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.
Highlights
Aging is the major risk factor for osteoarthritis (OA), but other factors have been recognized [1,2], including diabetes mellitus (DM), especially type 2 (DM2), and impaired glucose homeostasis
Chronic low grade inflammation is a major OA driver, being associated with other mechanisms involved in OA pathogenesis, including cell senescence, oxidative stress, mitochondrial dysfunction, and impaired autophagy [5,6], which together contribute to cell depletion and imbalanced anabolic and catabolic responses, especially in chondrocytes [7]
Since Nuclear Factor-κB (NF-κB) activation is required for the expression of pro-inflammatory and catabolic genes in human chondrocytes, we evaluated the expression of IL-1β, Tumor Necrosis Factor (TNF)-α, and inducible nitric oxide synthase (iNOS) in chondrocytes cultured in regular (10 mM) and high (30 mM) glucose containing media as readouts of NF-κB activity
Summary
Aging is the major risk factor for osteoarthritis (OA), but other factors have been recognized [1,2], including diabetes mellitus (DM), especially type 2 (DM2), and impaired glucose homeostasis. Chronic low grade inflammation is a major OA driver, being associated with other mechanisms involved in OA pathogenesis, including cell senescence, oxidative stress, mitochondrial dysfunction, and impaired autophagy [5,6], which together contribute to cell depletion and imbalanced anabolic and catabolic responses, especially in chondrocytes [7]. All the above mechanisms have been associated with the effects of hyperglycemia and have been implicated in the pathogenesis of DM and its complications. All these mechanisms have been related to inflammation, which is currently accepted as an important effector mechanism by which DM2 causes cell and tissue damage [8]
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