ATGL and CGI-58 mRNA expression in human adipose tissue and cell fractions
ATGL and CGI-58 mRNA expression was determined in human adipose tissue, isolated human adipocytes and the SVF from adipose tissue, and in skeletal muscle, and cultured human adipocytes. As shown in , ATGL was expressed in the adipocyte fraction was 23-fold higher than in the SVF. ATGL was expressed in skeletal muscle, but to a lesser extent compare to adipose tissue. CGI-58 was also expressed in adipose tissue; however there was no significant difference between adipocytes and the SVF. Both ATGL and CGI-58 were expressed at a higher level in SGBS adipocytes when compared to preadipocytes. LPL and leptin expression was measured in these samples as a control, and as expected, both were associated with the adipocyte fraction. To examine the expression of ATGL and CGI-58 in different adipose depots, ATGL and CGI-58 expression were measured in paired SAT and VAT samples obtained from 14 subjects, as described in the Methods. As shown in , ATGL expression was significantly higher in SAT than in VAT, while there was no difference in CGI-58 expression in the two depots. Leptin expression has previously been demonstrated to be higher in SAT.
ATGL and CGI-58 expression in human adipose tissue and culture
ATGL gene expression in relation to obesity and insulin resistance
ATGL deficient mice had increased insulin sensitivity and glucose tolerance due to the decreased availability of FFAs for oxidation [5
]. To determine whether ATGL was linked to obesity and/or insulin resistance, we measured ATGL mRNA levels in adipose tissue (n=86) and muscle (n=74) in Group 1 subjects using real-time RT-PCR. As described in Methods, the subjects covered a wide range of BMI and SI
and the relationships between ATGL, BMI, insulin sensitivity (SI
), and other parameters were studied using Pearson's correlation coefficients. As shown in , ATGL mRNA was not associated with BMI or SI
in either adipose tissue or muscle. In addition, there was no significant association between ATGL mRNA expression in VAT and BMI (n=14)(data not shown).
Correlation coefficient of adipose and muscle of ATGL mRNA with different variables in plasma, muscle and adipose tissue
To further examine ATGL in obesity, we measured adipose ATGL protein using Western blot in 15 subjects who were representative of the larger group, ranging in BMI from 22 to 40 kg/m2. The protein level was determined with densitometry analysis and normalized with β-actin protein level (). In contrast to adipose ATGL mRNA, adipose ATGL protein was negatively correlated with BMI (p<0.02) () and positively with SI (p<0.02)(). Thus, adipose tissue ATGL protein is higher in lean, insulin sensitive subjects, although this association was not reflected at the mRNA level.
Figure 1 ATGL protein levels in relation to obesity and insulin resistance. Human adipose ATGL protein levels were determined from the adipose tissue of 15 subjects as described in the Methods. ATGL protein level was expressed as its density ratio to actin. A. (more ...)
Muscle ATGL expression is associated with muscle fatty acid oxidation
Because ATGL is associated with muscle fatty acid oxidation in mice, we further examined the relationship between ATGL and CGI-58 gene expression and muscle triglyceride content and markers of muscle triglyceride metabolism. ATGL mRNA expression was not associated with IMCL (). However, the expression of muscle CGI-58 was negatively associated with IMCL in both type 1 (r=−0.35, p<0.02) and type 2 fibers (r=−0.40, p<0.01) (). ATGL mRNA level and CGI-58 mRNA levels were highly correlated with each other (), both in adipose tissue (r=0.71, p<0.001) and in muscle (r=0.49. p<0.001). In rodents, ATGL has been demonstrated to enhance muscle triglyceride oxidation, which is an important determinant of IMCL [5
Figure 2 Muscle CGI-58 and muscle ATGL mRNA expression are related to muscle fatty acid oxidation. A. Muscle CGI-58 was inversely related to IMCL in type 1 fibers (r=−0.35, p<0.02, n=45).B. Muscle CGI-58 was inversely related to IMCL in type 2 (more ...)
Hence, we examined the relationship between ATGL and other genes related to fatty acid oxidation. As shown in , ATGL gene expression in muscle was strongly associated with fatty acid oxidation genes, such as muscle adiponectin receptor 1 (AdipoR1, r=0.71, p<0.001), adiponectin receptor 2 (AdipoR2, r=0.74, p<0.001), and with carnitine-palmitoyl transferase 1 (CPT-1, r=0.82, p<0.001). CGI-58 mRNA levels also were correlated with the muscle adiponectin receptors 1 (r=0.35, n=74, p<0.01), adiponectin receptor 2 (r=0.42, n=74, p<0.01), and CPT1 (r=0.35, n=74, p<0.01), but these relationships were not as strong as with ATGL.
Pioglitazone increased adipose ATGL expression
Previous studies showed that ATGL was regulated by PPARγ activation in rodent adipose tissue and cell culture. To study the effect of insulin sensitizers on ATGL and CGI-58 in humans, IGT subjects were randomized to treatment with either pioglitazone or metformin, as described in Methods. Metformin treatment did not affect SI, whereas pioglitazone resulted in an increase in SI from 1.44 ± 0.13 × 10−5 to 2.21 ± 0.21 × 10%−5 min%−1/(μU/ml), p<0.05. As shown in , pioglitazone treatment resulted in a 31% increase of adipose ATGL expression (p<0.03), while there was no significant change in muscle ATGL. There was no change in CGI-58 expression in adipose tissue or muscle, and metformin had no effect on either ATGL or CGI-58 (data not shown). To determine the effect of pioglitazone on ATGL in vitro, human adipocytes were derived through the differentiation of stem cells, as described in methods, and were then treated with pioglitazone (1.5μM) for 48 hours, followed by measurement of ATGL mRNA. As shown in , ATGL expression was increased more than 2 fold after treatment with pioglitazone, indicating a direct effect on ATGL expression in adipose tissue.
Figure 4 Effects of pioglitazone on ATGL expression. Human subjects' adipose tissue (n=18) and muscle (n=14) were obtained through biopsy before and after 10 weeks of pioglitazone or metformin treatment. ATGL expression in adipose tissue and muscle was determined (more ...)
Both adipose tissue and muscle contain lipid droplets, and the initial step in the hydrolysis of triglyceride involves ATGL, which hydrolyses FFA from triglyceride, leaving the diacylglycerol for subsequent hydrolysis by hormone sensitive lipase [29
]. Subsequent studies of ATGL have suggested very different roles in adipose tissue and muscle. Based on observations in the ATGL deficient mouse, adipocytes are dependent on ATGL for lipolysis, and the absence of ATGL results in obesity and low plasma FFA levels. Absence of ATGL in muscle, however, results in muscle triglyceride accumulation, decreased use FFA for energy and decreased circulating FFA, followed by an increased dependence on carbohydrates as fuel, leading to increased glucose utilization [5
]. CGI-58 is an essential cofactor for ATGL-mediated triglyceride hydrolysis [8
], and mutations of both ATGL and CGI-58 have been found in humans with neutral lipid storage disease, suggesting that these proteins are also important in human tissues for the release of neutral lipid [31
In this study, we examined ATGL expression profiles in different tissues and cells. ATGL expression was significantly higher in SAT compared to VAT, yet there was no difference in CGI-58 expression between depots. ATGL was highly expressed in adipocytes from either adipose tissue or cultured adipocytes, with lower expression in the stromal fraction and cultured preadipocytes, and ATGL expression was induced after adipocyte differentiation [3
]. In contrast, CGI-58 expression in the adipocyte fraction was similar to the stromal fraction, which contains preadipocytes and other cell types. CGI-58 may have other functions, in addition to the activation of ATGL, as suggested by the different human syndromes caused by ATGL and CGI-58 deficiency [31
]. Whereas ATGL knockout mice become obese, CGI-58 knockout mice die within 16 hr of birth with severe hepatic steatosis and dehydration from severe impairment of their skin permeability barrier [11
]. Indeed, patients with CGI-58 mutations have prominent ichthyosis, perhaps suggesting some other role of CGI-58 in the dermis, perhaps to act as a cofactor for another neutral lipase [11
ATGL mRNA levels in adipose tissue were not significantly associated with BMI or SI
, although ATGL protein levels were lower with obesity and insulin resistance. Several studies have examined the physiologic regulation of ATGL or CGI-58 in human adipose tissue, and variable relationships with obesity or insulin resistance have been found [16
]. Differences in degree of extraction of ATGL from the lipid droplet could explain some of these differences, although this is hard to discern, since every study involving extraction of ATGL from adipose tissue used different methods. With obesity or insulin resistance, several studies noted either an increase or minimal change in ATGL mRNA yet a decrease in ATGL protein [17
], suggesting some level of posttranscriptional relationship. We also found no relationship between CGI-58 mRNA and obesity, which corresponds to a previous study [18
]. Some of these inconsistent findings could be due to different study populations and different methods for obtaining adipose tissue. Our primary study group (group 1) consisted of subjects with a wide range of BMI and insulin sensitivities and biopsies were obtained by incision, whereas some other studies concentrated on obese subjects, or used surgical fat. Based on these data, the primary regulatory forces behind ATGL are not clear. Insulin inhibits ATGL mRNA levels in 3T3 L1 adipocytes [7
], and obese subjects are hyperinsulinemic and insulin resistant. On the other hand, the obese environment is associated with adipose tissue macrophages [24
], and TNFα decreases ATGL mRNA levels in adipocytes [35
]. Since our data and others find evidence for posttranscriptional regulation, however, the regulation of ATGL is likely more complex and further studies are needed.
Previous studies in mice have demonstrated an important role for ATGL in muscle TAG lipolysis, fatty acid oxidation, and prevention of lipotoxicity [5
]. In mice, treadmill exercise training did not affect ATGL mRNA or protein levels, however ATGL knockout mice demonstrated a reduced exercise capacity [37
]. Fewer studies have been performed in human muscle, with one study demonstrating an increase in ATGL (but not CGI-58) following a period of training [38
]. Although there was no association with BMI or SI
, there were significant correlations relating either ATGL or CGI-58 expression to different elements of muscle fatty acid oxidation. There was a significant negative correlation between muscle CGI-58 expression and IMCL content in both type 1 and type 2 fibers. ATGL proteins have been shown to be exclusively expressed in human slow-twitch oxidative type 1 muscle fibers [20
]. In line with those findings, we found a strong correlation between muscle ATGL expression and genes involved with fatty acid oxidation, including CPT-1, AdipoR1, and AdipoR2. Furthermore, we observed that muscle ATGL gene expression along with other oxidative genes was increased in subjects following weight loss with exercise training, where the need for muscle fatty acid oxidation increases. Thus, these data are consistent with the mouse data that suggest an important role of ATGL-CGI-58 mediated intramyocellular TAG lipolysis, which then leads to muscle fatty acid oxidation. It is not clear why muscle ATGL does not increase with exercise in mice [37
], but could relate to other compensatory mechanisms for provision of FFA in mice, or perhaps the need for weight loss in addition to exercise. This increase in muscle lipid oxidation helps minimize muscle lipotoxicity, and is part of the response to weight loss and increased physical activity.
It is noteworthy that elements of muscle fatty acid oxidation (e.g. CPT-1, AdipoR1, AdipoR2) were associated with ATGL, but IMCL was associated with CGI-58. To our knowledge, no other study has identified regulation of muscle CGI-58 in humans. The precise mechanism for the interaction between ATGL and CGI-58 is yet to be understood. In adipocytes, CGI-58 binds to perilipin A and is released following hormonal stimulation to activate ATGL [39
], however it is not known whether this precise mechanism is operative in muscle.
In previous studies, PPARγ agonists has been shown to directly increase ATGL mRNA and protein levels in pre-adipocytes and mature adipocytes in vivo
and in vitro
], but no similar human data have been reported. In this study, adipose ATGL expression was increased by pioglitazone in human subjects, and this appeared to be a direct effect on adipocytes, rather than an indirect effect of improved insulin sensitivity, since the addition of pioglitazone to differentiated adipocytes induced ATGL mRNA level in cell culture. The magnitude of the increase in ATGL was higher in vitro, which could be due to compensatory changes induced in vivo that attenuate the effects of the thiazolidinedione, or to lower concentrations of drug achieved in vivo. Pioglitazone improves insulin sensitivity and results in an overall reduction in plasma FFA, along with an increase in expression of many adipocyte lipogenic enzymes [42
]. The impact of the ATGL increase, with no increase in CGI-58, following pioglitazone treatment is not clear.
It is possible that pioglitazone positively regulates ATGL as part of an overall increase in adipocyte gene expression, yet the improved adipocyte insulin sensitivity may downregulate overall lipolytic activity. Pioglitazone had no effect on muscle ATGL expression. Although muscle expresses PPARγ, the level of expression is low compared to adipose tissue [43
] and previous changes in IMCL from pioglitazone have not been accompanied by changes in muscle oxidative capacity [21
]. Pioglitazone improves insulin sensitivity, but this change in insulin sensitivity is not sufficient to alter muscle ATGL expression. Future study is needed to define the role of ATGL in the balance of adipose and muscle triglyceride metabolism.
In summary, this study examined the regulation of ATGL and CGI-58 in human adipose and muscle, and in response to training and pioglitazone treatment. Adipose ATGL protein was decreased with obesity and insulin resistance, even though there was no change in ATGL mRNA levels, and pioglitazone increased adipose ATGL mRNA. Muscle ATGL and CGI-58 were associated with other features of muscle fatty acid oxidation, and ATGL increased in muscle with a training/weight loss program. These data indicate that both ATGL and CGI-58 may be important components of adipose tissue and muscle control of insulin resistance.