis. Informed consent was obtained from all subjects after
explanation of purposes, nature, and potential risks of the study. The
protocol was approved by the ethical committee of the Istituto
Scientifico H San Raffaele.
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Subjects were instructed to consume an isocaloric diet (
250 g
carbohydrate/day) and to abstain from exercise activity for 3 wk before
the studies. Women were studied between days 3–10 of the menstrual
cycle. Subjects were studied by means of the
euglycemic-hyperinsulinemic clamp and indirect calorimetry to assess,
respectively, glucose metabolism and resting energy expenditure (REE),
and glucose and fat oxidation (after correction for the urinary
nitrogen excretion) after a 10-h overnight fast period and during the
insulin clamp. Within 2–3 days they were studied by means of
1H NMR spectroscopy to assess IMCL content. The
NMR session was performed at the Division of Diagnostic Radiology of
the Istituto Scientifico H San Raffaele between 0700 and 0900 h
after a 10-h overnight fast period. Within 2–3 days they were also
studied by means of DEXA to assess body composition. DEXA was performed
at the Department of Science, Nutrition, and Microbiology, Nutrition
Section, Università degli Studi di Milano.
Euglycemic-hyperinsulinemic clamp
Subjects were admitted to the Metabolic Unit of the Division of Internal Medicine I of the Istituto Scientifico H San Raffaele at 0700 h after a 10-h overnight fast. A Teflon catheter was inserted into an antecubital vein for infusions, and an additional catheter was inserted retrogradely into a wrist vein for blood sampling. The hand was kept in a heated box (50 C) throughout the experiment to allow sampling of arterialized venous blood. A bolus (5 mg/kg BW) followed by a 300-min period of continuous infusion (0.05 mg/kg BW·min) of [6,6-2H2]glucose obtained from massTrace (Woburn, MA) was administered. Basal blood samples for glucose and tracer enrichment were collected on four occasions before insulin infusion during the last 45 min of the 150-min tracer equilibration period (-45, -30, -15, and 0 min); samples for FFA, lipid profile, insulin, C peptide, tumor necrosis factor receptor 2 (TNF-R2), and leptin were drawn twice in the same interval (-30 and 0 min). After the 150-min tracer equilibration period a euglycemic/hyperinsulinemic clamp was performed as previously described (21). Human recombinant insulin was infused at 1 mU/kg·min to reach plasma insulin levels of approximately 350 pmol/L, and the plasma glucose level was kept at about 5 mmol/L for additional 150 min by means of a variable 20% dextrose infusion. Samples for plasma hormones, substrates, and tracer enrichment were drawn every 15 min during the study.
Indirect calorimetry
Indirect calorimetry was performed continuously for 45 min during the basal equilibration period and at the end of the euglycemic-hyperinsulinemic clamp to measure O2 consumption and CO2 production to calculate glucose and fatty acid oxidation.
1H NMR spectroscopy
1H NMR spectroscopy was performed on a GE Signa 1.5 Tesla scanner (General Electric Medical Systems, Milwaukee, WI) using a conventional linear extremity coil as previously described (21). Briefly, high resolution T1 weighted images of the right calf were obtained before the spectroscopic acquisitions to localize the voxel of interest. The voxel shimming was executed to optimize the homogeneity of the magnetic field within this volume. Two 1H spectra were collected from a 15 x 15 x 15-mm3 volume within the soleus and tibialis anterior muscles, respectively. A PRESS pulse sequence (TR = 2000 ms and TE = 60 ms) was used, and 128 averages were accumulated for each spectrum, with a final acquisition time of 4.5 min. The water signal was suppressed during the acquisition because it would dominate the other metabolite’s peak signals of interest. A third 1H spectrum of a triglyceride solution inside a glass sphere, positioned within the extremity coil next to the calf, was also obtained during the same session to have an external standard acquired in the same conditions as the subject’s spectra. Postprocessing of the data, executed with Sage/IDL software (General Electric Medical Systems), consisted in high pass filtering, spectral apodization, zero filling, Fourier transformation, and phasing of the spectra. The integral of the area under the peak was calculated using a Marquardt fitting with Lorentzian functions of the peaks of interest. The integral of the methylene signal (-CH2) at 1.35 parts/million was used to calculate IMCL content, expressed in arbitrary units (AU) as a ratio with the integral of the peak of the external standard x 1000.
Body composition
DEXA was performed with a Lunar Corp.-DPX-IQ scanner (Madison, WI). A different scan mode was chosen with respect to each subject’s body size, as suggested by the manufacturer’s operator manual. For regional analysis, three-compartment processing was performed in the arms, trunk, and legs (24). Fat content is expressed as kilograms of fat mass and as percentage of the tissues.
Analytical procedures
Plasma glucose was measured with a Beckman Coulter, Inc., glucose analyzer (21). Plasma FFA and plasma total cholesterol, high density lipoprotein (HDL) cholesterol, and triglycerides were measured as previously described (21). Low density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Plasma insulin was measured with a microparticle enzyme immunoassay technology (25) with no cross-reactions with proinsulin, C peptide, and glucagon (IMx insulin assay, Abbott Laboratories, Rome, Italy); the microparticle enzyme immunoassay sensitivity is 6 pmol/L, and coefficients of variation were less than 3.9% (intraassay, 3.1%; interassay, 3.9%) for the postabsorptive levels and less than 3.7% (intraassay, 3.3%; interassay, 3.7%) for the insulin clamp levels. C Peptide was measured with a RIA using a double antibody (21). Plasma leptin concentrations were determined as previously described by RIA with a human kit (Linco Research, Inc., St. Charles, MO) (21) and are presented as absolute plasma concentrations; the ratio between the plasma concentrations and kilograms of fat mass is also presented. TNFR-2 was measured with an enzyme immunoassay following the manufacturer’s (Immunotech Beckman Coulter, Inc., Marseilles, France) recommendations. The d2-glucose enrichment was measured by gas chromatography-mass spectrometry as previously described (26).
Calculations
Glucose turnover was calculated in the basal state by dividing
the [6,6-2H2]glucose
infusion rate by the steady state plateau of plasma
[6,6-2H2]glucose
enrichment achieved during the last 45 min of the basal period. Glucose
kinetics during the insulin clamp were calculated using Steele’s
equations for the nonsteady state (27). A steady state of
plasma enrichments was reached in the study groups during the last 30
min of the insulin clamp. Endogenous glucose production (EGP) was
calculated by subtracting the glucose infusion rate (GIR) from the rate
of glucose appearance measured with the isotope tracer technique. Total
body glucose uptake was determined during the clamp by adding the rate
of residual EGP to the GIR. MCRs were calculated as the ratio between
total body glucose uptake and plasma glucose levels x 100.
Insulin sensitivity (SIp(clamp)) was obtained as
follows: 
Rd/(I x G), where Rd is the increment of total
glucose uptake, I is the increment in the plasma insulin
concentration (both calculated at basal and clamp steady state
conditions), and G is the plasma glucose concentration during the clamp
(28, 29). Rates of glucose oxidation were calculated from
the nonprotein respiratory quotient (30) using the tables
of Lusk (31). Nonoxidative glucose disposal was calculated
by subtracting the glucose oxidation rate from the total glucose
uptake. Protein oxidation was estimated from urinary nitrogen excretion
(32).
Statistical analysis
All data are presented as the mean ± SEM. The steady state for plasma glucose 6,6-2H2 enrichment was defined as a nonsignificant correlation with time (P > 0.05) using standard linear regression. Comparisons among groups were performed using the ANOVA with Scheffé’s post-hoc testing when appropriate. Forward and backward stepwise regression analyses were performed using the F ratio to remove of 4 and the F ratio to enter of 3.996 to assess the useful variables to predict the insulin sensitivity. Simple regression analysis was performed to assess relationships of IMCL content and postabsorptive lipid oxidation to other variables.
Results
Anthropometric characteristics (Table 1
)
Men and women with intact hormonal cycles were matched for age and ideal body weight; women using OSC and women with intact hormonal cycles were comparable with respect to anthropometry. The anthropometric parameters were typical for nonobese subjects; in women, the percent body fat content was increased and prevalently localized in the legs compared with that in men, where it was more uniformly distributed. Habitual physical activity was comparable among the groups. REE was lower in women, but similar to that in men when normalized to kilograms of body weight or kilograms of lean body mass.
Plasma insulin and C peptide concentrations
Postabsorptive plasma insulin concentrations were comparable in
the study groups (Table 2;
P = 0.55) and similarly increased during the insulin
clamp (Table 2; P = 0.22). Postabsorptive plasma C
peptide concentrations were also similar among the groups (0.45 ±
0.03, 0.51 ± 0.05, and 0.45 ± 0.04 nmol/L;
P = 0.48, respectively, in men, women using OSC, and
women with intact hormonal cycles), and its concentration similarly
dropped during the clamp (57 ± 5%, 54 ± 7%, and 44
± 6; P = 0.31).
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)Postabsorptive plasma glucose levels, EGP, and MCR were similar in men and women with intact hormonal cycles. In women using OSC, a slight reduction of postabsorptive plasma glucose and EGP was observed. The contribution of glucose oxidation to the REE was comparable among the study groups. In men and women with intact hormonal cycles, insulin-stimulated total glucose uptake was comparable for both oxidative and nonoxidative glucose metabolism; on the contrary, it was reduced in women using OSC due to a defective stimulation of nonoxidative glucose disposal (P < 0.01); meanwhile, the oxidative disposal was preserved.
Fatty acid metabolism in the postabsorptive state and during the
insulin clamp (Table 2)
In the postabsorptive state the plasma lipid profile was
comparable among the groups; nevertheless, women using OSC had a trend
for increased levels of total cholesterol (P = 0.05),
LDL cholesterol (P = 0.07), triglycerides
(P = 0.05), and HDL fraction (P = 0.06)
compared with women with intact hormonal cycles. Plasma FFA (Fig. 1A) and ß-hydroxybutyrate
concentrations were significantly higher in women than in men
(P < 0.05) and significantly higher in women using OSC
than in women with intact hormonal cycles (P < 0.05).
Whole body lipid oxidation was reduced in women with intact hormonal
cycles (0.81 ± 0.05) and women using OSC (0.79 ± 0.09
mg/kg·min) compared with that in men (1.01 ± 0.07 mg/kg·min;
P < 0.05), but it was similar when normalized to
kilograms of lean body mass. Nevertheless, the contribution of lipid
oxidation to the REE was reduced in the two groups of women
(P < 0.05). During the insulin clamp, plasma FFA and
glycerol concentrations similarly dropped in the study groups. Lipid
oxidation was also similarly suppressed in men and women with intact
hormonal cycles; on the contrary, the suppression was significantly
impaired in women using OSC (P = 0.05).
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)1H NMR spectroscopy of the calf muscle showed that soleus IMCL content was increased in women with intact hormonal cycles (58.0 ± 5.9 AU; P < 0.05) and women using OSC (60.6 ± 6.3 AU; P < 0.01) compared with that in men (40.8 ± 8.9 AU); tibialis anterior IMCL content was increased in women using OSC (21.4 ± 2.2 AU; P < 0.05) compared with that in women with intact hormonal cycles (14.7 ± 1.4 AU) and men (12.1 ± 1.6 AU; P = 0.19 vs. women with intact hormonal cycles).
Plasma leptin concentration
The postabsorptive plasma leptin concentration was reduced in men (3.69 ± 0.37 ng/mL) compared with that in women with intact hormonal cycles (7.38 ± 0.63 ng/mL; P = 0.05) and women using OSC (9.61 ± 0.97 ng/mL; P = 0.01) and was also reduced when corrected to kilograms of fat mass (0.23 ± 0.02 in men vs. 0.41 ± 0.06 in women with intact hormonal cycles and 0.63 ± 0.06 in women using OSC; P = 0.03). This ratio was increased in women using OSC compared with that in women with intact hormonal cycles (P = 0.05).
Plasma soluble TNF
-R2 concentration
The postabsorptive plasma TNF-R2 concentration was comparable
in the study groups (1.27 ± 0.78, 1.34 ± 0.18, and
1.53 ± 0.82 ng/mL in men, women with intact hormonal cycles, and
women using OSC, respectively; P = 0.36).
Regression analysis
To address whether insulin sensitivity was differently
modulated in study groups by parameters of fatty acids metabolism, a
stepwise regression analysis was performed using alternatively total
glucose uptake, GIR, and SIp(clamp) as the
dependent variables and age, body mass index, percent fat content, lean
body mass, lipid oxidation and its insulin-dependent suppression, REE
(corrected for kilograms of body weight or lean mass), IMCL content
(soleus or tibialis anterior muscles), postabsorptive plasma FFA
levels, plasma total cholesterol (or HDL/LDL fractions) and
triglycerides, and postabsorptive plasma leptin levels (or leptin
levels corrected to kilograms of fat mass). Soleus IMCL content was
inversely related to all parameters of insulin-stimulated glucose
metabolism in all study groups; total glucose uptake had a more
significant relationship (Fig. 2A;
r2 = 0.49; P < 0.01). In men and
women with intact hormonal cycles, but not in women using OSC,
postabsorptive plasma FFA was inversely related to total glucose uptake
and SIp(clamp) (r2 = 0.48;
P < 0.01). In men and women using OSC the percentage
of insulin-dependent suppression of lipid oxidation was directly
related to total glucose uptake (r2 = 0.23;
P = 0.03). In men, the soleus IMCL content was
inversely associated with the postabsorptive lipid oxidation
(r2 = 0.44; P = 0.01); on
the contrary, in women, the soleus IMCL content was
directly proportional to the body fat mass
(r2 = 0.22; P = 0.05). The
soleus IMCL content appeared to be associated with the postabsorptive
leptin levels in a comparable fashion in all the groups
(r2 = 0.27; P = 0.05).
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Insulin sensitivity is related to plasma and intramyocellular fatty acid concentrations
In healthy nonobese men and women, the postabsorptive IMCL content and the plasma FFA levels were the most important modulators of insulin sensitivity, as shown by stepwise regression analysis, confirming more extensively previous works (19, 20, 21, 22). Recently, it was demonstrated that diet- and drug-induced modulation of IMCL content impaired insulin-mediated glucose disposal in rats via a mechanism related to IMCL accumulation (33). This relationship never failed to be found in Europeans; the exception was South Asian men, in whom other mechanisms underlie the high insulin resistance of this population (34).
Nonobese women are as insulin sensitive as men, notwithstanding higher postabsorptive circulating FFA and muscle fatty acid content
In this study we demonstrated that young healthy and nonobese
women, notwithstanding an increased amount of body fat, plasma FFA,
circulating ß-hydroxybutyrate, and IMCL content, maintained
normal insulin sensitivity with respect to men. A gender difference in
serum lipids and plasma FFA was observed in four large European
communities (35, 36, 37), but in those studies insulin
sensitivity was not properly controlled. These results suggest a
protective role of the feminine hormonal milieu against the detrimental
effects of higher postabsorptive circulating and tissue-stored fatty
acids on insulin sensitivity. Estradiol was shown to modulate lipid
homeostasis in peroxisome proliferator-activated receptor -deficient
mice during pharmacological inhibition of fatty acid oxidation when
they developed severe alterations of glucose and lipid homeostasis
(hypoglycemia and massive hepatic and cardiac lipid accumulation) that
induced death in 100% of male mice but only in 25% of female mice,
and males pretreated with ß-estradiol were rescued (38).
In the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen
production, liver messenger ribonucleic acid expression of enzymes
involved in peroxisomal and mitochondrial ß-oxidation was suppressed,
and the activities of these enzymes were reduced compared with those in
control animals (39). In parallel, the animals developed
hepatic steatosis, which was reversed to wild-type levels along with
enzyme expression and activity when animals were treated with
17ß-estradiol (39). The researchers concluded that fatty
acid metabolism may be orchestrated by an estrogen receptor-mediated
signaling pathway. In women the lack of impact of higher postabsorptive
tissue-stored fatty acid levels on insulin sensitivity may be secondary
to body fat distribution rather than due to a direct effect of estrogen
per se. Trunkal or central adiposity, which is typical in
men, is associated with the metabolic syndrome (40, 41, 42),
probably because of different responses of the visceral vs.
sc adipose cells to insulin and other hormones (43, 44, 45).
In this study it is difficult to assess visceral vs. sc fat
in our subjects because we assessed body composition by means of DEXA,
which cannot distinguish visceral adipose tissue from sc abdominal
adipose tissue (46).
OSC induces insulin resistance and abnormal fatty acid metabolism
Women using OSC exhibited 40% reduction of insulin sensitivity associated with increased plasma FFA, ß-hydroxybutyrate, cholesterol, and triglycerides levels and smaller increments in IMCL content. Women using OSC were taking monophasic estro-(ethinyl estradiol, ranging from 20–30 µg) progestins (Gestodene or desogestrel, ranging from 75–150 µg), which induces a drop in circulating estradiol levels. The reduced estradiol levels may be responsible for the loss of protection against the deleterious effects of higher postabsorptive tissue fatty acid levels on insulin sensitivity. Steroidal contraceptives can reduce HDL concentrations (47), raise triglyceride levels (47), induce hyperinsulinemia (48) and glucose intolerance (49), and elevate blood pressure (50), all observations common to the insulin resistance syndrome. OSC is believed to induce a 30–40% reduction of insulin sensitivity (51), as found in our study, and more recently in a large population of unrelated Caucasian subjects, a woman’s use of OSC at doses comparable to those used in this study explained a significant part of the variation in insulin sensitivity (52), but relationships to altered fatty acid metabolism was lacking. Low diabetogenic activity of OSC used to avoid pregnancy is in contrast with the present data; this may be explained by the fact that its administration is limited to a very short period of a woman’s lifetime, and its effects are reversible. Nevertheless, it would be important to assess 1) the reversibility of the effects of OSC on insulin action after withdrawal, and 2) eventual additive effects to genetic or environmental factors impacting insulin sensitivity; for example, in first degree relatives of type 2 diabetic parents and obese women.
Lipid oxidation is a determinant of IMCL content in nonobese men
In addition to the increased parameters of fat storage in adipose and muscle tissues, women were characterized by reduced whole body lipid oxidation (normalized to kilograms of body weight or to the REE) with respect to men. The regression analysis suggested that nonobese men may maintain low IMCL content due to a greater ability to metabolize fatty acids in the oxidative pathways; meanwhile, in women the impact of fatty acid oxidative disposal may be underscored because of the different sex hormone environment that is protective against IMCL accumulation or its effects. In this regard we must emphasize that the above-described inverse relationship between IMCL content and lipid oxidation in men is probably limited to nonobese subjects, because unpublished results in our laboratory show that in overweight subjects the relationship become proportional, suggesting that in conditions of increased IMCL content, lipid oxidation acts to limit further intramyocellular accumulation.
Circulating leptin and TNFR-2 concentrations
In animal models leptin overexpression was found to increase fat
oxidation (53, 54). In our study its level was higher in
women than in men, and this fact could not be completely ascribed to
the increased body adiposity, because when concentrations were
normalized to kilograms of fat mass a significant difference persisted.
This finding was paralleled by a higher ratio in women using OSC than
in women with intact hormonal cycles. This may be the effect of an
increased production due to leptin resistance, as suggested for obesity
(55, 56). The postabsorptive leptin levels were found to
correlate with body fat content in all groups (r = 0.92;
P < 0.01), but they were also directly proportional to
IMCL content. This association does not demonstrate that leptin has a
direct impact on muscle fat metabolism and might represent a secondary,
noncausative relationship. We also measured the TNF-R2 circulating
levels because it has been proposed that the pathogenic mechanism of
the obesity-related insulin resistance may be due to higher adipose
tissue expression of TNF (57); the soluble plasma
receptor (TNFR2) is a circulating modulator of TNF action
(58), and we found that its plasma level was similar among
women, suggesting that TNF system activity is not involved in the
OSC-induced insulin resistance even if paracrine effects of the
cytokine can not be excluded.
This study was performed in nonobese young healthy subjects and showed that in women higher postabsorptive plasma FFA and tissue-stored fatty acids than in men are not associated with the deleterious effects on insulin sensitivity; additional studies are necessary to test whether different gender-related susceptibilities may be involved in the pathogenesis of type 2 diabetes, obesity, and body fat distribution. Another finding of this work is that OSC-induced insulin resistance was associated with abnormal fatty acid metabolism due to the lack of ovarian estrogen effects on lipid homeostasis.
Acknowledgments
We thank Mr. Van Chuong Phan, Mrs. Paola Sandoli, and Mrs. Sabrina Costa for the skilled work of hormone and substrate assessments, and Antonella Scollo, R.N., of the Metabolic Unit of the Istituto Scientifico H San Raffaele for nursing assistance.
Footnotes
1 This work was supported by Istituto Scientifico H San Raffaele
(PZ709 and PZ806), grants from Italian Minister of Health
(030.5/RF96.305 and 030.5/RF98.49) and the Italian National Research
Council (CNR 97.00485.CT04), and Telethon-Italy (1032C). 
2 Recipient of a grant from the Associazione Italiana Ricerca
Cancro.
Received September 18, 2000.
Revised February 5, 2001.
Revised March 21, 2001.
Accepted March 26, 2001.
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 P. Shah, A. Vella, A. Basu, R. Basu, A. Adkins, W. F. Schwenk, C. M. Johnson, K. S. Nair, M. D. Jensen, and R. A. Rizza Elevated Free Fatty Acids Impair Glucose Metabolism in Women: Decreased Stimulation of Muscle Glucose Uptake and Suppression of Splanchnic Glucose Production During Combined Hyperinsulinemia and Hyperglycemia Diabetes, January 1, 2003; 52(1): 38 - 42. [Abstract] [Full Text] [PDF]
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G. Perseghin, P. Scifo, M. Danna, A. Battezzati, S. Benedini, E. Meneghini, A. Del Maschio, and L. Luzi Normal insulin sensitivity and IMCL content in overweight humans are associated with higher fasting lipid oxidation Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E556 - E564. [Abstract] [Full Text] [PDF]
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B. D. Hegarty, G. J. Cooney, E. W. Kraegen, and S. M. Furler Increased Efficiency of Fatty Acid Uptake Contributes to Lipid Accumulation in Skeletal Muscle of High Fat-Fed Insulin-Resistant Rats Diabetes, May 1, 2002; 51(5): 1477 - 1484. [Abstract] [Full Text] [PDF]
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M. Krebs, M. Krssak, E. Bernroider, C. Anderwald, A. Brehm, M. Meyerspeer, P. Nowotny, E. Roth, W. Waldhausl, and M. Roden Mechanism of Amino Acid-Induced Skeletal Muscle Insulin Resistance in Humans Diabetes, March 1, 2002; 51(3): 599 - 605. [Abstract] [Full Text] [PDF]
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, Men; •, women with intact hormonal cycles; 
, women using OSC.
*, P < 0.05 vs. men; 
,
P < 0.05 vs. women with intact
hormonal cycles.