Protein-tyrosine phosphatase 1B expression is induced by inflammation in vivo

Abstract

Protein-tyrosine phosphatase 1B (PTP1B) is a major negative regulator of insulin and leptin sensitivity. PTP1B overexpression in adipose tissue and skeletal muscle of humans and rodents may contribute to insulin resistance and obesity. The mechanisms mediating PTP1B overexpression in obese and diabetic states have been unclear. We find that adipose tissue inflammation and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) regulate PTP1B expression in vivo. High fat feeding of mice increased PTP1B expression 1.5- to 7-fold in adipose tissue, liver, skeletal muscle, and arcuate nucleus of hypothalamus. PTP1B overexpression in high fat-fed mice coincided with increased adipose tissue expression of the macrophage marker CD68 and TNFalpha, which is implicated in causing obesity-induced insulin resistance. TNFalpha increased PTP1B mRNA and protein levels by 2- to 5-fold in a dose- and time-dependent manner in adipocyte and hepatocyte cell lines. TNFalpha administration in mice increased PTP1B mRNA 1.4- to 4-fold in adipose tissue, liver, skeletal muscle, and hypothalamic arcuate nucleus and PTP1B protein 2-fold in liver. Actinomycin D treatment blocked, and high dose salicylate treatment inhibited by 80%, TNFalpha-induced PTP1B expression in adipocyte cell lines, suggesting TNFalpha may induce PTP1B transcription via nuclear factor kappaB (NFkappaB) activation. Chromatin immunoprecipitation from adipocyte cell lines and liver of mice demonstrated TNFalpha-induced recruitment of NFkappaB subunit p65 to the PTP1B promoter in vitro and in vivo. In mice with diet-induced obesity, TNFalpha deficiency also partly blocked PTP1B overexpression in adipose tissue. Our data suggest that PTP1B overexpression in multiple tissues in obesity is regulated by inflammation and that PTP1B may be a target of anti-inflammatory therapies.

Figures

FIGURE 1.

PTP1B protein overexpression in insulin-target tissues is not regulated by obesity, insulin resistance, or diabetes per se. a–c, PTP1B protein levels in liver, skeletal muscle, and perigonadal white adipose tissue (WAT) of 8- to 10-week old ob/ob mice (a), diabetic db/db mice (b), and Zucker fa/fa rats (c) and their respective lean controls were determined by immunoblotting. Results are means ± S.E. (n = 4–7 per group). *, p ≤ 0.05 compared with WT animals by t test. d, serum insulin levels in 11-week-old female FVB mice treated with the indicated concentration of dexamethasone daily for 4 days. Results are means ± S.E. (n = 3 per group). *, p ≤ 0.05 compared with untreated mice by one-way ANOVA. e, PTP1B protein levels in liver, skeletal muscle, and perigonadal WAT of dexamethasone-treated mice were determined by immunoblotting. Results are means ± S.E. (n = 3 per group). *, p ≤ 0.05 compared with control or untreated mice by one-way ANOVA. For all experiments, PTP1B protein amount was normalized to the amount of ERK1/2 or other control proteins. Proteins were quantified by densitometry of x-ray films.

FIGURE 2.

Overexpression of PTP1B protein in insulin-target tissues and the arcuate nucleus of hypothalamus of mice with DIO coincides with inflammation in adipose tissue. a, PTP1B protein levels in liver and perigonadal WAT of female FVB mice fed a low fat chow diet or a high fat diet from weaning until 15 weeks of age were determined by immunoblotting. PTP1B levels were normalized to ERK1/2 levels. Proteins were quantified by densitometry of x-ray films. Results are means ± S.E. (n = 8–9 per group). b, PTP1B protein levels in liver, skeletal muscle, perigonadal white adipose tissue (WAT), arcuate nucleus of hypothalamus, medial or lateral hypothalamus, of female FVB mice fed a low fat chow diet or a high fat diet from weaning until 20 weeks of age were determined by immunoblotting. Lateral hypothalamus, medial hypothalamus, and arcuate nucleus of the hypothalamus were dissected from a coronal brain slice. PTP1B levels were normalized to ERK1/2 levels for all tissues except WAT. For WAT, ERK1/2 levels were different between groups, and PTP1B levels were normalized to actin. PTP1B, ERK1/2, and actin were quantified by densitometry of x-ray films. Results are means ± S.E. (n = 7–8 per group). *, p ≤ 0.05 compared with chow-fed animals by t test. c, CD68 protein levels in adipose tissue of female FVB mice fed a low fat chow diet or a high fat diet from weaning until 20 weeks of age. Proteins in perigonadal adipose tissue (WAT) lysates were detected by immunoblotting with polyclonal antibodies specific for mouse CD68. Each lane represents one animal.

FIGURE 3.

Increased TNFα expression in adipose tissue accompanies PTP1B overexpression in insulin- and leptin-target tissues of mice with DIO. PTP1B, TNFα, and CD68 mRNA levels from perigonadal adipose tissue of female FVB mice fed a low fat chow diet or a high fat diet from weaning until 15 weeks (a) or 20 weeks (b) of age. PTP1B, TNFα, and CD68 mRNA amounts were normalized to HPRT mRNA. PTP1B, TNFα, CD68, and HPRT mRNAs were measured by quantitative real-time reverse transcription-PCR. Results are means ± S.E. (n = 7–9 per group). *, p ≤ 0.05 compared with chow-fed animals by t test.

FIGURE 4.

TNFα treatment increases PTP1B protein expression in multiple cultured cell types. PTP1B protein amount in lysates of three different cell types treated with TNFα was measured by immunoblotting. a and b, 14–21-day post-differentiation 3T3-L1 adipocytes were incubated with the indicated concentrations of TNFα for 16 h (a, top panel and b, left panel) or with 1 nm TNFα for the indicated times (a, bottom panel and b, right panel). Representative immunoblots for PTP1B (a) and means ± S.E. (n = 2–3 per condition) (b) are shown. c, PTP1B protein from H-4-II-E hepatoma cells incubated with the indicated concentrations of TNFα for 16 h. Results are means ± S.E. (n = 3 per condition). d, PTP1B protein from HeLa cells incubated with the indicated concentrations of TNFα for 15–16 h (left) or with 3 nm TNFα for the indicated times (right). Results are means ± S.E. (n = 3 per condition). For all experiments, PTP1B and ERK1/2 proteins were quantified by densitometry of x-ray films or direct chemiluminescence detection and PTP1B was normalized to ERK1/2. *, p ≤ 0.05 compared with cells without TNFα by one-way ANOVA.

FIGURE 5.

TNFα treatment increases PTP1B mRNA expression in cultured cells via transcriptional transactivation. a, PTP1B mRNA from 14–21-day post-differentiation 3T3-L1 adipocytes incubated with 0.2 nm TNFα for the indicated times is shown. Results are means ± S.E. (n = 2 per condition). b, PTP1B mRNA from HeLa cells incubated with the indicated concentrations of TNFα for 15 h (left) or with 3 nm TNFα for the indicated times (right) is shown. Results are means ± S.E. (n = 3 per condition). *, p ≤ 0.05 and &, p ≤ 0.1 compared with untreated cells by one-way ANOVA. c and d, PTP1B mRNA from 14–21-day post-differentiation 3T3-L1 adipocytes (c) or HeLa cells (d) treated with actinomycin D (aD)(2 μg/ml) or without inhibitor for 1 h before incubation with 1.2 nm TNFα or without cytokine for 4 h. Results are means ± S.E. (n = 6 in c and n = 12 in d, per condition). *, p ≤ 0.05 compared with the corresponding condition without TNFα; and #, p ≤ 0.05 compared with the corresponding condition without actinomycin D by two-way ANOVA. For all experiments, PTP1B mRNA and control 18S rRNA were measured by real-time quantitative reverse transcription-PCR and PTP1B mRNA was normalized to 18S rRNA.

FIGURE 6.

TNFα regulates PTP1B expression in part via transcription in 3T3-L1 adipocytes. a, PTP1B mRNA from 14-day post-differentiation 3T3-L1 adipocytes incubated with sodium salicylate (sal, 5 mm), PD98059 (PD, 10 μm), SB202190 (SB, 10 μm), SP600125 (SP, 5 μm), or without inhibitor for 1 h, followed by incubation with 1.2 nm TNFα or without cytokine for 4 his shown. Results are means ± S.E. (n = 6 per condition). *, p ≤ 0.05 compared with the corresponding condition without TNFα and #, p ≤ 0.05 compared with the corresponding condition without inhibitor by one-way ANOVA. b, data from a represented as the increment of increase in PTP1B expression with TNFα treatment in the presence of inhibitor divided by the increment in the absence of inhibitor. PTP1B mRNA and control 18S rRNA were measured by real-time quantitative PCR, and PTP1B mRNA was normalized to 18S rRNA.

FIGURE 7.

Acute TNFα treatment increases PTP1B expression in insulin- and leptin-target tissues of mice. a and b, FVB mice were injected intravenously with saline or TNFα (3.3 μg per mouse) and sacrificed 4 h later. PTP1B mRNA, suppressor of cytokine signaling 3 (SOCS-3) mRNA, and 18S rRNA from liver, gastrocnemius skeletal muscle, perigonadal adipose tissue (WAT), and arcuate nucleus of hypothalamus were measured by real-time quantitative reverse transcription-PCR. SOCS-3 (a) or PTP1B (b) mRNA was normalized to 18S rRNA. Results are means ± S.E. (n = 6 per condition). c, FVB mice were injected intravenously with saline or 3.3 μgof TNFα, followed by a second intravenous injection of the same 5 h later. Tissue was harvested from mice 9.5 h after the initial TNFα injection. PTP1B and ERK1/2 protein amount in liver was determined by immunoblotting. PTP1B and ERK1/2 proteins were quantified by densitometry of x-ray films or direct chemiluminescence detection and PTP1B amount was normalized to the amount control proteins ERK1/2. Results are means ± S.E. (n = 6 per condition). *, p ≤ 0.05 compared with untreated mice by t test.

FIGURE 8.

TNFα induces NFκB subunit p65 binding to the mouse PTP1B promoter in 3T3-L1 adipocytes in vitro and mouse liver in vivo. a, the sequence of the mouse PTP1B promoter from –1000 to –501 bp upstream of the start site of transcription is shown. The positions of the putative NFκB p65 binding site and primers used for ChIP analysis are indicated. ChIP was performed on chromatin from 3T3-L1 adipocytes (b) and liver of mice (c) treated with saline (basal) or TNFα for 4 h. Chromatin was immunoprecipitated (IP) with antibodies specific for the NFκB subunit p65 or control antibodies (ctl Ab). PCR products were amplified from input or immunoprecipitated samples with primers encompassing –933 to –626 of the PTP1B promoter or control primers specific for sequences in the first intron of PTP1B. Shown are duplicate PCR products, separated by agarose gel electrophoresis and visualized by ethidium bromide staining. d, quantitation of the results presented in c. Results are means ± S.E. of immunoprecipitated PCR products normalized to input PCR products for each sample (n = 4 per group). *, p ≤ 0.05 compared with saline-treated mice.

FIGURE 9.

TNFα deficiency blunts PTP1B overexpression in adipose tissue of mice with diet-induced obesity (DIO). TNFα+/+ (wild-type, WT) and TNFα–/– mice were fed either a low fat chow diet or a high fat diet from 6 weeks of age for 38 weeks for female mice or 26 weeks for male mice. Body weight (a), random-fed blood glucose (b), and serum insulin (c) were measured. Results are means ± S.E. (n = 6–8 per group). *, p ≤ 0.05 compared with WT chow-fed mice by one-way ANOVA. d and e, PTP1B protein was measured in perigonadal (P), perirenal (R), and subcutaneous (S) adipose tissue of chow-fed mice and mice with DIO. PTP1B was detected by immunoblotting and quantified by densitometry. Results are means ± S.E. of PTP1B levels in perigonadal, subcutaneous, or perirenal adipose tissue depots (n = 5–8 per group for each adipose depot). Because diet or TNFα deficiency altered levels of several internal control proteins, PTP1B levels were measured from equivalent amounts of protein, which were verified by Ponceau S staining of immunoblots. *, p ≤ 0.05 compared with WT chow-fed mice and #, p ≤ 0.05 compared with WT DIO mice. Symbols above bars indicate differences determined by one-way ANOVA. Symbols above lines indicate differences determined by two-way ANOVA, with mouse group and adipose depot being the two factors.