ER Stress, Autophagy, And Insulin Resistance

NAFLD refers to the spectrum of liver diseases in which steatosis can, in some humans, progress to more significant pathology including NASH and NASH-related cirrhosis. In one way or another each of these fatty acid reservoirs is a consequence of a diet high in sugar (e.g. high fructose corn syrup) or fatty acids that are highly saturated or have an all trans- structure. This diet results in both peripheral and hepatic insulin resistance. Recent data, however, daily dietary supplement indicate that the production of triglycerides may be a safety valve for hepatocytes prone to danger from lipotoxicity, the term used to describe the potential harm of long chain, saturated fatty acids. In this study we investigated whether the GLP-1 analogues could augment protective mechanisms in human hepatocytes that accumulated specific fatty acids; or in a mouse-feeding model with a diet as described above and consumed by Westerners. Both our in vitro and in vivo results here concur with their observations.



As evident from Figure 2A, different fatty acids impart differential viability to hepatocytes, suggesting that all fatty acids are not equally toxic. GLP-1 reduces the free fatty acid burden in human cells. In vitro experiments presented here revealed that saturated, cis- and trans-unsaturated fatty acids have a variable effect on hepatocyte survival. Cell viability was reduced following fat loading and correlatively cell death was an outcome of apoptosis, as observed by an increase in cleaved caspase 3 and DAPI imaging. We also observed a marked reduction in cleaved caspase 3 following treatment with exendin-4 in cells containing fat when compared to non-fat loaded hepatocytes. We speculated that cell survival was the consequence of enhanced capacity to handle the unfolded protein response (UPR) and thereby prevent hepatocyte cell death via apoptosis. We observed that exendin-4 induced the expression of GRP78 in hepatocytes and liraglutide did the same in mouse livers fed a high fat high fructose diet.



These data suggest that while free fatty acids result in an increase in unfolded protein accumulation, there is an insufficient or effective level of GRP78 to abate the unfolded protein response. Exendin-4 on the other hand increased the amount of GRP78. Furthermore, if ER stress persists; C/EBP homologous protein (CHOP) activation can promote an imbalance between survival and apoptosis in favor of the latter. CHOP is ubiquitously expressed at very low levels. As we observed in vitro, FFA induced stress leads to an accumulation of CHOP. This observation is in contrast to that of Puri et al. CHOP expression was suppressed in NAFL and NASH in human liver samples. We demonstrated here that CHOP levels were increased in hepatocytes that showed high steatosis with either of the fatty acid types. This expression is reversed following exendin-4 treatment. These data were also seen following liraglutide therapy in the ALIOS-fed mice. Taken together, increased availability of the chaperone GRP78 and reduction in CHOP expression provides stress-relief to hepatocytes, suppressing apoptosis and promoting hepatocyte survival.



We are currently investigating the specificity of this effect of GLP-1 via upstream mechanisms related to the G-protein coupled receptor MedicGLP Supplement (GPCR). Immunohistochemical staining of liver sections for GRP78 and MedicGLP Supplement CHOP in mice given ALIOS diet and liraglutide suggest that GLP-1 analogs impart a strong affect in liver; though reduction in hepatic steatosis could as well be a result of whole body response to liraglutide, since GLP-1 receptors are present in other organs also. GRP78 leads to a reduction in autophagosome formation, though the conversion of LC3-I to LC3-II was not affected. Autophagy has also been implicated in cell death via apoptosis. Most of the studies investigating autophagy have used starvation as a source of its induction. Only recently studies by Singh et al. Although we had no prior information suggesting that GLP-1 proteins promote autophagy, the disappearance of fatty acids following in vitro exendin-4 treatment prompted us to examine this process.



Exendin-4 increased the rate of autophagosome and autophagolysosome formation or the autophagic flux. Furthermore, exendin-4 induced key protein makers of autophagy both at the mRNA and protein levels. These data were identical in both the in vitro and in vivo models employed here. Beclin and LC3B were increased in mice treated with liraglutide. High fat diet suppressed the expression of these genes and consequently their proteins. These observations are consistent with those made by Liu et al. Additionally, we observed an increase in LAMP2A. This protein is associated with the lysosomal membrane and is a key component of chaperone mediated autophagy (CMA). While we did not investigate CMA mechanisms it is possible that CMA is also activated in response to exendin-4 along with macroautophagy. Investigations by Koga et al. In our studies the high fat diet also suppressed markers of autophagy. These markers, however, were substantially increased transcriptionally in GLP-1 analog treated mice.