Suppression of gp130 attenuated insulin-mediated signaling and glucose uptake in skeletal muscle cells (2024)

In glucose transporter-4 (GLUT-4) regulation, insulin upregulates insulin-binding receptor substrate (IRS) and protein kinase B (Akt) via phosphatidylinositol 3-kinase (PI3-kinase) in the skeletal muscle¹. Bjoprnholm et al. (1997) reported that insulin-stimulated PI3-kinase and Akt activations impaired and subsequently decreased GLUT-4 translocation in patients with type 2 diabetes and obesity². Furthermore, a previous study showed that the inflammatory markers interleukin-6 (IL-6) and C-reactive protein (CRP) within the highest quartiles were associated with an increased risk of developing type 2 diabetes over a four years period compared with those in the lowest quartile in middle-aged healthy individuals³. In individuals with obesity, the increased release of adipocyte-derived metabolites, such as fatty acids and various inflammatory cytokines, has been linked to the development of insulin resistance. Therefore, patients with obesity show increased production of inflammatory cytokines, impaired activation of PI3-kinase and Akt signaling, and GLUT-4 translocation in skeletal muscles, resulting in the development of type 2 diabetes. Several studies have revealed the relationship between inflammatory cytokines, especially Oncostatin M (OSM), and glucose metabolism in humans. Serum OSM concentration correlated with body weight and insulin, and was inversely correlated with the glucose disposal rate, as measured by a hyperinsulinemic–euglycemic clamp⁴. Additionally, the OSM mRNA levels were increased in white adipose tissues from patients with obesity compared to those from healthy controls, and its mRNA expression was elevated in patients with obesity who had hyperglycemia compared to those who had obesity with euglycemic conditions⁵. Therefore, the OSM levels could be a novel biomarker for obesity and type 2 diabetes and may be useful for the screening other metabolic disorders in patients with obesity.

The OSM is a member of the interleukin-6 (IL-6) family of cytokines and can bind to two different receptors, Leukemia inhibitory factor receptor (LIFR) and Oncostatin M receptor (OSMR), through a complex containing common glycoprotein 130 (gp130) subunit. Additionally, the OSM modulates a variety of biological processes such as liver development and regeneration⁶, hepatic insulin resistance and steatosis⁷, inflammation⁸, and cardiomyocyte dedifferentiation and remodeling⁹. Several studies have investigated the effect of OSM on glucose metabolism and tolerance. Previously, increased expression of OSM in white adipose tissue has been correlated with markers of metabolic diseases, including GLUT4 expression, hyperglycemia, hyperinsulinemia, and increased HOMA-IR in patients with obesity¹⁰. Henkel et al. (2011). determined the effects of OSM on hepatic glucose metabolism and found that the addition of OSM, IL-6, or prostaglandin E2 (PGE2) attenuated Akt phosphorylation and insulin-dependent gluco*kinase induction in hepatocytes⁷. Therefore, the OSM released in response to PGE2 in macrophages in the intra-abdominal adipose tissue and peritoneal macrophages may be additional relevant sources for inducing hepatic insulin resistance. In contrast, the knockdown of hepatic gp130 protects against the development of steatosis and insulin resistance in the liver¹¹. Therefore, different responses in hepatocyte glucose metabolism were observed with the addition of OSM or gp130 knockdown. However, the effect of gp130 knockdown on insulin-mediated glucose metabolism-related signaling in skeletal muscles remains unclear.

Thus, the aim of the present study was to clarify the effect of gp130 on insulin signaling and glucose uptake in the skeletal muscle. We hypothesized that gp130 knockdown in skeletal muscle cells might impair the activation of insulin-mediated glucose metabolism signaling, resulting in the attenuation of glucose uptake. To test this hypothesis, we disrupted gp130 subunit in the skeletal muscle cell and investigated the effect of gp130 on insulin-mediated glucose metabolism signaling such as IRS-1 phosphorylation, PI3-kinase activity, Akt phosphorylation and glucose uptake rate with 2-deoxy-glucose.

IL6ST mRNA and protein expression, and STAT 3 phosphorylation

The mRNA level of gp130, encoded by IL6ST mRNA levels were significantly decreased in gp130 knockdown cells (gp130^−/−) compared to the control (gp130^+/+) (P < 0.01; Fig.1A). The phosphorylation of STAT 3 was significantly lower in gp130^−/− cell (P < 0.01; Fig.1B). Additionally, no significant changes were seen in STAT 3 phosphorylation by insulin stimulation in both gp130^−/− and gp130^+/+ cells.

Glucose metabolism-regulated signaling and glucose uptake

There was no significant difference in the phosphorylation of IRS-1 Ser 1101 between gp130^−/− and gp130^+/+ cells without insulin stimulation, and phosphorylation of IRS-1 Ser 1101 in gp130^+/+ was significantly increased by insulin stimulation compared to gp130^−/− (P < 0.05; Fig.2A). Similarly, no significant differences were observed in PI3-kinase activity and Akt Thr 308 phosphorylation in both gp130^−/− and gp130^+/+ cells without insulin stimulation. However, PI3-kinase activity and Akt Thr 308 phosphorylation were significantly increased by insulin stimulation in gp130^+/+ cells compared to gp130^−/− cells (P < 0.05; Fig.2B,C). Figure3A showed luminescence which was the raw luminescence from the glucose uptake assay. The glucose uptake rate was slightly lower in gp130^−/− cells at baseline without insulin stimulation but was not statistically different from that in gp130^+/+ cells. However, a significant difference was observed after insulin stimulation; the insulin-stimulated increase in the glucose uptake rate was significantly attenuated in gp130^−/− cells compared to that in gp130^+/+ cells with or without insulin (P < 0.05; Fig.3A,B).

OSM and IL-6 levels in cell culture supernatant

The level of OSM in culture supernatant was significantly lower in gp130^+/+ compared to gp130^−/− cells (P < 0.05; Fig.4A), and did not change by insulin stimulation in both gp130^+/+and gp130^−/− cells. IL-6 level in culture supernatant significantly higher in gp130^+/+ compared to gp130^−/− (P < 0.05; Fig.4B), and no significantly changes with insulin stimulation both gp130^+/+and gp130^−/− cells.

In the present study, gp130 knockdown caused a decrease in STAT 3 phosphorylation and resulted in attenuation of insulin-mediated glucose metabolism-regulated signaling, including phosphorylation of IRS-1Ser 1101, Akt Thr 308, and PI3-kinase activity in skeletal muscle cells. Additionally, this attenuation induced a decrease in the glucose uptake rate in gp130-knockdown skeletal muscle cells. Thus, the lack of gp130 increases the extracellular OSM and decreased IL-6 levels, which might attenuate insulin-mediated glucose uptake through the impairment of glucose metabolism-regulated signaling pathway activation in the skeletal muscle.

OSMR is a receptor that mediates signaling of cytokines such as OSM and interleukin-31 (IL-31)¹². Additionally, OSM induced biological effects by activating functional receptor complexes of the common signal transducing component gp130 and OSMRβ or LIFR, which are mainly involved in chronic inflammatory and cardiovascular diseases, cancer and rheumatoid arthritis¹³. These cytokines are involved in various cellular processes, and the OSMR receptors, especially gp130, are primarily associated with the JAK-STAT, ERK, and PI3/Akt signaling cascades. Previously, disruption of gp130 signaling lead to changes in adipose tissue function, including increased release of pro-inflammatory adipokines, as well as exhibition of significantly elevated levels of uncoupling protein 1 (UCP1) and other browning markers such as peroxisome proliferator-activated receptor γ coactivator-α (PGC-1α)¹⁴. Additionally, in hepatic cells, gp130 knockdown animals show an imbalanced inflammatory response with increased hepatic tumor necrosis factor-alpha and decreased adiponectin messenger RNA levels, resulting in rapidly elevated fasting blood glucose, serum insulin levels, and transaminases¹¹. Therefore, the effects of the gp130 knockout and knockdown are complex and depend on the site and degree of disruption and/or suppression. The present study found that disrupted gp130 induced attenuation of insulin-mediated glucose metabolism regulated signaling which included PI3-kinase/Akt signaling with increasing extracellular OSM levels. Previously, increasing OSM levels impaired insulin signaling pathways in the liver and adipose tissue, leading to reduced glucose uptake and impaired insulin action¹⁵. Moreover, the OSM addition to 3T3-L1 cells blunted the insulin-induced phosphorylation of Akt, suggesting that the OSM affects insulin signaling in white adipocytes¹⁴. Additionally, previous studies reported IL-6 had insulin-like effects that were enhancing insulin action, increasing glucose uptake and fatty acid oxidation in skeletal muscle^16,17. In the present study, extracellular IL-6 level was decreased by gp130 knockdown, and it would also one of the mechanisms that decreased extracellular IL-6 level suppressed insulin-mediated increase in glucose metabolism signaling and glucose uptake in skeletal muscle cell. Another mechanism shows that disruption of gp130 attenuated PI3-kinase/Akt signaling through impairment phosphorylation of STAT3. A previous report demonstrated that the activation of the JAK2/STAT3 cascade was significantly inhibited by gp130 ablation in peritoneal macrophages, and STAT3 has important role in various aspects of cytokine and growth factor signaling in different tissues and cell types. For instance, in macrophages and neutrophils, the loss of STAT3 suppresses the overshooting of inflammatory stimulus-induced pro-inflammatory responses¹⁸. STAT3 is required to mediate both IL-6- and lipopolysaccharide-induced acute-phase gene expression in the liver¹⁹. Importantly, loss of STAT3 in the hypothalamus interferes with normal body weight homeostasis and glucose metabolism²⁰. Therefore, decreased STAT3 phosphorylation may attenuate PI3-kinase/Akt signaling and glucose uptake in skeletal muscle cells. However, further studies are needed to investigate the effects of genetically overexpressed OSM or the addition of OSM to skeletal muscle cells on the STAT3 phosphorylation, glucose metabolism signaling pathway, and glucose uptake.

Few studies have evaluated the effect of OSMR knockout and knockdown on glucose metabolism-regulated signaling, and the effects of increased OSMR and OSM expression on glucose metabolism in skeletal muscle cells have not been fully elucidated. Generally, physiological responses that increase the OSM depend on the tissue and physiological context. Responses to the OSM include the production of inflammatory chemokines and cytokines, expression of leukocyte adhesion factors, expression of extracellular matrix (ECM) components and ECM remodeling factors, and alterations in cell proliferation and differentiation²¹. The OSM is appropriately regulated, contributing to hom*oeostasis and tissue repair from any damage in the joints, skin, and bones²² as well as the enhancement of adipocyte and mature adipocyte differentiation in adipose tissue, endothelial and smooth muscle cell proliferation, and production of vascular endothelial growth factor (VEGF) in the vasculature, heart, lungs, and joints²³. In contrast, aberrant OSM levels promote inflammatory pathology and tissue/organ dysfunction, such as fibrosis in the liver, lungs, and bone marrow. Significant positive correlation has been found between OSM levels and BMI in humans, whereas, UCP1 was negatively correlated with the OSM levels¹⁴. Moreover, elevated OSM mRNA levels have been observed in the serum and adipose tissues of patients with type 2 diabetes, and an excessive increase in circulating OSM causes the development of insulin resistance^4,24. Moreover, people with obesity and hyperglycemia have significantly higher OSM mRNA expression in both subcutaneous and visceral white adipose tissues, resulting in the inhibition of human adipogenesis, reduction of GLUT4 expression, and induction of an inflammatory state in human adipocytes¹⁰. Additionally, increased mRNA expression of OSM in white adipose tissues correlates with markers of metabolic diseases, such as hyperglycemia, hyperinsulinemia, and increased HOMA-IR in patients with obesity¹⁰. Taken together, excessive levels of extracellular and circulating OSM and decreasing IL-6 suppress insulin-mediated glucose metabolism-regulated signaling and blunt insulin action, which might result in the development of insulin resistance, and increased or aberrant OSM levels could be a marker of obesity and type 2 diabetes.

While the phosphorylation of IRS-1 on tyrosine residue is required for insulin-stimulated responses, the phosphorylation of IRS-1 on serine residues has a dual role, either to enhance or to terminate the insulin effects, and the activation of Akt in response to insulin propagates insulin-mediated signaling and promotes the phosphorylation of IRS-1 on serine residue in turn generating a positive-feedback loop for insulin action²⁵. In the present study, only IRS-1 Ser 1101 phosphorylation was determined, however, it should determine the other Ser of Tyr sites of IRS-1 to clarify the effect of gp130 knockdown on insulin action, as well as insulin-mediated signaling in future study. In conclusion, our data suggested that suppression of gp130 induces suppression of insulin-mediated PI3-K/Akt signaling and glucose uptake in skeletal muscle with increasing extracellular OSM and decreasing IL-6 levels. Excessive increase in extracellular OSM and decreased IL-6 levels may cause blunted insulin action in skeletal muscle cells.

Incubated skeletal muscle cells

Mouse C2C12 skeletal muscle cell (American Type Culture Collection: ATCC, Manassas, VA, USA) was used for the present study. The cell was grown in growth medium (Ham’s F-12 medium; Fujifilm Wako Chemicals, Tokyo, Japan) containing 10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO₂. After confluent proliferation, myoblast cells were washed with PBS, and the culture medium was replaced with differentiation medium (Dulbecco’s modified Eagle’s medium; Fujifilm Wako Chemicals, Tokyo, Japan) containing 2% horse serum (BioWest, Nuaillé, France) and 1% penicillin/streptomycin and also incubated at 37°C in a humidified atmosphere with 5% CO₂. The cells were then differentiated to develop myotubes and used for further experiments. At day 7, the cells formed myotubes and were incubated for 1h in serum-free medium containing insulin (1μg/mL; Eli Lilly, Kobe, Japan).

siRNA-mediated gp130 knockdown

siRNA experiments were conducted according to previous study²⁶. A day before transfection, 500μL of growth medium without antibiotics such that cells were 60% confluent at the time of transfection. Transfection was conducted manufacture’s instruction with using Gibco Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA, USA) and Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Cells were treated with one small interfering RNA or 100nmol/L control siRNA (Thermo Fisher Scientific, Waltham, MA, USA) in the presence of 8μL/well Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) or 15μL/well Oligofectamine (Thermo Fisher Scientific, Waltham, MA, USA) in a 6-well plate. The siRNA targeted gp130 (sense, 5′-GGCAUGCCUAAAAGUUACYTT-3′; antisense, 5′-UGAAUAGUUUCCAGAGUCGTG-3′; Thermo Fisher Scientific). Silence^® Negative control siRNA (Thermo Fisher Scientific) was used as a control siRNA. The medium was replaced at 24h after transfection, and silencing efficacy was determined by qRT-PCR 48h after transfection. The insulin (1μg/mL; Eli Lilly) was incubated before 1h for harvested the cells.

For determination of IL6ST (encoded gp130) mRNA expression, RNA from cell lines was reverse transcribed, and the cDNA was amplified by PCR using a primer set spanning exons 1 to 3 of the IL6ST gene, (F: 5′-ATTTGTGTGCTGAAGGAGGC-3′, and R: 5′-AAAGGACAGGATGTTGCA-3′).

Immunoblot analysis

The cells were hom*ogenized with 20mM Tris–HCl (pH 7.8), 300mM NaCl, 2mM DTT, 2% Nonident P-40, 2mM EDTA, 0.2% SDS, 0.5mM phenylmethylsulfonyl fluoride, 0.2% sodium deoxycholate, 60μg/mL aprotinin and 1μg/mL leupeptin. The hom*ogenates were softly rotated for 30min at 4°C, and the protein concentration of the resulting supernatant was determined. Thereafter, 10μg of protein samples were subjected to heat denaturation at 96°C for 7min with Laemmli buffer. Protein concentration was analyzed using Bradford protein assay reagent (Fujifilm Wako Chemicals, Tokyo, Japan) with bovine serum albumin as a standard. Western Blot analyses of phosphorylated IRS-1 serine 1101, Akt threonine 308, total IRS-1, Akt and Phosphorylated signal transducer and activator of transcription 3 (STAT3) proteins were performed as previously described, with minor modifications^27,28,29,30. Briefly, each sample was separated on SDS-PAGE, transferred onto 10% gels, and then, transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan) at 15V for 60min. The membrane was treated with blocking buffer which contained 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 for 1h at 4°C. The membrane was proofed with polyclonal Thr 308-phosphorylated Akt (1:1000 dilution with blocking buffer; Cell Signaling, Beverly, MA, USA), Total Akt (1:1000 dilution with blocking buffer; Cell Signaling), phosphorylated STAT3 (1:1000 dilution with blocking buffer; Cell Signaling), and total STAT3 (1:1000 dilution with blocking buffer; Cell Signaling) for 24h on rotation at 4°C. The membrane was washed three times with PBS-Tween 20 (PBS-T) and incubated for 1h with a horseradish peroxidase-conjugated secondary antibody, anti-rabbit or anti-mouse immunoglobulin, at a 1:3000 dilution in blocking buffer (Cell Signaling). Subsequently, the membranes were washed again thrice with PBS-T. Finally, phosphorylated IRS-1 Ser 1101, Akt Thr 308, phosphorylated STAT3, total IRS-1, Akt, and STAT3 levels were measured using an Enhanced Chemiluminescence system with LuminoGraph One (Atto Corporation, Tokyo, Japan).

Sandwich enzyme immunoassay

PI3-kinase activity was evaluated using an ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA). Additionally, the OSM and IL-6 levels in cell culture supernatants were evaluated using an ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). Immobilized polyclonal antibodies were used against the OSM and IL-6, whereas the secondary HRP-coupled antibodies were monoclonal antibodies. The optical density was determined at 450nm using a microplate reader (Thermo Fisher Scientific, Multiskan FC, Yokohama, Japan). All samples were assayed in duplicates.

Glucose uptake assay

The level of glucose uptake was measured using the glucose uptake-Glo Assay, which is a non-radioisotope (RI) assay, according to previous studies^31,32. Briefly, glucose uptake was initiated by the addition of 1mM 2-deoxyglucose and was allowed to proceed for 20min at 37°C. Cellular 2-deoxyglucose uptake in total cell lysates was measured using the Glucose Uptake-Glo kit (Cat No. J1341: Promega, Madison, WI, USA) and calculated the glucose uptake rate from luminescence (RLU) according to the manufacturer’s instructions.

Statistical analysis

All values were expressed as means ± SE. Statistical evaluation of the data was performed using one-way ANOVA. When the analysis revealed significant differences, a post-hoc comparison test was used to correct for multiple comparisons (Bonferroni/Dunn test). P < 0.05 was considered significant for ANOVA and P < 0.01 for post-hoc test.

All relevant data can be found within the supplementary files.

OSM:

Oncostatin M

OSMR:

Oncostatin M receptor

Gp130:

Glycoprotein130

STAT3:

Signal transducer and activator of transcription 3

Akt:

Protein kinase B

IRS-1:

Insulin receptor substrate-1

IL-6:

Interleukin-6

Authors and Affiliations

1. Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada-ku, Kobe, Hyogo, 657-8501, Japan

   Koji Sato

Contributions

K.S. researched data, wrote the manuscript and researched data, contributed to discussion and reviewed/edited manuscript.

Corresponding author

Correspondence to Koji Sato.

Competing interests

The author declares no competing interests.

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