Homocysteine Upregulates Resistin Production From Adipocytes In Vivo and In Vitro

MnTMPyP, N-acetyl-cysteine (NAC), RO318220, calphostin C, 3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione, and MG132 were purchased from CalBiochem (La Jolla, CA). Anti–insulin receptor-β, anti–insulin receptor substrate (IRS)-1, anti-phospho-IRS-1 (Ser307), anti-Akt, anti–phospho-Akt (Ser473), anti–phospho-protein kinase C (PKC) (pan) (βII Ser660), anti–phospho-PKC-α/βII, anti–phospho-IκB kinase (IKK)-α/β (Ser180/181), and anti–nuclear factor-κB (NF-κB) p65 antibodies, all from rabbit source, were purchased from Cell Signaling Technology (Beverly, MA). Mouse anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit anti–IκB-α and mouse anti–β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-murine resistin antibody was purchased from CytoLab (Rehovot, Israel). IRDye-conjugated affinity-purified anti-rabbit, -mouse, and -goat IgGs were purchased from Rockland (Gilbertsville, PA). 2-deoxy-d-[1-³H]glucose was purchased from GE Healthcare (Buckinghamshire, UK). All other chemicals and drugs were purchased from Sigma Chemical (St. Louis, MO).

Male C57BL/6J mice, 6 weeks old, were fed standard mouse chow with or without 1.8 g/l DL-Hcy added to the drinking water for 4 weeks, as previously described (17). For the oral glucose tolerance tests, mice were fasted for 12 h before the gastric administration of glucose (3 g/kg body wt) by gavage. For insulin tolerance test, mice were fasted for 4 h, followed by intraperitoneal injection of 2 IU/kg. Blood was drawn from a cut at the tip of the tail at 0, 30, 60, 90, and 120 min, and blood glucose concentrations were detected immediately. After 3 days’ adaptation, mice were killed; blood and epididymal fat pads were taken. All animal protocols were approved by the Animal Care and Use Committee of Peking University.

Mouse epididymal adipose tissue (∼200 mg) was homogenized as previously described (25); the tissue lysate was centrifuged at 12,000 rpm for 15 min twice at 4°C. The clear lysate fraction was collected for Hcy and protein measurements. Total Hcy level in plasma and adipose tissue extracts was quantified by gas chromatography–mass spectrometry (17).

Triglycerides, total cholesterol and high-density lipoprotein cholesterol contents in plasma were measured using commercial kits from Biosino Biotechnology and Science (Beijing, China). Total blood glucose content was measured with Glucotrend from Roche Diagnostics (Mannheim, Germany). Resistin levels in plasma were assayed using the murine resistin EIA kit from Cayman Chemical (Ann Arbor, MI), which cross-reacts at 100% with murine resistin and shows <0.01% cross-reactivity with recombinant murine RELM-α and -β (data from Cayman Chemical). Insulin levels in plasma were assayed with the ELISA insulin kit from Linco Bioscience Institute (St. Charles, MO). Malondialdehyde contents in plasma and adipose tissue extracts were measured using commercial kits from Tianlai Biotechnology and Science (Beijing, China).

Mature adipocytes were isolated from epididymal fat pads of Sprague-Dawley rats (aged 6–8 weeks; 160–200 g) as previously described (26). Packed adipocytes were diluted in serum-free Dulbecco’s modified Eagle’s medium (DMEM) to generate a 10% (vol/vol) cell suspension. After being incubated at 37°C for 1 h, adipocytes were treated with Hcy (30–1,000 μmol/l) for 8–24 h. For the inhibition experiments, primary rat adipocytes were pretreated with the indicated inhibitors for 1 h before stimulation with Hcy at 500 μmol/l for 24 h.

Adipose precursor cells were isolated from epididymal fat pads of 6-week-old Sprague-Dawley rats (∼150 g) and differentiated into adipocytes for 3 days in serum-free DMEM-F12 (1:1) medium supplemented with 5 μg/ml insulin, 33 μmol/l biotin, and 200 pmol/l triiodothyronine (26). Differentiated adipocytes were then incubated in serum-free DMEM-F12 (1:1) medium for another 2 days before treatment.

Following treatment, differentiated rat adipocytes were glucose starved for 30 min. Insulin (100 nmol/l) was then added for 30 min, followed by the addition of labeled glucose (0.33 μCi of 2-deoxy-d-[1-³H]glucose/ml) in the continued presence of insulin for another 2 h. After terminating the reaction with three washes of ice-cold PBS, the plate was set in the Filtermate Havester (Packard Bioscience, Meriden, CT) and tritium counts were obtained.

Total RNA from primary rat adipocytes or mouse epididymal adipose tissue was isolated by use of Trizol reagent (Promega, Madison, WI) and reverse transcribed using the reverse transcription system (Promega, Madison, WI). Then the reaction mixture underwent PCR. The amount of PCR products formed in each cycle was evaluated on the basis of SYBR Green I fluorescence. Primers for the rat and mouse resistin, adiponectin, leptin, TNF-α, IL-6, PAI-1, and β-actin are in Table 1. All amplification reactions were performed using the Mx3000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA). Results were analyzed with Stratagene Mx3000 software.

Following treatment, primary rat adipocytes were packed and homogenized in ice-cold fractionation buffer (26). The cell lysate was incubated on ice for 15 min and then centrifuged at 20,000g for 30 min at 4°C. The cytosolic fraction was collected. Nuclear proteins were extracted with use of NE-PER reagents (Pierce, Rockford, IL).

Proteins were subjected to SDS-PAGE with a 10% running gel and then transferred to a polyvinylidene fluoride membrane. The membrane was incubated successively with 0.1% bovine serum albumin in tris-Tween-buffered saline at room temperature for 1 h, with different antibodies at 4°C for 12 h, and then with IRDye-conjugated second antibody for 1 h. A specific immunofluorescence band was detected by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Following treatment, differentiated rat adipocytes were incubated with a reactive oxygen species (ROS) probe dye 2′, 7′-dichlorofluorescin diacetate (20 μmol/l). Determination of intracellular oxidant production was based on the oxidation of dichlorofluorescin diacetate by intracellular ROS, resulting in the formation of the fluorescent compound 2′, 7′-dichlorofluorescin (16). Dichlorofluorescin fluorescence was monitored by confocal laser scanning microscopy (Leica, Germany).

NF-κB DNA-binding activity was measured using a colorimetric transcription factor assay kit (Chemicon, Temecula, CA) according to the manufacturer’s instructions (17). In brief, double-stranded biotinylated oligonucleotide containing the flanked DNA binding consensus sequence for NF-κB was mixed with nuclear extract. The bound NF-κB subunit, p65, was detected with a specific primary antibody, a rabbit anti–NF-κB p65. The horseradish peroxidase–conjugated secondary antibody was then used for spectrophotometric detection.

Data are expressed as means ± SEM. Data analysis used GraphPad Prism software. One-way ANOVA, Student-Newman-Keul’s test (comparisons between multiple groups), or unpaired Student’s t test (between two groups) was used as appropriate. P < 0.05 was considered significant.

The HHcy mice model was confirmed by a significant increase of Hcy concentration (2.4-fold) in plasma, accompanied by an even higher increase in adipose tissue (7.4-fold) (Table 2). Homeostasis model assessment of insulin resistance, but not the body weight, plasma lipid, and insulin levels in HHcy mice, was significantly high (Table 2). Two weeks of Hcy supplementation significantly increased blood glucose levels during feeding, an effect observed up to 4 weeks, whereas blood glucose levels during fasting were not affected (Fig. 1A). We further examined insulin sensitivity by performing oral glucose tolerance test and insulin tolerance test. HHcy mice demonstrated severe hyperglycemia upon administration of glucose, as well as impaired glucose tolerance, with typical glucose disposal curves of insulin resistance (Fig. 1B). Similarly, the insulin-stimulated glucose disposal curves in HHcy mice markedly shifted to the right relative to normal mice (Fig. 1C).

We next examined whether the signaling capacity of insulin receptor in adipose tissues of HHcy mice was impaired. HHcy mice showed significant reduction in tyrosine phosphorylation of insulin receptor and IRS-1 (Fig. 1D and E), a significant increase in serine phosphorylation of IRS-1 (Fig. 1E), and a decrease in insulin-induced Akt phosphorylation, a downstream molecule of insulin receptor signaling (Fig. 1F). All these data suggest that HHcy may promote insulin resistance of adipose tissues in vivo.

We then examined the effect of Hcy on insulin resistance by assaying the insulin-stimulated glucose uptake in cultured differentiated rat adipocytes. No significant change was found in the lactate dehydrogenase release rate from the adipocytes in response to Hcy treatment (11.01 ± 1.21 vs. 11.59 ± 0.64%, P = 0.323), which indicated no significant cytotoxity. Hcy (100–1000 μmol/l) impaired insulin-mediated 2-deoxy-d-[1-³H]glucose uptake in a time- and concentration-dependent manner, with maximal inhibition at 24 h and maximal inhibition of ∼70% found with 1,000 μmol/l Hcy (Fig. 2A and B), which suggests that Hcy impairs insulin sensitivity of rat adipocytes in vitro.

To investigate whether Hcy interferes with insulin action, we then examine the effect of Hcy on insulin-induced phosphorylation events in primary rat adipocytes. Hcy significantly decreased insulin-stimulated tyrosine phosphorylation of insulin receptor and IRS-1 (Fig. 2C and D), increased the serine phosphorylation of IRS-1 (Fig. 2D), and inhibited the Akt phosphorylation (Fig. 2E).

To determine whether the Hcy-mediated impairment of insulin sensitivity in adipocytes is an autocrine/paracrine effect, primary rat adipocytes or Cos-7 cells were treated with Hcy for 24 h and then conditioned media were collected and used to treat differentiated rat adipocytes. After another 24 h incubation, adipocytes treated with the conditioned medium from Hcy-treated adipocytes showed significant reduction in insulin-stimulated glucose uptake (Fig. 2F), which indicates that Hcy promotes insulin resistance through autocrine or paracrine effects.

We next examined whether Hcy induces the expression of inflammation-related adipokines. Epididymal adipose tissue of HHcy mice showed increased mRNA and protein levels of resistin relative to that in controls (Fig. 3A and B), accompanied by elevated plasma resistin level (Table 2). The mRNA expression of TNF-α and PAI-1 in HHcy mice was higher but that of leptin was lower than that in control mice, while adiponectin and IL-6 mRNA expressions remained unchanged (Fig. 3A).

Exposure of primary rat adipocytes to Hcy (500 μmol/l) for 24 h also induced a significant increase in resistin mRNA (Fig. 3C). Contrary to the in vivo results, adiponectin expression decreased significantly, with leptin, TNF-α, IL-6, and PAI-1 expression showing no change (Fig. 3C). Hcy at 500 μmol/l induced resistin mRNA expression in a time-dependent manner in primary rat adipocytes. Elevated resistin mRNA level occurred as early as 8 h and remained increased up to 24 h in these adipocytes (Fig. 3D). Hcy treatment (300–1000 μmol/l) for 24 h also caused a concentration-dependent increase in resistin mRNA expression (Fig. 3E), with maximal induction of approximately fourfold found with 1,000 μmol/l Hcy. Considering that Hcy promoted insulin resistance through the autocrine or paracrine effect of the induced adipokine(s), resistin may be an important target for Hcy-induced insulin resistance.

ROS has been suggested to mediate the expression of a few cytokines induced by Hcy in monocytes (16,17). We therefore examined whether Hcy also induced ROS production in adipocytes. Since malondialdehyde is an index of lipid peroxidation, increase malondialdehyde content is usually used as a marker of oxidative injury. HHcy mice showed a significant increase of malondialdehyde level (1.9-fold) in plasma, accompanied by a similar increase in adipose tissue (Table 2). Increased intracellular ROS level in differentiated rat adipocytes occurred at 15 min and peaked at 60 min after the addition of 500 μmol/l Hcy (Fig. 4A). To determine whether ROS participated in Hcy-induced resistin expression, primary rat adipocytes were pretreated for 1 h with the ROS scavengers NAC (2 mmol/l) or MnTMPyP (50 μmol/l) and showed significantly attenuated resistin expression induced by 500 μmol/l Hcy for 24 h (Fig. 4B).

Adipocytes express all PKC isoforms except PKC-γ (27). HHcy mice showed significantly increased phosphorylation of PKCs in epididymal adipose tissue (Fig. 5A). Exposure of primary rat adipocytes to 500 μmol/l Hcy also caused a significant increased phosphorylation of PKC (Fig. 5C), especially the α/βII isoform (Fig. 5B and D). Treatment with the PKC activator phorbol myristate acetate (20 nmol/l) for 24 h directly elevated resistin expression in these cells (Fig. 5E). In addition, 500 nmol/l phorbol myristate acetate added to cells for 20 h to exhaust PKCs or pretreatment with the PKC inhibitors RO 318220 (1 μmol/l), calphostin C (200 nmol/l), or the specific PKC-βII inhibitor 3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione (1 μmol/l) for 1 h significantly attenuated the Hcy-induced resistin expression (Fig. 5F–H). Inhibition of cAMP-dependent protein kinase (rp-cAMPs) or cGMP-dependent protein kinase (KT 5823) produced no effect (Fig. 5G). Furthermore, Hcy-induced PKC activation was inhibited by pretreatment of cells with the ROS scavenger MnTMPyP (50 μmol/l) or NAC (2 mmol/l) for 1 h (Fig. 5I). These results strongly indicate that PKC-βII may act downstream ROS to regulate the Hcy-induced resistin expression in primary rat adipocytes.

Since the resistin promoter contains NF-κB binding sites and the activation of NF-κB might be regulated by ROS in adipocytes (28), we next studied whether NF-κB is involved in Hcy-induced resistin expression. HHcy mice significantly induced phosphorylation of IKKs (Fig. 6A) in epididymal adipose tissue, followed by decreased I-κB protein level (Fig. 6B). Exposure of primary rat adipocytes to 500 μmol/l Hcy caused the nuclear translocation of the p65 subunit of NF-κB between 1 h and 6 h (Fig. 6C). Hcy also significantly enhanced NF-κB DNA-binding activity (Fig. 6D). The NF-κB inhibitors MG 132 (10 μmol/l) and pyrrolidine dithiocarbamate (10 μmol/l) significantly attenuated Hcy-induced resistin mRNA expression in adipocytes (Fig. 6E). In addition, adipocytes infected with recombinant adenovirus-expressing I-κB showed a significant decrease in Hcy-induced resistin expression (Fig. 6F). Furthermore, pretreatment of adipocytes with MnTMPyP (50 μmol/l) or calphostin C (200 nmol/l) significantly attenuated the nuclear translocation (Fig. 6G) and DNA-binding activity of NF-κB (Fig. 6H), which indicates that the ROS-PKC–NF-κB pathway is involved in Hcy-induced resistin expression in primary rat adipocytes.

In the present study, we have demonstrated for the first time that Hcy increases resistin expression and secretion from adipocytes via the ROS-PKC–NF-κB pathway and subsequently promotes insulin resistance both in vitro and in vivo.

Insulin resistance is speculated to be associated with a low-grade systemic inflammatory condition (29). The close relation between inflammation and diabetes is supported by the observation that stimulation of the innate immune response by bacterial endotoxin results in insulin resistance (30,31). The interaction between inflammation and insulin signaling is also suggested by the ability of aspirin to improve insulin resistance, in part by preventing the antagonistic effects of fatty acids and cytokines (32). Hcy is considered to be a potent proinflammatory factor. In vitro studies in rat hepatoma cells transfected with human insulin receptors suggested that the oxidative stress produced by Hcy thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase activity and inhibition of glycogen synthesis, thereby impairing insulin signaling (23,24). Hcy is therefore proposed to be a new mediator of oxidative stress–induced insulin resistance.

In the present study, HHcy mice manifested glucose intolerance, insulin resistance, and impaired insulin signaling pathway, which is consistent with a previous report of Sprague-Dawley rats (33) and epidemiological studies (18–22), despite other epidemiological studies finding no association between plasma Hcy level and insulin resistance (34,35). Besides finding the direct impairment of Hcy on the conventional insulin signaling pathway in adipocytes, we found Hcy to induce insulin resistance in differentiated adipocytes possibly in an autocrine/paracrine manner: adipocytes treated with the conditioned medium from Hcy-treated adipocytes also showed a significant reduction in insulin-stimulated glucose uptake, which indicates that Hcy might promote insulin resistance through induced adipokine(s).

Resistin levels are increased in insulin-resistant mice and humans (36,37). Administration of endotoxin to human volunteers was associated with greatly increased circulating resistin levels (10,11); thus, resistin seems to act as a critical mediator of the insulin resistance associated with sepsis and possibly other inflammatory conditions. A strong increase of resistin mRNA by proinflammatory factors also supports the hypothesis that resistin may play a role in the link between inflammation and obesity-associated insulin resistance (5). Our research and other previous studies have demonstrated the effect of Hcy-induced chemokine formation in many cell types, such as monocytic cell lines (16,17), vascular smooth muscle cells (14), and endothelial cells (38). However, little is known about whether Hcy can induce the expression and secretion of adipokines. Here, we show that Hcy is highly accumulated in adipose tissue (3.1 times that in plasma); moreover, increased expression and secretion of resistin is observed in HHcy mice (19 μmol/l in plasma). Short-term treatment with Hcy (300–1,000 μmol/l for up to 24 h) markedly increased resistin expression in primary rat adipocytes. The plasma concentration of Hcy in patients with homozygous homocystinuria was as high as 500 μmol/l (39), and patients with plasma Hcy level of ∼15–30 μmol/l are considered to have moderate HHcy (40). Since this is the range used in the present in vitro and in vivo study, our results have important clinical implications.

The present study also addresses the signaling pathways responsible for Hcy-induced resistin expression in adipocytes. Oxidative stress was observed in HHcy mice, as manifested by increased malondialdehyde levels in both plasma and adipose tissue. Hcy also increased intracellular ROS significantly, and Hcy-induced expression of resistin was inhibited by the antioxidants in adipocytes, which is consistent with previous reports that exposure of adipocytes to hyperglycemic conditions reduces insulin sensitivity and increases ROS levels in vitro (41) and that Hcy induces ROS formation in monocytes followed by chemokine formation (16,17) but argues against other results that ROS is not involved in Hcy-induced secretion of chemokines from endothelial cells (38). Moreover, our results do not support the observation that oxidative stress, from either short-term exposure to high concentrations of H₂O₂ or sustained exposure to low concentrations of H₂O₂, significantly decreases resistin expression and secretion in 3T3-L1 adipocytes (42). Increased ROS levels are an important trigger for insulin resistance in numerous settings (43); adipocytes isolated from animals with high-fat diet–induced diabetes display significantly elevated ROS levels, together with reduced insulin-mediated glucose uptake (44). The fact that ROS mediates elevated resistin level may give a new mechanism for oxidative damage–induced insulin resistance.

Primary cultured rat adipocytes express all PKC isoforms except PKC-γ (27). Our results show that in adipocytes, PKCs, most likely PKC-βII, are significantly activated with Hcy stimulation both in vivo and in vitro. In addition, Hcy-induced resistin expression is PKC dependent, since resistin expression is stimulated directly by the PKC activator and significantly attenuated if PKCs are depleted or blocked. Furthermore, PKCs act downstream of ROS in the Hcy-responsive signal pathway in adipocytes, which agrees with their pattern in hepatoma cells, thus showing that ROS plays a central role in mediating 12-O-tetradecanoylphorbol-13-acetate–triggered PKC activation (45). Inactivation of PKCs, most likely PKC-βII, elevates glucose uptake (46), and release of PKC-λ from PKC-βII is an important mechanism in the regulation of glucose transporter distribution in 3T3-L1 adipocytes (47). Our results that PKCs participate in the Hcy-induced resistin expression suggest that PKCs are involved in insulin resistance.

The superoxide radical activates the redox-sensitive proinflammatory transcription factor NF-κB, which increases the transcription of most proinflammatory genes. We have reported a determinant role of oxidative stress in the stimulatory effect of Hcy on chemokine expression in human monocytes, most likely via activation of NF-κB (16,17). Hcy also activates NF-κB in endothelial cells via oxidative stress (48). NF-κB is one of the important signal transduction pathways linking inflammation and insulin resistance (49). Hcy significantly activates IKKs, which then phosphorylates the natural inhibitor of NF-κB, IκB, resulting in the degradation of IκB. Present in vitro results show that Hcy increases NF-κB nuclear translocation and DNA binding activity, and inhibition of NF-κB significantly attenuates Hcy-induced resistin expression in primary rat adipocytes. Elimination of ROS or inhibition of PKCs impairs the Hcy-induced NF-κB nuclear translocation and DNA-binding activity and thus affects resistin expression, suggests that NF-κB is downstream the Hcy-induced ROS-PKC pathway. These results confirm that resistin acts as an inflammatory adipokine that impairs the insulin action. Further study on the promoter activity of resistin will reveal the role of NF-κB in resistin transcription.

Significant induction of TNF-α and PAI-1 and inhibition of leptin was detected in HHcy mice, although we found no change in level of these adipokines under Hcy stimulation in vitro or secretion of these cytokines in vivo or in vitro (data not shown). Overexpression of resistin augments the secretion of inflammatory cytokines such as TNF-α, macrophage chemoattractant protein-1, and IL-6 in 3T3-L1 cells (50). Differences in cell types and experimental condition may account for these different observations, since we induced the intrinsic expression and secretion of resistin by Hcy rather than overexpression by lentivirus (50). The effects of these adipokines in Hcy-induced insulin resistance cannot be totally excluded. Experimental observations in mice do not always hold true for humans; therefore, further investigation is still needed.

In conclusion, this study demonstrates that Hcy significantly enhances adipose resistin production both in vitro and in vivo and, subsequently, promotes insulin resistance. The intracellular oxidative products and subsequent activation of multiple signaling mediators, including PKCs and NF-κB, are involved in Hcy effects in adipocytes. These findings reveal a novel target of Hcy-induced insulin resistance with the direct regulation of the expression and secretion of resistin from adipose tissue.

This work was supported by the National Natural Science Foundation of China (30700376), the Beijing Natural Science Foundation (7083110), the China Postdoctoral Science Foundation (2005038038), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (to Y.L.); the Major National Basic Research Program of China (2006CB503802), a grant from the National Natural Science Foundation of China (30730042), and the Program for Innovative Research Teams in Universities (to X.W.).