NFκB as a potent regulator of inflammation in human adipose tissue, influenced by depot, adiposity, T2DM status, and TNFα

NFjB as a Potent Regulator ofInflammation in Human Adipose Tissue,Influenced by Depot, Adiposity, T2DMStatus, and TNFaAlison L. Harte1, Gyanendra Tripathi1, Milan K. Piya1, Thomas M. Barber1,2, John C. Clapham3,Nasser Al-Daghri4, Dara Al-Disi1, Warunee Kumsaiyai1, Ponnusamy Saravanan1,2, Anne E. Fowler1,Joseph P. O.’Hare1,5, Sudhesh Kumar1 and Philip G. McTernan1

Objective: Central obesity and sub-clinical inflammation increase metabolic risk, this study examined the

intracellular inflammatory pathways in adipose tissue (AT) that contribute to this risk.

Design and Methods: This study therefore addressed the influence of NFjB and JNK activation in

human abdominal subcutaneous (AbdSc) and omental (Om) AT, the effect of adiposity, T2DM status and

the role of TNFa in vitro, using molecular biology techniques.

Results: Our data showed NFjB activity is increased in Om AT versus AbdSc AT (P<0.01), which was

reversed with respect to depot specific activation of JNK (P<0.01). However, T2DM status appeared to

preferentially activate NFjB (P<0.001) over JNK. Furthermore, in vitro studies showed recombinant

human (rh) TNFa treated AbdSc adipocytes increased NFjB activity over time (2-48 h, P<0.05) whilst

JNK activity reduced (2 h, 4 h, P<0.05); inhibitor studies supported a preferential role for NFjB as a

modulator of TNFa secretion.

Conclusions: These studies suggest distinct changes in NFjB and JNK activation, dependent upon AT

depot, adiposity and T2DM status, with in vitro use of rh TNFa leading to activation of NFjB.Consequently NFjB appears to play a central role in inflammatory mediated metabolic disease over JNK,

highlighting NFjB as a potential key target for therapeutic intervention.

Obesity (2013) 00, 0000-0000. doi:10.1002/oby.20336

IntroductionSeveral studies have identified increasing central adiposity as a criti-

cal site for metabolic dysfunction and inflammatory response and, as

such, adipose tissue (AT) is considered to integrate metabolic and

immune functionality (1). Our previous studies have demonstrated

that isolated human abdominal subcutaneous adipocytes (AbdSc)

possess many of the key proteins in the inflammatory pathway,

including nuclear factor kappa B (NFjB) (2,3); to date, most of our

current understanding as to the function of NFjB in AT is derived

through some key murine studies (4,5). Primarily, these studies high-

light that the NFjB activator, I-kappaB kinaseb (IKK-b), increasesadipokine production in AT and links inflammation with the onset

of diabetes; secondly, that inflammatory responses are markedly

attenuated in the IKK-b knock-out (KO) mouse (3) whilst further

studies implicate IKK-gamma (IKKc) as essential for IKK activity

and classical activation of the NFjB pathway (6).

Concurrently, the mitogen activated protein C-Jun N-terminal kinase

(JNK) is also implicated as a central mediator for insulin action and

inflammation in AT (7-9). JNK represents a family of serine/threo-

nine protein kinases, consisting of three distinct JNK genes JNK 1-3

which exist in mammals. JNK1 and JNK2 are expressed across

many tissues including mouse AT, liver, muscle, and macrophages,

whilst JNK3 is mainly expressed in the central nervous system

(CNS) (8,9). Genetic and biochemical studies have shown that,

within in vitro systems, JNK activation results in serine phosphoryl-

ation of the insulin receptor substrate-1 (IRS-1), thus impairing the

insulin signaling cascade (8,10). TNFa activates the JNK signaling

1 Division of Metabolic and Vascular Health, Warwick Medical School, University of Warwick, Coventry, UK. Correspondence: Alison L. Harte([email protected]); Philip G. McTernan ([email protected]) 2 Human Metabolism Research Unit, WISDEM, UHCW, Coventry, UK3 AstraZeneca R&D, CVGI Bioscience, Macclesfield, Cheshire, UK 4 Biomarkers Research Program, Biochemistry Department, College of Science, King SaudUniversity, Riyadh, Kingdom of Saudi Arabia 5 George Eliot Hospital, Nuneaton, Warwickshire, UK

The first two authors contributed equally to this work.

Disclosure: The authors declared no conflicts of interest.

Additional Supporting Information may be found in the online version of this article.

Received: 11 September 2012 Accepted: 11 December 2012 Published online 14 February 2013. doi:10.1002/oby.20336

www.obesityjournal.org Obesity | VOLUME 000 | NUMBER 000 | MONTH 2013 1

Original ArticleOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Obesity

pathway, as noted in both in vivo and in vitro studies, whilst TNFadeficient mice exhibit restored insulin signaling capacity (11,12).

Therefore, this suggests that JNK may mediate the regulation of

multiple intracellular events, including the expression of the TNFagene through AP-1 and the insulin signaling pathway (11,12). JNK

activity is also elevated in AT, liver and muscle tissues of obese

subjects (8), further implicating the JNK pathway as a link between

obesity, insulin action, and inflammation.

To date, the role of JNK1 in insulin resistance (IR) has been exclu-

sively defined through murine studies, whilst the role of JNK2 is

unclear, in part due to the regulatory crosstalk between the two iso-

forms (9). However, KO studies in mice indicate that JNK1 activity

is an important mediator of IRS-1 dysregulation, which appears

unrelated to JNK2 function (8). As such, JNK may influence the IR

state, as both JNK1 and TNFa can disrupt insulin signaling through

differential phosphorylation of the IRS (8,13).

Whilst a role for JNK and NFjB in mouse models has been

described, and its complexity and potential overlapping functionality

is ascertained, the functional role and significance in human AT has

not been addressed. Therefore, the aims of this study were to exam-

ine (i) the inflammatory intracellular signaling pathways involving

NFjB and JNK as potential mechanisms of adipocytokine activation

leading to inflammation in human AbdSc and omental (Om) AT (ii)

the relative JNK and NFjB activity in fat depots and the effect of

adiposity, as well as T2DM status (iii) finally, we addressed the

acute and chronic in vitro effects of TNFa on NFjB and JNK activ-

ity, in human AbdSc adipocytes.

Research Design and MethodsSubjectsHuman abdominal AT was collected from patients (age: 44.7 (mean

6 SD) 6 9.3 years; BMI: 27.9 (mean 6 SD) 6 7.3 kg/m2; (34

women, 7 men), fasted for 8 h) undergoing elective surgery with

informed consent, obtained in accordance with Local Research

Ethics Committee (LREC) guidelines and approval. AbdSc AT was

obtained from T2DM patients by needle biopsy (age: 59.168.2

years; BMI: 35.269.2 kg/m2, n¼8; 6 women and 2 men). Human

sternum muscle (Medical Solutions, Peterborough, UK) and white

blood cells were also collected following LREC approval. All tissue

samples were flash frozen and/or utilized for in vitro studies, as

detailed below. In total, 41 human, non-T2DM (ND) abdominal AT

samples were analyzed, which were sub-divided into: AbdSc (n¼23)

and Om (n¼18). Subjects providing fat samples were not on endo-

crine therapy or receiving any anti-hypertensive therapy.

Extraction of AT RNA for quantitative PCRRNA was extracted from AT using RNeasy Lipid Tissue Mini Kit

(Quiagen, Crawley, UK) and reverse transcribed (RT) using AMV

reverse transcriptase (Promega, Southampton, UK) according to

manufacturers’ instructions (14). Quantitative RT-PCR was carried

out as previously described (15) assessing JNK1, JNK2 and CD45

sequences (11,12,15).

Protein determination and Western blot analysisProtein concentrations were determined using the Bio-Rad Detergent

Compatible (DC) protein assay kit (Biorad, Hertfordshire, UK) and

quantified by Nanophotometer (Geneflow, Fradley, UK). Western

blot analysis was performed using a method previously described

(14). Protein expression in AT and/or AbdSc adipocytes utilized a

rabbit polyclonal anti-JNK 1 and 2 phosphospecific antibody (286

lg/mL for both antibodies) to assess JNK expression (Biosource

Nivelles, Belgium), as well as NFjB, IjBa, IjBa-phosphorylatedform (IjBa-P) (1-2lg/mL, TCS Cellworks, Buckingham, UK),

IKKb and IKKc (2 lg/mL, Abcam, Cambridge, UK), using mouse

monoclonal antibodies. Equal protein loading was confirmed by den-

sitometry using the a-tubulin antibody (2.04 lg/mL, Abcam, Cam-

bridge, UK). A chemiluminescent detection system ECL/ECLþ (GE

Healthcare, Little Chalfont, UK) enabled visualization after exposure

to X-ray film.

Isolation and cell culture of mature adipocytesAbdSc AT was digested with collagenase (2 mg/mL, Worthington

Biochemical, Reading, USA), as previously described (3). Following

isolation, mature AbdSc adipocytes were cultured in phenol red-free

DMEM: F12 medium containing glucose (5 mM/L), penicillin (100

units/mL) and streptomycin (100 lg/mL), ready for treatment. A

compacted aliquot of AbdSc mature adipocytes (200 ll; 5�105 per

mL) were treated with recombinant human (rh) TNFa (Sigma,

Poole, UK). Acute (2 h, 4 h) and chronic (48 h) effects of rh TNFatreatment (10, 50, and 100 ng/mL; Sigma, Poole, UK) on JNK and

NFjB activity were assessed. AbdSc adipose cells were also treated

with rh TNFa treatment (50 ng/mL) alone or in combination with

the TNFa antagonist, WP9QY (1lM; Calbiochem, Nottingham, UK)

for 2 h and 4 h. Adipocytes maintained in untreated media acted as

controls. For inhibitor studies, AbdSc adipocytes were treated with

NF-jB inhibitor (SN50, CalBiochem, Nottingham, UK) (50 lg/ml;

24 h) or a JNK inhibitor (SP600125, A.G. Scientific, Inc., San

Diego, CA) (10 lM/mL); conditions were based on previous data

(2,3). Viability of adipocytes was assessed as previously described

(15). In brief, trypan blue (Sigma, Poole, UK) is used as part of a

dye exclusion methodology for viable cell counting. The live viable

cells do not take up the dye whereas nonviable cells take up the

dye. Following treatment, the conditioned media and adipocytes

were separated by centrifugation (360�g for 2 min). Conditioned

media were removed, aliquoted and stored at �80�C. Protein from

the adipocytes was extracted (3).

Adipokine secretionConditioned media from the NFjB and JNK inhibitor studies were

assessed using the human IL-6, TNFa and resistin ELISAs from

R&D Systems (Abingdon, UK), intraassay variability <10%.

Assessment of JNK1 and 2 activityProtein extracted from human AbdSc AT, Om AT and AbdSc

adipocytes were assayed using an ELISA-based colorimetric kit

(Invitrogen, Paisley, UK); intraassay variability <10%.

Assessment of NFjB activityAT and AbdSc adipocyte whole cell extract was obtained according

to manufacturer’s instructions (matched paired AT: Om and AbdSc

lean: n¼8, BMI: 22.362.1 kg/m2, Age: 43.066.6 years; Om and

AbdSc Overweight and Obese: n¼8, BMI: 31.463.6 kg/m2, age:

50.168.5 years; all female subjects). NFjB activation was expressed

as percentages based on NFjB activity/total NFjB in AT. NFjB

Obesity NF�B and JNK Expression in Human Abdominal Fat Harte et al.

2 Obesity | VOLUME 000 | NUMBER 000 | MONTH 2013 www.obesityjournal.org

activity was assessed with Trans-AM NFjB p65 transcription factor

assay kit (Active Motif, Rixensart, Belgium; detection limit: <40 ng

of whole cell extract), as previously described (14,16).

ImmunohistochemistryAbdSc adipocytes, human sternum, and mononuclear blood cells

were incubated with a human JNK polyclonal primary antibody

(1 lg/mL, Biosource UK, Nivelles, Belgium). All the human tissue

slides were developed using peroxidase substrate kit (Vector Labora-

tories Ltd, Peterbrough, UK) and all slides were counter-stained

with Mayer’s hematoxylin.

Statistical analysisFor protein assessment, statistical analysis was undertaken using

ANOVA for comparison of AT depots with post hoc analysis

(Bonferroni). Statistical analysis was undertaken using a paired stu-

dent t-test (ANOVA, SPSS 17.0, Woking, UK) for comparison of

AT depots from T2DM versus ND subjects whilst matched for

age, BMI and gender. Gene expression data were analyzed using

an unpaired t-test (SPSS 17.0, Woking, UK). The threshold for

significance was P<0.05. Data in the text and figures are presented

as mean 6 standard error of mean (SEM), unless otherwise stated.

ResultsDepot specific protein expression of NFjB, NFjBactivity and associated intracellular molecules inhuman ATNFjB, IKKb, IKKc, IjBa and phosphorylated IjBa (IjBa-P) pro-tein expression were determined in Om AT and AbdSc depots.

The level of NFjB protein expression was similar in both the

Om AT and AbdSc depots from the obese subjects but signifi-

cantly higher in the Om AT from the lean subjects in comparison

with the lean AbdSc (Figure 1A). Protein expression of both

IjBa and IjBa-P were significantly increased in Om compared

with AbdSc AT (P<0.01) (Figure 1B), as were IKKb and IKKcprotein expression (P<0.001, P<0.01, respectively, Figure 1C and

1D).

NFjB activity was altered by AT depot and adiposity (Figure 1E).

In obese Om AT, NFjB activity (measured as NFjB activity/total

NFjB expressed as a percentage) was increased in comparison with

lean or obese AbdSc AT as well as with lean Om AT (P<0.05; Fig-

ure 1E). AbdSc AT NFjB activity was unaltered by adiposity (Fig-

ure 1E).

The effect of adiposity on NFjB expression,NFjB activity and associated intracellular mole-cules in human ATThe effect of adiposity on NFjB, IjBa, IKKb and IKKc protein

expression was also analyzed in both depots. Firstly, there was a sig-

nificant reduction in NFjB protein expression with increasing adi-

posity (Figure 1A). Furthermore, IjBa-P expression was increased

in both depots in the obese group compared with the lean subjects

(Figure 1B); IKKb was noted to increase in obesity in Om AT but

no effect was observed in AbdSc AT (Figure 1C). However, IKKcprotein expression, alone, remained unaffected by increasing adipos-

ity (Figure 1D). Om AT NFjB activity was higher in the obese sub-

jects compared with lean Om AT (P<0.05). Additionally, total JNK

protein expression, phosphorylated JNK1 and JNK2 (P-JNK1 and P-

JNK2), appeared unaffected by increasing adiposity (data not

shown).

Depot-specific total JNK and phosphorylatedJNK1 and JNK2 protein expression in human ab-dominal ATTotal JNK (JNK1&2 combined) expression was measured by the

relative fold increase in expression of total JNK in AT depots. Om

AT expressed the highest level of total JNK compared with the

AbdSc AT showing a 1.51 fold increase when compared with

AbdSc AT depot (P<0.05) (Figure 2A).

The relative fold increase in expression of P-JNK1 and P-JNK2

across the various AT depots was calculated in relation to AbdSc

AT, which was taken as 1. AbdSc AT expressed the highest levels

of P-JNK compared with Om AT (AbdSc versus Om JNK1: 1.73

fold increase, P<0.01; AbdSc Vs Om JNK2: 1.52 fold increase,

P<0.01) (Figure 2B).

JNK1 and JNK2 mRNA expression and JNKactivity in human ATJNK1 and JNK2 gene expression were assessed in Om and AbdSc

AT (DCt mean6SEM, Figure 2C) AT. There was no noted depot

specific effect of JNK1 or JNK2 expression (Om AT lean: JNK1

DCt 11.0660.16; JNK2: DCt 12.0460.18; AbdSc AT lean JNK1

DCt 11.2960.15, JNK2 DCt 11.8160.18). Assessment of the effect

of adiposity noted no differences in Om JNK expression (Om AT

Obese: JNK 1 DCt 11.1760.21; JNK2: DCt 12.1360.18) whilst in

AbdSc AT JNK1 expression between lean and obese subjects was

significantly altered (AbdSc obese: JNK1 DCt 11.5960.15, P<0.01)

but did not reach significance for JNK2 expression (AbdSc obese:

JNK2 DCt 12.0560.2; P¼NS).

CD45 represents a marker of macrophages and may represent an im-

portant site for JNK expression, thereby in order to ascertain that

the effect of JNK expression was not due to macrophage content we

assessed CD45 expression in the depots. However there was no sig-

nificant difference in expression observed between AT depots ana-

lyzed (Om CD45 lean DCt 20.4460.27; obese DCt 19.7960.31;

AbdSc DCt 20.0460.52).

AbdSc AT expressed the highest ratio of P-JNK to total JNK

expression (JNK activity/total JNK expression in AT; Figure 2D) in

comparison to Om AT. Furthermore, the difference in relative activ-

ity was significant when comparing Om versus AbdSc AT (P<0.05,

Figure 2D). We further examined the effect of adiposity in AbdSc

and Om AT. No statistically significant difference was observed

with increasing adiposity (Figure 2D).

Effect of T2DM status on JNK & NFjB activityAssessment of JNK activity in AbdSc AT from T2DM subjects

compared with AbdSc AT from nondiabetic (ND) subjects

showed a significant reduction due to T2DM status (JNK1:

P<0.001, JNK2: P<0.001; Figure 3A). Assessment of NFjB ac-

tivity determined that activity significantly increased in AbdSc

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY


FIGURE 1 (A) Protein expression of NFjB, (B) phosphorylated IkBa, (C) IKKc and (D) IKKb in human abdominal AT depots (AbdSc n¼10and Om n¼6, **P<0.01, ***P<0.001). A representative Western blot is shown above. (E) Relative expression of NFjB activity (NFjB ac-tivity expressed as a % of total NFjB expression) in human AT (n¼25: AbdSc n¼18 and Om n¼7). AbdSc Lean measurements werestandardized as 100% for relative expression of NFjB activity between the AbdSc and Om depots (*P<0.05, Vs AbdSc Lean). [Color fig-ure can be viewed in the online issue, which is available at wileyonlinelibrary.com]



AT from T2DM subjects compared with ND (P<0.001; Figure

3A).

Further analysis of P-JNK 1 and P-JNK 2 expression in AbdSc AT

from T2DM compared with AbdSc AT from ND subjects showed,

again, a significant reduction in JNK1 and JNK2 expression in

T2DM (P<0.01, P<0.001 respectively, Figure 3B).

The acute and chronic effects of rh TNFa onJNK1&2 activity and NFjB activity in AbdScadipocytesNFjB activity was altered by rh TNFa both over time and at a

range of concentrations (10, 50 and 100 ng/mL rh TNFa; n¼6) in

the in vitro treated AbdSc adipocytes.

An increase in NFjB activity was detected at 2 h and 4 h post-treat-

ment with rh TNFa 50 ng/mL concentration compared with control

untreated cells, whilst at a concentration of 10 ng/mL rh TNFa, onlya significant increase 4 h post-treatment was observed (P<0.01; Fig-

ure 4). JNK1 appeared to reduce % expression compared with con-

trol at 2 h (rh TNFa: 10 ng; P<0.05, P<0.01, respectively) and 4 h

treatment (rh TNFa: 50 ng; P<0.01, P<0.01, respectively). With

chronic treatment (48 h) NFjB activity remained elevated (P<0.05;

Figure 4).

Effects of rh TNFa on phosphorylated JNK1 andJNK2 protein expression with and without TNFaantagonistAbdSc adipocytes treated with rh TNFa led to a significant reduc-

tion in the P-JNK1 at both time points (P<0.05, P<0.01, n¼4, Fig-

ure 5). Concurrently, AbdSc adipocytes treated with TNFa antago-

nist (AGT, 1 ng) and rh TNFa (50 ng/mL) increased JNK1 protein

expression at 2 h and 4 h (P<0.01, Figure 5) whilst AGT alone led

to a reduction in JNK1 protein expression (P<0.01, Figure 5).

AbdSc adipocytes treated with rh TNFa only altered P-JNK2

protein expression at 4 h 100 ng/mL treatment (Figure 5; 4 h: TNFa100 ng/mL: 0.9760.71 ODU*) whilst rh TNFa in combination with

AGT led to an increase in P-JNK2 protein expression at 4 h (4 h

TNFa 50 ng/mL/TNFa AGT 1 ng: 10.2363.01 ODU:*), whilst

again AGT alone led to a reduction in JNK2 protein expression at

4 h (4 h TNFa AGT 1 ng: 2.1260.62 ODU;*; n¼4, Figure 5).

FIGURE 2 (A) Expression of total JNK (JNK1 and 2) in human AT depots (n¼25, AbdSc n¼18 and Om n¼7,*P<0.05 versus AbdSc); (B) Phosphospecific JNK ac-tivity protein expression of P-JNK1 (light gray) and P-JNK2 (dark gray) in human AT depots (n¼25: AbdSc, n¼18 and Om, n¼7; **P<0.01 versus AbdSc). Expres-sion is shown as relative fold difference compared with AbdSc depot; (C) JNK1 (black) and JNK2 (white) mRNA expression in Omental (Om) and abdominalsubcutaneous (AbdSc) adipose tissue (AT) depots. JNK1 and JNK2 expression is shown as relative fold difference standardized to Lean Om; (D) Relative expres-sion of JNK1 (light gray) and JNK2 (dark gray) activity (JNK activity expressed as a % of total JNK expression) in human AT (n¼25: AbdSc, n¼18 and Om, n¼7).AbdSc Lean measurements were standardized as 100% for relative expression of JNK1 and JNK2 activity between the AbdSc and Om depots (**P<0.01 versusAbdSc lean).



FIGURE 3 (A) Relative expression of NFjB, JNK1 and JNK2 activity in human AbdSc AT taken from subjects with type 2 diabetes mellitus(T2DM, n¼8) and ND (ND, n¼8), that were age, BMI and gender matched (***P<0.01 versus ND); (B) Phosphospecific JNK activity pro-tein expression of P-JNK1 and P-JNK2 in human Abd Sc AT taken from ND and T2DM subjects, also age, BMI and gender matched(**P<0.01 versus AbdSc).

FIGURE 4 Relative expression of NFjB, JNK1, and JNK2 activity in mature human AbdSc adipocytes (n¼6) treated for2, 4, and 48 h with and without (control) rh TNFa (10, 50 ng/mL; P-values are denoted: *P<0.05, **P<0.001). [Colorfigure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

Effects of JNK or NFjB inhibitor on adipokinesecretionAbdSc adipocytes treated with an NFjB inhibitor significantly

reduced IL-6, TNFa and resistin, whilst no change was noted in

JNK inhibitor treated AbdSc adipocytes (Table 1).

Immunohistochemical analysis of JNK inhuman ATPositive expression of JNK was identified in human AbdSc adipo-

cytes (Supplemental Figure 1).

DiscussionPrevious studies have implicated both NFjB and JNK inflammatory

pathways as potential therapeutic molecular targets for the treatment

of obesity mediated insulin resistance and T2DM (7,8,10,17-19).

This particular study addressed two fundamental questions: (1) is

one inflammatory pathway predominantly activated in AT from ei-

ther obese individuals or people with T2DM, contributing to meta-

bolic risk; (2) does one pathway appear to be preferentially activated

in vitro in response to proinflammatory mediators, such as rh TNFawhich is elevated in obesity and T2DM subjects (2-4,20,21).

From our ex vivo findings, AbdSc and Om AT from ND subjects

appear to have distinct inflammatory mechanisms. Increased IjBa,IKKb and IKKc protein expression were apparent in Om AT com-

pared with the AbdSc AT depot, which corresponded with increased

NFjB activity in Om AT compared with AbdSc AT. With

FIGURE 5 In vitro analysis of phospho-specific JNK protein expression (P-JNK1, P-JNK2) from human AbdSc mature adipocytes (n¼4) treated for 2, 4, and48 h with and without (control) rh TNFa (10, 50, 100 ng/mL) or treated with rh TNFa (50 ng) with TNFa antagonist (AGT, 1 ng), or TNFa AGT (1 ng) alone.Representative Westerns blot are shown. P-values are denoted versus control and respective JNK isoform: *P<0.05, **P<0.001.

TABLE 1 The level of adipokine secretion from untreatedAbdSc adipocytes (control) or cells treated with NFjB orJNK inhibitor (n 5 7; *P<0.05; **P<0.001)

Adipokine

secretion

Control

(untreated cells),

mean 6 SE

NFjB inhibitor,

mean 6 SE

JNK inhibitor,

mean 6 SE

TNFa (pg/ml) 184 6 38 102 6 31* 111 6 21

IL-6 (pg/ml) 2912 6 321 1781 6 211** 2563 6 311

Resistin (pg/ml) 124 6 32 65 6 24* 96 6 17



increasing adiposity, both IjBa and IKKb were upregulated in

AbdSc and Om AT, whilst NFjB protein expression decreased. This

may potentially arise due to the rapid turnover of NFjB mediated

by increased IjBa-P expression, which translocates NFjB to the nu-

cleus. This rapid turnover of NFjB was supported by our NFjB ac-

tivity data, which determined increased NFjB activity in Om AT

taken from obese patients compared with lean subjects.

Our studies further addressed the activity of NFjB in AbdSc AT

from T2DM subjects compared with age and BMI matched ND sub-

jects. Interestingly, NFjB activity increased substantially in AbdSc

fat taken from the T2DM subjects; this could arise due to the known

systemic proinflammatory milieu in such subjects, leading to a

continuing vicious cycle of inflammation from AbdSc AT. Such a

finding develops our understanding of NFjB in T2DM, with several

studies highlighting the importance of this inflammatory switch in

other tissues, such as the heart, liver and peripheral mononuclear

blood cells (22,23), as well as the relevance of therapeutic targeting

of NFjB activity (19,24).

Concurrent to NFjB analysis, our studies investigated JNK1 and 2.

Positive immuno-staining for JNK expression in AT was noted; mRNA

and protein expression studies affirmed the presence of JNK1 and 2 in

human ND AT, which appeared unrelated to macrophage presence.

Assessment of total JNK protein denoted increased expression in

Om AT compared with the AbdSc AT. Additionally, both P-JNK1

and P-JNK2 forms were also increased in Om AT. In order to exam-

ine the association of total JNK and P-JNK activity more accurately,

we evaluated the relative activity of JNK as a ratio (expressed as a

percentage of P-JNK versus total JNK) within a comparable quantity

of AT from each depot; a similar equation was utilized to assess

NFjB activity. Of note was that JNK1 and JNK2 % activity were

substantially less in the Om AT compared with AbdSc AT, in con-

trast to NFjB activity observed in the respective depots. Further-

more active JNK expression appeared unaffected by increasing

adiposity, whilst reduced in AT taken from T2DM subjects - in con-

trast to NFjB activity, as well as previous murine studies (25).

However, such findings may imply that NFjB is the more dominant

pathway and is preferentially activated over JNK pathways or that

JNK activation may occur under certain proinflammatory mediated

circumstances, such as T2DM. Further to this, studies by Sourris

et al. affirm JNK activation in AbdSc AT as a determinant of insulin

resistance (26).

Both obesity and T2DM are associated with increased circulating

TNFa levels. JNK has been shown to be activated by insulin and

TNFa in animal models and differentiated human cultured preadipo-

cytes (7,11,13,27). We therefore explored the direct action of rh

TNFa on the P-JNK and NFjB activity. Specifically, we examined

the effect of rh TNFa on P-JNK and NFjB activity in mature

AbdSc adipocytes as a stimulus for the inflammatory pathways. Our

own previous in vitro studies have shown that human AbdSc adipo-

cytes respond to various influences, such as lipopolysaccharide, in-

sulin, resistin and TNFa (2,3,28), with rh TNFa shown to stimulate

other adipokines, such as angiotensin II (29).

In contrast to previous murine studies (7,27,30), our findings demon-

strated that rh TNFa appeared to reduce JNK activation, which was

reversed with the use of a TNFa antagonist (AGT). Additionally, rh

TNFa was observed to mediate NFjB activation at 2, 4, and 48 h

post-treatment. These findings suggest that, within human AbdSc

adipocytes, TNFa may possess a dual influence on intracellular sig-

naling, leading to a reduction in JNK activity, predominantly influ-

encing JNK1 expression, whilst activating NFjB more effectively.

As such, a reduction in JNK1 expression may occur through cross-

talk between the JNK and IKK pathways, where both IKKa and

IKKb can phosphorylate the same site of IRS1 as JNK, whilst JNK

activation may also be inhibited by NFjB target genes (31,32). Fur-

thermore NFjB and JNK inhibitors were utilized to examine the

influence on TNFa release. NFjB blocker treated cells reduced

TNFa secretion and other proinflammatory cytokines. In contrast,

AbdSc adipocytes treated with a JNK inhibitor did not affect any

adipokine secretion, a similar finding being noted in cultured human

monocytes (33). Taken together, these data suggest that in AbdSc

AT, in T2DM subjects, TNFa may preferentially activate NFjB,which mediates a reduction in JNK activity, therefore NFjB acts as

the dominant intracellular inflammatory pathway (4,24). However,

JNK may still mediate an inflammatory response where NFjB target

genes are less dominant (26,27,33-36).

In conclusion, these studies demonstrate distinct depot specific dif-

ferences in AbdSc and Om AT with respect to expression and activ-

ity of NFjB and JNK. Furthermore these studies illustrate that JNK

phophoshorylation does not appear to be central in AbdSc AT, for

mediating inflammatory responses in the pathogenesis of obesity

mediated T2DM, as previously identified in rodent models. As such,

these studies, in conjunction with previous data, further strengthen

the role for NFjB as a potential key target for therapeutic drug

intervention in the management and prevention of obesity and

T2DM in humans.O

AcknowledgmentsWe acknowledge the British Heart Foundation for Dr. Alison

Harte’s Intermediate fellowship, research support from Birmingham

Science City and The Rowlands Trust. We thank the Thai Govern-

ment for Warunee Kumsaiyai PhD Sponsorship and the Saudi Ara-

bian Government for Dara Al-Disi’s PhD sponsorship. We also

thank the surgeons and theatre staff at UHCW Hospital, Coventry,

and Jane Starcynski for immunohistochemical support.

VC 2013 The Obesity Society

References1. Fogelstrand L, Hulthe J, Hulten LM, Wiklund O, Fagerberg B. Monocytic expres-

sion of CD14 and CD18, circulating adhesion molecules and inflammatory markersin women with diabetes mellitus and impaired glucose tolerance. Diabetologia2004;47:1948-1952.

2. Creely SJ, McTernan PG, Kusminski CM, et al. Lipopolysaccharide activates aninnate immune system response in human adipose tissue in obesity and type 2 dia-betes. Am J Physiol Endocrinol Metab 2006;292:E740-E747.

3. Kusminski CM, da Silva NF, Creely SJ, et al. The in vitro effects of resistin on theinnate immune signaling pathway in isolated human subcutaneous adipocytes.J Clin Endocrinol Metab 2007;92:270-276.

4. Arkan MC, Hevener AL, Greten FR, et al. IKK-beta links inflammation to obesity-induced insulin resistance. Nat Med 2005;11:191-198.

5. Dietze D, Ramrath S, Ritzeler O, Tennagels N, Hauner H, Eckel J. Inhibitor kappaBkinase is involved in the paracrine crosstalk between human fat and muscle cells.Int J Obes Relat Metab Disord 2004;28:985-992.

6. Makris C, Godfrey VL, Krahn-Senftleben G, et al. Female mice heterozygous forIKK gamma/NEMO deficiencies develop a dermatopathy similar to the humanX-linked disorder incontinentia pigmenti. Mol Cell 2000;5:969-979.

7. Aguirre V, Uchida T, Yenush L, Davis R, White MF. The c-Jun NH(2)-terminal ki-nase promotes insulin resistance during association with insulin receptor substrate-1and phosphorylation of Ser(307). J Biol Chem 2000;275:9047-9054.



8. Hirosumi J, Tuncman G, Chang L. A central role for JNK in obesity and insulinresistance. Nature 2002;420:333-336.

9. Tuncman G, Hirosumi J, Solinas G, Chang L, Karin M, Hotamisligil GS. Functionalin vivo interactions between JNK1 and JNK2 isoforms in obesity and insulin resist-ance. Proc Natl Acad Sci USA 2006;103:10741-10746.

10. Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF. Phosphoryla-tion of Ser307 in insulin receptor substrate-1 blocks interactions with the insulinreceptor and inhibits insulin action. J Biol Chem 2002;277:1531-1537.

11. Bennett BL, Satoh Y, Lewis AJ. JNK: A new therapeutic target for diabetes. CurrOpin Pharmacol. 2003;3:420-425.

12. Ofei F, Hurel S, Newkirk J, Sopwith M, Taylor R. Effects of an engineered humananti-TNF alpha antibody (CDP571) on insulin sensitivity and glycemic control inpatients with NIDDM. Diabetes 1996;45:881-885.

13. Hotamisligil G, Peraldi P, Budavari A, Ellis R, White M, Spiegelman B. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF alpha- andobesity-induced insulin resistance. Science 1996;2:665-668.

14. Baker AR, Harte AL, Howell N, et al. Epicardial adipose tissue as a source of nu-clear factor-kappaB and c-Jun N-terminal kinase mediated inflammation in patientswith coronary artery disease. J Clin Endocrinol Metab 2009;94:261-267.

15. McTernan PG, McTernan CL, Chetty R, et al. Increased resistin gene and proteinexpression in human abdominal adipose tissue. J Clin Endocrinol Metab 2002;87:2407-2410.

16. Renard P, Ernest I, Houbion A, et al. Development of a sensitive multi-well colori-metric assay for active NF{{kappa}}B. Nucl Acids Res 2001;29:e21-e26.

17. Coll T, Jove M, Rodriguez-Calvo R, et al. Palmitate-mediated downregulation ofperoxisome proliferator-activated receptor-gamma coactivator 1alpha in skeletalmuscle cells involves MEK1/2 and nuclear factor-kappaB activation. Diabetes2006;55:2779-2787.

18. Gaillard P, Jeanclaude-Etter I, Ardissone V, et al. Design and synthesis of the firstgeneration of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetoni-trile inhibitors of the c-Jun N-terminal kinase. J Med Chem 2005;48:4596-4607.

19. Shoelson SE, Lee J, Yuan M. Inflammation and the IKK beta/I kappa B/NF-kappaB axis in obesity- and diet-induced insulin resistance. Int J Obes Relat Metab Dis-ord 2003;27:S49-S52.

20. Harte AL, da Silva NF, Creely SJ, et al. Elevated endotoxin levels in non-alcoholicfatty liver disease. J Inflamm 2010;30:15-25.

21. Al-Attas OS, Al-Daghri NM, Al-Rubeaan K, et al. Changes in endotoxin levels inT2DM subjects on anti-diabetic therapies. Cardiovasc Diabetol 2009;15:20-30.

22. Aljada A, Ghanim H, Dandona P. Activation of nuclear factor-kappa B (NF-kappaB) in mononuclear cells (MNC). Methods Mol Biol 2002;196:105-110.

23. Ghanim H, Aljada A, Hofmeyer D, Syed T, Mohanty P, Dandona P. Circulatingmononuclear cells in the obese are in a proinflammatory state. Circulation 2004;110:1564-1571.

24. Cai D, Yuan M, Frantz DF, et al. Local and systemic insulin resistance resultingfrom hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005;11:183-190.

25. Pirola L, Johnston AM, Van Obberghen E. Modulation of insulin action. Diabetolo-gia 2004;47:170-184.

26. Sourris KC, Lyons JG, de Courten MP, et al. c-Jun NH2-terminal kinase activity insubcutaneous adipose tissue but not nuclear factor-kappaB activity in peripheralblood mononuclear cells is an independent determinant of insulin resistance inhealthy individuals. Diabetes. 2009;58:1259-1265.

27. Fern�andez-Veledo S, Vila-Bedmar R, Nieto-Vazquez I, Lorenzo M. c-Jun N-termi-nal kinase 1/2 activation by tumor necrosis factor-alpha induces insulin resistancein human visceral but not subcutaneous adipocytes: reversal by liver X receptoragonists. J Clin Endocrinol Metab 2009;94:3583-3593.

28. McTernan PG, Harte AL, Anderson LA, et al. Insulin and rosiglitazone regulationof lipolysis and lipogenesis in human adipose tissue in vitro. Diabetes 2002;51:1493-1498.

29. Harte A, McTernan P, Chetty R, et al. Insulin-mediated upregulation of the reninangiotensin system in human subcutaneous adipocytes is reduced by rosiglitazone.Circulation 2005;111:1954-1961.

30. Nguyen MT, Satoh H, Favelyukis S, et al. JNK and TNFa mediated free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. J Biol Chem 2005;280:35361-35371.

31. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr.Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest2003;112:1796-1808.

32. Xu H, Barnes GT, Yang Q. Chronic inflammation in fat plays a crucial role in thedevelopment of obesity-related insulin resistance. J Clin Invest 2003;112:1821-1830.

33. Olsnes C, Olofsson J, Aarstad HJ. MAPKs ERK and p38, but not JNK phosphoryla-tion, modulate IL-6 and TNF-a secretion following OK-432 in vitro stimulation ofpurified human monocytes. Scand J Immunol 2011;74:114-125.

34. McGee KC, Harte AL, da Silva NF, et al. Visfatin Is Regulated by Rosiglitazone inType 2 Diabetes Mellitus and Influenced by NFjB and JNK in Human AbdominalSubcutaneous Adipocytes. PLoS One 2011;6:e20287-e20296.

35. Gao Z, Hwang D, Bataille F, et al. Serine phosphorylation of insulin receptor sub-strate 1 by inhibitor kappa B kinase complex. J Biol Chem 2002;277:48115-48121.

36. Tang G, Minemoto Y, Dibling B, et al. Inhibition of JNK activation through NF-kappaB target genes. Nature 2001;414:313-317.



NFκB as a potent regulator of inflammation in human adipose tissue, influenced by depot, adiposity, T2DM status, and TNFα

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