In vivo effect of an antilipolytic drug (3,5'-dimethylpyrazole) on autophagic proteolysis and autophagy-related gene expression in rat liver

Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5′-dimethylpyrazole, DMP, 12 mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.     

Evidence for selective mitochondrial autophagy and failure in aging

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Starvation-induced autophagy is required for maintaining an amino acid pool for gluconeogenesis and for the synthesis of proteins essential to survival under starvation conditions. In addition, autophagy plays an important role in the degradation of excess or injured organelles, including mitochondria. To test the hypothesis of an involvement of a decrease in autophagy in the process of aging, we explored the antiaging effects of pharmacological stimulation of autophagy on the age-dependent accumulation of 8-OHdG-rich mitochondria in rat liver. Male 3-month and 16-month-old 24 hours-fasted Sprague Dawley rats were injected with the antilipolytic agent [3,5-dimethylpyrazole (DMP)] intraperitoneally. Results showed that drug injection rescued older cells from the accumulation of 8-OHdG in the mtDNA in less than 6 hours, but no significant decrease in the level of cytochrome c oxidase activity was observed. Together, these data provide indirect evidence that 8-OHdG might accumulate in a small pool of mitochondria with increasing age rather than be degraded by the autophagic machinery selectively.     

Studies on cellular autophagocytosis : Vinblastine-induced autophagy in the rat liver

Administration of vinblastine (5 mg/kg) in two doses intraperitoneally to rats induced a prominent formation of autophagic vacuoles in rat liver parenchymal cells. Four hours after the first injection of vinblastine, clearly recognizable organelles were seen inside these vacuoles, and by 12 h the cells were filled with residual body type of lysosomes. As compared with glucagon administration, vinblastine was followed by a far greater degree of autophagy, and therefore, offers an excellent model for future studies on autophagy.     

Evidence for Selective Mitochondrial Autophagy and Failure in Aging

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Starvation-induced autophagy is required for maintaining an amino acid pool for gluconeogenesis and for the synthesis of proteins essential to survival under starvation conditions. In addition, autophagy plays an important role in the degradation of excess or injured organelles, including mitochondria. To test the hypothesis of an involvement of a decrease in autophagy in the process of aging, we explored the antiaging effects of pharmacological stimulation of autophagy on the age-dependent accumulation of 8-OHdG-rich mitochondria in rat liver. Male 3-month and 16-month-old 24 hours-fasted Sprague Dawley rats were injected with the antilipolytic agent [3,5-dimethylpyrazole (DMP)] intraperitoneally. Results showed that drug injection rescued older cells from the accumulation of 8-OHdG in the mtDNA in less than 6 hours, but no significant decrease in the level of cytocrome c oxidase activity was observed. Together, these data provide indirect evidence that 8-OHdG might accumulate in a small pool of mitochondria with increasing age rather than be degraded by the autophagic machinery selectively.

Addendum to:

Stimulation of Macroautophagy Can Rescue Older Cells from 8-OHdG mtDNA Accumulation: A Safe and Easy Way to Meet Goals in the SENS Agenda

A. Donati, M. Taddei, G. Cavallini and E Bergamini

Rejuvenation Res 2006; 9:408-12     

Glycogen autophagy in the liver and heart of newborn rats. The effects of glucagon, adrenalin or rapamycin

The effects of glucagon, adrenalin or rapamycin on glycogen autophagy in the liver and heart of newborn rats were studied using biochemical determinations and electron microscopy. Glucagon or adrenalin increased autophagic activity in the hepatocytes and myocardiocytes, glycogen-hydrolyzing acid glucosidase activity in the liver and heart and degradation of glycogen inside the autophagic vacuoles. Glucagon or adrenalin also increased the maltose-hydrolyzing acid glucosidase activity in the liver, but not in the heart. Similar effects were produced in the newborn heart by rapamycin. These observations support previous studies suggesting that the cellular machinery which controls glycogen autophagy in the liver and heart of newborn animals, is regulated by the cyclic AMP and the mTOR pathways.     

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.     

Liver autophagy contributes to the maintenance of blood glucose and amino acid levels

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.     

Liver autophagy contributes to the maintenance of blood glucose and amino acid levels

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.     

Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats

Sequential carcinogen treatment (diethylnitrosamine/partial hepatectomy followed by 2-acetylaminofluorene (2-AAF] induced multiple hepatocarcinomas in rats with 100% certainty within a year. Enzyme-altered lesions, i.e. gamma-glutamyltranspeptidase (GGT)-positive and/or ATPase-negative cell foci, were numerous already at 8 weeks, and suspensions of purified hepatocytes isolated (by collagenase perfusion) at this time contained 30-40% GGT-positive cells. These hepatocyte suspensions were markedly deficient with respect to autophagic protein degradation (in comparison with cell suspensions from normal rats), and the cells lost less protein and survived much better than normal hepatocytes in culture under conditions of amino acid deprivation (which activates the autophagic mechanism). The anabolic advantage of reduced autophagy may possibly contribute to the selective outgrowth of preneoplastic cells during the earliest stage of liver carcinogenesis. Inclusion of the autophagy inhibitor 3-methyladenine in the culture medium elevated the survival of normal hepatocytes up to the level seen with hepatocytes from carcinogen-treated animals, suggesting that protection of normal cells by autophagy suppression may be a potentially interesting therapeutic principle.     

Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Effects of antilipolytic agents on rat liver peroxisomes and peroxisomal oxidative activities

The mechanisms involved in the inhibitory effects of antilipolytic agents on rat liver peroxisomal fatty acid oxidative activity have been explored. Treatment of fasting rats with antilipolytic drugs (either 3,5-dimethylpyrazole (12 mg/kg body weight) or Acipimox (25 mg/kg body weight)) resulted in a decrease in free fatty acid and glucose plasma levels within 5–10 and in a significant increase in the plasma glucagon to insulin ratio within 15. Changes in the fatty acid oxidative activity appeared with a 2.5–3 h delay and were then very rapid (a 30–40% decrease in the activity occured in additional 2 h). Many peroxisomal enzyme activities (including non-β-oxidative activities such as uricase and D-amino acid oxidase) exhibited similar changes with the same delay. Simultaneously with the enzyme changes, at the electron microscope level many autophagic vacuoles were detected in the liver cells, often containing peroxisomal structures. Glutamine, an inhibitor of proteolysis in vivo, prevented the decrease in enzyme activities. It was concluded that the decrease in peroxisomal enzyme activities may be the consequence of enhanced peroxisome degradation due to the stimulation of autophagic processes in liver cells.     

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.     

Age-related differences in the effect of glucagon on metabolism in rat liver.

The authors found differences in the metabolic response of 10- and 120-day-old rats to glucagon. In 10-day-old young, the administration of glucagon was followed in 5 min by an abrupt small increase in the blood sugar level, which continued to rise and attained the maximum 2 hours after the injection of glucagon. In adult rats there was an abrupt large increase in the blood sugar level in the first minutes after administering glucagon; after that the blood sugar level fell, but remained significantly higher than in the controls. In a series of experiments on the isolated perfused liver, no differences were found in glucose and protein release from the liver into the perfusion medium, but the protein concentration in the liver of the younger rats fell. The results show that the liver of young rats, after the injection of glucagon, draws on its own protein resources for the substrates needed for gluconeogenesis.     

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A₁ or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.     

Effect of dietary protein on the capacity of urea synthesis in rats

The in vivo capacity of urea nitrogen synthesis (CUNS) during alanine stimulation was measured within the blood amino acid concentration interval 7.3-11.6 mmol/l, where urea synthesis is at maximum and independent of substrate concentration. Three groups of rats were fed for 14 days, either a low protein diet (8%), a normal diet (17%), or a high protein diet (53%). Diet protein modified both CUNS and plasma glucagon concentration. CUNS was 5.86 +/- 2.93, 7.43 +/- 2.16, and 19.31 +/- 4.32 mumol/(min.100 g BW) (mean +/- SD, N = 6), respectively. The corresponding plasma glucagon concentrations after alanine stimulation were 222 +/- 400, 633 +/- 229, and 1700 +/- 627 ng/l, respectively. The in vivo kinetics of urea production is regulated by dietary protein, possibly via glucagon. This implies that the liver plays an active part in adaptation of whole body nitrogen homeostasis to dietary changes.     

Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.     

Sphingosine kinase 1 protects renal tubular epithelial cells from renal fibrosis via induction of autophagy

Autophagy is an important homoeostatic mechanism for the lysosomal degradation of protein aggregates and damaged cytoplasmic components. Recent studies suggest that autophagy which is induced by TGF-β1 suppresses kidney fibrosis in renal tubular epithelial cells (RTECs) of obstructed kidneys. Sphingosine kinase 1(SK1), converting sphingosine into endogenous sphingosine-1-phosphate (S1P), was shown to modulate autophagy and involved in the processes of fibrotic diseases. Since SK1 activity is also up-regulated by TGF-β1, we explored its effect on the induction of autophagy and development of renal fibrosis in this study. In vitro, SK1 expression and activity were markedly increased by TGF-β1 stimulation in a time and concentration dependent manner, and concomitant changes in autophagic response were observed in HK-2 cells. Further, knockdown of SK-1 led to a decrease of autophagy whereas overexpression of SK1 caused a greater induction of autophagy. In addition, overexpression of SK1 resulted in decreased of mature TGF-β levels through autophagic degradation. In vivo, SK1 enzymatic activity and autophagic response were both up-regulated in a mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO); meanwhile, increased of mature TGF-β1 and deposition of extracellular matrix (ECM) were observed in tubulointerstitial areas compared with sham-operated mice. However, aggravation of renal fibrosis was detected when SK1 inhibitor PF-543 was applied to suppress SK1 enzymatic activity in UUO mice. At the same time, autophagy was also inhibited by PF-543. Thus, our findings suggest that SK1 activation is renoprotective via induction of autophagy in the fibrotic process.     

Transcriptional and translational control of the biphasic increase in ornithine decarboxylase activity in liver

The requirements for protein and RNA synthesis for each of the two increases in liver ornithine decarboxylase activity after the injection of unoperated rats with a solution containing glucagon and after 70% hepatectomy were studied with cycloheximide and actinomycin D. Protein synthesis is required for both increases whereas RNA formation is essential for the first elevation only. The second increase appears to be dependent upon RNA that is made during the period of the first rise in activity.The two rises in the decarboxylase activity may be caused by different stimuli. After the injection of the mixture with glucagon, the first elevation is accompanied by increases in hepatic tyrosine aminotransferase activity and in the rate of transport into liver cells of the model amino acid, α-aminoisobutyrate. Neither an increase in the aminotransferase activity nor in amino acid uptake occurs, however, during the period of the second elevation in the decarboxylase activity.