The adipose triglyceride lipase, adiponectin and visfatin are downregulated by tumor necrosis factor-α (TNF-α) in vivo

Cytokine 45 (2009) 12–19

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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

The adipose triglyceride lipase, adiponectin and visfatin are downregulated by tumor necrosis factor-a (TNF-a) in vivo Ling Li a,*,1, Gangyi Yang b,1, Shaochuan Shi a, Mengliu Yang b, Hua Liu c, Guenther Boden d a

The Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education and Department of Clinical Biochemistry, Chongqing Medical University, 400016 Chongqing, China Department of Endocrinology, The Second Affiliated Hospital, Chongqing Medical University, 400010 Chongqing, China c Department of Pediatrics, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi, MS 39216-4505, USA d The Division of Endocrinology/Diabetes/Metabolism and The Clinical Research Center, Temple University School of Medicine, Philadelphia, PA, USA b

a r t i c l e

i n f o

Article history: Received 20 May 2008 Received in revised form 3 August 2008 Accepted 10 October 2008

Keywords: Inflammatory cytokine Insulin resistance The adipose triglyceride lipase(ATGL) Adiponectin Visfatin

a b s t r a c t Inflammatory cytokines have been linked to obesity-related insulin resistance. To investigate the effect of TNF-a, an inflammatory cytokine, on insulin action, C57BL/6J mice were treated with TNF-a for 7 days after which we examined the in vivo effects of TNF-a on glucose tolerance and insulin sensitivity with IV glucose tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, we analyzed the in vivo effect of TNF-a on several metabolism-related genes and adipocytokines implicated in the development of insulin resistance. TNF-a treatment resulted in markedly increased fasting blood glucose, insulin and free fatty acids (FFA) levels and reduced glucose tolerance. During the clamps, the rates insulinstimulated whole body (GRd) and skeletal muscle glucose uptake (MGU) and insulin’s ability to suppress hepatic glucose production (HGP) were decreased in TNF-a treated animals, indicating insulin resistance. In addition, both PPARc and ATGL mRNA expression in adipose tissues as well as ATGL protein levels in plasma were downregulated. Moreover, adipose mRNA expression and plasma protein levels of adiponectin and visfatin were significantly down-regulated. We conclude that the alterations of PPARc, ATGL, adiponectin and visfatin may contribute to the development of insulin resistance mediated by TNF-a. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Insulin resistance (IR) is a common feature of several disorders such as obesity, dyslipidemias, type 2 diabetes (T2DM), and hypertension, all of which are risk factors for cardiovascular diseases [1,2]. The precise cause of insulin resistance is yet to be determined in any of these disorders but many studies have suggested that inflammatory cytokines may play a critical role [3–5]. For example, TNF-a, a proinflammatory cytokine, is highly expressed in adipose tissues of obese animals and obese humans with T2DM [6,7], and obese mice lacking either TNF-a or TNF-a receptors are protected from developing insulin resistance [3,8]. At the cellular level, it has been shown that 3–5 days exposure of 3T3-L1 or 3T3-F442A adipocytes to TNF-a reduces insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation in response to a maximal dose of insulin [9,10]. Furthermore, TNF-a may induce insulin resistance by inhibiting GLUT4 gene expression in brown adipocytes [11] and by elevating plasma free fatty acids via stimulation of adipose tis-

* Corresponding author. Address: Department of Clinical Biochemistry and the Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education, Chongqing Medical University, Chongqing, China. Fax: +86 23 68 48 61 15. E-mail address: [email protected] (L. Li). 1 These authors contributed equally to this project. 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.10.006

sue lipolysis [12]. Nevertheless, the molecular mechanism of TNF-

a induced insulin resistance is still not completely understood. Although there are several studies showing effects of TNF-a on adipokines including adiponectin [13,14], we are not aware of studies examining the effects of TNF-a on PPARc, adipose triglyceride lipase (ATGL) and visfatin in vivo. In the present study, we, therefore, have examined the hypothesis that TNF-a may contribute to the development of IR by modifying PPARc and ATGL activities. 2. Materials and methods 2.1. Animals pretreatment Twenty three male C57BL/6J mice weighing between 26 and 32 g were purchased from the Experimental Animal Center of Chongqing University of Medical Sciences (Chongqing, China). The mice were housed in individual cages and subjected to an environmentally controlled room with a 12-h light/dark cycle, where they had free access to standard rat chow and water for 7 d. Mice were randomly assigned to one of four groups. The first group (H group, n = 21) were injected intraperitoneally with recombinant murine TNF-a (PeproTechAsia, Israel), 6 lg/kg/d in 100 ll of sterile saline. The second group (M group, n = 5), 3 lg/kg/d. The third

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L. Li et al. / Cytokine 45 (2009) 12–19

group (L group, n = 5), 1 lg/kg/d. And the fourth group (NC group, n = 21), only 100 ll sterile saline. Four days after treatment, mice were anesthetized with an intraperitoneal injection of Ketamine (100 mg/kg; Nembutal Abbott Laboratories, Abbott Park, IL). A catheter was inserted into the right internal jugular vein and extended to the level of the right atrium. Another catheter was advanced through the left carotid artery until its tip reached the aortic arch. The free ends of both catheters were attached to long segments of steel tubing and tunneled subcutaneously around the side of the neck to the back of the neck where they were exteriorized through a skin incision and then securely anchored to the skin by a standard wounded clip. At the end of the procedure, catheters were flushed with 300 ll of isotonic saline containing heparin (20 U/ml) and Ampicillin (5 mg/ml), and then filled with a viscous solution of heparin (300 U/ml) and 80% polyvinyl pyrolidone (PVP-10, Fisher Scientific, Pittsburgh, PA) to prevent refluxing of blood into the catheter lumen. All procedures were approved by the Chongqing Medical University Animal Care and Use Committee. 2.2. Intravenous glucose tolerance tests After seven days of TNF-a treatment, intravenous glucose tolerance tests (IVGTT) were performed with 20 mice (H group, n = 5; M group, n = 5; L group, n = 5; Controls, n = 5). The overnight-fasted mice were given i.v. glucose (1 g/kg body weight) and venous blood was collected before (time 0) and at indicated times after injection for measurement of glucose (Glucometer Elite; Bayer). 2.3. Hhyperinsulinemic–euglycemic clamp study After 7 days of TNF-a treatment, euglycemic–hyperinsulinemic clamps were performed with 20 mice as described [15]. Briefly, after an overnight fast, HPLC-purified [3-3H]glucose (Amersham, Los Angeles, CA) was infused through the jugular vein catheter starting at 0 min with a bolus (5 lCi) followed by a continuous infusion (0.05 lCi/min) in 20 mice (H group, n = 10; Controls, n = 10) for 90 min (basal period) to estimate the rate of basal glucose turnover. Following the basal period, a 2-h hyperinsulinemic–euglycemic clamp was conducted with a primed, continuous infusion of human insulin (5 mU kg1 min1) (Humulin; Eli Lilly and Co., Indianapolis, IN, USA) to raise plasma insulin levels, while plasma glucose was maintained at basal concentrations with variable rates of 20% glucose infusion. Blood samples (50 ll) were taken at 80, 90, 100, 120 min after the start of the clamps for the determination of plasma [3-3H]glucose concentrations. Additional blood samples (50 ll) were collected before the start and at the

NC group (n=21) saline

L group (n=5) TNF-α(1μg/kg)

end of clamps for measurement of plasma glucose, insulin, triglyceride (TG), total cholesterol (TC) and free fatty acids (FFA). To assess insulin-mediated glucose uptake in individual tissues, a 2-h hyperinsulinemic–euglycemic clamp was performed in another 12 mice (H group, n = 6; Controls, n = 6), and 2-deoxy-D[3H]glucose (2-DG, Amersham, Los Angeles, CA) was administered as a bolus (2 lCi) 45 min before the end of clamp experiments. Blood samples (50 ll) were taken at 5, 15, 30 and 45 min after the injection to determine the tracer disappearance curve. In this study, each blood sample was replaced by the same volume of fresh whole blood from a donor mice. At the end of the clamps, mice were anesthetized with sodium pentobarbital injection. Within 5 min, muscles from both hind limbs, epididymal white adipose tissue, and liver were taken. Each tissue, once exposed, was dissected out within 30 s, frozen immediately using liquid N2-cooled aluminum blocks, and stored at 70 °C for later analysis (Fig. 1). 2.4. Metabolite measurements For the determination of plasma [3-3H]glucose and 2-DG concentrations, plasma was deproteinized with ZnSO4 and Ba(OH)2, dried to remove 3H2O, resuspended in water, and counted in scintillation fluid (Ultima Gold; Packard Instrument Co., Meriden, Connecticut, USA). For the determination of tissue 2-DG-6-phosphate (2-DG-6-P) content, tissue samples were homogenized, and the supernatants were subjected to an ion-exchange column to separate 2-DG-6-P from 2-DG, as described previously [16]. Plasma FFA was determined spectrophotometrically using an acyl-CoA oxidase-based colorimetric kit (Wako Pure Chemical Industries, Osaka, Japan). Plasma Insulin concentrations were measured by radioimmunoassay using kits from Linco Research (St. Charles, MO). Both plasma TG and TC concentrations were measured using enzymatic colorimetric methods (Sigma, St. Louis, MO). Adiponectin was evaluated using a commercially available ELISA (Phoenix Pharmaceuticals, Belmont, CA). 2.5. RT-PCR analysis of gene expression For analysis of gene expression, total RNA was isolated from adipose, liver or muscle tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was quantified by A260 and its integrity verified by agarose gel electrophoresis using ethidium bromide for visualization. The PCR mixture contained in a final volume of 2 lL of the first strand cDNA, 10 pmol of the specific primers, 500 lM dNTP, 2 lL 10 buffer, 1.25 U Taq DNA polymerase, 3.5 mM MgCl2, and 0.5 ll 10  SYBR Green I. After an initial denaturation step (120 s, 95 °C), reactions were performed in a 40-ll

M group (n=5) TNF-α(3μg/kg)

H group (n=21) TNF-α(6μg/kg)

IVGTT(n=5 for each group)

Euglycemic–hyperinsulinemic clamp with [3-3H]glucose (NC and H groups, both n=10)

Euglycemic–hyperinsulinemic clamp with 2-deoxy- D[3H]glucose (NC and H groups, both n=6)

Fig. 1. Experimental protocol.

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L. Li et al. / Cytokine 45 (2009) 12–19

volume, including diluted cDNA sample (6 ll) and primers (4 ll). Samples were incubated for an initial denaturation at 94 °C for 3 min 1 PCR cycle. In following cycles, each cycle was consisted of 94 °C 30 s for degeneration, 55 °C 30 s for annealing, 72 °C 40 s for extension, and different cycles for each gene. Eventually, one PCR cycling was performed at 72 °C 5 min for intense extension. The intensities of PCR bands were quantified with a Bio Imaging System Densitometer (Bio-Rad). The sequences, product lengths, and annealing temperatures of the primers are shown in Table 1. 2.6. Western blot analysis Adipose tissues were homogenized in 20 mM MOPS, 2 mM EGTA, 30 mM sodium fluoride, 40 mM b-glycerophosphate. Protein concentration was measured by use of a BCA protein quantification kit (Pierce, Rockford, Illinois, USA). For the determination of plasma visfatin and adipose ATGL protein, One microliter of plasma and tissue extracts (60 lg) were separated by SDS–polyacrylamide gel electrophoresis (10% resolving gel) and transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 1 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. PVDF membranes were then blocked in TBS containing 0.1% Tween-20 and 5% BSA for 2 h. After three washes with TBS– 0.1% Tween, the PVDF membranes were incubated with primary goat-anti-rat antibody for ATGL (Ann Arbor Inc., MI, USA) [1:200 dilution] and primary goat-anti-rat antibody for visfatin (Santa Cruz Biotechnology, Inc. CA, USA) [1:5000 dilution] overnight at 4 °C. The membranes were washed thoroughly for 60 min with TBS-0.1% Tween before incubation with the goat anti-rabbit HRPconjugated secondary antibodies (Santa Cruz Biotechnology, Inc. CA, USA) [1:5000] for 1 h at room temperature. Quantification of antigen–antibody complexes was performed using Quantity OneÒ 1-D analysis software (Bio-Rad). Optical density units are expressed as adjusted volume [Adj. Vol. OD, sum of pixels inside the volume boundary  area of a single pixel (in mm2) minus the background volume]. Differences in loading were adjusted to b-actin protein levels.

Gene

Forward and reverse primers

bp

Annealing temperature (°C)

PPARc

50 -GATGACCACTCCCATTCCTTTG-30 50 -GATGCTTTATCCCCACAGACTC-30 50 -GGCTCTGGCCGCAATGTA-30 50 -TGACCGAGG AGCG TGA GT-30 50 -CCTGCGTGTCCCTGGTCCTA- 30 50 -CTTTGGGTTACTGGGTTTG G-30 50 - GACCTGATGACCACCCTTTC-30 50 -CAGATACTGGCAGATGCTACC-30 50 -CG CCTTACGGAGTCTATGC-30 50 -GCTGTCTGATGGCTCTGAGTT-30 50 -GAACTCAGGGCCTCTGTCTG-30 50 -GAAACCATGCGTGTATCCCT-30 50 -GAGCGACTCTTCAATACTTC-30 50 -CTCTGCGTTTATGCCTATC-30 50 -ATTCCCGCCACAGTATCT-30 50 -TCCCGATTGAAGTAAAGG-30 50 -CTCTTAATCCTGCCCAGTCAT-30 50 -GAGGCTCACCTTCACACATCTTT-30 50 -CCACTGCCGCATCCTCTTCCTC-30 50 -TCCTGCTTGCTGATCCACATCT-30 50 -GCTGTCCCTGTATGCCTCT-30 50 -GATGTCACGCACGATTTCC-30

281

55

263

53

222

53

169

55

161

53

188

53

492

55

337

55

507

55

400

55–53

220

55–53

HMGCR ATGL HSL LDLr CPT-1 Visfatin Adiponectin b-Actin1 b-Actin2

Glucose rates of appearance (GRa) were determined with 3-[3H] glucose as described [17]. GRa and the glucose rate of disappearance (GRd) were calculated using the non-steady-state equation of Steele et al. [18]. The distribution volume for glucose was assumed to be 150 g/kg. Hepatic glucose production (HGP) was calculated as the difference between the isotopically determined GRa and the rate of glucose infused (GIR) to maintain euglycemia. Endogenous glucose production (EGP) = HGP = GRa-GIR. Glucose uptake in individual tissues was calculated from plasma 2-DG profile, which was fitted with a double exponential curve using MLAB (Civilized Software, Bethesda, MD, USA) and tissues 2-DG-6-P content [19]. 2.8. Statistical analysis Data presented as means ± SE. A repeated measures analysis of variance (repeated-measures ANOVA) was used to assess the results measured at consecutive multiple time points. A two-way design was used to incorporate additional effects of different experimental groups followed by a post hoc (PLSD) test to compare two individual groups. Within-group comparisons were made using the paired Student’s t test. Differences were statistically significant at P < 0.05. All analyses were performed using SPSS (SPSS graduate pack; SPSS, Chicago, IL).

3. Results 3.1. Basal metabolic parameters There were no significant differences in body weight among the four groups. Interestingly, plasma FFA, insulin and fasting blood glucose levels were significantly higher in the H group than in the NC group (P < 0.05 and P < 0.01). Plasma triglyceride and total cholesterol were slightly increased in H group. However, this increase did not reach statistical significance (Table 2). 3.2. Effect of TNF-a on IV glucose tolerance testing

Table 1 Characteristics of the specific used for RT-PCR analysis.

SREBP-2

2.7. Calculations

PPARc, Peroxisome proliferator-activated receptor-c; SREBP-2, Sterol regulatory element binding proteins 2; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A reductase; ATGL, Adipose triglyceride lipase; HSL, Hormone-sensitive lipase; LDLr, LDL receptor; CPT-1, Carnitine palmitoyltransferase-1.

The peak glucose levels achieved over the first 10 min of the IVGTT were significantly higher in the H group than in the L and NC groups (Fig. 2). Plasma glucose levels returned to baseline within 40–60 min in the L, M and NC groups while the return to baseline was delayed in the H group. Insulin responses to IV glucose were drastically reduced in the H group compared to L and NC groups with intermediate values in the M group (Fig. 2). 3.3. Effects of TNF-a on glucose and lipid metabolism during the hyperinsulinemic–euglycemic clamping We measured the effects of TNF-a on whole body insulin sensitivity in both NC and H groups after 7 days of TNF-a administration, using hyperinsulinemic–euglycemic clamps in combination with [3-3H]glucose. During the clamps, plasma glucose was clamped at approximately 5.8 mM in both groups. Plasma insulin concentrations were raised to approximately 7–10 times of basal values. Despite identical insulin infusion rates (5 mU kg1 min1) plasma insulin levels were higher in the H groups than in the controls (P < 0.01). Plasma FFA and TG were significantly suppressed during the clamp procedures in both groups, but remained higher in the H than in the NC group (P < 0.05 and P < 0.01, Table 2). The rate of glucose infusion needed to maintain euglycemia increased rapidly in the control and H groups and reached a steady state within 60 min. The GIR in the H group was markedly lower than

15

L. Li et al. / Cytokine 45 (2009) 12–19 Table 2 Plasma parameters and glucose turnover data in two groups during insulin clamping. NC group (n = 10)

H group (n = 10)

Index

Basal

Steady-state

Basal

Steady-state

FBG (mmol/L) Insulin (mU/L) TG (mmol/L) TC (mmol/L) FFA (mmol/L) GIR (mg kg1 min1) GRd (mg kg1 min1) HGP (mg kg1 min1)

5.58 ± 0.33 12.4 ± 1.12 0.57 ± 0.05 3.65 ± 0.30 1.54 ± 0.11 — 13.8 ± 0.7 13.8 ± 0.7

5.75 ± 0.78 84.7 ± 5.5* 0.13 ± 0.02* 2.13 ± 0.18* 0.43 ± 0.07* 54.2 ± 2.2 53.9 ± 2.0* 0.3 ± 0.8*

7.86 ± 0.40*** 32.4 ± 1.9** 0.62 ± 0.04 3.85 ± 0.32 1.78 ± 0.05** — 21.6 ± 0.9*** 21.6 ± 0.9***

5.83 ± 0.61 141.7 ± 17.7*,*** 0.21 ± 0.07*,** 2.58 ± 0.16* 0.82 ± 0.03*,*** 39.1 ± 2.3*** 47.9 ± 0.8*,** 8.9 ± 1.7*,***

Data are means ± SE. vs. basal values. * P < 0.01; vs. controls. ** P < 0.05. *** P < 0.01.

30 ##** #*

##**

15

NC group L group M group H group

#*

50

Insulin (mU/L)

Glucose(mmol/L)

25

20

60

NC group L group M group H group

#*

40 ##** #* 30 ##** 20

10 ##*

10

5 0

20

40

60

80

100

0

120

20

40

60

80

100

120

Fig. 2. Intravenous glucose tolerance test (IVGTT). (A) Glucose curves in four groups. (B) Insulin curves in four groups. Values are presented as means ± SE, vs. NC group, # P < 0.05, ##P < 0.01; vs. L group, *P < 0.05, **P < 0.01.

in the controls (39.1 vs. 54.2 mg kg1 min1, P < 0.01, Fig. 3A). Isotopically determined insulin-stimulated whole body glucose uptake (GRd) was also lower in the H group compared with the controls (P < 0.05), indicating insulin resistance (Table 2 and Fig. 3B). Insulin-stimulated glucose uptake in skeletal muscle was decreased by 24% in the H group (28.4 ± 3.1 vs. 37.6 ± 4.5 mg kg1 min1 in controls; P < 0.01)(Fig. 3B). Thus, most of the decrease in insulin-stimulated whole body glucose uptake could be attributed to a decrease in insulin-stimulated glucose uptake in skeletal muscle. In addition, insulin’s ability to suppress

hepatic glucose production (HGP) during clamps was significantly impaired in the H group compared with the controls (P < 0.01) (Table 2 and Fig. 3B). 3.4. Effects of TNF-a on PPAR-c, SREBP-2, HMG-CoA, HSL, CPT-1 and LDLr mRNA expression The mRNA expression of PPARc in adipose tissue was significantly down-regulated in mice after TNF-a treatment compared with controls (0.83 ± 0.06 vs. 1.07 ± 0.07 arbitrary units, P < 0.05). 60

NC group H group

60 **

**

**

**

mg.kg-1.min-1

GIR(mg.kg-1.min-1)

55

50

45

40

NC group H group

*

50

**

** **

30

20 40

**

10 35 0 0

40

60

80

100

120

GIR

G Rd

HGP

MGz

Time (min) Fig. 3. Whole body and skeletal muscle metabolic parameters during hyperinsulinemic–euglycemic clamps in awake mice. (A) The time course of glucose infusion rate. (B) Glucose turnover in vivo. GIR, glucose infusion rates; GRd, the glucose rate of disappearance; HGP, hepatic glucose production; MGU, muscle glucose uptake. vs. controls, * P < 0.05, **P < 0.01.

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L. Li et al. / Cytokine 45 (2009) 12–19

However, treatment with TNF-a did not significantly alter the mRNA expression of SREBP-2, HMG-CoA, HSL, CPT-1 and LDLr (Fig. 4). 3.5. TNF-a down-regulates ATGL mRNA expression and plasma protein levels

HMG-CoA mRNA(arbitrary units)

Adiponectin mRNA content in adipose tissues and plasma protein levels were lower in the TNF-a treatment rats than in the controls (0.48 ± 0.04 vs. 0.95 ± 0.12 arbitrary units, P < 0.01 and 16.43 ± 0.35 vs. 21.94 ± 0.59 lg/ml, P < 0.01, Fig. 6). In rats treated with TNF-a, plasma visfatin was significantly lower than in the controls (0.90 ± 0.05 vs. 1.14 ± 0.13 arbitrary

1.2 SREBP-2 mRNA(arbitrary units)

1.2 P<0.05

1.0 .8 .6 .4 .2 0.0

3.6. TNF-a down-regulates adiponectin and visfatin mRNA expression and plasma protein levels

1.0

.8 .6 .4 .2

H

NC

1.6

P>0.05

.8

.6

.4

.2

P>0.05

1.4 1.2 1.0 .8 .6 .4 .2

0.0 NC

1.6

0.0

H

NC

P>0.05

1.2 1.0 .8 .6 .4 .2 0.0 NC

H

1.2

1.4

LDLr mRNA(arbitrary units)

CPT1 mRNA(arbitraryunits)

P>0.05

1.0

0.0

H

NC

HSL mRNA(arbitrary units)

PPAR γ mRNA(arbitrary units)

ATGL mRNA expression in adipose tissue was significantly lower in mice after TNF-a treatment (6 lg/kg/d) compared with controls (0.85 ± 0.09 vs. 1.37 ± 0.12 arbitrary units, P < 0.01). The differences at the mRNA level were also reflected at the protein level in TNF-a treated mice, who had significantly lower ATGL pro-

tein levels in plasma (0.53 ± 0.03 vs. 0.65 ± 0.05 arbitrary units in controls, P < 0.05)(Fig. 5).

H

P>0.05

1.0 .8 .6 .4 .2 0.0

NC

H

Fig. 4. Effects of TNF-a on gene expression in adipose and liver tissue. (A) PPARc mRNA expression in adipose tissue. (B) SREBP-2 mRNA expression in liver tissue. (C) HMGCoA mRNA expression in liver tissue. (D) HSL mRNA expression in liver tissue. (E) CPT-1 mRNA expression in liver tissue. (F) LDLr mRNA expression in liver tissue.

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L. Li et al. / Cytokine 45 (2009) 12–19

.8 ATGL protein (arbitrary units)

ATGLmRNA (arbitraryunits)

1.6 1.4 P <0.01

1.2 1.0 .8 .6 .4 .2 0.0

.6

.4

.2

0.0

H

NC

P<0.05

H

NC

Fig. 5. Effects of TNF-a on ATGL mRNA expression and protein levels in adipose tissue. (A) ATGL mRNA expression. (B) ATGL protein levels.

25

20 Adiponectin protein (mg/L)

Adiponectin mRNA(arbitrary units)

1.2

P <0.01

1.0 P<0.01

.8 .6 .4

15

10

5

.2

0 H

NC 0.0 H

NC

Fig. 6. Effects of TNF-a on adipose adiponectin mRNA expression and plasma protein levels. (A) Adiponectin mRNA expression in adipose tissue. (B) plasma adiponectin protein levels.

1.2

1.2

Visfatin protein (arbitraryunits)

Visfatin mRNA(arbitrary units)

1.4 P<0.05

1.0 .8 .6 .4

1.0 P< 0.01

.8

.6

.4

.2

.2

0.0

0.0 NC

H

NC

H

Fig. 7. Effects of TNF-a on adipose visfatin mRNA expression and plasma protein levels. (A) Visfatin mRNA expression in adipose tissue. (B) plasma visfatin protein levels.

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units, P < 0.05). To examine whether TNF-a suppressed visfatin protein level by down-regulating its mRNA level, we examined visfatin mRNA expression in adipose tissues and found it to be significantly down-regulated (0.74 ± 0.03 vs. 0.92 ± 0.06 arbitrary units, P < 0.01, Fig. 7). 4. Discussion In addition to its antitumor and proinflammatory actions, TNF-a also modulates adipocyte biology and affects systemic glucose and lipid metabolism. These metabolic functions include regulation of lipogenesis and lipolysis and blocking the action of insulin [3,8]. Although a substantial amount of data demonstrate a role of TNFa in inducing insulin resistance via inhibition of insulin signaling [7,20], the in vivo effects of this cytokine on other adipokines are less well established. In this study, we have investigated the effects of TNF-a treatment on insulin action and on expression and protein levels of several adipokines in C57BL/6J mice. Treatment for 7 days with TNF-a (6 lg/kg/d) in C57BL/6J mice increased fasting plasma FFA, insulin and blood glucose levels and decreased IV glucose tolerance. The fact that insulin responses to IV glucose were reduced in the H group despite higher glucose levels suggested a suppressive effects of TNF-a on beta cell function. We also assessed the impact of TNF-a administration on overall glucose metabolism and insulin action using the euglycemic–hyperinsulinemic clamp technique. There, despite identical insulin infusion rates, plasma insulin, FFA and TG levels were higher in the TNF-a treated animals than in the controls indicating decreased insulin clearance, perhaps in an effort to compensate for TNF-a induced IR. Insulin-stimulated whole-body glucose turnover and glucose uptake in skeletal muscle were decreased by approximately 35% and 24%, and insulin’s ability to suppress HGP was significantly impaired in mice following TNFa treatment. These data indicate that TNF-a treatment resulted in insulin resistance in all three major insulin target tissues: muscle, liver, and fat. These findings are similar to most [21,22], but not all [23] previously reported in vivo and in vitro studies. To investigate the molecular mechanism of TNF-a induced dyslipidosis, we examined in vivo effects of TNF-a on expression and plasma levels of several metabolism-related compounds. TNF-a treatment did not alter mRNA expression of PEPCK, and SREBP-2, HMG-CoA, CPT-1 and LDLr in adipose or liver tissue. PEPCK is the enzyme that catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, the first committed step in hepatic gluconeogenesis [24]. CPT-1 is integral to fatty acid metabolism and governs a ratelimiting step of fatty acid transport into the mitochondria for its oxidation [25]. SREBP-2 regulates HMG-CoA reductase, LDL receptor and other genes directly involved in cholesterol homeostasis [26,27]. Inasmuch as TNF-a treatment did not alter cholesterol concentrations in plasma and the expression of genes involved in cholesterol homeostasis (SREBP-2, HMG-CoA and LDLr) the data from the present study suggested that TNF-a had no effect on cholesterol metabolism. However, TNF-a treatment significantly downregulated the adipose tissue mRNA expression and plasma protein levels of ATGL confirming previously reported in vitro studies [28] which have shown that genetic inactivation of ATGL in mice increases adipose mass and leads to triacylglycerol deposition in multiple tissues [29]. Thus, it can be speculated that TNF-a could contribute to insulin resistance, through a down-regulation of ATGL resulting in fat accumulation in liver and skeletal muscle. We also found that adipose tissue PPARc mRNA expression was significantly down-regulated by TNF-a treatment. This finding is in agreement with in vitro findings by others [30]. The parallel suppression of PPARc and ATGL mRNA expression supports the possibility first proposed by Kershaw et al. [31] that ATGL might be a

PPARc target gene, and suggests that the downregulation of ATGL and PPARc transcripts might be one mechanism of TNF-a induced insulin resistance. To further explore the effects of TNF-a treatment, we measured adiponectin and visfatin mRNA expression and their plasma protein levels. Our results showed that both adiponectin and visfatin mRNA expression and plasma protein levels were down-regulated by TNF-a. Our adiponectin mRNA expression and protein levels data are similar to in vitro data reported by Hector et al. but our visfatin mRNA expression data are different [32]. Adiponectin and visfatin are two recently identified adipocytokines which were proposed to be links between obesity and insulin resistance. Adiponectin reduces endogenous glucose production by increasing hepatic insulin sensitivity [33], increases glucose uptake in adipocytes and myocytes [34], and enhances fatty acid oxidation in muscle [35]. Adiponectin has also been reported to modulate the endothelial inflammatory response and to exert a direct anti-atherogenic effects [36–38]. Visfatin is also an adipose-derived hormone proposed to exert insulin-mimicking effects and to play a role in attenuating insulin resistance [39]. In the present study, we have demonstrated for the first time an inhibitory effect of TNF-a in vivo on adiponectin and visfatin mRNA and protein expression. In conclusion, we have shown that short-term TNF-a treatment in vivo not only downregulated adipose tissue PPARc and ATGL mRNA expression, and ATGL plasma protein levels but also reduced adipose tissue adiponectin and visfatin mRNA expression and their plasma protein levels. These results reveal what we believe to be novel actions of TNF-a and suggest that these alterations may contribute to TNF-a mediated insulin resistance. Acknowledgments This work was supported by research Grants from the National Natural Science Foundation of China (30771037, 30871199), Chongqing Municipal Education Commission (JK 050304), Chongqing medical university (XBZD200704) and the National Institutes of Health (R01-DK 58895 to G.B.). References [1] Reaven GM, Laws A. Insulin resistance, compensatory hyperinsulinemia, and coronary heart disease. Diabetologia 1994;37:948–52. [2] Hollenbeck CB, Chen YD, Reaven GM. A comparison of the relative effects of obesity, and NIDDM on in vivo insulin-stimulated glucose utilization. Diabetes 1984;33:622–6. [3] Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS. Protection from obesity-induced insulin resistance in mice lacking TNF-a function. Nature 1997;389:610–4. [4] Cheung AT, Ree D, Kolls JK, Fuselier J, Coy DH, Bryer-Ash M. An in vivo model for elucidation of the mechanism of tumor necrosis factor-a (TNF-a) - induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-a. Endocrinology 1998;139:4928–35. [5] Togashi N, Ura N, Higashiura K, Murakami H, Shimamoto K. Effect of TNF-aconverting enzyme inhibitor on insulin resistance in furctose-fed rats. Hypertension 2002;39:578–80. [6] Hotamisligil GS. Mechanisms of TNF-alpha-induced insulin resistance. Exp Clin Endocrinol Diabetes 1999;107:119–25. [7] Xu H, Hirosumi J, Uysal KT, Guler AD, Hotamisligil GKS. Exclusive action of transmembrane TNF-a in adiposetissue leads to reduced adipose mass and local but not systemic insulin resistance. Endocrinology 2002;143:1502–11. [8] Uysal KT, Wiesbrock SM, Hotamisligil GS. Functional analysis of tumor necrosis factor (TNF) receptors in TNF-a-mediated insulin resistance in genetic obesity. Endocrinology 1998;139:4832–8. [9] Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 1994;91:4854–8. [10] Guo D, Donner DB. Tumor necrosis factor promotes phosphorylation and binding of insulin receptor substrate 1 to phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. J Biol Chem 1996;271:615–8. [11] Bruce CR, Dyck DJ. Cytokine regulation of skeletal muscle fatty acid metabolism: effect of interleukin-6 and tumor necrosis factor-a. Am J Physiol Endocrinol Metab 2004;287:E616–21.

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