Hyperglycemia‐ and hyperinsulinemia‐induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle

Abstract

Insulin resistance is a well-known risk factor for obesity, metabolic syndrome (MetSyn) and associated cardiovascular diseases, but its mechanisms are undefined in the lymphatics. Mesenteric lymphatic vessels from MetSyn or LPS-injected rats exhibited impaired intrinsic contractile activity and associated inflammatory changes. Hence, we hypothesized that insulin resistance in lymphatic muscle cells (LMCs) affects cell bioenergetics and signaling pathways that consequently alter contractility. LMCs were treated with different concentrations of insulin or glucose or both at various time points to determine insulin resistance. Onset of insulin resistance significantly impaired glucose uptake, mitochondrial function, oxygen consumption rates, glycolysis, lactic acid, and ATP production in LMCs. Hyperglycemia and hyperinsulinemia also impaired the PI3K/Akt while enhancing the ERK/p38MAPK/JNK pathways in LMCs. Increased NF-κB nuclear translocation and macrophage chemoattractant protein-1 and VCAM-1 levels in insulin-resistant LMCs indicated activation of inflammatory mechanisms. In addition, increased phosphorylation of myosin light chain-20, a key regulator of lymphatic muscle contraction, was observed in insulin-resistant LMCs. Therefore, our data elucidate the mechanisms of insulin resistance in LMCs and provide the first evidence that hyperglycemia and hyperinsulinemia promote insulin resistance and impair lymphatic contractile status by reducing glucose uptake, altering cellular metabolic pathways, and activating inflammatory signaling cascades.-Lee, Y., Fluckey, J. D., Chakraborty, S., Muthuchamy, M. Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle.

Insulin resistance disrupts cell integrity, mitochondrial function, and inflammatory signaling in lymphatic endothelium

“…Cellular glycolytic rates in LECs were estimated by measuring ECAR, as described in our previous study . The LECs were treated as described above (n = 4 per group).…”

“…The cDNA was mixed with SYBR Green PCR master mix (ThermoFisher Scientific). Primers specific to IL‐1β, IL‐4, IL‐6, iNOS, TNF‐α, MCP1, and MIP2, CXCR2, CFOS, c‐Jun were designed and used for real‐time PCR . RPL19 amplification was used as a control for all samples, and the differences in the relative expression of cytokines between groups were calculated using the 2 −ΔΔCt method.…”

“…Immunofluorescence experiments were performed as described earlier to determine β‐catenin and NF‐κB localization . In brief, the treated LECs were fixed with 2% paraformaldehyde.…”

“…Further, the lymphatic vessels from MetSyn rats showed a significant decrease in the relative levels of eNOS that could be associated with endothelial dysfunction . We also showed that insulin resistance directly altered cellular bioenergetics and inflammatory signaling, coupled with impairment of a key contractile regulatory molecule in LMCs . We hypothesized that insulin resistance in LECs would decrease eNOS activity and would activate mitochondrial dysfunction and pro‐inflammatory signaling pathways, consequently disrupting barrier integrity and increasing permeability.…”

In vitro model reveals a role for mechanical stretch in the remodeling response of lymphatic muscle cells

“…Higher expression of signaling pathways associated with oxidative phosphorylation and insulin‐like growth factor‐1 in WL LMCs, regardless of stretch, indicates that the differentiation of the WL LMCs in response to a local injury persists 6 weeks after the initial insult and is maintained even after several passages in vitro (Figure C). Both of these pathways have been found to be critical to the growth, proliferation, and survival of VSMCs . This, in part, may explain the increased sensitivity of CL LMCs to tissue degeneration and apoptosis in response to stretch (Figure ).…”