In accordance with a growing literature showing that Toll-like receptors exhibit functions in the homeostasis of several types of non-immune cells [13–20], here we show that β-cells harvested from human non-diabetic brain-dead organ donors also express variable levels of TLR4. Our results indicate that there could be a role for TLR4 in β-cell homeostasis, possibly related to cell viability or insulin production.
In contrast with a previous study , we showed that LPS not only increases TLR4 expression and diminishes cell viability, but, also, leads to loss of β-cell insulin content. In both non-diabetic human and murine β-cells, TLR4 as well as CD14 transcripts were identified, with LPS (50 ng/mL) leading to a concentration-dependent increase in their gene expression levels, in parallel with lowered cell viability and decreased insulin content and mRNA transcription. These results are in agreement with a study showing that blocking TLR4 expression in murine islets by carbon monoxide treatment leads to increased survival of islet implants in an allogenic murine model 
In a recent study  with the BRIN BD11 rat clonal cell line, the authors showed decreased insulin secretion upon culturing in the presence of LPS, but no change in insulin content. The authors also observed a reversal of the effect on insulin secretion upon removal of LPS. The observed lack of change in insulin content may be explained by the fact that cells were incubated with 100 ng/mL LPS only for 24h, in contrast to our incubation period for up to 48h, but using half the dose of LPS. Indeed, in our study, we also observed a low effect upon insulin content when cells were incubated for 24h (data not shown). Taken together, these findings suggest that effects on β-cell insulin content and cell viability require more time to take place.
In addition to the classical activation, via pathogen-associated molecular patterns, TLR expression in monocytes has been shown to be regulated by glucose . High glucose levels induced TLR2 and TLR4 expression while Mannitol did not, excluding an osmotic effect as the cause for observed results. Increased TLR4 expression occurred due to PKC-δ-mediated activation and stimulated p47Phox-dependent NADPH oxidase activity, establishing a link between high glucose, NF-κB-mediated inflammatory cytokine production, and TLR expression. In another study, with type 2 diabetic patients, in which TLR expression in mononuclear cells was monitored in response to low-dose insulin infusion, a further relationship between TLR2 and TLR4, and insulin homeostasis was identified . Steady-state infusion during a short 4h period caused an increase of approximately 2.5 fold in insulin plasma levels while maintaining the patient's glycemia, and led to decreased TLR expression, which occurred independently of blood glucose levels. The effect of this short-term experiment, however, was more pronounced on TLR2 than on TLR4 expression. It is possible that increasing the period of infusion would have increased the effect on TLR4 expression.
Our study suggests a further link between insulin homeostasis, cell viability, and TLR4 expression. Although we did not measure apoptosis-related molecules, we used TMRE as an indirect marker for apoptosis. TMRE is a dye with a high degree of membrane permeability that accumulates in viable mitochondria, thereby marking only cells that are not undergoing apoptosis. In the presence of LPS-induced TLR4 expression, in both human and murine β-cells, insulin mRNA, insulin secretion and insulin content were diminished, and β-cell viability was decreased (from 100 to approximately 60%, Figure 3b).
Endogenous ligands for TLR4 have been described, most of which have been proven to exert their effect independently of an eventual experimental LPS contamination . The list includes heat shock proteins 60 and gp96 [27, 28], fibronectin type III extra domain A , hyaluronan , saturated fatty acids , advanced glycation end-products  and heme . In other words, molecules produced or circulating during abnormal situations such as tissue damage and ischemia are capable of triggering TLR4- dependent pathways. These conditions are commonly present in brain-dead organ donors, being potential causes of engraftment failure as well as setting the conditions for inappropriate expression of TLR4 on non-immune cells. It remains to be seen how many of these potentially damaging factors are present before and during the long-lasting islet isolation procedures, and which, by favoring TLR4 expression, may further contribute to diminished insulin content and lower islet yield.
We can also envisage that increased glucose plasma levels not only induce insulin secretion, triggering insulin gene transcription and replacement of the intracellular stocks, but also increase surface expression of TLR4 molecules. The release of endogenous TLR4 ligands during tissue damage and inflammation followed by docking onto β-cells, could then result in loss of the ability of these β-cells to respond normally to glucose, contributing to the extra load of circulating plasma glucose and worsening of the glycemia control observed in response to TNF-α and adrenalin, as well as other hormones secreted under stressful situations.