Starvation-induced microautophagic vacuoles in rat myocardial cells

During prolonged starvation the heart atrophies and loses protein mass. Debate lingers over the basic mechanisms in the production of negative cardiac protein balance during starvation. The extent to which cardiac proteolysis takes place within the lysosomal vacuolar system is unknown. The present communication examines the starvation-induced changes within the lysosomal system of rat myocardial cells, as studied by means of conventional electron-microscopic techniques. Special attention has been paid to the occurrence of microautophagic vacuoles. It is concluded that during prolonged starvation microautophagic vacuoles appear in rat myocardial cells, suggesting the induction of a microautophagic pathway of lysosomal proteolysis.     

Naphthol-yellow-S protein content of individual rat hepatocytes as related to food intake and short-term starvation

With regard to the protein content, as analysed cytophotometrically, of hepatocytes from rats kept under a 12L 12D photoperiod (photophase 7:00-19:00), the following facts have been established: 1) Hepatocytes of different classes of ploidy all demonstrate, more or less equally, daily variations in protein content and also its reduction after 24-h fasting. 2) With computer analysis of data obtained at eight time points during a period of 24 h, a sinusoidal curve of the protein content of individual mononuclear tetraploid hepatocytes throughout the day could be demonstrated with a maximum at 6:20 and a minimum at 18:20. 3) Animals, fed with meals via a dispensing machine from 23:00 to 24:00 only, show a similar sinusoidal curve but with higher amplitude, and a virtually identical mean value as those fed ad libitum. The maximum was found at 10:40, revealing a time lag of 12 h after food intake, the minimum at 22:40. 4) Trained animals deprived of food during the standardized feeding time revealed a moderate reduction of their hepatocyte protein content in the first 6 h, then a 6-h period with a steep fall followed by a slower reduction. After 24 h, the mean hepatocyte protein mass had decreased to 72% of that at the commencement of fasting at 23:00.     

Studies on vinblastine-induced autophagocytosis in mouse liver. III. A quantitative study

The microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection. The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.     

Regulation of microautophagy and basal protein turnover in rat liver. Effects of short-term starvation

Basal rates of long-lived (resident) protein degradation in rat liver, measured during perfusion after amino acid suppression of macroautophagy, were shown to be strongly regulated by caloric deprivation, decreasing 70% over 48 h in animals fed a high protein diet and 50% in normal controls. Intralysosomal pools of degradable protein correlated directly with basal turnover over this range, yielding a slope (0.09 min-1) that was virtually identical with previous estimates of macroautophagic turnover. The specific radioactivity of valine released from lysosomes in previously labeled livers was the same as that in plasma in both basal and deprivation-induced states. Quantitative electron microscopy revealed a significant decrease with starvation in the absolute volume of a class of secondary lysosome (type A) previously associated with basal or microautophagy. By contrast, the volumes of other microautophagic forms, which comprised roughly 10% of the total, did not change. Taking 0.087 min-1 as the turnover constant of degradable intralysosomal protein and assuming that the concentration of sequestered protein was the same in all vacuoles as that in cytoplasm, we obtained close agreement between predicted and observed rates of basal protein turnover over the range of regulation. The results support the view that the lysosomal system is the final step in the basal degradation of long-lived proteins in the hepatocyte and that a specific class of secondary lysosome (type A) plays a direct role in its regulation during caloric starvation.     

Impact of fasting time on hepatic lipid metabolism in nutritional animal studies

Many animal studies on improvement of lipid metabolism, using dietary components, fast the animals on the final day of the feeding. Although fasting has a significant impact on lipid metabolism, its time-dependent influence is not fully understood. We examined the effects of several fasting times on lipid metabolism. Rats fed with a semisynthetic diet for 2 wk were killed after 0 (9:00?am), 6 (7:00?am–1:00?pm), 9 (0:00?am–9:00?am), and 13?h (8:00?pm–9:00?am) of fasting. Compared to the 0?h group, marked reduction of liver weight and hepatic triacylglycerol content was observed in the 9 and 13?h groups. Activities of hepatic enzymes involved in fatty acid synthesis gradually decreased during fasting. In contrast, drastic time-dependent reduction of gene expression, of the enzymes, was observed. Expression of carnitine palmitoyltransferase mRNA was higher in the fasting groups than in the 0?h group. Our study showed that fasting has a significant impact on several parameters related to lipid metabolism in rat liver.     

Isolation and characterization of autophagic vacuoles from rat kidney cortex

The number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues. The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparent vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.     

Regulation of lipogenesis in isolated hepatocytes by triglyceride-rich lipoproteins.

Very low density lipoproteins, chylomicrons, and remnants caused, within an hour, significant inhibition of fatty acid synthesis but not cholesterol synthesis in hepatocytes isolated from meal-fed rats. In contrast, low density lipoproteins, high density lipoproteins, and the serum fraction of density greater than 1.21 failed to significantly inhibit either fatty acid or cholesterol synthesis within 1 h. The Scatchard plots of specific binding showed that rat and human very low density lipoproteins interact with the high affinity sites on the hepatocytes with the apparent dissociation constants of 64 and 106 nM, respectively. These data also indicated that each hepatocyte was capable of binding 6 X 10(5) molecules of very low density lipoproteins.     

Time relationship between circadian variation of serum levels of leptin, insulin and cortisol in healthy subjects.

BACKGROUND: Leptin is involved in the regulation of eating behavior. Its serum levels are determined by fat mass but a diurnal rhythm is also described. It is not clear whether leptin levels are also controlled in vivo by hormonal stimuli, like insulin or cortisol. METHODS AND RESULTS: This possible temporal relation was investigated by serial measurements during 24 h (group A) and 46 h (group B) in 15 healthy volunteers and another 10 subjects (group C) while fasting for 72 h. Maximal leptin levels were observed at 4:00 a.m. and 4:00 p.m. in subjects on a normal diet. During 24 h starvation (group B), there was a 40% decrease of mean leptin concentration when compared to baseline values. In group C, the leptin concentration under starvation dropped to 25% of basal levels after 72 h. Pooled data from group A and the nonfasting data from group B showed an insulin increase preceding leptin increase by 6 h (r = 0.405, p < 0.0001), while cortisol decreased 4 h (r = 0.361, p < 0.001) after leptin decrease. CONCLUSION: Starvation results in a fall of circulating leptin, ending leptin rhythmicity. Food intake is causally involved in the fluctuation of leptin levels in serum. Presumably this effect is mediated by insulin, while cortisol does not seem to affect leptin release directly in vivo.     

Decrease in the Time-Dependent Difference in Urinary Excretion of Furosemide with Age

The influence of age on the time-dependent difference in urinary excretion of furosemide, a loop diuretic agent, was examined in this longitudinal study. Male Wistar rats were maintained under conditions of light from 07:00 to 19:00 h and dark from 19:00 to 07:00 h. Furosemide (30 mg/kg) was given orally at 12:00 h (day trial) or 00:00 h (night trial) to rats at 3 months of age, and urine was collected for 8 h after dosage. Thereafter, the identical protocol was repeated using the same animals at 6, 9, 12, 15, 18, and 21 months of age. The urinary excretion of furosemide was significantly greater in the day than in the night trial at 3 months of age. Such a time-dependent difference was observed for up to 15 months, but disappeared at 18 and 21 months of age. The time-dependent difference in urinary excretion of furosemide (day trial — night trial) decreased gradually throughout the observation period of the study. These results suggest that the time-dependent difference in the urinary excretion of furosemide diminishes during the aging process and disappears by 18 months of age in male Wistar rats.     

The effects of starving versus fasting on blood composition in larval house crickets

Larval crickets (Acheta domesticus) starved for 2 days during the growth phase of the instar consumed twice as much water as larvae that ceased feeding of their own accord during the last 2 days of the last instar. The behaviour of drinking more water during starvation may compensate for dry weight loss and prevent the larvae from missing the critical weight required to initiate the next moult. During starvation the plasma volume increased while the tissue volume remained constant, which produced a shift in both organic and inorganic solutes from the tissues into the plasma. During fasting there was no change in tissue or plasma volume, therefore large osmotic adjustments were unnecessary, and the only change in plasma solutes noted was a decline in plasma proteins.The titres of proteins, lipids and amino acids remained constant during 2 days of starvation, though the amount of each increased because of the increased plasma volume. Although both the titre and the amount of plasma sugar sharply declined during starvation, there was no change in the sugar titre when the insects fasted. There was some evidence that prior to fasting the programmed gradual decline in food intake matched the decline in metabolic rate, which permitted a plasma sugar stability not evident in starved larvae. The decline in plasma proteins during the fasting phase appeared due to the removal of a larval specific protein and not a direct result of fasting.     

Division of Physarum mitochondria during starvation

Microplasmodia of Physarum polycephalum used in this study form spherules after 18 h of starvation. Stereological morphometry revealed that between the 2nd and the 5th hour of starvation the number of mitochondria in 1 mm3 of cytoplasm rises from about 12 to 24 millions and the mean volume of mitochondria drops from circa 4.6 to 3.0 microns3. This denotes the synchronous division of mitochondria. The daughter mitochondria show an increase in density of the matrix and a decrease in condensation of the net of tubular cristae. The mitochondrial division, decrease in activity of the respiratory chain and maximum of its cyanide resistance occur at the same time.     

Cytochemical determination of acid phosphatase activity in isolated rat hepatocytes during starvation-induced proteolysis

Summary Acid phosphatase activity in isolated rat liver parenchymal cells has been investigated with quantitative histochemical means during short-term starvation, which leads to a considerable loss in protein mass in the parenchyma. Animals trained to a meal-feeding regime in which food was available during 1 h only per 24 h (using an automatic food dispensing machine), were sacrified 6, 12, 18, 24 and 36 h after food had been withheld at the time point (23.00 h) of meal feeding. Acid phosphatase activity was analysed cytophotometrically in isolated hepatocytes incorporated into polyacrylamide gels before the enzyme reaction technique with the post-azo coupling was carried out. No indication could be found for any significant changes in the amount of acid phosphatase activity per individual hepatocyte during the entire period of fasting, as compared with two time points (11.00 and 23.00 h) before the theoretical onset of fasting. It is concluded that the considerable enhancement of protein degradation in the lysosomal apparatus during fasting is not reflected by changes in the cellular acid phosphatase activity.     

The vacuolar transporter chaperone (VTC) complex is required for microautophagy

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.     

Some physiological and biochemical features of starvation and refeeding in small wild rodents (Microtinae)

Blood glucose and leucocytes and liver glycogen and lipids were investigated in Cl. rutilus and Cl. rufocanus fasted for 0, 6, 12, 18 and 22 hr. It was observed: 1. In both species blood glucose content drops (1.3-1.5 times) by 6 hr, slowly rises by 12 hr and then progressively and strongly declines up to the end of starvation (36% and 27% of control for Cl. rutilus and Cl. rufocanus respectively). 2. Liver glycogen was depleted by 6 hr of fasting while lipids accumulate in liver during starvation in a large quantity (3.4-3.6 times at the end). 3. White blood cells content in fasted animals decreases. At one end of starvation it equals 23% and 16% of control for Cl. rutilus and Cl. rufocanus, respectively. 4. Hypoglycemia in Microtinae during starvation is pronounced compared with that in Muridae. Leucopenia and accumulation of lot of lipids in liver are new phenomena for fasting. 5. After refeeding hyperglycemia develops, the liver accumulates large quantities of glycogen. Recovery of all indexes slow. Complete normalization does not occur by 16 hr of refeeding.     

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.     

Comparison of tissue pyruvate dehydrogenase activities on re-feeding rats fed ad libitum or meal-fed rats with a chow-diet meal.

Meal-fed rats and rats fed ad libitum had similar rates of hepatic glycogenesis at 60 min after the initiation of re-feeding a chow meal after 22 h starvation, but hepatic PDHa (active form of pyruvate dehydrogenase) activities were 4-fold higher in the meal-fed group. In heart, PDHa activities were 3-fold higher before re-feeding and 2-fold higher after re-feeding in the meal-fed group compared with the group fed ad lib. The blood metabolite profile suggested diminished fat oxidation in starved meal-fed rats and accelerated flux through PDH in meal-fed re-fed rats compared with the group fed ad lib.     

Urinary free cortisol and cortisone excretion in healthy individuals: influence of water loading

The influence of water loading on urinary excretion of free cortisol and cortisone was investigated in healthy men. The results were as follows: water loading tests (intake of 0.25-1.5 L) in a single individual showed that a water load of 1.5 L reliably increased the excretion of urine, free cortisol and cortisone (p < 0.01). Regression analyses gave significant correlations of urine volume with free cortisol and free cortisone, and of free cortisol and free cortisone. Corresponding results were obtained when water loading tests were performed in males who ingested 1.5 L of water (n = 8): the excretion of urine, free cortisol and free cortisone were significantly augmented; correlated was urine volume with free cortisol and free cortisone, and free cortisol with free cortisone. In a third set of tests, volunteers collected one 5 h urine (10:00-15:00 h) after the intake of 3 x 0.1 or 0.5 L at 11:00, 12:00 and 14:00 h. Excretion of urine, free cortisol and free cortisone in males of the low water loading group (3 x 0.1 L) was 0.59 mL/min, and 8.2 or 15.0 microg/5 h; corresponding values in individuals ingesting 3 x 0.5 L of water were 1.5 mL/min (p < 0.01), 12.3 microg/5 h (p > 0.05) and 26.3 microg/5 h (p < 0.02). In summary, urinary free cortisol and cortisone excretion in healthy men depends on urine volume, especially during water diuresis. Thus, interpretation of free cortisol and especially of free cortisone excretion is only possible if subjects strictly control their fluid intake and if urine volume is considered an important pre-analytical parameter-otherwise, interpretation of urinary free cortisol results is difficult and of urinary free cortisone data remains tenuous at best.     

Routine overnight starvation and immunocytochemistry of hepatocyte albumin content

Summary The indirect immunoperoxidase method was used to identify albumin in hepatocytes of rats before and after periods of starvation. All hepatocytes in fed rats contained a relatively large amount of nascent albumin. Overnight fasting reduced the number of hepatocytes with a large amount of albumin to primarily those surrounding terminal hepatic venules. These were estimated to be about 30% of the population. The other cells had only a slight amount of albumin. After 48 h of fasting all hepatocytes contained a low level of albumin.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

西伯利亚鲟在高温下饥饿后的补偿生长

在高温(29±1)℃下将西伯利亚鲟幼鱼(21.61±0.03)g饥饿0(对照)、6、12和18d后恢复摄食3周, 研究摄食、生长和鱼体组成的变化。结果表明, 经过不同程度饥饿的鱼体重均显著低于对照组(P0.05), 而饥饿18d(S18组)的鱼体重显著低于对照(P0.05)。在饥饿过程中,鱼体脂肪含量和肝脏肝糖原含量下降的同时, 各饥饿组的灰分含量上升, 但仅S18组与对照差异显著(P0.05)。结果表明, 西伯利亚鲟在高温下表现出完全补偿现象, 且是通过同时提高摄食率和饲料效率来实现补偿生长的, 因此在夏季高温时对鲟鱼进行一段时间适度的饥饿可以在不影响生长和体成分的前提下节约饲料成本, 减少因过量投饵而引起的环境污染。