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Elevated Adipose Triglyceride Lipase in Newly Diagnosed Type 2 Diabetes Mellitus With Hypertension
CLINICAL INVESTIGATION
Elevated Adipose Triglyceride Lipase in Newly Diagnosed Type 2 Diabetes Mellitus With Hypertension Shengnan Xu, MD, Gangyi Yang, MD, Mengliu Yang, MD, Shengbing Li, MD, Hua Liu, MD and Ling Li, MD
Abstract: Introduction: Adipose triglyceride lipase (ATGL) is a recently identified triacylglycerol lipase responsible for adiposity lipolysis. Its pathophysiologic role in humans remains unknown. Material and Methods: In this study, the authors investigated the levels of plasma ATGL among patients with type 2 diabetes mellitus (T2DM), patients with T2DM and hypertension and control subjects. They also assessed the association between plasma ATGL and body composition and metabolic parameters. Results and Conclusions: Plasma ATGL levels significantly increased in patients with T2DM and hypertension compared with those with T2DM (78.3 ⫾ 23.4 versus 65.1 ⫾ 22.8 g/L, P ⬍ 0.01). No gender differences were found among plasma ATGL levels. Furthermore, they found that the plasma ATGL level was positively correlated with total cholesterol (r ⫽ 0.17, P ⬍ 0.05) and high-density lipoprotein C (r ⫽ 0.16, P ⬍ 0.05) in simple regression analysis of pooled data, whereas, in multiple stepwise regression analysis, diastolic blood pressure, total cholesterol and homeostasis model assessment of insulin resistance were independently related factors with plasma ATGL levels (Y ⫽ ⫺13.662 ⫹ 0.343 ⫻ waist ⫹ 0.268 ⫻ diastolic blood pressure ⫹ 0.053 ⫻ 2hPin ⫹ 0.966 ⫻ homeostasis model assessment of insulin resistance). This work indicates the potential link of ATGL with the pathogenesis of insulin resistance and T2DM. Key Indexing Terms: Adipose triglyceride lipase; Type 2 diabetes mellitus; Hypertension. [Am J Med Sci 2011;342(6):452–455.]
I
n 2004, a novel key enzyme capable of hydrolyzing triacylglycerols (TG) was identified by 3 independent groups. The TG hydrolase was named adipose triglyceride lipase (ATGL), desnutrin or phospholipase A2.1–3 ATGL is highly expressed in adipose tissue, and its expression markedly increases during 3T3-L1 adipocyte differentiation.1,3–5 It specifically hydrolyzes the first fatty acid (FA) from TG, resulting in the formation of diacylglycerols and FA and exhibits a 10-fold higher substrate specificity for TG over diacylglycerols. Furthermore, the enzyme shows no hydrolytic activity when other lipid substrates such as cholesteryl esters or retinyl esters are used.3 Several observations underscore the importance of this enzyme in energy homeostasis and metabolic regulation: Overexpression of ATGL enhances lipolysis in 3T3-L1 adipocytes, which can
From the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education and Department of Clinical Biochemistry (SU, LL), Chongqing Medical University, Chongqing 400016, China; Department of Endocrinology (GY, MY, SL), the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China; and Department of Pediatrics (HL), University of Mississippi Medical Center, Jackson, Mississippi 39216-4505. Submitted August 5, 2010; accepted in revised form February 28, 2011. The first 2 authors contributed equally to this project. This study was supported by the National Natural Science Foundation of China grants 30871199, 81070640, 30971388 and 30771037. Correspondence: Ling Li, MD, Department of Clinical Biochemistry, Chongqing Medical University, Chongqing 400016, China (E-mail: [email protected]).
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be inhibited by antisense technologies against ATGL1; pronounced up-regulation of ATGL in fasting mice and downregulation after refeeding2,4; reduction of mRNA in adipose tissue of genetically obese mice2; down-regulation in animal models for insulin resistance (IR; ob/ob and db/db)6 and increased ATGL mRNA expression by treatment of the peroxisome proliferator-activated receptor (PPAR)-␥ agonist rosiglitazone in mice.7 These data suggest the pathophysiological significance of ATGL in energy homeostasis and IR. However, the essential role of ATGL has not been elucidated especially in IR. Although the change of plasma ATGL has been investigated in patients with type 2 diabetes mellitus (T2DM),3 plasma ATGL levels in patients with T2DM and hypertension and the association between ATGL and IR have not been demonstrated. In this study, we have measured plasma ATGL levels in patients with T2DM and hypertension and newly diagnosed T2DM, which closely correlate with IR. We also assessed the association of plasma ATGL with body composition and several metabolic parameters in these subjects.
MATERIALS AND METHODS Subjects Eighty-one subjects with newly diagnosed T2DM (42 men, 39 women, age 53 ⫾ 12 years, T2DM group), 33 newly diagnosed patients with T2DM and hypertension (18 men, 15 women, age 60 ⫾ 8 years, T2DMH group) and 60 normal control subjects(22 men, 38 women, age 51 ⫾ 12 years, NGT group) participated in the study. The diagnosis of T2DM was based on oral glucose tolerance test and World Health Organization criteria (1999),8 and that of hypertension was based on World Health Organization criteria (1999).9 Patients with type 1 diabetes, macrovascular or microvascular complications, urinary tract infections, urolithiasis, liver cirrhosis, congestive heart failure, overt proteinuria, obstructive sleep apnea or other known major diseases were excluded. All patients were newly diagnosed, and none of them were treated with oral hypoglycemic agents or diet control. Subjects without clinical evidence of major disease or family history of T2DM or hypertension were recruited from an unselected population that underwent routine medical checkups and were used as the controls. None of the control subjects were taking medications known to affect glucose tolerance. The study was approved by the human research ethics committee of Chongqing Medical University, and informed consent was obtained from all patients and controls. Anthropometry and Plasma Samples Anthropometric parameters measured included the body mass index, the waist-to-hip ratio and blood pressure (BP). Waist and hip circumferences were measured to the nearest 0.1 cm at the narrowest point between the lowest rib and the uppermost lateral border of the right iliac crest. The hips were
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measured at their widest point. Seated BP was taken by a trained nurse after the subjects had rested for 10 minutes. Plasma Biochemical Parameters and ATGL Blood samples were drawn after an overnight fast, and plasma samples were kept at ⫺80°C until assayed. Plasma insulin was measured in deproteinized serum by radioimmunoassay using human insulin as standard (Linco, St. Charles, MO). Free FA (FFA) was measured in plasma with a kit (Randox Laboratories, Antrim, United Kingdom). Plasma glucose was assayed using the glucose oxidase method. Glycosylated hemoglobin (HbA1c) was measured by isoelectric focusing. Plasma triglyceride (TG), cholesterol (TC), low-density lipoprotein C (LDL-C) and high-density lipoprotein C (HDL-C) concentrations were determined enzymatically. Plasma ATGL levels were evaluated using a commercially available enzyme-linked immunosorbent assay (Phoenix Pharmaceuticals, Belmont, CA). The intra-assay coefficients of ATGL were less than 5%, and the interassay coefficients were less than 12%. The linear ranges of the assays were 1.5 to 300 ng/mL for ATGL. The homeostasis model assessment of IS (HOMAIR) and the homeostasis model assessment of -cell insulin secretion (HOMAIS) were calculated from fasting insulin and glucose levels with the following equations: HOMAIR ⫽ insulin [U/mL] ⫻ glucose [mmol/L]/22.5 and HOMAIS ⫽ [20 ⫻ Insulin (U/mL)]/[fasting blood glucose (FBG, mmol/ L) ⫺ 3.5].9 Statistical Analyses The data are shown as the mean ⫾ standard deviation. All statistical analyses were performed using SPSS 8.0 software (SPSS, Chicago, IL). Baseline characteristics of case and control subjects were compared by 1-way, Wilcoxon rank sum test or 2 test. The general linear modeling function analysis was used to control for potential confounders. As the distributions of plasma insulin and HOMAIR values were skewed, logarithmically transformed values were used for statistical analysis. As our primary approach, we included plasma ATGL levels as continuous independent variables in the multivariable models. Simple and multiple regression analyses were used to examine the association between plasma ATGL levels and the values of other biomarkers.
RESULTS The clinical characteristics of our subjects are shown in Table 1. After glucose overload, the T2DMH group had higher weight, systolic BP, diastolic BP (DBP), body mass index, waist-to-hip ratio, LDL-C, FBG and 2 hours plasma insulin (2 hours Ins) than that of the T2DM and control groups, whereas HOMAIS was lower than the NGT group. The T2DM group had higher TG, free FA, FBG, plasma glucose after glucose load, HbA1C, HOMAIR and lower HOMAIS than the controls (Table 1). Plasma ATGL levels were significantly increased in T2DMH compared with T2DM (78.3 ⫾ 23.4 versus 65.1 ⫾ 22.7 g/L, P ⬍ 0.01, Figure 1). We found that plasma ATGL levels in T2DM group had an elevating tendency compared with NGT groups (78.3 ⫾ 23.4 versus 70.9 ⫾ 22.5 g/L) but no significant difference. The data also revealed that the difference in age and gender was of little statistical significance. Bivariate correlation analyses were performed to assess the relationship of plasma ATGL concentrations with body composition and metabolic parameters. Plasma ATGL levels correlated positively with TC (r ⫽ 0.17, P ⬍ 0.05) and HDL-C (r ⫽ 0.16, P ⬍ 0.05, Table 2). Multiple regression analysis showed that DBP, TC and HOMAIR were independently re© 2011 Lippincott Williams & Wilkins
TABLE 1. Clinical characteristics of study subjects Factor
NGT
T2DM
T2DMH
N 60 81 33 Age (yr) 50.7 ⫾ 12.3 52.7 ⫾ 11.5 60.4 ⫾ 8.1a,b SBP (mm Hg) 122.9 ⫾ 12.8 119.9 ⫾ 14.8 134.1 ⫾ 18.5a,b DBP (mm Hg) 76.7 ⫾ 7.3 76.8 ⫾ 8.5 85.8 ⫾ 9.9a,b 2 BMI (kg/m ) 23.3 ⫾ 3.4 24.3 ⫾ 3.0 26.2 ⫾ 3.7a,b WHR 0.85 ⫾ 0.08 0.89 ⫾ 0.94 0.95 ⫾ 0.17a,b FAT (%) 29.5 ⫾ 9.2 32.2 ⫾ 10.7 34.7 ⫾ 9.3c a FBG (mmol/L) 5.4 ⫾ 0.4 10.5 ⫾ 4.5 9.3 ⫾ 4.3a a 2hPBG (mmol/L) 6.1 ⫾ 1.1 19.2 ⫾ 7.8 17.4 ⫾ 7.2a FINS (mU/L) 8.9 ⫾ 4.5 10.1 ⫾ 7.3 11.9 ⫾ 6.3c 2hPINS (mU/L) 36.2 ⫾ 22.5 50.0 ⫾ 40.1c 69.8 ⫾ 68.4c HOMA–IR 2.2 ⫾ 1.1 4.5 ⫾ 3.4a 4.9 ⫾ 3.5a a HbA1c (%) 5.6 ⫾ 0.4 8.9 ⫾ 2.9 7.7 ⫾ 2.5a TG (mmol/L) 1.16 ⫾ 0.50 1.80 ⫾ 1.07a 2.24 ⫾ 1.33a TC (mmol/L) 4.40 ⫾ 0.86 4.78 ⫾ 1.11 5.27 ⫾ 1.92a HDL–C (mmol/L) 1.31 ⫾ 0.32 1.24 ⫾ 0.31 1.60 ⫾ 0.21 LDL–C (mmol/L) 2.56 ⫾ 0.67 2.71 ⫾ 0.95 3.18 ⫾ 1.80c,d FFA (mol/L) 0.61 ⫾ 0.27 0.73 ⫾ 0.31c 0.84 ⫾ 0.37a Data are mean ⫾ SD or frequency (percentage). a P ⬍ 0.01 compared with NGT. b P ⬍ 0.01 compared with T2DM. c P ⬍ 0.05 compared with NGT. d P ⬍ 0.05 compared with T2DM. NGT, normal glucose tolerance; T2DM, type 2 diabetes mellitus; T2DMH, T2DM with hypertension; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WHR, waist hip ratio; FAT %, visceral fat %; FBG, fasting blood glucose; 2hPBG, 2 h plasma glucose after glucose load; FINS, fasting plasma insulin; 2hPINS, 2 h plasma insulin after glucose load; HOMA-IR, HOMA-insulin resistance index; FFA, free fatty acids; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin A1c.
lated factors with plasma ATGL levels (Y ⫽ ⫺13.662 ⫹ 0.343Xwaist ⫹ 0.268XDBP ⫹ 0.053X2hPin⫹ 0.966XHOMA-IR, Table 2). Increasing levels of ATGL showed a significant linear trend and were independently associated with T2DMH, especially when concentrations were analyzed both by tertile and by a continuous variable (Table 3).
FIGURE 1. The change of ATGL levels in subjects studied.
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TABLE 2. Linear and multiple regression analysis of variables associated with plasma ATGL in subject studied Simple Variable Age SBP DBP BMI WHR FAT (%) TG TC HDL–C LDL–C FFA FBG 2hPBG FINS 2hPINS HbA1c HOMA–IR
Multiple
Estimate
P
Estimate
P
0.195 0.119 0.265 0.341 0.239 0.298 0.253 0.066 ⫺0.024 0.048 0.163 0.166 0.209 0.183 0.205 0.188 0.214
0.011 0.122 0.001 0.001 0.002 0.001 0.001 0.391 0.755 0.537 0.035 0.030 0.006 0.017 0.011 0.014 0.005
— — 0.268 — — — — — — — — — — — 0.053 0.966 —
— — — — — — — — — — — — — — 0.042 0.015 —
In multiple linear stepwise regression analysis, values included for analysis were age, gender, BMI, WHR, SBP, DBP, FBG, 2hPBG, HbA1c, INS, HOMA-IR, FFA, TC, HDL-C, LDL-C,TG and FAT %. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WHR, waist hip ratio; FAT %, visceral fat %; FBG, fasting blood glucose; 2hPBG, 2 h plasma glucose after glucose load; FINS, fasting plasma insulin; HOMA-IR, HOMA-insulin resistance index; FFA, free fatty acids; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin A1c.
DISCUSSION IR plays a primary role in the development of T2DM10 and is a characteristic feature of other health disorders including obesity, dyslipidemias, hypertension and cardiovascular disease.11 The molecular basis of IR has not been fully elucidated, but secreted proteins are central regulators of metabolism and play key roles in food intake, insulin sensitivity and
energy metabolism. ATGL was recently discovered as a novel lipase in adipose tissue contributing to adipocyte lipolysis.3 Previous experiments have demonstrated an important role of ATGL in TG lipolysis and energy metabolism in cultured adipocytes and in weight maintenance of mice and other lower-order organisms.1,2,12 However, its importance in the pathogenesis of obesity and IR has not been evaluated. It was demonstrated that ATGL was essential for efficient TG metabolism, which altered fatty acid storage and oxidation. Rather, excessive TG degradation may promote IR when fatty acid oxidation cannot match intracellular production, resulting in the accumulation of bioactive FA metabolites.13 IR plays a primary role in the development of T2DM.This study is the first to report plasma ATGL levels in patients with T2DM and hypertension and their relationship with body composition and metabolic parameters. The results of the current study showed that plasma ATGL concentrations were significantly increased in T2DMH compared with T2DM (78.3 ⫾ 23.5 versus 65.1 ⫾ 22.8 g/L, P ⬍ 0.01). Therefore, it seems that BP exerts an important effect on elevating plasma ATGL levels. Furthermore, we found that plasma ATGL levels correlated positively with TC, HDL-C and DBP. Multiple regression analysis showed that TC, DBP and HOMA-IR were independently related factors with plasma ATGL levels. There was a decreasing trend of plasma ATGL levels in T2DM compared with the NGT group (65.1 ⫾ 22.8 versus 70.9 ⫾ 22.6 g/L) but not statistically significant. A plausible interpretation is that this reduced lipolytic response served as an appropriate downregulation of lipolysis per unit of fat mass to prevent excessive FA outflow from the expanded fat mass and to prevent worsening of the IR state.6 Collectively, these data support that insulin, blood glucose and lipids may influence circulating ATGL levels by regulating its expression and secretion. BP could be another parameter affecting ATGL concentrations. ATGL may play a role in the pathogenesis of IR and T2DM, and this role may be at least partially mediated via PPAR-␥ signals.4 Results of genetic studies indicate the involvement of PPAR-␥ in glucose homeostasis. Consistent with this, several dominant-negative mutations in PPAR-␥ in humans have been shown to cause partial lipodystrophy, marked IR, diabetes and hypertension.14 Also, it has been demonstrated that induction of ATGL in adipogenesis was in parallel with increasing PPAR-␥ and its target gene expression, suggesting a potential role of
TABLE 3. General liner and logistic analysis of the impact of ATGL level on T2DM and T2DMH Tertiles of ATGL Factor All subjects No. GT/T2DM/T2DMH (IGR/T2DM/NGT) Cutoff ATGL (g/L) T2DM Univariate Multivariatea T2DMH Univariate Multivariatea
Q1 (95% CI)
Q2 (95% CI)
Q3 (95% CI)
P
18/34/6 ⬍56.8
19/28/11 56.8–78.3
23/19/16 ⬎78.3
0.045
1.00 1.00
0.780 (0.345–1.764) 0.977 (0.362–2.639)
0.437 (0.190–1.007)) 0.406 (0.138-1.193)
0.586 0.467
1.00 1.00
1.737 (0.531–5.683) 1.764 (0.344–9.033)
2.087 (0.679–6.414) 1.075 (0.214-5.406)
0.061 0.648
Values shown are cutoffs of plasma ATGL levels in all subjects and odds ratios with 95% confidence intervals. a Adjusted for age, gender, body mass index, waist to hip ratio, visceral fat %, systolic blood pressure, diastolic blood pressure, total cholesterol, triglyceride, high-density lipoprotein- and low-density lipoprotein-cholesterol, and free fatty acids. ATGL, adipose triglyceride lipase; NGT, normal glucose tolerance; T2DM, type 2 diabetes mellitus; T2DMH, T2DM with hypertension.
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PPAR-␥ for regulating ATGL expression in adipogenesis. Several recent studies7,15 have demonstrated increased ATGL mRNA expression in adipose tissue of rodents treated with rosiglitazone, which served as PPAR-␥ agonist and insulin sensitizer. Furthermore, PPAR-␥ activation increases ATGL mRNA expression in fully differentiated 3T3-L1 adipocytes and adipose tissues. Also, the interactions between ATGL and PPAR-␥ activation were further confirmed by a significant reduction of ATGL mRNA and protein levels after RNAimediated PPAR-␥ knockdown.16 Therefore, the ATGL gene may be a direct transcriptional target of the key adipocyte transcription factor PPAR-␥. Taken together, the regulation of ATGL by PPAR-␥ may constitute a potential therapeutic target for the treatment of IR and T2DM. In summary, we have for the first time demonstrated that plasma ATGL levels are significantly increased in T2DMH compared with a T2DM group. However, more work is needed to clearly define the functional consequences of hypertensioninduced plasma ATGL level increasing.
lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 2006;55:148 –57. 5. Kim JY, Tillison K, Lee J, et al. The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3–L1 adipocytes and is a target for transactivation by PPARgamma. Am J Physiol Endocrinol Metab 2006;291:E115–27. 6. Jocken JW, Langin D, Smit E, et al. Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state. J Clin Endocrinol Metab 2007;92:2292–9. 7. Festuccia WT, Laplante M, Berthiaume M, et al. PPARgamma agonism increases rat adipose tissue lipolysis, expression of glyceride lipases, and the response of lipolysis to hormonal control. Diabetologia 2006;49:2427–36. 8. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. 1. Diagnosis and classification of diabetes mellitus, provisional report of a WHO consultation. Diabet Med 1998;15:539 –53. 9. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9.
ACKNOWLEDGMENTS The authors thank Amelia Griggs, University of Mississippi Medical Center, for technical editing of the manuscript.
10. DeFronzo RA. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 1988;37:667– 87.
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12. Soni KG, Lehner R, Metalnikov P, et al. Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase. J Biol Chem 2004;279:40683–9.
1. Zimmermann R, Strauss JG, Haemmerle G, et al. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 2004;306:1383– 6. 2. Villena JA, Roy S, Sarkadi-Nagy E, et al. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis. J Biol Chem 2004;279:47066 –75. 3. Jenkins CM, Mancuso DJ, Yan W, et al. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J Biol Chem 2004;279: 48968 –75. 4. Kershaw EE, Hamm JK, Verhagen LA, et al. Adipose triglyceride
© 2011 Lippincott Williams & Wilkins
11. Reaven GM, Hollenbeck C, Jengk CY, et al. Measurement of plasma glucose, free FA, lactate and insulin for 24 hours in patients with NIDDM. Diabetes 1988;37:1020 – 4.
13. Watt MJ. Storing up trouble: Does accumulation of intramyocellular triglyceride protect skeletal muscle from insulin resistance? Clin Exp Pharmacol Physiol 2009;36:5–11. 14. Barroso I, Gurnell M, Crowley VE, et al. Dominant negative mutations in human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 1999;402:880 –3. 15. Shen WJ, Patel S, Yu Z, et al. Effects of rosiglitazone and high fat diet on lipase/esterase expression in adipose tissue. Biochim Biophys Acta 2007;1771:177– 84. 16. Kershaw EE, Schupp M, Guan HP, et al. PPAR-␥ regulates adipose triglyceride lipase in adipocytes in vitro and in vivo. Am J Physiol Endocrinol Metab 2007;293:E1736 – 45.
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Elevated Adipose Triglyceride Lipase in Newly Diagnosed Type 2 Diabetes Mellitus With Hypertension Shengnan Xu, MD, Gangyi Yang, MD, Mengliu Yang, MD, Shengbing Li, MD, Hua Liu, MD and Ling Li, MD
Abstract: Introduction: Adipose triglyceride lipase (ATGL) is a recently identified triacylglycerol lipase responsible for adiposity lipolysis. Its pathophysiologic role in humans remains unknown. Material and Methods: In this study, the authors investigated the levels of plasma ATGL among patients with type 2 diabetes mellitus (T2DM), patients with T2DM and hypertension and control subjects. They also assessed the association between plasma ATGL and body composition and metabolic parameters. Results and Conclusions: Plasma ATGL levels significantly increased in patients with T2DM and hypertension compared with those with T2DM (78.3 ⫾ 23.4 versus 65.1 ⫾ 22.8 g/L, P ⬍ 0.01). No gender differences were found among plasma ATGL levels. Furthermore, they found that the plasma ATGL level was positively correlated with total cholesterol (r ⫽ 0.17, P ⬍ 0.05) and high-density lipoprotein C (r ⫽ 0.16, P ⬍ 0.05) in simple regression analysis of pooled data, whereas, in multiple stepwise regression analysis, diastolic blood pressure, total cholesterol and homeostasis model assessment of insulin resistance were independently related factors with plasma ATGL levels (Y ⫽ ⫺13.662 ⫹ 0.343 ⫻ waist ⫹ 0.268 ⫻ diastolic blood pressure ⫹ 0.053 ⫻ 2hPin ⫹ 0.966 ⫻ homeostasis model assessment of insulin resistance). This work indicates the potential link of ATGL with the pathogenesis of insulin resistance and T2DM. Key Indexing Terms: Adipose triglyceride lipase; Type 2 diabetes mellitus; Hypertension. [Am J Med Sci 2011;342(6):452–455.]
I
n 2004, a novel key enzyme capable of hydrolyzing triacylglycerols (TG) was identified by 3 independent groups. The TG hydrolase was named adipose triglyceride lipase (ATGL), desnutrin or phospholipase A2.1–3 ATGL is highly expressed in adipose tissue, and its expression markedly increases during 3T3-L1 adipocyte differentiation.1,3–5 It specifically hydrolyzes the first fatty acid (FA) from TG, resulting in the formation of diacylglycerols and FA and exhibits a 10-fold higher substrate specificity for TG over diacylglycerols. Furthermore, the enzyme shows no hydrolytic activity when other lipid substrates such as cholesteryl esters or retinyl esters are used.3 Several observations underscore the importance of this enzyme in energy homeostasis and metabolic regulation: Overexpression of ATGL enhances lipolysis in 3T3-L1 adipocytes, which can
From the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education and Department of Clinical Biochemistry (SU, LL), Chongqing Medical University, Chongqing 400016, China; Department of Endocrinology (GY, MY, SL), the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China; and Department of Pediatrics (HL), University of Mississippi Medical Center, Jackson, Mississippi 39216-4505. Submitted August 5, 2010; accepted in revised form February 28, 2011. The first 2 authors contributed equally to this project. This study was supported by the National Natural Science Foundation of China grants 30871199, 81070640, 30971388 and 30771037. Correspondence: Ling Li, MD, Department of Clinical Biochemistry, Chongqing Medical University, Chongqing 400016, China (E-mail: [email protected]).
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be inhibited by antisense technologies against ATGL1; pronounced up-regulation of ATGL in fasting mice and downregulation after refeeding2,4; reduction of mRNA in adipose tissue of genetically obese mice2; down-regulation in animal models for insulin resistance (IR; ob/ob and db/db)6 and increased ATGL mRNA expression by treatment of the peroxisome proliferator-activated receptor (PPAR)-␥ agonist rosiglitazone in mice.7 These data suggest the pathophysiological significance of ATGL in energy homeostasis and IR. However, the essential role of ATGL has not been elucidated especially in IR. Although the change of plasma ATGL has been investigated in patients with type 2 diabetes mellitus (T2DM),3 plasma ATGL levels in patients with T2DM and hypertension and the association between ATGL and IR have not been demonstrated. In this study, we have measured plasma ATGL levels in patients with T2DM and hypertension and newly diagnosed T2DM, which closely correlate with IR. We also assessed the association of plasma ATGL with body composition and several metabolic parameters in these subjects.
MATERIALS AND METHODS Subjects Eighty-one subjects with newly diagnosed T2DM (42 men, 39 women, age 53 ⫾ 12 years, T2DM group), 33 newly diagnosed patients with T2DM and hypertension (18 men, 15 women, age 60 ⫾ 8 years, T2DMH group) and 60 normal control subjects(22 men, 38 women, age 51 ⫾ 12 years, NGT group) participated in the study. The diagnosis of T2DM was based on oral glucose tolerance test and World Health Organization criteria (1999),8 and that of hypertension was based on World Health Organization criteria (1999).9 Patients with type 1 diabetes, macrovascular or microvascular complications, urinary tract infections, urolithiasis, liver cirrhosis, congestive heart failure, overt proteinuria, obstructive sleep apnea or other known major diseases were excluded. All patients were newly diagnosed, and none of them were treated with oral hypoglycemic agents or diet control. Subjects without clinical evidence of major disease or family history of T2DM or hypertension were recruited from an unselected population that underwent routine medical checkups and were used as the controls. None of the control subjects were taking medications known to affect glucose tolerance. The study was approved by the human research ethics committee of Chongqing Medical University, and informed consent was obtained from all patients and controls. Anthropometry and Plasma Samples Anthropometric parameters measured included the body mass index, the waist-to-hip ratio and blood pressure (BP). Waist and hip circumferences were measured to the nearest 0.1 cm at the narrowest point between the lowest rib and the uppermost lateral border of the right iliac crest. The hips were
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ATGL in T2DM With Hypertension
measured at their widest point. Seated BP was taken by a trained nurse after the subjects had rested for 10 minutes. Plasma Biochemical Parameters and ATGL Blood samples were drawn after an overnight fast, and plasma samples were kept at ⫺80°C until assayed. Plasma insulin was measured in deproteinized serum by radioimmunoassay using human insulin as standard (Linco, St. Charles, MO). Free FA (FFA) was measured in plasma with a kit (Randox Laboratories, Antrim, United Kingdom). Plasma glucose was assayed using the glucose oxidase method. Glycosylated hemoglobin (HbA1c) was measured by isoelectric focusing. Plasma triglyceride (TG), cholesterol (TC), low-density lipoprotein C (LDL-C) and high-density lipoprotein C (HDL-C) concentrations were determined enzymatically. Plasma ATGL levels were evaluated using a commercially available enzyme-linked immunosorbent assay (Phoenix Pharmaceuticals, Belmont, CA). The intra-assay coefficients of ATGL were less than 5%, and the interassay coefficients were less than 12%. The linear ranges of the assays were 1.5 to 300 ng/mL for ATGL. The homeostasis model assessment of IS (HOMAIR) and the homeostasis model assessment of -cell insulin secretion (HOMAIS) were calculated from fasting insulin and glucose levels with the following equations: HOMAIR ⫽ insulin [U/mL] ⫻ glucose [mmol/L]/22.5 and HOMAIS ⫽ [20 ⫻ Insulin (U/mL)]/[fasting blood glucose (FBG, mmol/ L) ⫺ 3.5].9 Statistical Analyses The data are shown as the mean ⫾ standard deviation. All statistical analyses were performed using SPSS 8.0 software (SPSS, Chicago, IL). Baseline characteristics of case and control subjects were compared by 1-way, Wilcoxon rank sum test or 2 test. The general linear modeling function analysis was used to control for potential confounders. As the distributions of plasma insulin and HOMAIR values were skewed, logarithmically transformed values were used for statistical analysis. As our primary approach, we included plasma ATGL levels as continuous independent variables in the multivariable models. Simple and multiple regression analyses were used to examine the association between plasma ATGL levels and the values of other biomarkers.
RESULTS The clinical characteristics of our subjects are shown in Table 1. After glucose overload, the T2DMH group had higher weight, systolic BP, diastolic BP (DBP), body mass index, waist-to-hip ratio, LDL-C, FBG and 2 hours plasma insulin (2 hours Ins) than that of the T2DM and control groups, whereas HOMAIS was lower than the NGT group. The T2DM group had higher TG, free FA, FBG, plasma glucose after glucose load, HbA1C, HOMAIR and lower HOMAIS than the controls (Table 1). Plasma ATGL levels were significantly increased in T2DMH compared with T2DM (78.3 ⫾ 23.4 versus 65.1 ⫾ 22.7 g/L, P ⬍ 0.01, Figure 1). We found that plasma ATGL levels in T2DM group had an elevating tendency compared with NGT groups (78.3 ⫾ 23.4 versus 70.9 ⫾ 22.5 g/L) but no significant difference. The data also revealed that the difference in age and gender was of little statistical significance. Bivariate correlation analyses were performed to assess the relationship of plasma ATGL concentrations with body composition and metabolic parameters. Plasma ATGL levels correlated positively with TC (r ⫽ 0.17, P ⬍ 0.05) and HDL-C (r ⫽ 0.16, P ⬍ 0.05, Table 2). Multiple regression analysis showed that DBP, TC and HOMAIR were independently re© 2011 Lippincott Williams & Wilkins
TABLE 1. Clinical characteristics of study subjects Factor
NGT
T2DM
T2DMH
N 60 81 33 Age (yr) 50.7 ⫾ 12.3 52.7 ⫾ 11.5 60.4 ⫾ 8.1a,b SBP (mm Hg) 122.9 ⫾ 12.8 119.9 ⫾ 14.8 134.1 ⫾ 18.5a,b DBP (mm Hg) 76.7 ⫾ 7.3 76.8 ⫾ 8.5 85.8 ⫾ 9.9a,b 2 BMI (kg/m ) 23.3 ⫾ 3.4 24.3 ⫾ 3.0 26.2 ⫾ 3.7a,b WHR 0.85 ⫾ 0.08 0.89 ⫾ 0.94 0.95 ⫾ 0.17a,b FAT (%) 29.5 ⫾ 9.2 32.2 ⫾ 10.7 34.7 ⫾ 9.3c a FBG (mmol/L) 5.4 ⫾ 0.4 10.5 ⫾ 4.5 9.3 ⫾ 4.3a a 2hPBG (mmol/L) 6.1 ⫾ 1.1 19.2 ⫾ 7.8 17.4 ⫾ 7.2a FINS (mU/L) 8.9 ⫾ 4.5 10.1 ⫾ 7.3 11.9 ⫾ 6.3c 2hPINS (mU/L) 36.2 ⫾ 22.5 50.0 ⫾ 40.1c 69.8 ⫾ 68.4c HOMA–IR 2.2 ⫾ 1.1 4.5 ⫾ 3.4a 4.9 ⫾ 3.5a a HbA1c (%) 5.6 ⫾ 0.4 8.9 ⫾ 2.9 7.7 ⫾ 2.5a TG (mmol/L) 1.16 ⫾ 0.50 1.80 ⫾ 1.07a 2.24 ⫾ 1.33a TC (mmol/L) 4.40 ⫾ 0.86 4.78 ⫾ 1.11 5.27 ⫾ 1.92a HDL–C (mmol/L) 1.31 ⫾ 0.32 1.24 ⫾ 0.31 1.60 ⫾ 0.21 LDL–C (mmol/L) 2.56 ⫾ 0.67 2.71 ⫾ 0.95 3.18 ⫾ 1.80c,d FFA (mol/L) 0.61 ⫾ 0.27 0.73 ⫾ 0.31c 0.84 ⫾ 0.37a Data are mean ⫾ SD or frequency (percentage). a P ⬍ 0.01 compared with NGT. b P ⬍ 0.01 compared with T2DM. c P ⬍ 0.05 compared with NGT. d P ⬍ 0.05 compared with T2DM. NGT, normal glucose tolerance; T2DM, type 2 diabetes mellitus; T2DMH, T2DM with hypertension; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WHR, waist hip ratio; FAT %, visceral fat %; FBG, fasting blood glucose; 2hPBG, 2 h plasma glucose after glucose load; FINS, fasting plasma insulin; 2hPINS, 2 h plasma insulin after glucose load; HOMA-IR, HOMA-insulin resistance index; FFA, free fatty acids; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin A1c.
lated factors with plasma ATGL levels (Y ⫽ ⫺13.662 ⫹ 0.343Xwaist ⫹ 0.268XDBP ⫹ 0.053X2hPin⫹ 0.966XHOMA-IR, Table 2). Increasing levels of ATGL showed a significant linear trend and were independently associated with T2DMH, especially when concentrations were analyzed both by tertile and by a continuous variable (Table 3).
FIGURE 1. The change of ATGL levels in subjects studied.
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TABLE 2. Linear and multiple regression analysis of variables associated with plasma ATGL in subject studied Simple Variable Age SBP DBP BMI WHR FAT (%) TG TC HDL–C LDL–C FFA FBG 2hPBG FINS 2hPINS HbA1c HOMA–IR
Multiple
Estimate
P
Estimate
P
0.195 0.119 0.265 0.341 0.239 0.298 0.253 0.066 ⫺0.024 0.048 0.163 0.166 0.209 0.183 0.205 0.188 0.214
0.011 0.122 0.001 0.001 0.002 0.001 0.001 0.391 0.755 0.537 0.035 0.030 0.006 0.017 0.011 0.014 0.005
— — 0.268 — — — — — — — — — — — 0.053 0.966 —
— — — — — — — — — — — — — — 0.042 0.015 —
In multiple linear stepwise regression analysis, values included for analysis were age, gender, BMI, WHR, SBP, DBP, FBG, 2hPBG, HbA1c, INS, HOMA-IR, FFA, TC, HDL-C, LDL-C,TG and FAT %. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WHR, waist hip ratio; FAT %, visceral fat %; FBG, fasting blood glucose; 2hPBG, 2 h plasma glucose after glucose load; FINS, fasting plasma insulin; HOMA-IR, HOMA-insulin resistance index; FFA, free fatty acids; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin A1c.
DISCUSSION IR plays a primary role in the development of T2DM10 and is a characteristic feature of other health disorders including obesity, dyslipidemias, hypertension and cardiovascular disease.11 The molecular basis of IR has not been fully elucidated, but secreted proteins are central regulators of metabolism and play key roles in food intake, insulin sensitivity and
energy metabolism. ATGL was recently discovered as a novel lipase in adipose tissue contributing to adipocyte lipolysis.3 Previous experiments have demonstrated an important role of ATGL in TG lipolysis and energy metabolism in cultured adipocytes and in weight maintenance of mice and other lower-order organisms.1,2,12 However, its importance in the pathogenesis of obesity and IR has not been evaluated. It was demonstrated that ATGL was essential for efficient TG metabolism, which altered fatty acid storage and oxidation. Rather, excessive TG degradation may promote IR when fatty acid oxidation cannot match intracellular production, resulting in the accumulation of bioactive FA metabolites.13 IR plays a primary role in the development of T2DM.This study is the first to report plasma ATGL levels in patients with T2DM and hypertension and their relationship with body composition and metabolic parameters. The results of the current study showed that plasma ATGL concentrations were significantly increased in T2DMH compared with T2DM (78.3 ⫾ 23.5 versus 65.1 ⫾ 22.8 g/L, P ⬍ 0.01). Therefore, it seems that BP exerts an important effect on elevating plasma ATGL levels. Furthermore, we found that plasma ATGL levels correlated positively with TC, HDL-C and DBP. Multiple regression analysis showed that TC, DBP and HOMA-IR were independently related factors with plasma ATGL levels. There was a decreasing trend of plasma ATGL levels in T2DM compared with the NGT group (65.1 ⫾ 22.8 versus 70.9 ⫾ 22.6 g/L) but not statistically significant. A plausible interpretation is that this reduced lipolytic response served as an appropriate downregulation of lipolysis per unit of fat mass to prevent excessive FA outflow from the expanded fat mass and to prevent worsening of the IR state.6 Collectively, these data support that insulin, blood glucose and lipids may influence circulating ATGL levels by regulating its expression and secretion. BP could be another parameter affecting ATGL concentrations. ATGL may play a role in the pathogenesis of IR and T2DM, and this role may be at least partially mediated via PPAR-␥ signals.4 Results of genetic studies indicate the involvement of PPAR-␥ in glucose homeostasis. Consistent with this, several dominant-negative mutations in PPAR-␥ in humans have been shown to cause partial lipodystrophy, marked IR, diabetes and hypertension.14 Also, it has been demonstrated that induction of ATGL in adipogenesis was in parallel with increasing PPAR-␥ and its target gene expression, suggesting a potential role of
TABLE 3. General liner and logistic analysis of the impact of ATGL level on T2DM and T2DMH Tertiles of ATGL Factor All subjects No. GT/T2DM/T2DMH (IGR/T2DM/NGT) Cutoff ATGL (g/L) T2DM Univariate Multivariatea T2DMH Univariate Multivariatea
Q1 (95% CI)
Q2 (95% CI)
Q3 (95% CI)
P
18/34/6 ⬍56.8
19/28/11 56.8–78.3
23/19/16 ⬎78.3
0.045
1.00 1.00
0.780 (0.345–1.764) 0.977 (0.362–2.639)
0.437 (0.190–1.007)) 0.406 (0.138-1.193)
0.586 0.467
1.00 1.00
1.737 (0.531–5.683) 1.764 (0.344–9.033)
2.087 (0.679–6.414) 1.075 (0.214-5.406)
0.061 0.648
Values shown are cutoffs of plasma ATGL levels in all subjects and odds ratios with 95% confidence intervals. a Adjusted for age, gender, body mass index, waist to hip ratio, visceral fat %, systolic blood pressure, diastolic blood pressure, total cholesterol, triglyceride, high-density lipoprotein- and low-density lipoprotein-cholesterol, and free fatty acids. ATGL, adipose triglyceride lipase; NGT, normal glucose tolerance; T2DM, type 2 diabetes mellitus; T2DMH, T2DM with hypertension.
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ATGL in T2DM With Hypertension
PPAR-␥ for regulating ATGL expression in adipogenesis. Several recent studies7,15 have demonstrated increased ATGL mRNA expression in adipose tissue of rodents treated with rosiglitazone, which served as PPAR-␥ agonist and insulin sensitizer. Furthermore, PPAR-␥ activation increases ATGL mRNA expression in fully differentiated 3T3-L1 adipocytes and adipose tissues. Also, the interactions between ATGL and PPAR-␥ activation were further confirmed by a significant reduction of ATGL mRNA and protein levels after RNAimediated PPAR-␥ knockdown.16 Therefore, the ATGL gene may be a direct transcriptional target of the key adipocyte transcription factor PPAR-␥. Taken together, the regulation of ATGL by PPAR-␥ may constitute a potential therapeutic target for the treatment of IR and T2DM. In summary, we have for the first time demonstrated that plasma ATGL levels are significantly increased in T2DMH compared with a T2DM group. However, more work is needed to clearly define the functional consequences of hypertensioninduced plasma ATGL level increasing.
lipase: function, regulation by insulin, and comparison with adiponutrin. Diabetes 2006;55:148 –57. 5. Kim JY, Tillison K, Lee J, et al. The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-alpha in 3T3–L1 adipocytes and is a target for transactivation by PPARgamma. Am J Physiol Endocrinol Metab 2006;291:E115–27. 6. Jocken JW, Langin D, Smit E, et al. Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state. J Clin Endocrinol Metab 2007;92:2292–9. 7. Festuccia WT, Laplante M, Berthiaume M, et al. PPARgamma agonism increases rat adipose tissue lipolysis, expression of glyceride lipases, and the response of lipolysis to hormonal control. Diabetologia 2006;49:2427–36. 8. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. 1. Diagnosis and classification of diabetes mellitus, provisional report of a WHO consultation. Diabet Med 1998;15:539 –53. 9. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–9.
ACKNOWLEDGMENTS The authors thank Amelia Griggs, University of Mississippi Medical Center, for technical editing of the manuscript.
10. DeFronzo RA. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 1988;37:667– 87.
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