XOMA 052, an anti-IL-1β monoclonal antibody, prevents IL-1β-mediated insulin resistance in 3T3-L1 adipocytes§


  • Disclosure: Masahisa Handa, Sandra Vanegas, Nerissa Mendoza, Shirley Zhu, and Amer Mirza were employees of XOMA during the preparation of this manuscript.

  • Present address of Masahisa Handa: Merck Research Labs, Palo Alto, California, USA.

  • §

    Present address of Shirley Zhu: Progenitor Cell Therapy LLC, Mountain View, California, USA.



Interleukin-1β (IL-1β) has recently been implicated as a major cytokine that is involved in the pancreatic islet inflammation of type 2 diabetes mellitus. This inflammation impairs insulin secretion by inducing beta-cell apoptosis. Recent evidence has suggested that in obesity-induced inflammation, IL-1β plays a key role in causing insulin resistance in peripheral tissues.

Design and Methods:

To further investigate the pathophysiological role of IL-1β in causing insulin resistance, the inhibitory effects of IL-1β on several insulin-dependent metabolic processes in vitro has been neutralized by XOMA 052. The role IL-1β plays in insulin resistance in adipose tissue was assessed using differentiated 3T3-L1 adipocytes and several parameters involved in insulin signaling and lipid metabolism were examined.

Results and Conclusion:

IL-1β inhibited insulin-induced activation of Akt phosphorylation, glucose transport, and fatty acid uptake. IL-1β also blocked insulin-mediated downregulation of suppressor of cytokine signaling-3 expression. Co-preincubation of IL-1β with XOMA 052 neutralized nearly all of these inhibitory effects in 3T3-L1 adipocytes. These studies provide evidence, therefore, that IL-1β is a key proinflammatory cytokine that is involved in inducing insulin resistance. These studies also suggest that the monoclonal antibody XOMA 052 may be a possible therapeutic to effectively neutralize cytokine-mediated insulin resistance in adipose tissue.


In most patients with type 2 diabetes mellitus (T2DM), hyperglycemia occurs when pancreatic beta-cells can no longer compensate for the insulin resistance present in peripheral tissues (1,2). Insulin resistance not only plays a role in T2DM but also is one of the clinical hallmarks of “metabolic syndrome” (1,3). The etiology of insulin resistance is complex and includes diet, excess body weight, stress, lack of exercise, and inflammation (1-5). Recently, studies have shown that chronic exposure of insulin-responsive cells to proinflammatory cytokines cause desensitization to insulin action (4,6).

One of the key cytokines involved in diabetic inflammation is interleukin-1β (IL-1β) (7). IL-1β is secreted by several different cell types and exerts robust proinflammatory activities as part of the innate immune response to pathogen infection. Several inhibitors of IL-1β are endogenously expressed to regulate the inflammatory response to prevent host tissue damage. Nonetheless, overproduction of IL-1β during diseased states can overcome the natural regulatory mechanisms to drive pathogenesis of inflammatory and autoinflammatory disorders (8), thus indicating there is a need for therapeutic intervention.

Previously, we demonstrated in vivo in a diet-induced obesity (DIO) mouse model of diabetes that a high-affinity humanized anti-IL-1β monoclonal antibody, XOMA 052, improved impaired insulin secretion and glycemic control. The data also suggested that XOMA 052 prevented insulin resistance and maintained normal lipid metabolism (9). To further investigate the ability of XOMA 052 to neutralize IL-1β-induced insulin resistance, we have now studied this antibody in vitro. We have used 3T3-L1 adipocytes, a model cell line whose insulin-responsive functions are inhibited by IL-1β (10). In these cells, XOMA 052 reversed IL-1β inhibition of insulin signaling actions.

Material and Methods


Following are the materials used and their manufacturers: insulin solution (Sigma-Aldrich, St. Louis, MO), IL-1β (R&D Systems, Minneapolis, MN), XOMA 052 (XOMA manufactured), [3H]-2-deoxy-D-glucose (2-DG) (PerkinElmer NEN Radiochemicals, Waltham, MA).


Cell culture

3T3-L1 cells were obtained from ATCC (Manassas, VA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% NCS (Invitrogen; Carlsbad, CA). Adipocyte differentiation was carried out using reagents from Zen-Bio (Research Triangle Park, NC). Assays were performed on day 10 differentiated adipocytes. To induce insulin resistance, cells were preincubated with 1 ng/ml IL-1β for 24 h in the presence or absence of 10 μg/ml XOMA 052. Studies indicated that this concentration of IL-1β maximally inhibited insulin signaling, and that 10 μg/ml XOMA 052 maximally neutralized IL-1β (vida infra). The molar ratio of XOMA 052 to IL-1β at these concentrations is ∼ 1,100:1.

Glucose uptake

Glucose uptake was measured in 3T3-L1 adipocytes differentiated in 24-well plates. After preincubation with IL-1β ± XOMA 052, cells were serum starved for 3 h in DMEM containing 0.1% BSA. Cells were then washed with prewarmed (37°C) transport buffer (TB; 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2.5 mM MgCl2, and 1 mM CaCl2) containing 0.2% BSA. Insulin-mediated 2-DG uptake was performed in TB for 20 min and the reaction was stopped by adding 200 mM glucose in PBS. Cells were solubilized and counted, and the incorporated radioactivity was measured as pmol/(mg min).

Fatty acid uptake

Fatty acid uptake was measured in 3T3-L1 adipocytes differentiated in 96-well plates using the QBT Fatty Acid Uptake Kit according to the manufacturer's specifications (Molecular Devices, Sunnyvale, CA). After preincubation with IL-1β ± XOMA 052, the cells were washed and serum starved in Hank's Balanced Salt Solution (HBSS) containing 0.2% fatty acid-free BSA for 1-2 h at 37°C. Insulin dilutions were added from a 10× stock and incubated at 37°C for 30 min to activate cells. Subsequently, an equal volume of QBT loading buffer was added and internalized fluorescent fatty acids were measured after 3 h.

Phospho-Akt detection

Phosphorylation of Akt on Ser-473 in 3T3-L1 cells was detected using a signaling detection kit from Meso Scale Discovery (Gaithersburg, MD). Adipocytes were treated under the conditions used for fatty acid uptake. Cell lysates were generated and both phospho-Akt (Ser-473) and total Akt were measured according to the manufacturers recommended protocol.

Suppressor of cytokine signaling-3 mRNA detection

Suppressor of cytokine signaling-3 (SOCS-3) mRNA was measured using real time PCR on 3T3-L1 adipocytes using the Roche LightCycler 480 system (Indianapolis, IN). 24 h prior to assay, cells were treated with either media, 10 nM insulin, 1 ng/ml IL-1β, insulin plus IL-1β, or insulin plus IL-1β, and 10 μg/ml XOMA 052. Cells were harvested and resuspended in Trizol Reagent (Invitrogen) to extract total RNA. cDNA was generated using the ABI High Capacity cDNA Reverse Transcription Kit (Foster City, CA). GAPDH was used for normalization.


We studied the effects of IL-1β on desensitizing four metabolically relevant insulin-mediated functions in an adipose tissue model system, 3T3-L1 adipocytes. The functions studied included fatty acid uptake, Akt phosphorylation, glucose transport, and SOCS-3 expression. XOMA 052 neutralized nearly all of these inhibitory effects in 3T3-L1 adipocytes.

Fatty acid uptake represents the first step involved in sequestration of circulating lipids. Insulin, at a concentration of 20 nM, induced maximal fatty acid uptake (approximately 2- to 3-fold above background) and preincubation with IL-1β decreased uptake by 30-40% (Fig. 1A). Consistent with previous studies, coincubation with XOMA 052 neutralized this effect. The dose response of IL-1β on this function was maximal between 0.5 and 2.0 ng/ml (Figure 1B). XOMA 052 at 10 μg/ml neutralized the effect of IL-1β used at 1 ng/ml (Figure 1C).

Figure 1.

XOMA 052 neutralization of IL-1β desensitization of insulin signaling in 3T3-L1 adipocytes. In these studies, cells were preincubated for 24 hours in either incubation media alone, media containing IL-1β, or media containing IL-1β plus XOMA 052. (A) After preincubation, cells were incubated with 20 nM insulin for 3 h and inhibition of fatty acid uptake by IL-1β at 1 ng/ml and reversal with XOMA 052 at 10 μg/ml was measured. (B) Effect of increasing concentrations of IL-1β on inhibition of insulin-stimulated fatty acid uptake. (C) Effect of increasing concentrations of XOMA 052 on reversing effects of 1 ng/ml IL-1β on fatty acid uptake. (D) Inhibition of phosphorylation of Akt was measured under the preincubation conditions in (A) followed by incubation with 20 nM insulin for 10 min. (E) Inhibition of glucose transport was measured under the preincubation conditions of (A) followed by incubation with 10 nM insulin for 10 min followed by a 20 min pulse with 2-DG. For (F), after cells were incubated for 24 h under the aforementioned conditions for (A), SOCS-3 mRNA was measured. (*P < 0.05, **P < 0.01, and ns: not significant; data represented are the mean ± SEM for three experiments).

Phosphorylation of Akt is a key signaling component downstream of insulin receptor (INSR) activation and mediates several functions relevant to lipid metabolism. Treatment of cells with 20 nM insulin increased Akt phosphorylation by approximately 7-fold (Figure 1D). Preincubation with IL-1β decreased phospho-Akt levels by ∼ 50% which was neutralized by coincubation with XOMA 052. Akt activation plays a major role in increasing cellular glucose transport in response to insulin. 3T3-L1 adipocytes showed maximal glucose transport at 10 nM insulin (approximately 2- to 3-fold above background) and preincubation with IL-1β decreased transport by ∼ 40% (Fig. 1E). Coincubation with XOMA 052 neutralized the inhibitory effect of IL-1β.

To further elucidate the mechanism of IL-1β-induced INSR desensitization, we examined SOCS-3 expression. SOCS-3 is a cytokine-induced inhibitor of INSR activity and has been shown to directly interact with the INSR leading to impairment of insulin-induced phosphorylation of several downstream signaling proteins including IRS-1, IRS-2, and Akt (11). Preincubation of 3T3-L1 adipocytes with 10 nM insulin for 24 h reduced SOCS-3 levels by ∼75% (Fig. 1F). IL-1β alone did not significantly increase SOCS-3 levels but when coincubated with insulin, prevented the downregulation of SOCS-3 expression. XOMA 052 neutralized the antagonistic effect of IL-1β on insulin-induced downregulation of SOCS-3.


Currently, there is a worldwide epidemic of T2DM and the “metabolic syndrome” (1-4). Critical factors in the etiology of these disorders are obesity and the accompanying insulin resistance. Lifestyle decisions such as diet and exercise are primary ways to prevent insulin resistance, but once these disorders occur, therapeutic intervention becomes necessary to help manage the progression of the diseases. Over the past decade, the role of inflammation has been studied as a factor underlying the mechanism by which obesity can lead to insulin resistance (4). A key peripheral tissue affected by chronic inflammation is fat. Adipose tissue plays a central role in maintaining normal insulin sensitivity and glucose homeostasis through two main processes: sequestration of circulating lipids to prevent systemic lipotoxicities and secretion of adipokines to maintain metabolic homeostasis (12). Under normal energy balance, adipocytes efficiently sequester postprandial increases in serum-free fatty acids (FFA) and convert them to triglycerides for future energy demands (i.e., fasting and exercise). Continual excess consumption of dietary fat can overload the storage capacity of adipocytes and lead to dysregulation of lipid metabolism and inflammation (13). The resultant dyslipidemia leads to high circulating levels of FFA and ectopic fat storage in other areas such as muscle and liver leading to insulin resistance in those tissues (13). Obese individuals can have as much as 40% of their adipose cellular mass made up of infiltrating adipose tissue macrophages, which secrete several proinflammatory cytokines, including IL-1β and Tumor Necrosis Factor-alpha (TNFα) (14).

In this study, we used XOMA 052 to further characterize IL-1β-induced insulin resistance in vitro. We studied differentiated 3T3-L1 adipocytes to elucidate the specific effects of IL-1β on adipose tissue by testing several metabolically relevant cellular processes downstream of INSR activation, including Akt phosphorylation and two functions involved in normal lipid metabolism, uptake of glucose to support esterification of FFA into triglycerides and uptake of FFA to sequester circulating lipids. IL-1β inhibited all three processes, and XOMA 052 neutralized nearly all the effects.

Cytokines such as interleukin-6 (IL-6) upregulate SOCS-3 expression through activation of the JAK/STAT pathway, and IL-1β is a primary inducer of IL-6 production (6). Conversely, insulin can inhibit IL-6-induced gene expression and serves as a negative regulator of SOCS-3 expression (15). To investigate the mechanism of INSR desensitization, we measured SOCS-3 mRNA and showed that IL-1β inhibited insulin-mediated downregulation of SOCS-3 expression. This effect would lead to elevated levels of SOCS-3 protein and, subsequently, prolonged inhibition of INSR signaling. XOMA 052 was also able to neutralize this effect.

The data presented here provide in vitro data supporting the pathophysiological role of IL-1β as a key cytokine involved in driving insulin resistance in adipose and other tissues. Moreover, these data suggest that the potential usage of antibodies to IL-1β, such as XOMA 052, may be a new therapeutic approach to reduce inflammation and insulin resistance.


We thank Alex Owyang for thoughtful discussions and review of the manuscript.