Sections of 1Nutrition/Metabolism and 2Organ Transplantation, Internal Medicine, 3Division of Nuclear Medicine, 4Division of Diagnostic Radiology, and 5Unit of Clinical Spectroscopy, Università Vita e Salute San Raffaele IRCCS H San Raffaele, 20132 Milan; and 6International Center for the Assessment of Nutritional Status, and 7Faculty of Exercise Sciences, Università degli Studi di Milano, 20100 Milan, Italy
Submitted 19 June 2003 ; accepted in final form 19 August 2003
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ABSTRACT |
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intramyocellular triglyceride content; leptin; magnetic resonance spectroscopy
Adiponectin, a plasma protein exclusively produced by the adipose tissue, has recently been suggested to be an insulin-sensitizing agent involved in the pathogenesis of insulin resistance (1, 5, 20, 44, 46). Whether an inappropriate accretion of the IMCL content and reduced circulating adiponectin levels might also be involved in the pathogenesis of insulin resistance in patients with T1DM is unknown. This study was designed to assess the relationships of insulin resistance to IMCL content and plasma adiponectin levels in patients with T1DM. T1DM patients had to be compared with T2DM patients, but because the anthropometric features of these two groups of diabetic individuals were completely different, a more appropriate comparison was also made with tightly matched insulin-resistant first-degree offspring of parents with T2DM (OFF), who are known to be characterized by alterations of insulin action similar to those of their parents (17, 32, 35), and with normal healthy (NOR) subjects.
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METHODS |
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Eighteen patients with T1DM and seven patients with recently diagnosed T2DM recruited at the outpatient service of the Diabetes Unit of the Istituto Scientifico H San Raffaele, and 30 healthy individuals with (OFF) or without (NOR) first-degree relatives with T2DM recruited at the Center of Nutrition/Metabolism, participated in the study. Among the 18 T1DM patients, five had proliferative retinopathy, four had nonproliferative retinopathy, and three had sensorymotor nuropathy. None of the patients with T2DM had signs of retinopathy, neuropathy, or nephropathy, but they were obviously older and fatter than the patients with T1DM. Individuals in the control groups (OFF and NOR) were selected to be tightly comparable to T1DM for anthropometric features and lifestyle habits. Habitual physical activity was assessed using a questionnaire (2). Body weights were stable for 612 mo. The three women with T2DM were in menopause. Two women of those with T1DM were taking oral steroidal contraception (OSC), and because of the possible effects of OSC on insulin sensitivity and IMCL content (36) two of the women in the OFF and NOR groups were selected because they were in OSC treatment. The anthropometric characteristics of the subjects are summarized in Table 1. All subjects were in good health as assessed by medical history, physical examination, hematological analysis, and urinalysis. Informed consent was obtained from all subjects after explanation of the purposes, nature, and potential risks of the study. The protocol was approved by the Ethics Committee of the Istituto Scientifico H San Raffaele.
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Experimental Protocol
Subjects were instructed to consume an isocaloric diet and to abstain from exercise activity for 3 wk before the studies. Women were studied between days 3 and 8 of the menstrual cycle. Subjects were studied by means of the euglycemic hyperinsulinemic clamp to assess whole body insulin sensitivity after a 10-h overnight fast and during the insulin clamp. Within 23 days, they were studied by means of 1H MRS to assess IMCL content. The MRS session was performed in the Division of Diagnostic Radiology of the Istituto Scientifico H San Raffaele between 0700 and 0900 after a 10-h overnight fast.
Euglycemic hyperinsulinemic clamp. Patients with T1DM were instructed to undergo the usual therapeutic regimen the day before the studies and to abstain from taking any subcutaneous insulin in the morning of the clamp and MRS studies. Patients with T2DM were not taking any drug and were only on a diet treatment. Subjects were admitted to the Metabolic Unit of the Division of Internal Medicine I of the Istituto Scientifico H San Raffaele the night before the clamp study. During the night, when capillary glucose concentration exceeded 11 mmol/l, an exogenous insulin infusion (0.1 mU · kg1 · min1) was started. Nondiabetic patients were admitted at 0700 after a 10-h overnight fast. A Teflon catheter was inserted into an antecubital vein for infusions, and an additional one was inserted retrogradely into a wrist vein for blood sampling. The hand was kept in a heated box throughout the experiment to allow sampling of arterialized venous blood. A bolus of 5 mg/kg body wt of [6,6-2H2]glucose was administered to the OFF and NOR groups; the bolus administered to T1DM patients was adjusted, as previously described (19), according to the fasting plasma glucose concentration. Immediately after administration of the bolus, a continuous infusion (0.05 mg · kg body wt1 · min1) of [6,6-2H2]glucose (MassTrace, Woburn, MA) was started. Blood samples for postabsorptive plasma glucose, total cholesterol, HDL-cholesterol, triglycerides, free fatty acids, insulin, C-peptide, tumor necrosis factor- receptor-2 (
-TNFR2), leptin, and adiponectin were performed in duplicate in the postabsorptive condition. After a 150-min tracer equilibration period, a euglycemic hyperinsulinemic clamp was performed as previously described (35, 36). Insulin was infused at 40 mU · m2 · min1 to reach a plasma insulin concentration of
350 pmol/l, and plasma glucose concentration was kept at
5 mmol/l for an additional 150 min by means of a variable infusion of 20% dextrose. When fasting hyperglycemia was present in T1DM and T2DM patients, the plasma glucose concentration was allowed to drop to the euglycemic level, and thereafter the attempt was made to maintain a plasma glucose concentration of 5 mmol/l as in the normoglycemic individuals. Blood samples for plasma hormones, substrates, and tracer enrichment were drawn every 15 min throughout the study.
1H MRS. 1H MRS 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 (35, 36). Briefly, high resolution T1-weighted images of the right calf were obtained before the spectroscopic acquisitions to localize the voxel of interest for the 1H spectroscopy study. Voxel shimming was executed to optimize the homogeneity of the magnetic field within the specific volume of interest. Two 1H spectra were collected from a 15 x 15 x 15-mm volume within the soleus and tibialis anterior muscles, respectively. A PRESS pulse sequence (TR = 2,000 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, as it would have dominated 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 acquire an external standard in the same conditions as the subject's spectra. Postprocessing of the data, executed with the Sage/IDL software (General Electric Medical Systems), consisted of 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 ppm was used to calculate IMCL content expressed in arbitrary units (AU) as a ratio with the integral of the peak of the external standard times 1,000.
Analytical Procedures
Plasma glucose was measured with a Beckman glucose analyzer (35). Plasma free fatty acids and plasma total cholesterol, HDL-cholesterol, and triglycerides were measured as previously described (35). LDL-cholesterol was calculated using the Friedewald formula. Plasma free insulin was measured with microparticle enzyme immunoassay technology (36) with no cross-reactions with proinsulin, C-peptide, and glucagon (IMx Insulin assay; Abbott Laboratories, Rome, Italy), and C-peptide was measured by RIA with a double antibody (36). Plasma leptin concentrations were determined, as previously described (36), by RIA with a human kit (Linco Research, St. Charles, MO) following the manufacturer's assay protocol [intra- and interassay coefficients of variation (CV) <5 and <10%, respectively]. -TNFR2 was measured with an enzyme immunoassay (Immunotech; Beckman Coulter, Marseille, France) following the manufacturer's recommendations (intra- and interassay CV <3% and 5%, respectively). Plasma adiponectin was measured using a commercially available RIA kit (catalog no. HADP-61HK; Linco Research) (21). The intra- and interassay CV for adiponectin were 4 ± 1 and 14 ± 3%, respectively. The titers of the immunologic markers of T1DM (anti-GAD, -ICA, and -IA2 antibodies) were measured as previously described (33). The [2H2]glucose enrichment was measured by GC-MS as previously described (3).
Calculations
Glucose kinetics were calculated using Steele's equations for the nonsteady state (40). Steady state of plasma dideuterated glucose enrichments was reached in the study groups during both the end of the equilibration period and the last 30 min of the insulin clamp. Endogenous glucose production was calculated by subtracting the glucose infusion rate 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 endogenous glucose production to the rate of glucose disappearance. The metabolic clearance rate (MCR) was calculated to normalize the glucose turnover rate to the plasma glucose concentration.
Statistical Analysis
All data are presented as means ± SE. Comparisons among groups were performed using the analysis of variance and Tukey's post hoc testing when appropriate. Simple regression analysis was performed to assess relationships between variables.
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RESULTS |
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Anthropometric parameters of the study subjects are summarized in Table 1. T1DM, OFF, and NOR subjects were tightly matched for age, sex, body mass index, and physical activity index; in contrast, T2DM subjects were older and fatter.
Glucose Metabolism in Postabsorptive State and During Insulin Clamp
Postabsorptive plasma glucose levels (Table 1) and endogenous glucose production rates were increased in T1DM (4.15 ± 0.34 mg · kg1 · min1; P < 0.001) compared with T2DM, OFF, and NOR groups (2.59 ± 0.08, 2.19 ± 0.06, and 2.39 ± 0.11 mg · kg1 · min1, respectively). During the insulin clamp, plasma glucose concentration was maintained at the fasting level in T2DM, OFF, and NOR subjects, whereas in T1DM patients it was allowed to drop to euglycemic levels, and thereafter it was clamped at a slightly higher level than in the OFF and NOR groups (Table 1; P = 0.05). During the clamp, T1DM patients showed a certain degree of insulin resistance with respect to the suppression of the endogenous glucose production (69 ± 5 vs. 79 ± 11, 86 ± 4, and 88 ± 8% in T1DM vs. T2DM, OFF, and NOR, respectively; P = 0.05), even if it must be stated that the insulin dose used in the present experimental protocol (40 mU · m2 · min1) was probably too high to detect more subtle differences at the level of the endogenous glucose production. Also, the insulin-stimulated glucose metabolism was impaired in T1DM (5.31 ± 0.46 mg · kg1 · min1, P < 0.01) as well as in T2DM (3.19 ± 0.78 mg · kg1 · min1, P < 0.01) and OFF subjects (4.76 ± 0.40 mg · kg1 · min1, P < 0.01) compared with the NOR group (7.54 ± 0.37 mg · kg1 · min1). Because fasting and clamp plasma glucose levels were slightly but significantly higher in T1DM and T2DM patients compared with the other groups, estimates of insulin-stimulated glucose metabolic rates had to be corrected for the circulating glucose levels. The glucose MCR was therefore calculated and found to be similarly reduced in the T1DM, T2DM, and OFF groups compared with the NOR group (5.1 ± 0.6, 3.51 ± 1.0, and 5.3 ± 0.4 ml · kg1 · min1, respectively, vs. 8.5 ± 0.5 ml · kg1 · min1, P < 0.001; Fig. 1, top).
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IMCL Content
1H MRS of the calf muscle showed that IMCL content was increased in T1DM (112 ± 15 AU), T2DM (108 ± 10 AU), and OFF (82 ± 13 AU) compared with NOR subjects (52 ± 7 AU, P < 0.05; Fig. 1, middle) in the soleus muscle, whereas in the tibialis anterior only T1DM patients showed a significant increment of IMCL (40 ± 15 AU vs. 23 ± 3, 19 ± 5, and 13 ± 2 AU, P = 0.05; Fig. 1, middle).
Plasma Adiponectin, Leptin and Soluble -TNFR2 Concentrations
Plasma adiponectin levels were reduced in T2DM (7 ± 1 µg/ml, P = 0.01 vs. NOR) and OFF subjects (11 ± 1 µg/ml, P = 0.03 vs. NOR) but not in T1DM patients (25 ± 6 µg/ml), whose plasma level was increased compared with the T2DM (P = 0.01), OFF (P = 0.03), and NOR groups (16 ± 2 µg/ml, P = 0.05; Fig. 2). At the end of the clamp study, plasma adiponectin concentration showed a nonsignificant decrement of 5 ± 5, 6 ± 9, 7 ± 10, and 6 ± 9% in the T2DM, OFF, T1DM, and NOR subjects, respectively. The lack of a significant decrement is in contrast to the work by Yu et al. (48), but this discrepancy is likely due to the different duration (300-min vs. 150-min clamps in the present study) and to the different insulin dose (80 vs. 40 mU · m2 · min1 in the present study) designed for these clamp studies. Fasting plasma leptin concentrations (5.0 ± 1.3, 6.2 ± 1.4, and 4.6 ± 0.7 ng/ml, P = 0.77) were similar in the T1DM, OFF, and NOR groups, respectively, and increased in T2DM patients (10.6 ± 2.0 ng/ml) according to the increased body mass index. -TNFR2 concentrations (1.4 ± 0.1, 1.5 ± 0.1, 1.4 ± 0.1, and 1.3 ± 0.1 ng/ml, P = 0.44) were not different in the T1DM, T2DM, OFF, and NOR groups, respectively.
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Effects of Metabolic Control
To test whether the above-described metabolic and endocrine parameters observed in patients with T1DM may be explained by a different metabolic control, analysis of data was performed segregating and comparing the patients in good (n = 5; Hb A1c <7.5%) and poor (n = 13; Hb A1c >7.6%) metabolic control (Table 2). Those in good metabolic control showed a higher insulin-stimulated glucose MCR (P < 0.05) in association with lower soleus IMCL content (P < 0.05). In contrast, no effect was observed on the plasma adiponectin concentration.
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Regression Analysis
The glucose MCR was inversely associated with the IMCL soleus content (Fig. 1, bottom, R2 = 0.27, P < 0.001) and plasma free fatty acid levels (R2 = 0.25, P < 0.01), and these relationships were best fitted with a logarithmic curve. The associations of glucose MCR with the soleus IMCL content and plasma free fatty acids were maintained separately in the study groups of normal-weight individuals (OFF, T1DM, NOR) but were not in the patients with T2DM (R2 = 0.15, P = 0.23). The association of Hb A1c with glucose MCR (R2 = 0.27, P < 0.04) and both soleus (Fig. 3: R2 = 0.57, P < 0.001) and tibialis anterior (R2 = 0.37, P < 0.02) IMCL content was found only in the group of patients with T1DM. No associations were found between parameters of insulin sensitivity and the titers of the immunologic markers of T1DM (anti-GAD, -ICA, and -IA2 antibodies). No association was found between adiponectin and the glucose MCR or the disappearance rate separately in each study group; a significant relationship was also lacking when individual data were plotted all together. When data regarding the T1DM patients were excluded and the analysis was limited to T2DM, OFF, and NOR subjects, a significant relationship was close to being reached (R2 = 0.21; P = 0.07). No association was found between adiponectin and endogenous glucose production in the fasting state or with the percent suppression of endogenous glucose production during the clamp study.
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DISCUSSION |
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Whole body insulin resistance was found to be associated with increased IMCL content in normal subjects (25, 42), offspring of T2DM parents (23, 35), individuals with HIV infection and lipodystrophy due to antiretroviral therapy (14, 27), and patients affected by T2DM and obesity (41). Insulin resistance is to some extent genetically inherited (38) and in the abovedescribed populations is strongly related to abdominal fat independent of total adiposity (29). In contrast, abdominal obesity, lipids, and androgens were not able to explain insulin resistance in young women with T1DM (15). The findings of this work therefore highlight the importance of IMCL content as a reliable marker of insulin resistance not only in T2DM but also in T1DM patients, as is also suggested by a previous study in which muscle triglyceride was assessed using the biopsy technique (12). In T2DM and obesity, the abnormal IMCL accretion may be determined by an unbalanced muscular fatty acid uptake, fatty acid oxidative disposal, and intramuscular lipogenic activity (24, 34) before the development of overt hyperglycemia. In T1DM, we found in this work a remarkable relationship between the soleus and tibialis anterior IMCL content and Hb A1c (Fig. 3). It may be speculated that, in these patients, the chronic hyperglycemic state may be relevant in the mechanisms of exaggerated IMCL accumulation. In the skeletal muscle cell, the sustained hyperglycemia (with or without high fatty acid availability) may determine a profound stimulation of acetyl-CoA carboxylase; this in turn determines a cytoplasmic accumulation of malonyl-CoA, which is a potent inhibitor of the carnitine palmitoyltransferase I system. This scenario previews a marked reduction of fatty acid transport inside the mitochondrion, a reduced fatty acid oxidative disposal, and a consequent IMCL accumulation (4), as has also been recently reported in normal healthy individuals (37) during moderately prolonged (5-h) experimental combined hyperglycemia (8.7 mmol/l) and hyperinsulinemia (258 pmol/l). The T1DM patients did not undergo a more intensive insulin therapy regimen to test whether, along with an improvement of the metabolic control and consequent improvement of peripheral insulin sensitivity, a reduction of the IMCL content was detectable. Nevertheless, we noticed that, when analysis of data was performed segregating the T1DM patients into good (n = 5; Hb A1c <7.5%) and poor (n = 13; Hb A1c >7.6%) metabolic control, those in good metabolic control showed a higher insulin-stimulated glucose MCR (P < 0.05) in association with lower soleus IMCL content (P < 0.05; Table 2). These findings further suggest a major role of chronic hyperglycemia in the induction of abnormal IMCL accretion and development of insulin resistance in T1DM and support the possibility that, when a good glucose homeostasis is achieved, these abnormalities may, at least partially, be reversed. Whether the IMCL content was the factor able to influence directly whole body insulin sensitivity in humans has recently been challenged by data published in athletes (16) and by unpublished observations in our laboratory in patients with myotonic dystrophy. These two groups of individuals showed increased or normal insulin sensitivity in association with increased rather than decreased IMCL content. Also in this study, we observed that, when data were plotted separately by groups, muscle IMCL content was strongly associated with insulin-stimulated glucose MCR in normal-weight individuals (T1DM, OFF, and NOR), whereas the association was less pronounced in the fatter, T2DM patients. In addition, the IMCL content was similar in patients with T2DM and T1DM despite a more severe degree of insulin resistance in T2DM; this fact may be explained by the effects of other well-known variables such as the different anthropometric features of these two groups of patients (29). It must also be stated that it is now believed that the accumulation of IMCL is not the direct cause of the development of insulin resistance but rather an inert marker of the presence of other lipid intermediates (diacylglycerol, fatty acyl-CoAs, or ceramide) that have been directly linked to defects in insulin signaling (13, 18, 30, 39, 43).
The second task of this work was to test whether plasma levels of adipocyte-derived peptides might be associated with the development of muscle insulin resistance, as recently suggested by several other investigators in T1DM. Measurements of fasting plasma adiponectin, leptin, and -TNFR2 were therefore performed in the study groups. Fasting plasma leptin and
-TNFR2 were comparable among the normal-weight study groups. Not surprisingly, plasma leptin was increased in T2DM patients in accordance with the higher body mass index. Plasma adiponectin concentration resulted in being reduced in patients with T2DM and in the OFF compared with the NOR groups in accord with other data (20). In contrast, its plasma concentration resulted in being increased in patients with T1DM compared with the T2DM and OFF subjects but also compared with the NOR group. Apparently the plasma levels of adiponectin were not different when measured in those patients with good compared with those in poor metabolic control (Table 2). Recently, increased plasma adiponectin concentrations were described in T1DM (22), but that report was characterized by a lack of assessment of insulin sensitivity in the study subjects. Supporting our finding of no major changes of plasma adiponectin levels depending on the metabolic control, Imagawa et al. (22) reported that intensive insulin treatment was not associated with a reduction of plasma adiponectin concentration. To our knowledge, the present work represents the first report in humans in which increased plasma adiponectin concentrations are associated with an insulin-resistant state in contrast with other classical insulin resistance conditions (T2DM and obesity), in which its level is reduced. Another condition in humans in which adiponectin showed unexpected behavior was recently reported in normal healthy individuals in whom the improvement of insulin sensitivity obtained by means of physical exercise training was not paralleled by an increment of the plasma adiponectin concentration (21). These findings therefore demonstrate that the role of adiponectin in the regulation of peripheral insulin sensitivity in vivo in humans is still unclear. Besides the relationship between adiponectin and peripheral insulin sensitivity, it is known that adiponectin has major effects at the liver site on glucose (7) and fatty acid (45) metabolism. With respect to glucose metabolism, the T1DM patients, along with increased circulating adiponectin levels, had increased fasting endogenous glucose production rates and a certain degree of resistance to the inhibitory effects of insulin, even if these parameters were not associated with the adiponectin levels and even if the insulin dose used in the present protocol was not appropriate for the assessment of hepatic insulin sensitivity. With respect to fatty acid metabolism, the intrahepatic fat content was not measured in these patients because the study was focused on peripheral insulin sensitivity. We cannot, therefore, exclude the possibility that the increased plasma adiponectin concentration found in T1DM patients may be explained by metabolic features at the liver site.
In conclusion, this work demonstrates that, in patients with T1DM, as well as in patients with T2DM and in individuals at higher risk of developing T2DM, increased IMCL content is a marker of peripheral insulin resistance, suggesting that inherited and/or acquired metabolic alterations may be predisposing factors for abnormal accretion of IMCL content and of specific IMCL intermediates able to induce insulin resistance. This study also suggests that maintenance of normal IMCL content is important for preserving insulin sensitivity, not only in patients with T2DM but also in patients with T1DM, and that recommendations for a proper lifestyle and possibly the use of drugs capable of reducing the IMCL content may represent a therapeutic tool useful to improve glucose homeostasis also in T1DM patients.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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