The autophagy-lysosomal pathway is involved in TAG degradation in the liver: the effect of high-sucrose and high-fat diet.

This study was designed to test the role of liver lipases in the degradation of liver triacylglycerols (TAG) and to determine the effect of dietary induced TAG accumulation in the liver on regulation of their lipolysis. Male Wistar rats were administered high-fat or high-sucrose diet for two weeks. Individual lipases (HL; TGH; LAL) were identified according to their different pH optimum. Administration of both diets resulted in liver TAG accumulation (HFD >>> HSD). The only lipase capable to hydrolyse intracellular TAG was LAL. On standard diet, LAL activity towards both endogenous and exogenous substrates was up-regulated in fasting and downregulated in fed state. The intensity of autophagy determined according to the LC3-II/LC3-I protein ratio followed a similar pattern. HFD led to an increase of this ratio, elevation of LAL activity in phagolysosomal fraction and abolishment of fasting/fed-dependent differences. LAL activity significantly correlated with ketogenesis in all groups (r = 0.86; P < 0.01). In the HFD group, we determined the enhanced release of lysosomal enzymes (glucuronidase, LAL) into the cytosol. Dgat-1 expression was up-regulated in HFD- and HSD-fed groups, which indicates increased FFA esterification. We demonstrated that LAL is a dominant enzyme involved in degradation of intracellular TAG in the liver and its translocation into the fraction of active (auto)phagolysosomes is stimulated by diet-induced TAG accumulation. Autophagy is stimulated under the same conditions as LAL and may represent the mechanism ensuring the substrate enzyme contact in autophagolysosomes. In fatty liver, destabilization of (auto)phagolysosomes may contribute to their susceptibility to further stress factors.

Lipasin/betatrophin is differentially expressed in liver and white adipose tissue without association with insulin resistance in Wistar and Goto-Kakizaki rats.

Lipasin is a recently identified lipokine expressed predominantly in liver and in adipose tissue. It was linked to insulin resistance in mice and to type 1 and type 2 diabetes (T1D, T2D) in humans. No metabolic studies concerning lipasin were performed yet in rats. Therefore, we used rat model of T2D and insulin resistance, Goto-Kakizaki (GK) rats, to determine changes of lipasin expression in liver and in white adipose tissue (WAT) over 52 weeks in the relation to glucose tolerance, peripheral tissue insulin sensitivity and adiposity. GK rats were grossly glucose intolerant since the age of 6 weeks and developed peripheral insulin resistance at the age of 20 weeks. Expression of lipasin in the liver did not differ between GK and Wistar rats, declining with age, and it was not related to hepatic triacylglycerol content. In WAT, the lipasin expression was significantly higher in Wistar rats where it correlated positively with adiposity. No such correlation was found in GK rats. In conclusion, lipasin expression was associated neither with a mild age-related insulin resistance (Wistar), nor with severe genetically-based insulin resistance (GK).

Formation of Cholangiogenic Cysts Following Intrahepatic Islet Transplantation in Streptozotocin Diabetic Rats.

Permanent hyperinsulinemia and the resulting overstimulation of the insulin receptor signaling pathway is suspected as a trigger of cancer genesis in the livers of type 2 diabetic patients. Liver tissue (LT) surrounding transplanted pancreatic islets (PI) can be permanently exposed to insulin in even higher concentrations than in type 2 diabetic patients. Therefore, this study examines the effect of PI transplantation (Tx) on LT in animals with streptozotocin (STZ)-induced diabetes mellitus. The suboptimal mass (400 or 1000) of isogeneic PI was transplanted into either the portal vein or under the kidney capsule of diabetic Brown Norway (BN) rats. Healthy BN rats treated with 400 isogeneic PI transplanted in the portal vein served as a control group. During the first 6 months after PI Tx, small and infrequent cystic lesions developed in animals with STZ diabetes, irrespective of the Tx site. In 10 months, frequent and complex cystic lesions appeared in these animals. In the control group, several small lesions were detected but not until 10 months after the PI Tx. In summary, STZ is the likely main inductor of hepatic cystic lesions, but the contribution of PI was not confirmed.

Important role of autophagy in regulation of metabolic processes in health, disease and aging.

Autophagy is the basic catabolic mechanism that involves degradation of dysfunctional cellular components through the action of lysosome as well as supplying energy and compounds for the synthesis of essential biomacromolecules. This process enables cells to survive stress from the external environment like nutrient deprivation. Autophagy is important in the breakdown of proteins, carbohydrates and lipids as well. Furthermore, recent studies have shown that autophagy is critical in wide range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including lysosomal storage disease, myopathies, neurodegeneration and various metabolic disorders. This review summarizes the most up-to-date findings on what role autophagy plays in metabolism.

Effect of short- and long-term high-fat feeding on autophagy flux and lysosomal activity in rat liver.

Autophagy-lysosomal pathway is a cellular mechanism ensuring degradation of various macromolecules like proteins or triacylglycerols (TAG). Its disruption is related to many pathological states, including liver steatosis. We compared the effect of short- and long-established steatosis on the intensity of autophagy-lysosomal pathway in rat liver. The experiments were carried out on 3-month old Wistar rats fed standard (SD) or high-fat diet for 2 (HF-2) or 10 (HF-10) weeks. HF diet administered animals accumulated an increased amount of TAG in the liver (HF-2->HF-10). Autophagy flux was up-regulated in HF-2 group but nearly inhibited after 10 weeks of HF administration. The expression of autophagy related genes was up-regulated in HF-2 but normal in HF-10. In contrast, total activities of two lysosomal enzymes, lysosomal lipase (LAL) and acid phosphatase, were unaffected in HF-2 but significantly increased in HF-10 groups. mRNA expression of lysosomal enzymes was not affected by the diet. We conclude that in a state of metabolic unbalance (steatosis), autophagy machinery and lysosomal enzymes expression are regulated independently. The accumulation of TAG in the liver is associated with the increase of total LAL activity and protein expression. In contrast, the autophagy response is bi-phasic and after rapid increase it is significantly diminished. This may represent an adaptive mechanism that counteracts the excessive degradation of substrate, i.e. TAG, and eliminate over-production of potentially hazardous lipid-degradation intermediates.

Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.