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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

Abdominal visceral tissue (VAT) and subcutaneous adipose tissue (SAT), comprised of superficial-SAT (sSAT) and deep-SAT (dSAT), are metabolically distinct. The antidiabetic agents thiazolidinediones (TZDs), in addition to their insulin-sensitizing effects, redistribute SAT suggesting that TZD action involves adipose tissue depot-specific regulation. We investigated the expression of proteins key to adipocyte metabolism on differentiated first passage (P1) preadipocytes treated with rosiglitazone, to establish a role for the diverse depots of abdominal adipose tissue in the insulin-sensitizing effects of TZDs. Adipocytes and preadipocytes were isolated from sSAT, dSAT, and VAT samples obtained from eight normal subjects. Preadipocytes (P1) left untreated (U) or treated with a classic differentiation cocktail (DI) including rosiglitazone (DIR) for 9 days were evaluated for strata-specific differences in differentiation including peroxisome proliferator-activated receptor-γ (PPAR-γ) and lipoprotein lipase (LPL) expression, insulin sensitivity via adiponectin and glucose transport-4 (GLUT4), glucocorticoid metabolism with 11β-hydroxysteroid dehydrogenase type-1 (11βHSD1), and alterations in the adipokine leptin. While depot-specific differences were absent with the classic differentiation cocktail, with rosiglitazone sSAT had the most potent response followed by dSAT, whereas VAT was resistant to differentiation. With rosiglitazone, universal strata effects were observed for PPAR-γ, LPL, and leptin, with VAT in all cases expressing significantly lower basal expression levels. Clear dSAT-specific changes were observed with decreased intracellular GLUT4. Specific sSAT alterations included decreased 11βHSD1 whereas secreted adiponectin was potently upregulated in sSAT with respect to dSAT and VAT. Overall, the subcompartments of SAT, sSAT, and dSAT, appear to participate in the metabolic changes that arise with rosiglitazone administration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

In the 1950s, Vague et al. were the first to suggest that regulation of the endocrine and metabolic functions of abdominal adipose tissue were controlled, in part, by the anatomical distribution of fat with android obesity associated to diabetes and atherosclerosis (1). It is now accepted that the anatomical distribution of abdominal adipose tissue is dependent on adipose tissue depots (2). These include visceral adipose tissue (VAT) centrally located and enclosed by the peritoneum (2) and the subcutaneous adipose tissue (SAT) located directly below the skin. Both VAT and SAT show subject-to-subject variations with respect to distribution and volume, being dependent on age, gender, nutritional intake, and the autonomic regulation of energy homeostasis (2). However, between the two, VAT accumulation is central to android obesity and is an independent risk factor for obesity-related metabolic and cardiovascular disorders (3), in particular insulin resistance and dyslipidemia (3).

An evaluation of the association of SAT accumulation with obesity-associated complications has generated many contradictory studies. SAT has been conventionally regarded as a homogenous depot; however, it is anatomically divided by the scarpas's fascia to form two subcompartments, the superficial-SAT (sSAT) directly below the skin and the deep-SAT (dSAT) that comes into contact with the preperitoneal adipose tissue (4). Each is histologically distinct (5), with conventional imaging procedures illustrating subject-to-subject variations in distribution, particularly in association with obesity and insulin resistance (4,6,7). In a previous investigation, cellular studies of biopsies from the two SAT subcompartments in lean subjects revealed a diverse pattern of expression of key proteins that have been associated with obesity and its complications, particularly glucose transport-4 (GLUT4), 11β-hydroxysteroid dehydrogenase type-1 (11βHSD1), resistin, and leptin (8). This raised the possibility that the SAT subcompartments may have distinct, yet unknown, metabolic activities, which may explain differences in the pattern of fat deposition and the controversial findings of differential lipolysis that has been observed between the sSAT and dSAT strata (7,9,10).

Thiazolidinediones (TZDs) are a family of antidiabetic pharmacological agents including rosiglitazone, pioglitazone, and troglitazone that have been proven to ameliorate insulin sensitivity in insulin-resistant subjects (11). Insulin resistance is a common complication of obesity, yet paradoxically TZDs are potent inducers of preadipocyte differentiation in both human (12) and murine (13) cell lines in vitro. The association of TZDs with preadipocyte differentiation can be explained by the fact that they are high-affinity ligands for peroxisome proliferator-activated receptors (PPARs), in particular PPAR-γ (14). The PPARs are nuclear hormone receptors abundantly expressed in adipose tissue and central to the regulation of preadipocyte differentiation through transcriptional control of adipocyte-specific genes (15). The insulin-sensitizing effects of TZDs have been shown to correlate to their ability to activate PPAR-γ (16). Interestingly, a number of in vivo human studies evaluating the effect of TZDs as a treatment for diabetes observed that although there was an improvement in glycemic control there was also a modest weight gain in the study subjects (17,18). This weight gain was reflected in a relative increase of abdominal SAT over VAT, suggesting that TZDs may elicit depot-specific effects promoting the redistribution of adipose tissue (17,18).

Previous studies evaluating the effects of rosiglitazone on SAT and VAT preadipocytes documented a depot-specific responsiveness, with SAT preadipocyte differentiation enhanced by the inclusion of the TZD, whereas VAT preadipocytes were resistant (12,19). In the studies described, the effects of TZDs were not tested on preadipocytes isolated from dSAT, a subcompartment of SAT that appears to have independent metabolic functions in a normal physiological state (8) and is correlated to insulin resistance and lipolysis (6,10). As dSAT is correlated to insulin resistance (6) and reflects physiologically a protein profile intermediate to both sSAT and VAT in lean subjects (8), we sought to explore the effect of TZDs, specifically rosiglitazone, on preadipocyte differentiation from sSAT, dSAT, and VAT and evaluate the expression of key proteins associated with differentiation and obesity-associated complications.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

Patients and sampling

Adipose tissue samples from the three anterior abdominal adipose depots sSAT, dSAT, and VAT were obtained, as previously described (8), from eight healthy normal subjects at the peri-umbilical level during abdominal surgery unrelated to inflammatory or neoplastic abdominal disorders. Of the subjects, five were postmenopausal women and three were men with a collective BMI 23.3 ± 0.65 kg/m2 and age 55.3 ± 4.9 years. At the time of surgery, fasting blood samples were collected for chemical analysis. All chemical measurements were performed using commercially available kits, as described previously (8). The study protocol was approved by the individual Institution Ethics Committees, with the aim and the design of the study explained to each subject, who in turn gave his/her informed consent.

Cell isolation and differentiation

Both adipocytes and preadipocytes were isolated as previously described (8). Adipocytes were stored directly at −80 °C, whereas first passage (P1) preadipocytes were left to reach confluency, which took between 4 and 6 days from the point of isolation. Differentiation was initiated on P1 preadipocytes that had clear cellular contact with an overnight incubation in serum-free medium (1:1 DMEM: Ham's-F12) containing 1× concentration of insulin-transferrin-selenium (Gibco BRL, Rockville, MD). Preadipocytes were then either untreated (U; serum-free medium + 1× insulin-transferrin-selenium) or treated for 3 days with one of two differentiation cocktails, dexamethasone with 3-isobuty-1-methylxanthine (DI; 1:1 DMEM: Ham's-F12, 1× insulin-transferrin-selenium, 1 μmol dexamethasone (Sigma Chemical, St Louis, MO) 100 μmol 3-isobuty-1-methylxanthine (Sigma)) or DI with 1 μmol rosiglitazone (DIR; rosiglitazone (GlaxoSmithKline, West Sussex, UK)). At 3 days the conditioned mediums (CMs) were collected and substituted with the DI and DIR cocktails minus 3-isobuty-1-methylxanthine. The CM collection was repeated at 6 days and on completion at 9 days when the cells were harvested for total RNA and protein as previously described (8).

Assessment of differentiation

Differentiation was assessed by cell morphology and triglyceride (TG) content using Oil Red O staining. At 9 days, cells were dehydrated with 60% isopropenol and stained in 1% Oil Red O (Sigma) in 60% isopropenol. Cells were washed in 60% isopropenol and left to dry for image capture or they were washed with water and treated with 100% isopropenol to release the TGs which were measured spectrophotometrically at A 500 normalized to an empty well. Results are given as optical density A 500.

Semiquantative reverse transcriptase-PCR

Semiquantitative reverse transcriptase-PCR was performed with as previously described (8) with hypoxanthine phosphoribosyl transferase serving as the internal control for the basal expression of PPAR-γ (forward: 5′-AGACAACAGACAAATCACCAT, reverse: 5′-CTTCACAGCAAACTCAAACTT) and lipoprotein lipase (LPL) (forward: 5′-GAGATTTCTCTGTATGGCACC, reverse: 5′-CTGCAAATGAGACACTTTCTC) in adipocytes and preadipocytes and glyceraldehyde-3-phosphate dehydrogenase (forward: 5′-AGCCTCAAGATCATCAGCAATG, reverse: 5′-ATGGACTGTGGTCATGAGTCCTT) serving as the internal control for differentiation studies. All samples were analyzed in duplicate and quantified by densitometry using QuantityOne software (Bio-Rad, Hercules, CA) normalized to the expression of hypoxanthine phosphoribosyl transferase or glyceraldehyde-3-phosphate dehydrogenase and a standard intra-experimental control. Results are presented as arbitrary units relative to the expression of hypoxanthine phosphoribosyl transferase or glyceraldehyde-3-phosphate dehydrogenase.

Protein evaluation

The CM collected at 3, 6, and 9 days of differentiation were centrifuged at 3,000 r.p.m. for 10 min at room temperature and stored at −20 °C for the evaluation of secreted proteins by western immunoblot. Protein evaluations were made on cell lysates and CM as previously described (8).

Statistical evaluation

Data are expressed as mean ± s.e.m. Differences and trends within each strata were evaluated using a repeated measures ANOVA with between strata differences evaluated using a one-way ANOVA, each with a Bonferroni post hoc test. Statistical significance was set at P < 0.05. Analyses were performed using Prism (Graphpad Sofware, San Diego, CA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

A metabolic evaluation of the study subjects showed insulin and glucose levels within expected ranges for nondiabetic and insulin-sensitive subjects (Table 1). Likewise, the obesity-associated serum factors adiponectin, leptin, TNF-α, and resistin were within the range of levels previously reported for normal subjects (20,21,22,23).

Table 1.  Mean baseline characteristics of study subjects
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Depot-specific differences with rosiglitazone administration

Preadipocytes isolated from three abdominal adipose tissue compartments were left untreated (U) or induced to differentiate with DI or DIR for 9 days. Preadipocyte differentiation at 9 days was assessed morphologically by the presence and the staining of fat droplets with Oil Red O (Figure 1) and were quantified spectrophotometrically (A 500) for released TGs (Table 2). Both the brightfield and the semiquantitative evaluation of Oil Red O levels revealed that preadipocytes from each depot treated with DI or DIR differentiated, with the inclusion of rosiglitazone inducing more potent effects (Figure 1; Table 2). While the effects of DI were similar, the presence of rosiglitazone enhanced markedly the differentiation of preadipocytes from the two SAT subcompartments, sSAT and dSAT, as opposed to VAT which resisted the effects of rosiglitazone (Figure 1; Table 2).

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Figure 1. Abdominal adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. Preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and a visceral adipose tissue (VAT) abdominal depot were left untreated (U) in serum-free medium or were treated with differentiation cocktails including (DIR) or not including rosiglitazone (DI) for 9 days. Differentiation was assessed morphologically and by triglyceride accumulation evaluated by Oil Red O staining with brightfield images taken at ×200 and the remainder ×100.

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Table 2.  Oil red O staining of sSAT, dSAT, and VAT preadipocytes
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PPAR-γ: no depot-specific effects by rosiglitazone

Earlier studies observed that PPAR-γ expression increased with differentiation and was enhanced by rosiglitazone in both SAT and VAT depots, with VAT preadipocytes expressing significantly lower levels with respect to SAT (12). With the division of SAT into sSAT and dSAT subcompartments, untreated sSAT preadipocytes expressed the highest levels of PPAR-γ mRNA and VAT the lowest, whereas dSAT was intermediate with statistically no difference in PPAR-γ mRNA expression between the two (Figure 2a). Under treatments, PPAR-γ mRNA expression was further enhanced by rosiglitazone to levels persistently higher in sSAT. An evaluation of basal PPAR-γ expression from adipocytes and preadipocytes confirmed that sSAT preadipocytes expressed the highest levels of PPAR-γ, VAT the lowest with dSAT intermediary (P = 0.0024; Figure 2b). Despite a similar pattern of expression with DI and DIR treatments between depots, overall higher PPAR-γ mRNA expression levels were observed for sSAT.

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Figure 2. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression during adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. (a) The expression of PPAR-γ mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) in serum-free medium or with differentiation cocktails with the inclusion (DIR) or exclusion of rosiglitazone (DI). PPAR-γ and GAPDH mRNA were analyzed by a semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 30 cycles when transcripts were within the linear range and are given in arbitrary units. (b) Basal expression of PPAR-γ mRNA normalized to hypoxanthine phosphoribosyl transferase (HPRT), from adipocytes and preadipocytes isolated from sSAT, dSAT, and VAT abdominal depots of lean subject.

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LPL: no depot-specific effects by rosiglitazone

The hydrolysis of TGs for adipose tissue storage is driven by the enzyme LPL, with its expression also an indicator of growth arrest during adipocyte differentiation. Untreated preadipocytes showed a decrease in LPL mRNA expression from sSAT through to VAT (Figure 3a). With differentiation, there was a universal increase in the expression of LPL mRNA in all three depots which was further amplified with rosiglitazone, whereas depot-specific differences in expression were sustained (Figure 3a). An evaluation of basal LPL mRNA expression levels revealed that VAT preadipocyte levels were significantly lower than sSAT and dSAT (P = 0.036; Figure 3b). As an indicator of growth arrest during adipocyte differentiation, LPL expression suggests that sSAT is more advanced in the stages of rosiglitazone-induced differentiation, results supporting the morphological evaluation and Oil Red O staining.

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Figure 3. Lipoprotein lipase (LPL) expression during adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. (a) The expression of LPL mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) or with DI or DIR were analyzed by semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 30 cycles when transcripts were within the linear range and are given in arbitrary units. (b) Basal expression of LPL mRNA normalized to hypoxanthine phosphoribosyl transferase, from adipocytes and preadipocytes isolated from sSAT, dSAT, and VAT abdominal depots of lean subjects.

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Glucose metabolism: depot-specific regulation of adiponectin and GLUT4 by rosiglitazone

Serum levels of adiponectin are strongly correlated to insulin resistance and atheroschlerosis with adiponectin synthesis almost exclusive to adipocytes. In all three depots, differentiation coexisted with an increase in the expression of adiponectin which was further amplified in the presence of rosiglitazone (Figure 4a). Between depots, no depot-specific changes were observed. An evaluation of adiponectin secretion, however, revealed a diverse profile, with a strata-specific expression profile most evident. With DI differentiation, adiponectin could be detected in the CM at 9 days, specifically to the sSAT cells (Figure 4b). This observation was further supported with the inclusion of rosiglitazone where adiponectin secretion was predominantly observed in sSAT cells as early as 6 days, while being evident at 9 days in dSAT cells and VAT cells with an overnight exposure (Figure 4b; data not shown).

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Figure 4. Adiponectin expression during adipose depot-specific preadipocyte differentiation and the effect of rosiglitazone. (a) The expression of adiponectin mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) in serum-free medium or with differentiation cocktails with the inclusion (DIR) or absence of rosiglitazone (DI). Adiponectin and GAPDH mRNA were analyzed by semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 30 cycles when transcripts were within the linear range and are given in arbitrary units. (b) In the same lean subjects, equal volume of the CM from 3, 6, and 9 days of U, DI, and DIR treated sSAT, dSAT, and VAT preadipocytes were run on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis to investigate the secretion of adiponectin by western immunoblot. Results shown are a representative short exposure of sSAT preadipocytes under the different treatments.

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Key to adipose tissue glucose response is the cell surface presence of GLUT4 and its subsequent internalization. With differentiation, there was an increase in GLUT4 mRNA expression, enhanced further in all three depots by rosiglitazone (Figure 5a). Depot-specific differences in GLUT4 expression were observed only for untreated preadipocytes with an increase in GLUT4 expression from sSAT through to VAT (Figure 5a). In contrast to mRNA expression, strata-specific differences in the endogenous protein expression of GLUT4 were evident with an increase in expression in rosiglitazone-treated sSAT and VAT preadipocytes, whereas for dSAT there was a loss of endogenous GLUT4 protein (Figure 5b).

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Figure 5. Expression of key proteins involved in glucose metabolism during adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. (a) The expression of glucose transport-4 (GLUT4) mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) or with DI or DIR. GLUT4 and GAPDH mRNA were analyzed by a semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 35 cycles when transcripts were within the linear range and are given in arbitrary units (AUs). (b) In the same lean subjects, 20 μg of cell lysate from 9 days of U, DI, and DIR treated sSAT, dSAT, and VAT preadipocytes were run on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis to investigate the endogenous expression of GLUT4. Endogenous levels of GLUT4 are normalized to α-tubulin and are given in AUs.

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Glucocorticoid metabolism: sSAT-specific downregulation of 11βHSD1 with rosiglitazone

Fundamental to glucocorticoid metabolism is the presence and activity of 11βHSD1, an enzyme central to cortisone:cortisol conversion in adipose tissue. With differentiation and inclusion of rosiglitazone, 11βHSD1 mRNA expression in sSAT differed from dSAT and VAT (Figure 6a). Differentiation decreased 11βHSD1 mRNA expression, whereas the inclusion of rosiglitazone appeared to restore or amplify 11βHSD1 expression in dSAT and VAT (Figure 6a). These results were further supported by the pattern of endogenous 11βHSD1 protein in VAT preadipocytes, whereas no significant changes were observed for dSAT (Figure 6b). In contrast to dSAT and VAT, 11βHSD1 mRNA expression in sSAT preadipocytes decreased significantly with differentiation and was further blunted by the inclusion of rosiglitazone (Figure 6a); results supported by the pattern of endogenous protein (Figure 6b) suggesting that between the three anterior abdominal depots, the downregulation of 11βHSD1 by rosiglitazone is specific to sSAT.

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Figure 6. Expression of 11β-hydroxysteroid dehydrogenase type-1 (11βHSD1) during adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. (a) The expression of 11βHSD1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) or with DI or DIR were analyzed by semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 32 cycles when transcripts were within the linear range and are given in arbitrary units (AUs). (b) In the same lean subjects, 20 μg of cell lysate from 9 days of U, DI, and DIR treated sSAT, dSAT, and VAT preadipocytes were run on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis to investigate the endogenous expression of 11βHSD1. Endogenous levels of 11βHSD1 are normalized to α-tubulin and are given in AUs.

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Adipocytokines: no depot-specific effects by rosiglitazone

The adipocytokine leptin, which is secreted predominantly by adipocytes, is a fundamental link between the nutritional status and the regulation of energy homeostasis and participates in hypothalamic-pituitary-gonadal and immune functions. With preadipocyte differentiation, leptin mRNA expression increased in all depots, with the inclusion of rosiglitazone having no significant additive effects (Figure 7). While depot-specific differences were observed with rosiglitazone treatment, a near significant identical pattern was also observed with differentiation (DI) suggesting that leptin regulation is not a principal target for rosiglitazone action (Figure 7; P = 0.054). The detection of leptin in the CM by western immunoblot was below the limits of this methodology.

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Figure 7. Expression of leptin during adipose tissue depot-specific preadipocyte differentiation and the effect of rosiglitazone. The expression of leptin mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from preadipocytes isolated from superficial-subcutaneous adipose tissue (sSAT), deep-SAT (dSAT), and visceral adipose tissue (VAT) abdominal depots following 9 days untreated (U) or with DI or DIR. Leptin and GAPDH mRNA were analyzed by a semiquantitative reverse transcriptase-PCR with specific primers as described in Methods and Procedures. Results were taken at 35 cycles when transcripts were within the linear range and are given in arbitrary units.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES

Abdominal SAT and VAT differentially regulate the endocrine and metabolic functions of adipose tissue (2). In a previous investigation, we observed that dSAT, anatomically and histologically distinct from the sSAT (4), displayed an independent protein profile, suggesting an abdominal adipose tissue strata with peculiar endocrine and metabolic characteristics (8). Although the accumulation of VAT has been thought to be exclusively associated with the characteristics of the metabolic syndrome (2,3), there is now evidence to support a role for the accumulation of dSAT in obesity, insulin resistance, and an increased lipolysis (6,10). Based on this evidence, we sought to analyze sSAT and dSAT and evaluate the effect of preadipocyte differentiation and the influence of the TZD rosiglitazone, an antidiabetic pharmacological agent shown previously to also alter the adipose tissue distribution (17,18). Clear depot-specific regulation of proteins fundamental to glucose metabolism and glucocorticoid metabolism by rosiglitazone suggest that sSAT and dSAT play a role in the metabolic changes that arise with rosiglitazone treatment.

Given the complications associated with visceral obesity, a redistribution in abdominal fat has been thought to be the mechanism by which TZDs elicit their antidiabetic function. To understand this effect, a number of groups evaluated the influence of TZDs on SAT and VAT (P2–P3) preadipocytes, where they collectively observed that SAT preadipocyte differentiation was enhanced with TZDs, whereas VAT preadipocytes were resistant (10,19). Likewise in the present investigation, VAT (P1) preadipocytes were resistant to differentiation with rosiglitazone. As an extension to previous investigations, we dissected the SAT strata into its substrata sSAT and dSAT and observed that sSAT as early as 9 days was more advanced in its differentiation stage with rosiglitazone, as opposed to dSAT. Overall, between the three major strata of abdominal adipose tissue, sSAT appeared to be the more TZD responsive tissue.

The TZDs are high-affinity ligands for PPAR-γ, a nuclear hormone receptor abundantly expressed in adipose tissue and central to the regulation of preadipocyte differentiation (15). The strata-specific differences in the response of human SAT and VAT preadipocytes to TZDs has been shown to correlate to PPAR-γ expression levels (19). In line with these studies, we observed that while the rosiglitazone mediated upregulation of PPAR-γ mRNA expression was similar between depots, VAT preadipocytes had lower basal PPAR-γ mRNA levels, while sSAT had the highest, suggesting that the potent TZD response by sSAT is due to higher basal PPAR-γ levels.

The association of VAT accumulation with metabolic complications has been proposed to be due in part to its higher lipolytic rate (24). With TZD treatment, a redistribution of adipose tissue to SAT is suggested to contribute to the whole body improvement in insulin sensitivity (17,18). Paradoxically, the contribution of SAT is unclear based on the observation that dSAT accumulation, like VAT, correlates to insulin resistance (6) due to a higher lipolytic activity (10). We opted to evaluate the potential strata-specific TG accumulation following TZD treatment by studying the enzyme LPL, which is central to the hydrolysis of TG from serum lipoproteins making them available for adipocyte storage. We observed that with differentiation and rosiglitazone treatment, the baseline to treatment increments of LPL mRNA expression was equal among depots, whereas strata-specific differences appeared to be a consequence of basal LPL mRNA levels. A combination of the redistribution of adipose tissue to SAT and an overall increase in LPL mRNA expression in all depots may contribute to improve insulin sensitivity with rosiglitazone treatment.

To assess the contribution of the SAT subcompartments in TZD-regulated insulin sensitization, we evaluated key adipose tissue-derived proteins to determine their potential depot-specific role. Based on our results, rosiglitazone-regulated effects can be divided into three groups: (i) strata-independent effects, (ii) dSAT-specific effects, and (iii) sSAT-specific effects. Although leptin expression increased with differentiation, no additive effects or depot-dependent effects following rosiglitazone treatment were observed, as previously described between omental and SAT preadipocytes (25). A number of studies examining adipose tissue biopsies have shown that SAT expresses higher leptin mRNA and secretes higher levels of leptin with respect to VAT (26,27). Our observations are in agreement with these studies as well as the strata-independent effects of rosiglitazone reported by Van Harmelen et al. (25). The additional observation from the present study is that dSAT leptin expression was intermediary to both sSAT and VAT.

A role for dSAT in obesity-associated metabolic complications is emerging with an extensive study by Kelley et al. (6) showing a strong association between dSAT accumulation and insulin resistance. When considering whole body glucose homeostasis, the importance of adipose tissue GLUT4 expression has been shown. Adipose-specific GLUT4 overexpression in muscle-specific GLUT4 knockout mice was able to restore whole body insulin sensitivity, accompanied by an alteration in the endocrine function of adipose tissue itself (28). In the present investigation, although changes in GLUT4 mRNA expression increased with differentiation and rosiglitazone treatment similarly across the three strata, the pattern of intracellular 45-kDa GLUT4 expression decreased in dSAT with differentiation and rosiglitazone compared with sSAT and VAT. Possible explanations for the discrepancy between GLUT4 mRNA and protein expression in the dSAT depot may include alterations in protein synthesis and/or turnover or differences in the cellular localization of GLUT4. In line with these results, the level of intracellular 45-kDa GLUT4 has previously been demonstrated to be lower in dSAT whole biopsies when compared with sSAT and VAT from lean subjects (8). Studies in preperitoneal adipocytes from obese subjects have shown that their greater response to insulin when compared with adipocytes from other depots may be due to a greater GLUT4 expression and/or altered GLUT4 translocation (29). It has also been suggested that VAT adipocytes from type 2 diabetic subjects have an altered GLUT4 subcellular distribution (30). While the exact mechanism(s) leading to our findings in dSAT remain to be established, its clarification may add evidence to the hypothesis that dSAT is a prominent target of insulin resistance (6) among the abdominal adipose tissue depots herein investigated.

Previous in vivo and in vitro investigations have indicated that there is a significant role for SAT in TZD therapy (12,17,18,19). When examining TZD actions on adipose tissue, it has been observed that TZDs and non-TZD PPAR-γ agonists reduce 11βHSD1 gene expression in 3T3-L1 cells (31,32), which further correlated to a decrease in 11βHSD1 activity (31). This finding led to the hypothesis that 11βHSD1 regulates the TZD adipogenic effect (32). Previously, 11βHSD1 expression and activity have been reported to correlate with the degree of obesity and insulin resistance, supporting its role in the onset/persistence of the metabolic syndrome (33,34). In the present investigation, rosiglitazone-induced adipose depot-specific effects in 11βHSD1 mRNA expression were evident. While observing an upregulation in 11βHSD1 mRNA in VAT in the presence of rosiglitazone, we also observed a significant downregulation exclusive to the human anterior abdominal sSAT, further implicating the participation of the sSAT depot in the insulin-sensitizing effects of rosiglitazone.

In contrast to 11βHSD1, TZDs upregulate the production of adiponectin, a step proposed to be key to the insulin-sensitizing effects of TZDs in diabetic subjects (35,36). With pioglitazone treatment in glucose-tolerant and glucose-intolerant subjects, results suggest that adiponectin regulation is posttranscriptional (37), data supported in the present investigation. We observed depot-specific differences at the posttranscriptional level, with substantially elevated secreted adiponectin levels indicating that sSAT may be key to the restoration of adiponectin secretion with rosiglitazone treatment and overall fundamental in the modulation of adiponectin production. The importance of such a finding is reflected by the function of adiponectin, which plays a key role in insulin sensitivity, anti-inflammatory and antiatherogenic functions. Such functions are reduced or lost with poor glycemic control and obesity due to significantly decreased adiponectin serum levels (38).

Based on the theory that VAT accumulation is an independent risk factor for obesity-related metabolic and cardiovascular disorders, in particular insulin resistance and dyslipidemia (2,3), it appears paradoxical that in the presence of the insulin-sensitizer rosiglitazone, VAT-specific changes were modest for the proteins investigated in the present investigation. Previous investigations have proposed that a redistribution in abdominal fat is the mechanism by which TZDs have their antidiabetic function, implicating an important role for the SAT depot in the insulin-sensitizing effects of TZDs (17,18). Although not excluding a role for VAT, in the present investigation we confirm a potentially important role for SAT. Through the evaluation of proteins key to adipocyte differentiation as well as adipocyte function, we propose that that the subcompartments of SAT, sSAT, and dSAT are key to metabolic changes that arise with rosiglitazone treatment, and that the independent metabolic activities of the SAT subcompartments are fundamental to the overall activity of abdominal adipose tissue.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Disclosure
  8. REFERENCES
  • 1
    Vague J. The degree of masculine differentiation of obesities: a factor determining predisposition to diabetes, atheroschelrosis, gout and uric calculous disease. Am J Clin Nutr 1956; 4: 2031.
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    Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 2000; 21: 697738.
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