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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 4 1753-1759
Copyright © 2004 by The Endocrine Society

Further Lowering of Muscle Lipid Oxidative Capacity in Obese Subjects after Biliopancreatic Diversion

R. Fabris, G. Mingrone, G. Milan, M. Manco, M. Granzotto, A. Dalla Pozza, A. Scarda, R. Serra, A. V. Greco, G. Federspil and R. Vettor

Endocrine-Metabolic Laboratory, (R.F., G.Mil., M.G., A.D.P., A.S., R.S., G.F., R.V.) Internal Medicine, Department of Medical and Surgical Sciences, University of Padova, 35128 Padova, Italy; and Department of Medicine, Catholic University (G.Min., M.M., A.V.G.), 8-00168 Rome, Italy

Address all correspondence and requests for reprints to: Dr. Roberto Vettor, Department of Medical and Surgical Sciences, University of Padova, via Ospedale 105, 35128 Padova, Italy. E-mail: roberto.vettor{at}unipd.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
A reduced lipid oxidative capacity is considered a risk factor for the development of obesity, but a further impairment of lipid oxidative capacity is observed after weight loss. We aimed to define the mechanisms underlying this phenomenon in skeletal muscle and in particular to study the mitochondrial and peroxisomal lipid oxidative pathways. Thus we measured intramyocellular triglyceride content (IMTG) and the expression of genes of lipid oxidation [peroxisome proliferator-activated receptor-{alpha}, carnitine palmitoyltransferase 1B, and acyl-coenzyme A (acyl-CoA) oxidase 1] and synthesis (acetyl-CoA carboxylase B) using RT-PCR analysis in muscle biopsies of morbidly obese patients before and after biliopancreatic diversion. Weight reduction significantly decreased IMTG while increasing insulin sensitivity, measured by euglycemic hyperinsulinemic clamp. Moreover, an increase in glucose and a decline in lipid oxidation, as assessed by respiratory chamber, were observed. Weight loss reduced the expression of peroxisome proliferator-activated receptor-{alpha} (–46.7%), carnitine palmitoyltransferase 1B (–43.1%), acyl-CoA oxidase 1 (–37.8%), and acetyl-CoA carboxylase B (–48.7%). Our results indicate that a defect of both peroxisomal and mitochondrial oxidative pathways at the muscular level may contribute to the reduced fat oxidation in obese subjects after biliopancreatic diversion. They also suggest that a depression of the de novo lipogenesis may account for IMTG depletion.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
A NUMBER OF factors are thought to be involved in weight gain or regain after weight loss; among them, much attention has been focused on lipid oxidation. In fact, a high respiratory quotient, indicating relatively low fat oxidation, has been recognized in the predisposition of weight gain in Pima Indians (1) and Caucasians (2).

Skeletal muscle plays an important role in whole body lipid oxidation. During fasting conditions, up to 90% of the energy requirements of resting skeletal muscle are obtained from fatty acid oxidation (3), and about 40% of fatty acids supplied are cleared by one circuit through the muscular capillary system (4). Obese subjects present an elevated intramyocellular triglyceride content (IMTG) with an apparent paradoxical reduction of muscle lipid oxidation (5, 6). Such a relationship is of importance because an increase in IMTG is linked with insulin resistance in obese individuals (5, 7). A reduced lipid oxidation in muscle may favor the partitioning of ingested nutrients toward storage in adipose tissue, thus promoting fat mass (FM) gain (8).

It has been recently observed that weight loss secondary to fat malabsorption after bariatric surgery in morbidly obese individuals increases both the respiratory quotient and insulin sensitivity (9). This is not only the expected consequence of IMTG depletion after weight loss, as observed in nondiabetic obese subjects and obese patients with type 2 diabetes (6, 10), but probably represents a further impairment of skeletal muscle lipid oxidative capacity (5). However, it is still a matter of debate whether the diminished muscle lipid oxidation may represent a primary defect or arises secondarily, after an individual has become obese. Another interesting issue is whether the decrease in ß-oxidation is prevalent at the mitochondrial or peroxisomal level.

Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a nuclear hormone receptor that is thought to play a key role in regulating muscle lipid utilization. In fact it has been shown that PPAR{alpha} regulates the expression of genes involved in mitochondrial ß-oxidation of fatty acids, such as carnitine palmitoyltransferase 1 (CPT1), whereas it seems to have little or no influence on the peroxisomal marker enzyme acyl-coenzyme A (acyl-CoA) oxidase 1, palmitoyl (ACOX1) (11). CPT1 is an enzyme that regulates the transport of long-chain fatty acids across mitochondrial membranes (12, 13) and therefore represents one of the possible candidates for explaining the defective lipid oxidation observed in obesity (14).

In contrast, the decreased lipid oxidation and IMTG depletion after weight loss could be the consequence of reduced triglyceride synthesis. In this respect a key role is played by acetyl-CoA carboxylase (ACAC), the rate-limiting enzyme in the synthesis of malonyl-CoA that concomitantly stimulates long-chain fatty acid synthesis and inhibits their oxidation (12).

Sterol regulatory element-binding factors (SREBFs) are master transcription factors involved in lipid metabolism regulation (15). In particular, SREBF1 plays a crucial role in most lipogenic genes, such as ACAC (16, 17), and can be up-regulated by hyperinsulinemia induced by overnutrition (18, 19), possibly facilitating ectopic lipogenesis in nonadipocytes. The increased lipogenic capacity observed in well trained mice is accompanied by an up-regulation of SREBF1c in skeletal muscle (20). Moreover, it has been recently shown that in addition to up-regulating SREBF1c gene expression in control and obese subjects (21) and animals (19, 22), insulin activates SREBF1c transcriptional activity (22), thus being involved in the regulation of genes implicated in the mechanism of action of insulin.

The aim of our study was therefore to investigate the changes in lipid synthesis and oxidation induced by weight loss in obese patients and their relationship with the muscular lipid content. In particular, we aimed to assess whether the IMTG depletion following weight loss may be the consequence of reduced lipid synthesis, and if the further reduction of lipid oxidation occurs at the mitochondrial or peroxisomal level.

To investigate these topics, we measured IMTG in muscle biopsies in a group of morbidly obese patients before and after weight reduction obtained by means of biliopancreatic diversion (BPD). In the same samples we evaluated the expression of genes of oxidative pathways, such as PPAR{alpha}, CPT1B (muscle isoform), and ACOX1, and the lipid synthesis pathway, such as ACACB (muscle isoform), and the expression of the transcription factor SREBF1. All patients were characterized before and after weight loss with respect to insulin sensitivity, by euglycemic hyperinsulinemic clamp, and glucose and lipid oxidation, as assessed by the respiratory chamber.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The study group included 10 morbidly obese patients (body mass index, >40 kg/m2) without impaired glucose tolerance, diabetes mellitus, or other endocrine or nonendocrine diseases. At the time of the baseline study and after BPD, all subjects were on a free diet (the Italian diet typically consists of 55% carbohydrate, 30% fat, and 15% protein). Patients were studied before and 18 ± 2 months after BPD operation, consisting of a partial gastrectomy with a distal Roux-en-Y reconstruction (23). Physical fitness testing was not performed before or after BPD. Two orthogonal ultrasound sensors allowed a rough evaluation of the spontaneous physical activity inside the respiratory chamber, which was increased after BPD (data not shown).

The study protocol was approved by the ethics committee of Catholic University of Rome (Rome, Italy). The nature and purpose of the study were carefully explained to all subjects before obtaining their written consent to participate.

Body composition

Anthropometric parameters were determined at 0 and 18 ± 2 months after surgery. Body weight (BW) was measured to the nearest 0.1 kg by a beam scale, and height was determined to the nearest 0.5 cm using a stadiometer (Holatin, Crosswell, Wales, UK). Total body water (TBW) was determined using 0.19 Bq tritiated water in 5 ml saline solution, administered as an iv bolus injection (24). Blood samples were drawn before and 3 h after the injection. Radioactivity was determined in duplicate on 0.5 ml plasma using a ß-scintillation counter (model 1600TR, Canberra-Packard, Meriden, CT). Corrections were made (5%) for nonaqueous hydrogen exchange (25); water density at body temperature was assumed to be 0.99371 kg/liter. TBW (kilograms) was computed as tritiated water dilution space (liters) x 0.95 x 0.99371. The within-subject coefficient of variation for this method is 1.5% (26). Fat-free mass (FFM; in kilograms) was obtained by dividing the TBW by 0.732 (27). FM was determined as the difference between body weight and FFM.

24-h energy expenditure (EE)

EE in the respiratory chamber was measured. Subjects entered the chamber in the morning after an overnight fast and remained there for 24 h. They were fed a standardized diet, with the amount of calories calculated according to previously determined equations to achieve energy balance. Meals were provided at 0800, 1200, 1700, and 2000 h. The rate of EE was measured continuously, calculated for each 15-min interval. Spontaneous physical activity was detected by radar sensors and was expressed as the percentage of time over the 24-h period in which activity was detected. Carbon dioxide production and oxygen consumption were calculated at 15-min intervals, summed for the 24 h in the chamber. The 24-h respiratory quotient was calculated as the ratio of 24-h carbon dioxide production and 24-h oxygen consumption and was adjusted for the 24-h energy balance (24-h energy intake – 24-h EE during the stay in the chamber) in a multiple regression analysis, as previously described (28). Substrate oxidation rates were calculated from oxygen consumption, carbon dioxide production, and nitrogen urinary excretion (29).

Hyperinsulinemic euglycemic clamp

Insulin sensitivity was determined in all subjects after an overnight fast by the euglycemic insulin clamp technique with a primed constant insulin infusion rate of 7 pmol/min·kg. After inserting a cannula in a dorsal hand vein for sampling arterialized venous blood and another one in the antecubital fossa of the contralateral arm for infusions, the subjects rested in a supine position for at least 1 h. They were placed with one hand warmed in a heated air box set at 60 C to obtain arterialized blood samples. The fasting plasma glucose concentration was maintained throughout the insulin infusion by means of a variable glucose infusion and blood glucose determinations every 5 min. Whole body glucose uptake (M value, milligrams per kilogram of BW per minute) was determined by averaging the exogenous glucose infusion rates during the last 40 min of the clamp and correcting them for the glucose space (30).

Biochemical analysis

Plasma glucose levels were measured by a glucose oxidase method (Beckman, Fullerton, CA). Nonesterified fatty acid (NEFA) levels were determined using a commercial kit (Roche, Tokyo, Japan). Serum triglycerides were determined by enzymatic colorimetric methods (Roche, Mannheim, Germany). Serum immunoreactive insulin was assayed using a microparticle enzyme immunoassay (Abbott Laboratories, Inc., Pasadena, CA). To avoid interassay variability, all specimens for a given substance were run in a single assay.

Muscle biopsy

Subjects were instructed not to perform strenuous physical exercise for 48 h before the muscle biopsy procedure. Biopsies were obtained in fasting conditions from the middle region of the quadricep muscles by a percutaneous needle, before and after BPD surgical treatment. Muscle specimens were trimmed accurately free of fat, immediately frozen in liquid nitrogen, and stored at –80 C. Moreover, leptin mRNA was undetectable in all muscle biopsies, thus indicating the absence of measurable contamination of muscle with adipose tissue (31).

Muscle lipid analysis

A specimen of 100 mg was taken, immediately placed into calcium-free Hanks’ solution with added EDTA, and bubbled with O2 95% and CO2 5%. The sample was washed and then immersed in a fresh Hanks’ solution with added collagenase type IV (50 mg) and calcium ions, then agitated in a Dubnoff water bath maintained at 37 C until the tissue appeared soft. At this point the specimen was gently removed, and cells were brushed with a blunted spatula, filtered, suspended in PBS, and centrifuged twice at 50 x g for 2 min each time. The supernatant was discarded, and the muscle cells were dried under a nitrogen stream. After protein precipitation with 5–10 mg trichloroacetic acid, lipids were extracted twice with 8 vol chloroform-methanol (2:1, vol/vol) stirring the solutions at 60 C for 15 min. The combined extracts were dried in a GyroVap apparatus (GV1, Gio. DeVita, Rome, Italy) at 60 C, coupled with a vacuum pump and a gas trap (FTS System, Stone Ridge, NY). The dry weight of lipid extracts was obtained by weighing the sample tube before and after drying the extracts. The above extracts were redissolved in chloroform-methanol (2:1, vol/vol) and fractionated into their various components by thin layer chromatography on standard thin layer plates (Stratocrom SI AP, Carlo Erba, Mila, Italy) coated with a 0.25-mm thick layer of silica gel and activated by heating at 120 C for 20 min. The plates were developed into successive solvent systems as described by Passi et al. (32). The area of silica gel corresponding to the ratio of fraction time of a triolein and tripalmitin standard mixture was scraped off and extracted with peroxide-free diethyl ether. The triglyceride fraction eluted from the thin layer chromatography plates was saponified by a treatment with 2 N KOH in methanol and successive acidification to pH 2–3 with 2 N HCl. NEFA were thus obtained and finally separated and measured according to a previously described method (32).

RNA extraction and RT-PCR assay

Only five of the 10 patients undergoing BPD had enough muscle tissue available to measure the expression of PPAR{alpha}, ACOX1, CPT1B, ACACB, and SREBF1 by RT-PCR analysis. Total RNA was isolated from the muscle tissue using the RNAzol method (Tel-Test, Inc. Friendswood, TX). The concentration and purity of the RNA were determined by absorbance at 260 and 280 nm. RNA was treated for 1 h at 37 C with 1 U RQ1 ribonuclease-free deoxyribonuclease/µg RNA in 10 µl buffer containing 40 mM Tris-HCl (pH 8.0), 10 mM MgSO4, and 1 mM CaCl2 (Promega Corp., Madison, WI). Two micrograms of total RNA were reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp.) and 20 U RNasin ribonuclease inhibitor in 50 µl buffer containing deoxy-NTP mix (0.5 mM each) and 100 ng oligo(deoxythymidine)15 primer (Promega Corp.) in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol. PCR was performed using Hot Star Taq DNA polymerase (Qiagen, Hilden, Germany) in 25 µl standard buffer with 1.5 mM MgCl2, 200 µM of each deoxy-NTP, and 0.5 µM of each sense- and antisense-specific oligonucleotide primer.

The primer sequences were designed using the Omiga 2.0 program (Accelrys Ltd., Cambridge, UK). The primers used for ß-actin were 5'-CAA AGA CCT GTA CGC CAA CAC AGT-3' and 5'-GAC GAT GGA GGG GCC GGA CTC GTC-3'; those used for PPAR{alpha} were 5'-CTG AGA AAG CAA AAC TGA AAG C-3' and 5'-TGG GAA GAG AAA GAT ATC GTC C-3'; those used for ACOX1 were 5'-TCA CCG GAT TAA CGA AGG-3' and 5'GCT GCA CGG AGT TTA TAT GC-3'; those used for CPT1B were 5'-GAA GCA CCA GAA TAT GTA CC-3' and 5'-GAA AAG ATC AGC AAT GTC C-3'; those used for ACACB were 5'-AAA ATG ACG GAC TCC AAG C-3' and 5'-TCA TCA AAA GAG CCC AGG-3'; and those used for SREBF1 were 5'-CGA TCT TGA CCC TAA GAC C-3' and 5'-TGC AAA AGG CAA AGT AGC-3'. HotStar Taq DNA polymerase was activated by a 15-min 95 C incubation step. After initial denaturation at 94 C for 40 sec, reactions were performed as follows: for ß-actin detection, 60 C for 30 sec, 72 C for 30 sec, 32 times (amplification product size, 241 bp); for PPAR{alpha} detection, 57.5 C for 40 sec, 72 C for 1 min, 43 times (amplification product size, 737 bp); for ACOX1 detection, 58 C for 40 sec, 72 C for 50 sec, 36 times (amplification product size, 420 bp); for CPT1B detection, 58.5 C for 40 sec, 72 C for 40 sec, 35 times (amplification product size, 382 bp); for ACACB detection, 60 C for 40 sec, 72 C for 50 sec, 40 times (amplification product size, 440 bp); and for SREBF1 detection, 60 C for 40 sec, 72 C for 50 sec, 32 times (amplification product size, 318 bp). At the end of each PCR reaction, a final extension step at 72 C for 7 min was performed. Ten microliters of amplification reaction were separated by electrophoresis (1% agarose gel in Tris-borate-EDTA buffer), visualized using ethidium bromide staining, revealed with Image Master VDS (Amersham Pharmacia Biotech Europe, Freiburg, Germany), and densitometrically analyzed with Image Master Total Lab 1.00 software (Amersham Pharmacia Biotech Europe).

The number of cycles for the semiquantitative RT-PCR analysis and the conditions of the reaction temperature were estimated to be optimal for a linear relationship between the amount of input template and the amounts of PCR product generated over a significant concentration range (20–100 ng for total RNA). In particular, the linearity of the RT-PCR amplifications for all genes tested was measured at 15, 30, and 40 cycles (data not shown). mRNA levels were expressed as the ratio of signal intensity for the target genes relative to that for ß-actin. The RT-PCR analyses were performed three times on the same sample; the intraassay coefficient of variation was less than 5%.

Statistical analysis

Data are given as the mean ± SEM. One-way ANOVA was used to compare before and after values (P < 0.05 was considered significant). A single linear regression analysis was also performed using the STATISTICA for Windows (StatSoft, Inc., Tulsa, OK) package.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The BPD surgical approach and the subsequent effect on weight reduction allowed us to study a group of 10 obese subjects after a substantial, long-lasting body weight decrease. After BPD, BW was significantly decreased compared with baseline values as was body mass index, with concomitant reductions of FM and FFM (Table 1Go). BPD significantly decreased plasma levels of glucose, insulin, NEFA, and triglycerides, as shown in Table 2Go. IMGT was also considerably reduced after BPD (Table 2Go).


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  		TABLE 1. Anthropometric characteristics of 10 obese subjects at the beginning of the study and after BPD

 

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  		TABLE 2. Effects of BPD on biochemical parameters and IMTG content of 10 obese subjects

 
Insulin resistance, assessed by the hyperinsulinemic euglycemic clamp technique, was fully reversed after BPD. The whole body glucose disposal (M) increased from 2.14 ± 0.31 to 7.28 ± 0.23 mg/kg BW·min (P < 0.001; Fig. 1Go). After weight loss, a slight increase in glucose oxidation (117.97 ± 9.84 vs. 140.99 ± 8.396 mg/min; P = 0.02) and a decline in lipid oxidation (295.47 ± 15.60 vs. 188.40 ± 6.99 mg/min; P < 0.0001) were observed (Fig. 1Go). Moreover, lipid oxidation data have been examined by dividing the day into daytime (from 0700–2300 h) and nighttime (2300–0700 h) periods. During the day, lipid oxidation was 208.27 ± 10.01 mg/min in subjects before BPD vs. 132.46 ± 4.36 mg/min after BPD (P < 0.01); nighttime lipid oxidation was 102.38 ± 5.10 mg/min in subjects before BPD vs. 55.94 ± 3.17 mg/min after BPD (P < 0.01).



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  		FIG. 1. Changes in metabolic characteristics before and after BPD. M values and glucose and lipid oxidation rates (mean ± SEM) of 10 subjects before ({blacksquare}) and after ({square}) BPD. Statistics were determined by one-way ANOVA.*, P < 0.001; **, P = 0.02.

 
Parallel to the observed decline in overall lipid oxidation, we found a reduced expression of genes involved in both mitochondrial and peroxisomal lipid oxidation (PPAR{alpha}, CPT1B, and ACOX1) in muscle (Fig. 2Go). The mRNA level of ACACB, the key enzyme of liposynthesis, was also significantly reduced, whereas SREBF1 mRNA expression in muscle did not change (Fig. 3Go).



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  		FIG. 2. Muscle mRNA levels of genes involved in lipid oxidation pathways in patients undergoing BPD. PPAR{alpha}, CPT1B, and ACOX1 mRNA levels of five patients before ({blacksquare}) and after ({square}) BPD are shown as the mean ± SEM normalized to the ß-actin mRNA content. Statistical analysis was performed by one-way ANOVA. *, P < 0.05; **, P < 0.01. The inset shows the RT-PCR products of two representative samples (referred to as PT1 and PT2) before (b) and after (a) BPD resolved on agarose gel together with the 100-bp molecular weight ladder (Mw 100 bp), the 500-bp band of which is present at triple the intensity of the other fragments.

 


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  		FIG. 3. ACACB and SREBF1 mRNA expression in muscle biopsies of patients undergoing BPD. ACACB and SRBF1 mRNA levels of five patients before ({blacksquare}) and after ({square}) BPD are shown as the mean ± SEM normalized to the ß-actin mRNA content. Statistical analysis was performed by one-way ANOVA. *, P = 0.05. The inset shows the RT-PCR products of two representative samples (referred to as PT1 and PT2) before (b) and after (a) BPD resolved on agarose gel together with the 100-bp molecular weight ladder (Mw 100 bp), the 500-bp band of which is present at triple the intensity of the other fragments.

 
A significant correlation between changes in IMTG content and the expression of PPAR{alpha} (r = 0.90; P < 0.01), CPT1B (r = 0.69; P < 0.05), ACOX1 (r = 0.78; P < 0.05), and ACACB (r = 0.67; P < 0.05) was detected in muscle biopsies. Insulin sensitivity was inversely related to IMTG in a linear fashion (r = –0.96; P < 0.001). Moreover, a significant indirect correlation was found between the insulin sensitivity index (M of clamp) and the expression of PPAR{alpha} (r = –0.86; P < 0.001), CPT1B (r = –0.77; P < 0.01), ACOX1 (r = –0.63; P < 0.05), and ACACB (r = –0.64; P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
BPD provoked a dramatic weight loss in all patients studied along with a significant improvement in several metabolic parameters as well as in insulin sensitivity, as measured by the euglycemic hyperinsulinemic clamp technique. High systemic concentrations of NEFAs are known to be associated with impaired insulin sensitivity (33). In contrast, the reversibility of insulin resistance after BPD probably depends on the lowering of NEFA (34).

As previously observed (7, 31), metabolic improvement was associated with IMTG depletion. It has been hypothesized that selective depletion of intracellular fat depots in skeletal muscle is the key metabolic change leading to reversal of insulin resistance, because the reduced flux of long-chain fatty acyl-CoA (and other signal molecules) emanating from intramyocellular fat stores may up-regulate insulin signaling and restore muscle insulin sensitivity (7).

Along with the metabolic changes and IMTG depletion, a concomitant increase in 24-h glucose oxidation and a decrease in 24-h lipid oxidation were observed after BPD. The amount of reduction in lipid oxidation was similar when divided into daytime and nighttime periods, suggesting that both fasting and postprandial lipid oxidation were equally affected. Prospective studies have shown that a low ability to oxidize fat is a risk factor for weight gain and obesity (1). Moreover, it has been reported that previously obese people show a persistent defect in the oxidative capacity of skeletal muscle that appears to be organized toward fat esterification rather than oxidation (5, 35), but to date the molecular mechanisms underlying this alteration have not been fully elucidated. Studies carried out in rats showed that a prolonged pharmacological inhibition of muscle CPT-1 was associated with IMTG accumulation and development of insulin resistance (36). These findings suggest that an impaired muscle fatty acid oxidation could be considered as a primary defect causing IMTG accumulation and muscle insulin resistance development in humans with obesity and type 2 diabetes (37).

The reduced mRNA levels of PPAR{alpha} and CPT1B we observed after BPD give further support to the hypothesis of impaired lipid oxidative capacity at the muscular level (14, 35) that is not restored by weight loss. It is important to consider that a perturbation of both structural and functional parameters of mitochondria has been found in the skeletal muscle of type 2 diabetics and, to a lesser extent, obese subjects, both related to the degree of insulin resistance (38). Moreover, the increased insulin resistance present in the elderly has recently been attributed to reduced insulin-stimulated muscle glucose metabolism associated with increased fat accumulation in muscle. In these patients, nuclear magnetic resonance spectroscopy revealed a 40% reduction in mitochondrial oxidative and phosphorylation activity (39). Recently, the gene expression profiling in muscle of nondiabetic and diabetic subjects showed reduced expression of several genes encoding key enzymes in oxidative metabolism and mitochondrial function and biogenesis (40).

All of these observations support the hypothesis that a decline in mitochondrial function may contribute to insulin resistance development and fat deposition inside muscle tissue. However, the muscle fat depletion following weight loss requires the presence of further mechanisms able to counteract fat deposition. First, we tested the possibility of a compensatory increase in peroxisomal oxidation, which appears to be regulated independently of the mitochondrial pathway (11). The pattern of ACOX1 gene expression we found clearly demonstrates the absence of any increase in the mRNA levels of this key step enzyme and thus tends to exclude any increase in the peroxisomal oxidative pathway, which, on the contrary, appears to be reduced, similar to what was observed for mitochondrial ß-oxidation. These data add further support to the view that other pathways, beyond mitochondrial ß-oxidation, may be implicated in the decreased ability to oxidize lipids that characterizes human obesity.

In the presence of decreased lipid oxidation throughout the ß-oxidative and peroxisomal pathways, the depletion of muscle triglyceride content could be attributed to at least two other factors: 1) a decrease in lipid fuels entering muscle cells and the subsequent decrease in triglyceride synthesis, and 2) a decrease in the de novo fatty acid synthesis catalyzed by ACACB. It is well known that ACACB catalyzes the synthesis from acetyl-CoA of malonyl-CoA (41), which, in turn, controls mitochondrial fatty acid ß-oxidation through the inhibition of CPT1 (13). Thus, the reduction in ACACB we found should lead to an increase in CPT1B expression and probably its activity, with an increment in the mitochondrial oxidation of fatty acids. This biochemical assumption appears to be in contrast with our present data [and previous data reported by our group (42)] that show a further depression of CPT1 expression and activity after weight loss in obese subjects.

In our study the reduction of ACACB mRNA is directly correlated to the decrease in IMTG content and the improvement in insulin sensitivity. The decrease in de novo fatty acid synthesis therefore justifies the lowering of IMTG depot despite the further decrease in muscle lipid oxidation capacity. The lowering of IMTG and the decrease in lipid oxidation account for the improvement of insulin-mediated glucose disposal.

Recent data showed that insulin is able to up-regulate SREBF1c gene expression (19, 22) and its transcriptional activity (22), which results in the activation of lipogenic enzymes such as ACAC (43). It has also been observed that exercise training up-regulates SREBF1c and lipogenic genes in skeletal muscle, decreases plasma insulin levels, and increases insulin sensitivity and lipid oxidation (20).

We found that SREBF1 mRNA expression is unchanged in the skeletal muscle of obese subjects after weight loss and the subsequent muscle fat depletion with improved insulin sensitivity. Our present results lead to the conclusion that this gene does not play a predominant role in the improvement of insulin sensitivity and the depletion of fat stores after weight loss. The lack of any variations in the expression of SREBF1 may depend on some divergent phenomena affecting its expression in an opposite manner: the improvement of insulin resistance (which could be linked with its increase) and the lipid stores depletion (which could be linked with a reduction of its expression). However, one can speculate that the lack of an up-regulation of SREBF1, contrary to what happens after exercise training, could justify the decreased lipogenic enzyme gene expression.

Increased physical activity can influence muscle gene expression. None of the subjects involved in our study performed regular physical exercise, either before or after BPD. Nevertheless, a rough evaluation of spontaneous physical activity in the respiratory chamber showed an increase after BPD (data not shown). It is difficult to precisely assess the impact of this increase on the expression of the different genes we considered; however, it would have been expected to increase, rather than decrease, lipid oxidation. In fact, it has been demonstrated that exercise is able not only to improve insulin sensitivity, but also to enhance fat oxidation (44). The fact that weight loss per se is able to reduce insulin resistance without improving fat oxidative capacity emphasizes the importance of adding an exercise program to diet in the integrated management of obesity.

In summary, our results obtained in a group of obese subjects after weight loss show that muscle fat was depleted, and this was accompanied by an improvement of insulin sensitivity, an increase in glucose oxidation, and a further worsening of lipid oxidation. Moreover, they show that skeletal muscle continues to be inefficient in the oxidation of fat, and that the defect in fatty acid oxidation is located at both the mitochondrial ß-oxidation (PPAR{alpha} and CPT1B) and peroxisomal oxidative (ACOX1) pathways. These findings suggest that impaired muscle fatty acid oxidation is the primary defect causing IMTG accumulation and muscle insulin resistance in patients with obesity. The depletion of IMTG in patients with obesity probably occurs in association with elevated muscle triglyceride lipolysis and depression of the de novo fatty acid synthesis catalyzed by ACACB. Finally, the reversal of hyperinsulinemia and the reduced availability of lipid fuels after weight loss may have affected im lipid synthesis without causing any changes in SREBF1 gene expression.


   Acknowledgments
 
The excellent technical assistance of Ms. Marilena Tormene and Sonia Leandri is greatly appreciated.


   Footnotes
 
This work was supported by grants from Ministero della Ricerca Scientifica e Tecnologica (no. 2001065883_003) and Ministero della Salute, Ricerca Finalizzata: OBEIR.

Abbreviations: ACAC, Acetyl-coenzyme A carboxylase; ACOX1, acyl-coenzyme A oxidase 1, palmitoyl; BPD, biliopancreatic diversion; BW, body weight; CoA, coenzyme A; CPT1, carnitine palmitoyltransferase 1; EE, energy expenditure; FFM, fat-free mass; FM, fat mass; IMTG, intramyocellular triglyceride content; NEFA, nonesterified fatty acid; PPAR{alpha}, peroxisome proliferator-activated receptor-{alpha}; SREBF, sterol regulatory element-binding factor; TBW, total body water.

Received August 4, 2003.

Accepted January 20, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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