FA1 Induces Pro-Inflammatory and Anti-Adipogenic Pathways/Markers in Human Myotubes Established from Lean, Obese, and Type 2 Diabetic Subjects but Not Insulin Resistance

Abstract

Aims: Delta like 1/fetal antigen 1 (Dlk1/FA1) is a protein secreted by hormone producing cells in adult human and mice that is known to inhibit adipogenesis. Recent studies demonstrated the role of Dlk1/FA1 in inducing insulin resistance in mice. To investigate the involvement of circulating Dlk1/FA1 in insulin resistance and type 2 diabetes in human subjects, we studied the effects of chronic FA1 on the intermediary metabolism in myotubes established from lean, obese, and type 2 diabetic (T2D) subjects.Methods: Myotube cultures were established from lean and obese control subjects, and obese T2D subjects and treated with soluble FA1 for 4 days supplemented with/without palmitate (PA). Lipid- and glucose metabolism were studied with labeled precursors while quantitative expression of genes was analyzed using real-time PCR.Results: Diabetic myotubes express significantly reduced insulin stimulated glucose metabolism compared to lean myotubes and a significantly decreased basal PA oxidation. Chronic FA1 exposure did not affect the intermediary metabolism in myotubes. Insulin sensitivity of glucose and lipid metabolism was not affected by chronic FA1 exposure in myotubes established from lean, obese, and T2D subjects. Instead, chronic FA1 exposure induced pro-inflammatory cytokines expression (IL-6 and CCL2) in association with reducing adipogenic markers (ADD1, AP2, CD36, and PPARg2) in myotubes. Consistent with this observation, addition of FA1 to cultured myotubes was show to significantly inhibit their differentiation into adipocyte.Conclusion: Our results exclude direct effects of FA1 on glucose and lipid metabolism in cultured myotubes established from lean, obese, and T2D subjects. Therefore, the pathogenesis of FA1-induced IR might mainly be mediated via the FA1-induced stimulation of pro-inflammatory cytokines, which on turn inhibit adipogenesis in human myotubes.

Altered Myokine Secretion Is an Intrinsic Property of Skeletal Muscle in Type 2 Diabetes

“…The picture regarding IL6 is more mixed. While we found IL6 secretion to be higher in T2D hSMC, as did Bouzakri et al after TNFa treatment of ND myotubes [ 11 ], others found no difference from ND [ 14 , 35 ] or even decreased release [ 34 ]. The observations that MCP-1 mRNA is elevated in T2D hSMC [ 12 , 36 ] and after TNFa treatment, and that secretion is also elevated in the later case [ 11 ], would be congruent with our results regarding MCP-1 release.…”

“…Furthermore, the current report looks at only a small number of potential myokines. The decision was made to take a targeted approach, focusing on a limited number of factors, including several that have been under study by others [ 11 – 14 , 33 – 36 ], as well as factors for which there is evidence that they could possibly play roles in the diabetic phenotype.…”

“…Cultured myotubes from T2D subjects displayed elevated mRNA content for TNFa and MCP-1 compared to cells from non-diabetic (ND) individuals [ 12 ]; we reported previously that release of TNFa protein was also elevated [ 13 ]. Meanwhile, IL6 mRNA and secretion did not differ between T2D and ND myotubes [ 12 , 14 ]. However, caution must be exerted while determining the significance of changes in mRNA content, as there are multiple examples of discrepancies between mRNA content and expression and/or secretion of the same protein [ 15 ].…”

The diabetic phenotype is preserved in myotubes established from type 2 diabetic subjects: a critical appraisal

“…Comparing results from various metabolic assays conducted at three different days in myotubes established from the same donors in accordance with our protocols (73,92,144) shows that day-today variation is high, while within-day variation is small. Absolute rates of glucose uptake and glycogen synthesis can differ twofold between studies.…”

“…In addition to studies on primary insulin resistance in diabetic myotubes, various aspects of insulin resistance induced by hyperinsulinaemia (48,52,68,165), hyperglycaemia (48,66,73,(165)(166)(167)(168), hyperlipidaemia (67,73,93,155) and other agents (92,144,169) have been investigated in human myotubes. Please refer to the relevant literature for further reading, as induced insulin resistance in skeletal muscle/myotubes is beyond the scope of the present review.…”