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. 2015 Mar 3;29(4):558–570. doi: 10.1210/me.2014-1287

11β-HSD1 Modulates LPS-Induced Innate Immune Responses in Adipocytes by Altering Expression of PTEN

Wenfang Lai 1,*, Xue Tian 1,*, Qing Xiang 1, Kedan Chu 1, Yicong Wei 1, Jingti Deng 1, Shaoping Zhang 1, John Brown 1, Guizhu Hong 1,✉
PMCID: PMC5414746  PMID: 25734515

Abstract

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) represents a therapeutic target for treating hyperglycemia in type 2 diabetes. Here, we investigate the effects of 11β-HSD1 on the innate immune response of adipocytes to produce proinflammatory cytokines. The 11β-HSD1 inhibitor emodin, or 11β-HSD1-targeted small interfering RNA, dose dependently suppressed IL-6, IL-1β, and TNF-α expression in lipopolysaccharide-treated 3T3-L1 adipocytes. Inhibiting 11β-HSD1 also reduced phosphatase and tensin homologue (PTEN) expression, a negative regulator of phosphatidylinositol 3-kinase effects, whereas 1pM cortisone or dexamethasone induced IL-6 and PTEN levels. PTEN-targeted small interfering RNA decreased IL-6, IL-1β, and TNF-α without affecting 11β-HSD1 levels. Correspondingly, emodin increased phosphorylated protein kinase B (p-PKB) (Ser473) to PKB ratio but not p-PKB (Thr308) to PKB ratio. Emodin did not increase the p-PKB (Ser473) to PKB ratio when the rapamycin-insensitive companion of mTOR was depleted, further supporting the involvement of mammalian target of rapamycin complex 2 in PKB phosphorylation. Moreover, emodin suppressed phosphorylated inhibitor of κB α (p-IκBα) to IκBα ratio and reduced nuclear factor κ B subunit p50 in the nuclear fraction. In contrast, 1pM cortisone or dexamethasone decreased p-PKB (Ser473) to PKB ratio, increased p-IκBα to IκBα ratio, and increased nuclear NF-κB subunit p50. Additionally, wortmannin had similar effects on IL-6, p-PKB (Ser473) to PKB ratio, and p-IκBα to IκBα ratio as 1pM cortisone or dexamethasone. Finally, emodin treatment of streptozotocin diabetic rats on a high-fat diet reduced levels of IL-6, PTEN, Cluster of Differentiation 68, and the ratio of p-IκBα to IκBα in visceral fat, indicating that our findings in vitro may also apply to visceral fat in vivo. Together, these results suggest that inhibiting 11β-HSD1 reduces lipopolysaccharide-induced proinflammatory innate immune responses in adipocytes by down-regulating PTEN expression, leading to activation of the PI3K/PKB pathway.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is thought to play a critical role in the development of metabolic disease (1–4) by catalyzing the conversion of inactive cortisone to active cortisol, which then mediates local glucocorticoid actions, notably in liver and adipose tissues. The enzyme is expressed in mature adipocytes, including mature 3T3-L1 cells in vitro (5), and is selectively increased in adipose tissue, but not in the liver, in obese humans and obese rodents (4, 6, 7). Insulin resistance, visceral obesity, dyslipidaemia, and hypertension occur when 11β-HSD1 is transgenically overexpressed in adipocytes of mice (2, 4). Conversely, inhibition or knockout, or adipocyte-specific inactivation of 11β-HSD1 (3, 9, 10), or reduction of the levels of adipocyte glucocorticoids by other means (11), leads to improved insulin sensitivity, glucose and lipid profiles, reduced visceral obesity, and arteriosclerosis. Thus, inhibition of 11β-HSD1 represents an attractive therapeutic target for the treatment of the metabolic syndrome of insulin resistance, dyslipidaemia, and atherosclerosis, and recent reports of the results of early clinical trials of 11β-HSD1 inhibitors have been promising (12).

Inflammatory mediators are released by adipose tissue from early in the course of metabolic syndrome, and these are also thought to contribute significantly to its pathogenesis (13, 14). At an intracellular level, insulin resistance is associated with the activation of nuclear factor κB (NF-κB) in adipocytes, which is under the control of surface receptors such as Toll-like receptor 4 (TLR4) and triggers production of a number of proinflammatory adipokines, including IL-6, TNF-α, and IL-1β (15). Among other agonists, TLR4 is activated by bacterial lipopolysaccharides (LPSs), and circulating LPS and IL-6 correlates with insulin resistance and the risk of developing diabetes in patients (16, 17). However, despite this, and the despite the evidence that adipocyte 11β-HSD1 is important to the control of glucose homeostasis, much less is known of the actions of adipocyte 11β-HSD1 on inflammation. Homozygous deletion of 11β-HSD1 in mice suppresses chronic inflammation in adipose tissue but such knockout also affects nonadipocytic cells, such as macrophages, which themselves express 11β-HSD1 and may have secondary effects on the production of proinflammatory mediators by adipocytes (18, 19). Therefore, we have examined the direct effects of inhibiting the action of 11β-HSD1, using both a selective inhibitor, emodin (20), and small interfering RNA (siRNA) technology, on the levels of inflammatory cytokines in LPS-treated 3T3-L1 adipoctyes. We have also examined the intracellular mechanisms of these effects and provide evidence that they are strongly dependent on phosphatase and tensin homologue (PTEN) and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) downstream signaling. Finally, we have shown that emodin produces a corresponding reduction in PTEN and in the inflammatory markers IL-6, phosphorylated inhibitor of κB (p-IκB), and Cluster of Differentiation 68 (CD68) in visceral fat when given to type 2 diabetic rats, suggesting that the antiinflammatory mechanisms we identified in vitro may also apply in vivo.

Materials and Methods

Culture and differentiation of 3T3-L1 cells

3T3-L1 cells were obtained from American Type Culture Collection and maintained in DMEM supplemented with 100-U/mL penicillin, 100-μg/mL streptomycin, and 10% fetal bovine serum (FBS) (corticosteroid < 10−10M) (HyClone Laboratories) (21) at 37°C in a tissue culture incubator (5% CO2). For adipocyte differentiation, cells were seeded in 24-well plates (5 × 104 cells/well) in complete medium and were cultured to 100% confluence. After an additional 48 hours, adipocyte differentiation was induced by replacing the medium with high glucose DMEM supplemented with 10% FBS, 0.5mM 3-isobutyl-1-methylxanthine, 10-μg/mL insulin, and 1μM dexamethasone (all purchased from Sigma-Aldrich). After another 48 hours, cells were cultured in high glucose DMEM supplemented with 10% FBS (and 10-μg/mL insulin for 13 d). Medium was changed every other day.

Treatment of 3T3-L1 adipocytes

Differentiated adipocytes were cultured in DMEM/1% FBS overnight and were then treated with vehicle (0.1% dimethyl sulfoxide [DMSO]) or 10μM emodin in the absence or presence of vehicle, 10-ng/mL LPS, or 250μM palmitate (Sigma-Aldrich) for 24 hours. Cell culture medium was collected, and levels of IL-6 were measured by ELISA. The cells were also harvested for analysis by real-time RT-PCR or Western blotting. For the dose response studies, cells were treated with vehicle (0.1% DMSO) or various concentrations of emodin, cortisone, or dexamethasone (Sigma-Aldrich) as indicated, in the presence of 10-ng/mL LPS for 24 hours. Cell culture medium was collected, and levels of IL-6 were measured. The cells were also harvested for analysis by real-time RT-PCR and Western blotting.

To investigate the effects of combination treatment of emodin and glucocorticoids, cells were incubated with 10μM emodin, 1pM cortisone or dexamethasone, or 10μM emodin, 1pM cortisone, or dexamethasone plus emodin in the presence of 10-ng/mL LPS. After 24 hours, cell culture medium was collected, and levels of IL-6 were measured. The cells were also harvested for analysis by real-time RT-PCR and Western blotting. For wortmannin treatment, cells were incubated with 200nM wortmannin (Sigma-Aldrich) for 30 minutes before the addition of 10μM emodin, 1pM cortisone, or 1pM dexamethasone in the presence of 10-ng/mL LPS. After 24 hours, cell culture medium was collected, and levels of IL-6 were measured. The cells were also harvested for analysis by real-time RT-PCR and Western blotting.

11β-HSD1 activity assay

Differentiated 3T3-L1 cells were treated with various concentrations of emodin in the presence of 10-ng/mL LPS. After 24 hours, 100μM reduced nicotinamide adenine dinucleotide phosphate and 1μM cortisone were added. After 3 hours, cell culture medium was collected, and cortisol production was assayed using a Cortisol EIA kit (Enzo Life Sciences), as per the manufacturer's instructions. Cortisol was undetectable in the absence of added cortisone (result not shown), so cortisol production in our assay system reflects 11β-HSD1 activity in converting exogenous cortisone to cortisol (22). The results are expressed as the fold-change relative to the control after adjustment for cell numbers.

Transfection of siRNA

Differentiated 3T3-L1 cells were transfected with 2 different 11β-HSD1-targeted siRNA sequences (catalog numbers SI00173880 and SI02687041; QIAGEN) at 1nM or 5nM, using RNA interference (RNAi) Human/Mouse Starter kit (QIAGEN), according to the manufacturer's instructions. AllStars Control siRNA (QIAGEN), which is a well-established control siRNA and did not affect expression of the genes tested in our laboratory, such as IL-6, IL-1β, TNF-α, 11β-HSD1, and PTEN (data not shown), was included in the experiments. The mRNA and protein levels of 11β-HSD1 were measured 24 hours after each transfection. A dose-dependent decrease in 11β-HSD1 expression was achieved with both 11β-HSD1-targeted siRNA sequences compared with the control siRNA. However, the 11β-HSD1-targeted siRNA sequence, SI00173880, at 5nM was used for all subsequent experiments. Two hours after transfection with 11β-HSD1-targeted siRNA or control siRNA, cells were treated with 10μM emodin or vehicle (0.1% DMSO) in the presence of 10-ng/mL LPS. After 24 hours, cell culture medium was collected for IL-6 assay, and cells were harvested for analysis by RT-PCR and Western blotting.

PTEN knockdown was achieved using 2 different siRNA sequences (catalog numbers SI02734494 and SI04920783; QIAGEN) at 1nM or 5nM. The control siRNA described above was also included in the experiment. A dose-dependent decrease in PTEN levels was observed for both PTEN-targeted siRNA sequences compared with the control siRNA. However, the SI02734494 siRNA at 5nM was used for subsequent experiments. A similar approach was applied to knockdown Rictor using rapamycin-insensitive companion of mTOR (Rictor)-targeted siRNA (QIAGEN). Two hours after transfection, cells were treated with 10μM emodin or vehicle (0.1% DMSO) in the presence of 10-ng/mL LPS for 24 hours and then collected for Western blot analysis.

Enzyme-linked immunosorbent assay

IL-6 secretion into cell culture medium was measured using a mouse IL-6 ELISA kit (BoShiDe Biotechnology), according to the manufacturer's instructions. The results are expressed as fold-change relative to the control.

Quantitative real-time RT-PCR (qRT-PCR)

Total RNA was extracted from 3T3-L1 adipocytes or visceral fat using the RNeasy Mini kit (QIAGEN), according to the manufacturer's instructions. Briefly, first-strand cDNA was synthesized from total RNA using the Superscript First-Strand Synthesis System (Life Technologies), according to the manufacturer's instructions. Then, mRNA levels of 11β-HSD1, IL-6, IL-1β, TNF-α, and PTEN were quantified in 3T3-L1 adipocytes or IL-6, PTEN, and CD68 in visceral fat tissues using real-time RT-PCR, as previously described (23). The results were normalized to levels of 18S RNA (the internal control), and fold-change relative to the control is reported. Ready-to-go primers and probes for the real-time PCR assays were purchased from Life Technologies.

Western blot analysis

Western blot analysis was performed as previously described, with some modifications (23). Briefly, cells or tissues were lysed in radio-immunoprecipitation assay buffer (50mM Tris-Cl [pH 7.5], 150mM NaCl, 0.5% Nonidet P-40, and 0.1% sodium dodecyl sulfate) plus 1% protease inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifugation at 14 000g for 10 minutes at 4°C. Cytosolic and nuclear fractions were separated using the Ne-PER Nuclear protein Extraction kit (Thermo Scientific), according to the manufacturer's instructions. Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were subsequently incubated with various antibodies. Antibodies purchased from Cell Signaling Technology included: antiphosphorylated PKB antibody (Ser473, 1:500), antiphosphorylated PKB antibody (Thr308, 1:500), anti-IκBα antibody (1:800), and antiphosphorylated IκBα antibody (Ser32/36, 1:500). Antibodies purchased from Santa Cruz Biotechnology, Inc included: anti-11β-HSD1 antibody (1:1000), anti-PTEN antibody (1:1000), anti-PKB antibody (1:500), anti-Rictor antibody (1:800), anti-NF-κB p50 antibody (1:1000), and anti-proliferating cell nuclear antigen (PCNA) antibody (1:1000). Anti-β-actin antibody (1:1000) was purchased from Beyotime.

After incubation with horseradish peroxidase-conjugated secondary antibodies, immunoreactive protein signals were detected using enhanced chemiluminescence AB reagent (Beyotime). A ChemiDoc XRS+ imaging system (Bio-Rad) was used to image stained membranes, with the bands of target proteins quantified using ImageJ software. β-actin and PCNA were used as internal loading controls for whole-cell lysates/cytosolic fractions and nuclear fractions, respectively. The results are expressed as fold-change relative to the controls, after data were normalized to β-actin or PCNA levels.

Animal experiments

Male Wister rats (n = 20) weighing 180–220 g were purchased from the Slac Laboratory Animal Co Ltd. Animals were housed under controlled temperatures (21°C–23°C), 55%–75% humidity, and with a 12-hour light, 12-hour dark cycle.

All animal experiments were performed in accordance with the guidelines of the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. The procedures for this study were approved by the Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine.

Rats were fed with a high-fat diet (HFD) followed by injection of streptozotocin (STZ) as previously described (24). Briefly, rats received a chow diet consisting of 5% fat, 53% carbohydrate, and 23% protein, with a total caloric value of 25 kJ/kg (Xiewei Experimental Animal Service), for 1 week before dietary manipulation. Animals were subsequently fed a HFD, which consisted of 22% fat, 48% carbohydrate, and 20% protein, with a total caloric value of 44.3 kJ/kg (Xiewei Experimental Animal Service). The rats received the HFD for 6 weeks, then were administered an ip injection of STZ (25 mg/kg). Seventy-two hours later, fasting blood glucose (FBG) levels were measured. Rats with a FBG les than 7.8 mmol/L received another dose of STZ (25 mg/kg). After 4 weeks of STZ injections, animals with a FBG more than 7.8 mmol/L were considered diabetic.

The HFD/STZ-induced diabetic rats were randomly divided into a treatment group and a control group (n = 8 per group), and fasting plasma was collected from the tail vein of each animal. For the treatment group, emodin (100 mg/kg body weight) was administered by oral gavage as a mixture with 0.5% sodium carboxyl methyl cellulose at a dose of 0.5 ml/kg body weight, once daily for 21 days. The control group received vehicle (0.5% sodium carboxyl methyl cellulose) alone. Body weight and food uptake for both groups were recorded daily. Twenty-one days after treatment, fasting plasma was collected, and levels of glucose, insulin, triglycerides, and cholesterol were assayed as previously described (25). Animals were then sacrificed and visceral fat samples were dissected for qRT-PCR and Western blot analysis.

Statistical analysis

The results are the mean ± standard error of the mean (SEM). Statistical analyses were performed using one-way ANOVA followed by multiple comparisons with post hoc Bonferroni test (SPSS 8.0), or 2-tailed Student's t test where stated. P < .05 was considered statistically significant.

Results

Inhibition of 11β-HSD1 suppresses proinflammatory cytokine expression in LPS-treated 3T3-L1 adipocytes

The 11β-HSD1 inhibitor emodin dose dependently reduced 11β-HSD1 activity in 3T3-L1 adipocytes (Figure 1A). Over the same concentration range, emodin also dose dependently suppressed the increase in IL-6, IL-1β, and TNF-α transcripts, as well as the level of IL-6 induced by 10-ng/mL LPS (Figure 1, B and C, and Supplemental Figure 1, A–D). Emodin also inhibited IL-6 production in the cells treated with 250μM palmitate, but it failed significantly to decrease basal IL-6 level in the absence of LPS or palmitate (Figure 1C). The inhibitory effects of emodin were not due to generalized cellular toxicity, because emodin in the absence of LPS or palmitate did not alter the basal levels of IL-6, and MTT assay revealed that cell viability was not significantly altered by emodin concentrations up to 50μM whether or not LPS or palmitate were present (data not shown).

Figure 1. Inhibiting 11β-HSD1 suppresses proinflammatory cytokines in LPS-treated 3T3-L1 adipocytes.

Figure 1.

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A, Emodin dose dependently inhibits 11β-HSD1 activity in 3T3-L1 adipocytes. Cells were treated with 10-ng/mL LPS in the presence or absence of emodin at the indicated concentrations for 24 hours, followed by incubation with 100μM reduced nicotinamide adenine dinucleotide phosphate and 1μM cortisone. Cortisol production in the supernatant was assayed using a Cortisol EIA kit. Cortisol was undetectable in the absence of exogenous cortisone, so cortisol production reflects conversion of exogenous cortisone to cortisol by 11β-HSD1 activity. B, Emodin inhibits IL-6, IL-1β, and TNF-α transcripts in 10-ng/mL LPS-treated 3T3-L1 cells. LPS-treated 3T3-L1 cells were incubated with vehicle or 10μM emodin for 24 hours. The levels of mRNA were measured by qRT-PCR and normalized to18S RNA. C, Emodin inhibits LPS or palmitate-induced IL-6 production but not the basal level. Left panel, Cells were treated with vehicle or 10-ng/mL LPS in the absence (■) or presence (□) of 10μM emodin. Right panel, Cells were incubated with vehicle or 10μM emodin in the presence of 250μM palmitate. After 24 hours, IL-6 secretion was assayed by ELISA. The data are mean ± SEM and are expressed as fold-changes relative to basal production of IL-6 (left panel) or control (right panel). D, The inhibitory effects are shown of 10μM emodin, 5nM 11β-HSD1-targeted siRNA, or a combination of both, on IL-6 production. An unrelated control RNAi had no significant effect on IL-6 production. Cells were transfected with 11β-HSD1-targeted siRNA or a control RNAi (control) in the presence or absence of emodin for 24 hours, and IL-6 production was measured by ELISA. E, Cortisone induces IL-6 production and that is reversed by emodin. LPS-treated cells were incubated with vehicle (control), or 10μM emodin, or cortisone (1pM) with or without 10μM emodin. After 24 hours, IL-6 production was assayed by ELISA. Unless otherwise specified, all values are mean ± SEM and are expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

Previous studies have shown that LPS-mediated activation of TLR4 signaling plays an important role in mediating the inflammatory state associated with diabetes (26, 27). Therefore, we sought to confirm the role of 11β-HSD1 in the control of proinflammatory cytokines in response to LPS by transfecting 11β-HSD1-targeted-specific siRNA into 3T3-L1 adipocytes. This transfection decreased both mRNA and protein levels of 11β-HSD1 in a dose-dependent manner, compared with cells transfected with control siRNA (Supplemental Figure 2A), and resulted in reduction of IL-6 levels in response to LPS (Figure 1D). Combined treatment with emodin and 11β-HSD1-targeted siRNA was not additive (Figure 1D), suggesting that both mechanisms of inhibiting the enzyme might reduce IL-6 production through common downstream effects. In addition, 11β-HSD1-targeted siRNA also inhibited IL-6, IL-1β, and TNF-α mRNA levels in LPS-treated 3T3-L1 adipocytes (Supplemental Figure 2, B–D).

We then investigated the effects of cortisone, a glucocorticoid that is inactive at the glucocorticoid receptor until converted to cortisol by 11β-HSD1 (28), on IL-6 production in LPS-treated 3T3-L1 adipocytes. We found 1pM cortisone increased levels of IL-6 in response to LPS, and that this effect of cortisone was blocked by emodin (Figure 1E). In contrast, a high concentration of cortisone (1μM) suppressed IL-6 production (Supplemental Figure 2E). The does-dependent differential effects of glucocorticoid on IL-6 were also observed in the cells treated with dexamethasone (Supplemental Figure 2E), a glucocorticoid that does not require modification by 11β-HSD1 to bind to the glucocorticoid receptor and has little affinity for the mineralocorticoid receptor (28). As with cortisone, low concentrations of dexamethasone augmented the IL-6 response to LPS, whereas high concentrations of dexamethasone inhibited it. These results indicate that proinflammatory effects of cortisone require the activity of 11β-HSD1 to convert cortisone to cortisol and are mediated through activation of the glucocorticoid receptor rather than the mineralocorticoid receptor.

Expression of proinflammatory cytokines mediated by 11β-HSD1 is modified by PTEN/PI3K/PKB signaling

It has previously been shown that the PI3K pathway can regulate NF-κB and IL-6 production in mature 3T3-L1 cells (29). We therefore investigated whether the PI3K pathway may also be implicated in the stimulation of IL-6 production by LPS through the activity of 11β-HSD1 in these cells. We found that emodin did indeed increase the ratio of p-PKB (Ser473) to PKB in LPS-treated 3T3-L1 adipocytes (Figure 2A). In contrast, 1pM cortisone or 1pM dexamethasone decreased the ratio of p-PKB (Ser473) to PKB, and these inhibitory effects were reversed by emodin (Figure 2, B and C, and Supplemental Figure 3A). Wortmannin, as expected, decreased the ratio of p-PKB (Ser473) to PKB, and this decrease was overcome by emodin (Figure 2, A and B). Moreover, wortmannin enhanced IL-6 levels and this effect was also overcome by emodin (Figure 2, D and E). These results suggest that changes in PI3K/PKB signaling are associated with the modulation of IL-6 production by 11β-HSD1.

Figure 2. Effects of emodin, cortisone and wortmannin (Wort.) on p-PKB and IL-6 production.

Figure 2.

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A–C, The ratio of p-PKB to PKB is increased by emodin but decreased by cortisone or Wort. LPS-treated 3T3-L1 adipocytes were incubated with (A) vehicle (control), 10μM emodin, 200nM Wort., or a combined treatment of emodin and Wort.; or (B) with vehicle (control), 1pM cortisol, 200nM Wort., or a combination of cortisone and Wort.; or (C) with vehicle (control), 10μM emodin, 1pM cortisol, or a combined treatment of emodin and cortisol. After 24 hours, proteins were measured by Western blotting and normalized to β-actin, and the results presented are the ratio of p-PKB (Ser473) to PKB. D and E, IL-6 production is inhibited by emodin but enhanced by cortisone or Wort. LPS-treated 3T3-L1 adipocytes were incubated with (D) vehicle (control), 10μM emodin, 200nM Wort., or a combined treatment of emodin and Wort. or (E) with vehicle (control), 1pM cortisol, 200nM Wort., or a combination of cortisone and Wort. After 24 hours, IL-6 production was measured by ELISA. All values are mean ± SEM and expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

We then investigated whether PTEN, a key negative regulator of PI3K/PKB downstream signaling that specifically catalyzes the conversion of phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol 4,5-bisphosphate (30, 31), was involved in the emodin-mediated activation of PI3K/PKB signaling by LPS. We found that inhibition of 11β-HSD1 by emodin suppressed both mRNA and protein levels of PTEN in LPS-treated 3T3-L1 cells (Figure 3A). siRNA specifically directed against 11β-HSD1 also suppressed the levels of PTEN mRNA and protein (Figure 3B). Moreover, cells treated with 11β-HSD1-targeted siRNA were not able to respond to emodin by further blockade of the level of PTEN (Figure 3B). In contrast, 1pM cortisone or dexamethasone increased the level of PTEN and these increases were blocked by emodin (Figure 3C and Supplemental Figure 3B). These results suggest that the inhibitory effects of emodin on PTEN were mediated by reducing 11β-HSD1 activity. To examine whether down-regulation of PTEN had a critical role in the suppression of IL-6 production mediated by 11β-HSD1 inhibition, IL-6 was assayed in PTEN-depleted cells. Transfecting LPS-treated 3T3-L1 adipocytes with a PTEN-targeted siRNA lowered mRNA and protein levels of PTEN (Supplemental Figure 4A) and reduced production of IL-6 without significantly affecting the level of 11β-HSD1 protein, compared with cells that were transfected with a control siRNA (Figure 3, D and E). Furthermore, cells treated with PTEN-targeted siRNA were not able to respond to emodin by further blockade of IL-6 production (Figure 3D). Additionally, PTEN-targeted siRNA reduced the mRNA levels of IL-6, IL-1β, and TNF-α in LPS-treated 3T3-L1 adipocytes (Supplemental Figure 4, B–D). Taken together, these results suggest that PTEN is involved in the downstream pathway of control of proinflammatory cytokine expression by 11β-HSD1 in 3T3-L1 cells treated with LPS. Similarly, emodin inhibited the levels of PTEN and IL-6 in 3T3-L1 adipocytes treated with palmitate, another agonist of TLR4 (29, 32). Depleting PTEN by siRNA inhibited IL-6 production, whereas emodin could not further inhibit IL-6 production in response to palmitate in 3T3-L1 cells in which PTEN had been depleted (Supplemental Figure 5).

Figure 3. Inhibition of IL-6 production caused by blocking 11β-HSD1 in LPS-treated 3T3-L1 adipocytes depends on reduction of PTEN.

Figure 3.

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A, Emodin reduces the level of PTEN. LPS-treated 3T3-L1 adipocytes were incubated with vehicle or 10μM emodin for 24 hours. PTEN mRNA (left panel) was measured using qRT-PCR and normalized to18S RNA, and PTEN protein (right panel) was measured by Western blotting and normalized to β-actin. B, 11β-HSD1-targeted siRNA reduces PTEN expression. LPS-treated 3T3-L1 adipocytes were transfected with a control RNAi, or 5nM 11β-HSD1-targeted siRNA, 10μM emodin, or a combination of both. PTEN mRNA (left panel) was measured using qRT-PCR and normalized to18S RNA. PTEN protein (right panel) was measured by Western blotting and normalized to β-actin levels. C, Cortisone increases the level of PTEN. LPS-treated 3T3-L1 adipocytes were incubated with vehicle, 1pM cortisone, 10μM emodin, a combination of both for 24 hours. PTEN protein was measured by Western blotting and normalized to β-actin. D and E, Depletion of PTEN reduces IL-6 production but does not affect the level of 11β-HSD1 in LPS-treated 3T3-L1 adipocytes. Cells were treated with a control RNAi, 5nM PTEN-targeted siRNA, 10μM emodin, or a combination of both. IL-6 production was assayed by ELISA (D). 11β-HSD1 protein was measured by Western blotting and normalized to β-actin levels (E). All values are mean ± SEM and are expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

PKB is activated by PIP3-dependent phosphorylation at either Ser473, typically by mammalian target of rapamycin complex 2 (mTORC2), or at Thr308 by PDK1 (33), and so the effects of 11β-HSD1 through modulation of PTEN might involve either phosphorylation. We actually found that emodin did not alter the ratio of p-PKB (Thr308) to PKB (Figure 4A) but only altered phosphorylation at Ser473 as reported above. We therefore sought to confirm the involvement of mTORC2 in the action of emodin. We found that knocking down levels of Rictor, one of the core complements of mTORC2 (33), by target-specific siRNA not only reduced the ratio of p-PKB (Ser473) to PKB, and increased IL-6 production, but also prevented emodin from increasing the ratio of p-PKB (Ser473) to PKB and from suppressing IL-6 production (Figure 4, B–D). These data suggest that emodin-mediated phosphorylation of PKB is indeed dependent on mTORC2, rather than PDK1.

Figure 4. Phosphorylation of PKB and inhibition of IL-6 by emodin depends on mTORC2.

Figure 4.

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A, No effect of emodin on the p-PKB (Thr308) to PKB ratio. LPS-treated 3T3-L1 adipocytes were incubated with vehicle, 10μM emodin, 200nM wortmannin (Wort.), or a combination of both. After 24 hours, proteins were measured by Western blotting and normalized to β-actin, and the results presented are the ratio of p-PKB (Thr308) to PKB. B, Rictor-targeted siRNA reduces the level of Rictor. LPS-treated 3T3-L1 cells were transfected with 5nM Rictor-targeted siRNA, or a control RNAi for 24 hours. Protein was measured by Western blotting and normalized to β-actin levels. C and D, Effects of Rictor-targeted siRNA and emodin on the ratio of p-PKB (Ser473) to PKB and IL-6 production. LPS-treated 3T3-L1 adipocytes were incubated with vehicle (control siRNA), 5nM Rictor-targeted siRNA, 10μM emodin, or a combination of both. After 24 hours, proteins were measured by Western blotting and normalized to β-actin, and the results presented in C are the ratio of p-PKB (Ser473) to PKB. IL-6 production was measured by ELISA assay (D). All values are mean ± SEM and expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

PTEN/PI3K/PKB signaling mediated by 11β-HSD1 is associated with activation of NF-κB

To understand how activation of PI3K/PKB signaling might suppress proinflammatory cytokine production in LPS-treated 3T3-L1 cells, we measured the levels of IκBα and p-IκBα (Ser32/36). IκBα forms an inhibitory complex with NF-κB in the cytoplasm, and phosphorylation of IκBα marks IκBα for degradation, freeing activated NF-κB, which translocates to the nucleus (34). We found that wortmannin increased the ratio of p-IκBα (Ser32/36) to IκBα in LPS-treated cells, whereas emodin decreased the ratio of p-IκBα (Ser32/36) to IκBα and also abolished the effect of wortmannin (Figure 5A). In contrast, 1pM cortisone increased the ratio of p-IκBα (Ser32/36) to IκBα (Figure 5, B and C), whereas this effect of cortisone was blocked by emodin (Figure 5C). Similarly, 1pM dexamethasone increased the ratio of p-IκBα to IκBα (Supplemental Figure 6A). Measurement of NF-κB revealed that emodin reduced the level of the p50 subunit of NF-κB in the nuclear fraction relative to the cytosolic fraction from LPS-treated 3T3-L1 cells (Figure 5D), whereas 1pM cortisone or 1pM dexamethasone increased the level of p50 subunit of NF-κB in the nuclear fraction relative to the cytosolic fraction (Figure 5E and Supplemental Figure 6B). These results indicate that the activation of PI3K that is mediated by inhibiting 11β-HSD1 is associated with suppressed NF-κB signaling in LPS-treated 3T3-L1 adipocytes.

Figure 5. Effects of emodin, cortisone and wortmannin (Wort.) on IκBα and NF-κB in LPS-treated 3T3-L1 adipocytes.

Figure 5.

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A–C, p-IκBα to IκBα ratio is decreased by emodin, but increased by cortisone or Wort. LPS-treated 3T3-L1 adipocytes were incubated with (A) vehicle (control), 10μM emodin, 200nM Wort., or a combination of both 10μM emodin and 200nM Wort.; or (B) vehicle (control), 1pM cortisone, 200nM Wort., or a combination of both 1pM cortisone and 200nM Wort.; or (C) vehicle (control), 10μM emodin, 1pM cortisone, or a combination of both 10μM emodin and 1pM cortisone. After 24 hours, proteins were measured by Western blotting and normalized to β-actin, and the results are presented as the ratio of p-IκBα to IκBα. D and E, The nuclear NF-κB p50 subunit is reduced by emodin but enhanced by cortisone. LPS-treated 3T3-L1 adipocytes were incubated with vehicle (control) or indicated concentrations of emodin (D); or the cells were incubated with vehicle (control) or 1pM cortisone (E). After 24 hours, levels of NF-κB p50 subunit were measured in the nuclear (left panel) and cytosolic fractions (right panel) by Western blotting and were normalized to PCNA and β-actin, respectively. All values are mean ± SEM and expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

PTEN and proinflammatory mediators are suppressed in visceral adipose tissues of diabetic rats administered the 11β-HSD1 inhibitor emodin

To determine whether the inhibition of 11β-HSD1 leads to reduce PTEN and IL-6 in adipose tissue in vivo, 100-mg/kg emodin or vehicle was administered as a daily oral gavage to HFD/STZ-induced diabetic rats for 21 days. Table 1 summarizes the metabolic parameters for the diabetic rats that received emodin or vehicle treatment. The emodin-treated group showed a significant reduction in FBG and fasting insulin levels, and in mRNA levels for IL-6, CD68, and PTEN, when compared with the vehicle-treated group (Figure 6, A–C). Treatment with emodin also resulted in lowering the protein level of PTEN (Figure 6D) and the ratio of p-IκBα to IκBα (Figure 6E). However, body weight, food uptake, and plasma lipid profiles were similar for both groups.

Table 1.

Metabolic Parameters of Treated and Untreated Rat Groups

Metabolic Parameters Control Group
 Emodin Treatment Group
T = 0 T = 21 d T = 0 T = 21 d
Food uptake (g per rat/d) 20.1 ± 2.18 23.3 ± 3.53 19.2 ± 3.92 24.2 ± 2.7
Body weight (g) 367 ± 8 376 ± 9 378 ± 11 362 ± 15
FBG (mmol/L) 14.69 ± 0.92 13.75 ± 0.9 15.85 ± 1.34 9.98 ± 1.44a
Fasting insulin (μIU/mL) — 45.21 ± 4.27 — 20.89 ± 2.26b
Triglyceride (nmol/L) — 0.92 ± 0.05 — 1.00 ± 0.06
Cholesterol (mmol/L) — 2.00 ± 0.07 — 2.10 ± 0.11
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All values are expressed as the mean ± SEM (n = 8 per group). The dashes means Not Measured.

a

.01 < P < .05, according to Student's t test.

b

P < .01, according to Student's t test.

Figure 6. Emodin treatment decreases levels of IL-6, CD68, PTEN, and the ratio of p-IκBα to IκBα in visceral fat tissue.

Figure 6.

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HFD/STZ-induced diabetic rats were administered emodin (100 mg/kg · d) or vehicle for 21 days. mRNA levels of IL-6 (A), CD68 (B), and PTEN (C) were then measured in resected visceral fat using qRT-PCR and normalized to18S RNA. Protein levels of PTEN, p-IκBα, and IκBα (presented as the ratio of p-IκBα to IκBα) were measured by Western blotting and normalized to β-actin (D and E). Results are the mean ± SEM and fold-change values relative to the controls are shown (n = 4 per group). *,.01 < P < .05; **, P < .01, Student's t test.

Discussion

Activation of TLR4 on adipocytes by LPS or saturated fatty acids is known to trigger innate immune responses, and the resulting increase in IL-6 and other proinflammatory cytokines is thought to contribute in vivo to insulin resistance and other aspects of the metabolic syndrome (32, 35, 36). Here, we provide evidence, for the first time, that this response of 3T3-L1 adipocytes to LPS, and to palmitate, is greatly reduced in the presence of emodin, a specific inhibitor of 11β-HSD1, and we have confirmed the effect of 11β-HSD1 by showing that siRNA specifically directed against 11β-HSD1 also blocks the proinflammatory cytokine response of 3T3-L1 adipocytes to LPS. It has been noticed previously that adipose tissues from 11β-HSD1−/− homozygous mice fed a basal diet can show less basal production of IL-6 (19). However, this difference in basal production does not hold under all circumstances, and the responses of tissues from 11β-HSD1−/− homozygous mice to LPS may be modified by chronic changes that are secondary to the homozygous knockout, such as changes in tissue vascularity and oxygenation (37). By contrast, the effects we report here in cultured 3T3-L1 cells are evidence for direct autocrine/paracrine effects of 11β-HSD1 activity in the LPS-stimulated innate immune response of these cells to produce proinflammatory cytokines, including IL-6.

It has previously been shown that wortmannin can activate basal NF-κB and increase basal IL-6 production and acts additively to the stimulation of NF-κB and IL-6 production by palmitate, another agonist of TLR4, in mature 3T3-L1 cells, implicating PI3K in the control of the innate immune response of adipocytes (29). We confirmed this by showing that wortmannin stimulated IL-6 production, decreased the ratio of p-PKB (Ser473) to PKB, and increased the ratio of p-IκBα (Ser32/36) to IκBα in 3T3-L1 adipocytes in response to LPS. Because it is well established that phosphorylation of IκBα triggers nuclear translocation of NF-κB and contributes to activation of NF-κB signaling, these results with wortmannin indicate that inhibition of the activity of PI3K can augment the innate immune response to LPS stimulation in 3T3-L1 cells at least in part by increasing nuclear translocation of NF-κB. Consistent with this, it has been reported that PI3K/PKB signaling inhibits inflammation in monocytes and macrophages in response to Gram-negative bacteria or LPS (38, 39). One possible mechanism linking activity of PI3K/PKB to the nuclear translocation of NF-κB is that activation of PI3K/PKB phosphorylates glycogen synthase kinase 3β at the residue Ser9 and inactivates the kinase, resulting in negatively regulated NFκB, including inhibition of nuclear translocation of p65 (39). Moreover, it is already known that emodin or its derivatives inhibit glycogen synthase kinase 3β activity in various cell types (40, 41). However, PTEN deletion can suppress LPS-induced IL-6 production in the monocyte/macrophage lineage, without changing LPS-induced degradation of IκBα or nuclear translocation of NF-κB (42). Further work is therefore required to investigate the significance of this apparent difference of NF-κB control after activation of PI3K/PKB signaling in the monocyte/macrophage lineage compared with 3T3-L1 adipocytes.

The results with wortmannin do not address how PI3K/PKB signaling is controlled endogenously in 3T3-L1 cells. However, our further observations implicate adipocytic 11β-HSD1 and PTEN in this. We found that LPS increases the expression of PTEN in 3T3-L1 cells and that this effect is abolished by emodin (Supplemental Figure 7A). Moreover, we found that inhibition of 11β-HSD1, either with emodin or with 11β-HSD1-specific siRNA, inhibits PTEN expression in LPS-treated 3T3-L1 cells. This depletion of PTEN caused by inhibiting the activity of 11β-HSD1 is likely to be responsible for the associated reduction of proinflammatory cytokines because treatment with PTEN-specific siRNA, which reduced PTEN to a similar extent as emodin, strongly inhibits expression of IL-6, IL-1β, and TNF-α in response to LPS by 3T3-L1 cells, without significantly affecting the protein level of 11β-HSD1. Furthermore, emodin could not further inhibit IL-6 production in response to LPS in 3T3-L1 cells in which PTEN had already been inhibited by specific siRNA. These results suggest that inhibition of 11β-HSD1, either by emodin or siRNA, reduces IL-6 production via suppression of PTEN in LPS-treated 3T3-L1 adipocytes.

In contrast to the inhibitory effects of emodin on LPS-induced PTEN levels and IL-6 production, emodin did not alter the basal level of either IL-6 or PTEN (Supplemental Figure 7A). Nevertheless, IL-6 is still under the control of PTEN levels even under basal conditions because we found that depleting PTEN with targeted siRNA also inhibited basal IL-6 production in the absence of LPS (Supplemental Figure 7, B and C). Our finding of this basal suppression is also consistent with the previous finding that the activity of PI3K can increase basal NF-κB and basal IL-6 production in mature 3T3-L1 cells (29). The activation of TLR4 by LPS entrains complex and specific intracellular cascades of messengers that are not significantly activated in the absence of TLR4 stimulation (43). These results suggest that 1 or more of the intracellular cascade(s) entrained on activating TLR4 in 3T3-L1 cells can regulate PTEN and, thereby, control IL-6 production, that these cascade(s) can be significantly modulated by the activity of 11β-HSD1, but that these intracellular cascade(s) are not significant activated in the absence of stimulation of TLR4. Other agents are also known to abolish IL-6 production in response to LPS without affecting basal IL-6 levels in 3T3-L1 (44) and other cells (45), consistent with the idea that there are probably several ways in which the principal controls of basal IL-6 production differ from those after LPS stimulation.

PTEN controls the supply of PIP3, which is a substrate for the activation of PKB, and emodin increased the ratio of p-PKB (Ser473) to PKB but did not alter the ratio of p-PKB (Thr308) to PKB in LPS-treated 3T3-L1 adipocytes, indicating that the phosphorylation of PKB in response to emodin involves mTORC2 rather than PDK1 (33). This was further supported by fact that emodin was not able to increase the p-PKB (Ser473) to PKB ratio in Rictor-depleted cells. Further studies are required to investigate the potential effects of emodin on other components of mTORC2, such as protein observed with rictor-1, mammalian stress-activated protein kinase interacting protein 1, mammalian lethal with SEC13 protein 8, DEP domain-containing mTOR-interacting protein, and mTOR. Comparable results have been reported in cells and tumors of skeletal muscle origin, where Ser473, but not Thr308, is the key target residue through which PTEN modulates PKB phosphorylation and activation (46). In contrast, overexpression of PTEN decreases phosphorylation of PKB at Thr308 in HT-29 epithelial cells (47), or at both Ser473 and Thr308 residues in U87 glioblastoma cells and in A6 epithelial cells (48, 49). Whether the target residue by which PTEN modulates PKB activation depends on cell type remains unknown.

Inhibition of 11β-HSD1-mediated activation of the PI3K/PKB pathway was associated with inhibition of NF-κB signaling in 3T3-L1 cells responding to LPS. The cytokine response to activation of TLR4 is only partly dependent of the activation of NF-κB (43), and NF-κB can be activated through the binding of IL-1β and TNF-α to their own receptors, as well as through activation of TLR4 (15). Further work is therefore necessary to determine how LPS might control the activity of NF-κB through the modulation of PTEN by 11β-HSD1, and whether other proinflammatory transcription factors, apart from that of NF-κB, can be regulated by 11β-HSD1 in adipocytes. Nevertheless, our results suggest for the first time that inhibition of 11β-HSD1 in adipocytes inhibits levels of PTEN activates the PI3K signaling pathway and inhibits the innate immune response to LPS stimulation and that this occurs in association with a reduction in NF-κB activation. In addition, we found that emodin inhibited the increased level of IL-6 stimulated by treating 3T3-L1 cells with palmitate, another TLR4 agonist that induces NF-κB signaling in adipocytes (29). Moreover, emodin reduced the level of PTEN in palmitate-treated 3T3-L1 cells and could not further decrease IL-6 levels in 3T3-L1 cells in which PTEN had been depleted. These results suggest that inhibition of 11β-HSD1 also reduces proinflammatory effects caused by palmitate in adipocytes, possibly through inhibition of PTEN levels, just as we found for the LPS-driven proinflammatory effects in these cells.

It is likely that the effects of inhibition of 11β-HSD1 on PTEN, the PI3K signaling pathway and IL-6 production depend on activation of the adipocyte glucocorticoid receptor, because we found that picomolar concentration of cortisone, or of dexamethasone, which is a glucocorticoid receptor-selective agonist (28), stimulated IL-6 production and PTEN expression, reduced p-PKB to PKB ratio, and increased p-IκBα (Ser32/36) to IκBα ratio and nuclear NF-κB subunit p50 in 3T3-L1 cells. At present, it is unclear how activation of the glucocorticoid receptor in adipocytes links to increases in PTEN levels. However, the control of PTEN levels by 11β-HSD1 may not only modulate the innate immune response of adipocytes, but may also partly mediate the prodiabetic effects of these cells, because the binding of insulin to its receptor activates the PI3K pathway, mediating effects on glucose homeostasis (50), and adipocyte-specific deletion of PTEN increases insulin sensitivity and the resistance to STZ-induced diabetes in mice (51).

Our evidence for a proinflammatory effect of endogenous glucocorticiods generated by the activity of 11β-HSD1 in 3T3-L1 adipocytes may seem counter to a general view of glucocorticoids as antiinflammatory agents. However, at the level of gene expression, glucocorticoids have both stimulatory and inhibitory effects on lymphoid cells and macrophages (52–54), and it has been proposed that glucocorticoids within the normal diurnal concentration range are permissive or stimulate host defenses through both glucocorticoid and mineralocorticoid receptors, whereas higher concentrations dampen host defense mechanisms through the glucocorticoid receptor to prevent stress-activated overstimulation of host defenses (55, 56). Our data with dexamethasone and cortisone, that low concentrations enhanced LPS-stimulated IL-6 release whereas higher concentrations suppressed it, support this idea in the context of the innate immune response of adipocytes. Moreover, we found that low concentrations of cortisone or dexamethasone had opposite effects to those of emodin on PTEN levels, p-PKB to PKB ratio, p-IκB to IκB ratio, and the nuclear translocation of NFκB p50 subunit in LPS-stimulated 3T3-L1 cells. Our results with low concentrations of cortisone, which requires conversion to cortisol by 11β-HSD1 in order to bind and activate the corticosteroid receptors (1, 28), and our results using low concentrations of dexamethasone, therefore together support the idea that low concentrations of endogenous glucocorticoids produced by 11β-HSD1 are proinflammatory in 3T3-LI adipocytes through actions to control PTEN/PKB and IκB/NFκB signaling.

Consistent with its effect as a selective inhibitor of 11β-HSD1, emodin is known to lower FBG and insulin levels (57). In addition to these effects, we found that emodin decreased levels of PTEN, IL-6, and the ratio of p-IκBα to IκBα in visceral fat tissues from HFD/STZ diabetic rats. These decreases are consistent with the decreases we observed in the response of 3T3-L1 adipocytes to LPS in vitro. Moreover, we observed that emodin treatment reduced the mRNA levels of CD68 (a macrophage marker) in visceral fat tissues in these rats, indicating that macrophage infiltration was suppressed. Previous studies have shown that emodin reduces the levels of serum glucose, circulating inflammatory cytokines and acute phase proteins in rats fed with HFD/high-fructose diet (58) and in KKAy diabetic mice (8). Further work is now required to confirm that emodin treatment reduces circulating inflammatory mediators in other models of diabetes. However, our data support the hypothesis that inhibiting endogenous 11β-HSD1 in visceral fat reduces inflammation in visceral fat from HFD/STZ diabetic rats and can do so, at least in part, by modulating PTEN-dependent inflammatory adipokine production.

Conclusions

The results of the present study establish that proinflammatory responses of 3T3-L1 adipocytes to LPS depend significantly on the activity of 11β-HSD1 in these cells and that inhibition of 11β-HSD1 reduces LPS-induced proinflammatory innate immune in adipocytes by down-regulating PTEN expression, leading to activation of the PI3K/PKB pathway via mTORC2, in association with an attenuation of the ratio of p-IκBα to IκBα and a decrease of nuclear translocation of NF-κB subunit p50 (Figure 7). The proinflammatory increase in IL-6 levels produced by palmitate in 3T3-L1 cells is similarly associated with an increase in PTEN levels, and these effects of palmitate can also be blocked by inhibiting 11β-HSD1 activity. Further studies are required to define the exact roles of the modulation by 11β-HSD1 of the effects of proinflammatory mediators such as LPS and palmitate under particular diabetic conditions. However, our data suggest that therapeutic inhibition of 11β-HSD1 may help to ameliorate the chronic inflammation occurring in the adipose tissue of type 2 diabetes.

Figure 7. A schematic diagram illustrating that 11β-HSD1 modulates LPS-induced innate immune response in adipocytes.

Figure 7.

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Emodin or 11β-HSD1-trageted siRNA (RNAi) inhibits LPS-induced PTEN level and its downstream effects on cytokine production. Bolded arrows indicate the direction of the effects of emodin and/or RNAi. The zigzag line represents the signaling cascades entrained by activating TLR4, and + indicates that LPS/TLR4 signaling induces PTEN.

Acknowledgments

This work was supported by the Collaborative Innovation Center for Rehabilitation Technology of Fujian University of Traditional Chinese Medicine and the Rehabilitation Research Center of the State Administration of Traditional Chinese Medicine of the People's Republic of China SATCM.

Disclosure Summary: The authors have nothing to disclose.

Funding Statement

This work was supported by the Collaborative Innovation Center for Rehabilitation Technology of Fujian University of Traditional Chinese Medicine and the Rehabilitation Research Center of the State Administration of Traditional Chinese Medicine of the People's Republic of China SATCM.

Footnotes

Abbreviations:
CD68
cluster of differentiation 68
DMSO
dimethyl sulfoxide
FBS
fetal bovine serum
FBG
fasting blood glucose
HFD
high-fat diet
11β-HSD1
11β-hydroxysteroid dehydrogenase type 1
LPS
lipopolysaccharide
mTORC2
mammalian target of rapamycin complex 2
NF-κB
nuclear factor κB
p-IκB
phosphorylated inhibitor of κB
PIP3
phosphatidylinositol (3,4,5)-triphosphate
PCNA
proliferating cell nuclear antigen
PI3K
phosphatidylinositol 3-kinase
PKB
protein kinase B
PTEN
phosphatase and tensin homologue
qRT-PCR
quantitative real-time RT-PCR
Rictor
rapamycin-insensitive companion of mTOR
RNAi
RNA interference
siRNA
small interfering RNA
STZ
streptozotocin
TLR4
Toll-like receptor 4.

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