Hyperglycemia and Hyperinsulinemia-Like Conditions Independently Induce Inflammatory Responses in Human Chondrocytes

Abstract

To elucidate the mechanisms by which type 2 Diabetes Mellitus (DM2) constitutes a risk factor for the development and progression of osteoarthritis (OA), this work determined whether high glucose and/or high insulin, the hallmarks of DM2, are capable of activating the transcription factor, Nuclear Factor-κB (NF-κB), which plays a critical role in OA by inducing the expression of pro-inflammatory and catabolic genes. For this, we analyzed NF-κB activation by measuring the nuclear levels of p65 by western blot. As readouts of NF-κB activity, Interleukin-1β, Tumor Necrosis Factor-α, and inducible nitric oxide synthase (iNOS) expression were analyzed by real time RT-PCR and western blot. Culture of the human chondrocytic cell line, C28-I2, in high glucose (30 mM) increased nuclear NF-κB p65 levels in a time-dependent manner, relative to cells cultured in medium containing 10 mM glucose (regular culture medium). High glucose-induced NF-κB activation was inhibited by co-treatment with its specific inhibitor, Bay 11-7082, 5 µM. Culture of primary human chondrocytes under high glucose for 24 h increased IL-1β and TNF-α mRNA levels by 97% (p = 0.0066) and 85% (p = 0.0045), respectively, while iNOS mRNA and protein levels and NO production increased by 61% (p = 0.0017), 148% (p = 0.0089), and 70% (p = 0.049), respectively, relative to chondrocytes maintained in 10 mM glucose. Treatment of chondrocytic cells with 100 nM insulin was also sufficient to increase nuclear NF-κB p65 levels, independently of the glucose concentration in the culture medium. This study shows that hyperglycemia and hyperinsulinemia are independently sufficient to induce inflammatory responses in human chondrocytes, namely by activating NF-κB. This can be a relevant mechanism by which DM type 2 and other conditions associated with impaired glucose and insulin homeostasis, like obesity and the metabolic syndrome, contribute to the development and progression of OA.

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“…Further, subclinical inflammation is involved in all stages of the atherosclerotic process, from the initiation of fatty streaks to the development of plaque rupture and thrombus formation 111,152. As mentioned before, hyperinsulinemia,117,118 the SNS119 and the RAS109 are causally related to the development of subclinical inflammation.…”

“…On the one hand, chronic inflammation causes OS, on the other hand, OS plays a crucial role in the development and perpetuation of inflammation, indicating that inflammation and OS are pathophysiological events that are closely related 116. In addition to OS, hyperinsulinemia,49,117,118 ANG II109 and activation of the SNS119 are also causally related to the development of chronic subclinical inflammation, linking insulin-resistant states like obesity and T2DM to inflammation. WDs were shown to reprogram the innate immune system and induce low-grade inflammation 21,120.…”

“…In addition to its role in atherogenesis and hypertension, the RAS plays a critical role in the pathogenesis of many other types of CVDs including cardiomyopathy, valvular heart disease, aneurysms, infarction, stroke and renal disease 109,172. Hyperinsulinemia and low-grade inflammation have been implicated in the development of osteoarthritis 117…”

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“…Hyperinsulinemia has been shown to promote adipose tissue inflammation in mice, whereas a decrease in circulating insulin levels lead to a decrease in the expression of pro-inflammatory markers and macrophage content in adipose tissue ( Pedersen et al, 2015 ). Hyperinsulinemia and hyperglycemia were (independently) shown to induce inflammatory responses in human chondrocytes in vitro , namely by activating NF-κB ( Rufino et al, 2017 ).…”