The Role Of Autophagy In Maintaining The Rat Liver Energy Metabolism During Acute Cold Exposure

BackgroundCold exposure is a common specific environmental health risk factor. Wehave found that0.5h of acute cold exposure at-15℃could lead to the changeof liver energy and relative cell signal pathways. Our work also shows thatcompound preparation can significantly relieve the falling of the rectaltemperature. However, the mechanisms are still not clear. Autophagy is thecommon physiological cellular process which could remove injured cellularorganelles, provide metabolism substrate, and promote cell survival. Duringenergy stress, activated autophagy could maintain cellular ATP level. Whetherautophagy participates in acute cold exposure-caused liver response and theregulative mechanism should be elucidated. And whether compound preparationcould affect rat liver energy level and protect liver from apoptosis during acutecold exposure and whether the protect function was mediated by the activation of autophagy also need to be clarified.AimsWith an established rat acute cold exposure model, we observed the effect ofacute cold exposure on apoptosis and cellular energy level and the role ofautophagy in hepatocytes during acute cold exposure. We further determined therole of autophagy in the regulatory effects of compound preparation on livercellular energy and apoptosis level in response to acute cold exposure. This couldhelp to clarify the regulatory mechanism of energy regulation during coldexposure and find out the effective target for compound preparation to promotecold resistant ability.Methods1. Acute cold exposure animal model: rats were exposed to cold chamber for4h.2. Liver glycogen assessment: liver tissue glycogen level was assessed by adetection kit.3. Liver cellular energy level: liver ATP level was assessed by an ATP detectionkit.4. Liver cell apoptosis: the cleavage of caspase-3was detected by western blot.And TUNEL assay kit was also used to determine the cell apoptosis.5. Autophagy activity: the expression of autophagy marker LC3-I and LC3-IIwas determination by western blot, and immunology method was used todetermine the autophagic vacuoles using MDC staining.6. The effect of autophagy inhibitor: the inhibitor was injected byintraperitoneal injection to reduce autophagy level. Then, its impact onenergy metabolism in the liver tissue in response to cold was observed.7. The effects of compound preparation on animals: the compound preparation was given to animals by intragastric administration. Then the change ofenergy and autophagy was determined.Results1. The effect of acute cold exposure on cell apoptosis in liver4h of acute cold exposure at-15℃caused significant cell apoptosis in liver.2. The effects of acute cold exposure on liver ATP and the level of liverglycogenThe exposure under-15℃for4h resulted the significant down-regulationof cellular ATP and glycogen in liver. The level of liver glycogen was5.03±0.86mg/g in control group and2.24±1.03mg/g in cold-exposed animals, while thelevel of ATP was0.293±0.018μmol/mg in control group and0.195±0.044μmol/mg in cold exposed animal. Statistical significance was observed both forATP and glycogen （p＜0.05）.3. The effect of acute cold exposure on autophagy in liverMDC staining results show that exposure under-15℃for4h lead to thesignificant increase of autophagic vacuoles in hepatocytes compare with that ofcontrol group（p＜0.05）. Western blot results show that the expression of LC3-IIwas significantly enhanced after acute cold exposure （p＜0.05）. Our resultssuggest that acute cold exposure lead to the significant enhancing of autophagy inrat liver.4. The effect of autophagy inhibiton on cell apoptosis and energy level inhepatocytes of rats exposed to acute cold exposureThe intraperitoneal injection of autophagy inhibitor wortmannin significantlyreduced the level of autophagy and the ATP content in hepatocytes（p＜0.05）.The addition of inhibitor caused significant increase of cell apoptosis and reducedATP level of cold exposure group from0.193±0.047μmol/mg to0.146±0.018 μmol/mg. And there is no observed significance of liver glycogen level betweenthese two groups.5. The effect of compound preparation on autophagy, cellular energy leveland cell apoptosis in hepatocytesThe intragastric administration of compound preparation for3dayssignificantly enhanced both ATP and liver glycogen and reduced the level ofapoptosis; it also caused decrease of autophagy in rats exposed to acute exposure.The ATP level was0.236±0.018μmol/mg in acute cold exposure plus compoundpreparation group and187±0.030μmol/mg in acute cold exposure group （p＜0.05）. The liver glycogen level was2.20±1.24mg/g in acute cold exposure pluscompound preparation group and1.38±0.97mg/g in acute cold exposure group（p＜0.05）.Conclusion[1] Acute cold exposure could cause rat liver cellular ATP and glycogendepletion and apoptosis.[2] The activation of autophagy during acute cold exposure could help tomaintain cellular ATP and energy level, and protect cell from apoptosis.[3] Compound preparation could protect liver cell from apoptosis bymaintaining liver cellular ATP level and liver glycogen level, and reducedcellular autophagy level, suggesting that compound preparation could reducethe damage on liver to a certain extent caused by acute cold exposure.