The protective effects of GLP-1 and liraglutide on insulin resistant 3D kidney spheroids.

Increased plasma levels of non-esterified fatty acids (NEFAs) through their over-consumption in the diet, link obesity with the onset of type 2 diabetes mellitus (T2D). Increased NEFA concentration is implicated in the development of insulin resistance (IR) in insulin sensitive tissues. Patients with T2D have shown impaired cardiac remodeling following an ischemic event, where hypoxia-inducible factor1 alpha (HIF-1α) activation is diminished due to the presence of increased long chain fatty acids. Glucagon-like peptide-1 (GLP-1) receptor agonist; GLP-1 (7-36) and incretin mimetic; liraglutide, have been implicated in their ability to reverse or diminish the effect of IR in multiple organ types by increasing cellular viability. We aim to induce IR in human embryonic kidney cells (HEK293T) using the three most abundant NEFAs present in T2D patients; oleate, stearate and palmitate. Furthermore, we aim to distinguish the protective effects of GLP-1 and liraglutide, in multiple organ types that are affected by IR under an ischemic event. Traditional in vitro 2D monolayer studies will be further compared to 3D spheroid studies, to demonstrate physiological relevance in 3D models over traditional methodology. Statistical significance was calculated using a one-way ANOVA and post-hoc Tukey HSD test (p ≤ 0.05). Palmitate has been shown to induce IR in multiple cell lines however, to ensure a more physiologically relevant profile of NEFAs, a relevant ratio of 4:4:1 was used combining palmitate, oleate and stearate (POS), respectively. A viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) indicated that 500 µM palmitate concentration in normoxic and hypoxic conditions diminished cellular viability significantly (p ≤ 0.05), where POS revealed similar results (p ≤ 0.05)(n=6). Western blot analysis showed that POS and palmitate at 500 µM induced IR in HEK293T cells compared to controls, decreasing levels of insulin stimulated p-Akt (ser473) alongside additional markers of IR (n=4). 3D spheroids of HEK293Ts (100 cells) were used to better stimulate the in vivo microenvironment. MitoTracker red CMXRos, Caspase 3/7 reagent and DAPI were used to determine spheroid viability, mitochondrial protection and apoptosis. Fluorescence studies showed that at 500 µM POS, cellular viability was diminished. However upon treatment with GLP-1 and liraglutide, this was attenuated significantly (p ≤ 0.05)(n=4). In conclusion, the presence of NEFAs palmitate, oleate and stearate, in HEK293T kidney cells decreased cellular viability and induced IR. GLP-1 and liraglutide indicated an ability to diminish these effects by increasing cellular viability. The data observed in monolayer studies, appeared to be replicated within 3D spheroid models. However further work is essential to characterize this and to determine the mode of action of GLP-1 in protection.