Energy reserves during food deprivation and compensatory growth in juvenile roach: the importance of season and temperature

The effect of 21 days of starvation, followed by a period of compensatory growth during refeeding, was studied in juvenile roach Rutilus rutilus during winter and summer, at 4, 20 and 27° C acclimation temperature and at a constant photoperiod (12L : 12D). Although light conditions were the same during summer and winter experiments and fish were acclimated to the same temperatures, there were significant differences in a range of variables between summer and winter. Generally winter fish were better prepared to face starvation than summer fish, especially when acclimated at a realistic cold season water temperature of 4° C. In winter, the cold acclimated fish had a two to three‐fold larger relative liver size with an approximately double fractional lipid content, in comparison to summer animals at the same temperature. Their white muscle protein and glycogen concentration, but not their lipid content, were significantly higher. Season, independent of photoperiod or reproductive cycle, was therefore an important factor that determined the physiological status of the animal, and should generally be taken into account when fish are acclimated to different temperature regimes. There were no significant differences between seasons with respect to growth. Juvenile roach showed compensatory growth at all three acclimation temperatures with maximal rates of compensatory growth at 27° C. The replenishment of body energy stores, which were utilized during the starvation period, was responsible for the observed mass gain at 4° C. The contribution of the different energy resources (protein, glycogen and lipid) was dependent on acclimation temperature. In 20 and 27° C acclimated roach, the energetic needs during food deprivation were met by metabolizing white muscle energy stores. While the concentration of white muscle glycogen had decreased after the fasting period, the concentrations of white muscle lipid and protein remained more or less constant. The mobilization of protein and fat was revealed by the reduced size of the muscle after fasting, which was reflected in a decrease in condition factor. At 20° C, liver lipids and glycogen were mobilized, which caused a decrease both in the relative liver size and in the concentration of these substrates. Liver size was also decreased after fasting in the 4° C acclimated fish, but the substrate concentrations remained stable. This experimental group additionally utilized white muscle glycogen during food deprivation. Almost all measured variables were back at the control level within 7 days of refeeding.     

The effects of long-term food deprivation on respiration and haematology of the neotropical fish Hoplias malabaricus

Growth of adult traíras Hoplias malabaricus ceased and body mass ( M ) decreased during starvation periods of 30, 60, 90, 150, 180 and 240 days. Hepatic reserves were mobilized in fish starved for 30 days, but liver mass of fish starved for longer periods was not significantly different from those starved for 30 days. Perivisceral fat bodies were consumed gradually, being completely exhausted after 240 days of food deprivation. Length of starvation was associated with a significant decrease in the oxygen uptake ( V o₂). In spite of this reduction, the respiratory frequency ( f _R) was kept nearly constant during the starvation periods. The haematocrit and the number of red blood cells decreased after 150 and 240 days of starvation, respectively. These parameters did not recover after refeeding (after 90 and 240 days of starvation). This hypometabolic state in response to food deprivation contributed to energy conservation during these periods. Traíras can survive food deprivation for periods of up to 180 days without reductions in metabolism and when they do become hypometabolic, normal metabolic rates are rapidly restored upon refeeding.     

Effect of fasting on glycogen metabolism and activities of glycolytic and gluconeogenic enzymes in the mudskipper Boleophthalmus boddaerti

During starvation, muscle glycogen in Boleophthalmus boddaerti was utilized preferentially over liver glycogen. In the first 10 days of fasting, the ratio of the active‘a’form of glycogen phosphorylase to total phosphorylase present in the liver was small. During this period, the active‘I’form of glycogen synthetase increased in the same tissue. In the muscle, the phosphorylase‘a’activity declined during the first 7 days and increased thereafter while the total glycogen synthetase activity showed a drastic decline during the first 13 days of fasting. The glycogen level in the liver and muscle of mudskippers starved for 21 days increased after refeeding. After 6 and 12 h refeeding, liver glycogen level was 8·5 ± 2·3 and 6·9 ± 4·5 mg·g wet wt ¹, respectively, as compared to 5·8 ± l·6mg·g wet wt ¹ in unfed fish. Muscle glycogen level after 6 and 12 h refeeding was 0·96±0·76 and 0·82 ± 0·50 mg·g wet wt ¹, respectively, as opposed to 0·21 ± 0·12 mg·g wet wt ¹ in the 21-days fasted fish. At the same time, activities of glycogen phosphorylase in the muscle and liver increased while the active‘I’form of glycogen synthetase showed higher activity in the liver. Since glycogen was resynthesized upon refeeding, this eliminated the possibility that glycogen depletion during starvation was due to stress or physical exhaustion after handling by the investigator. Throughout the experimental starvation period, the body weight of the mudskipper decreased, with a maximum of 12% weight loss after 21 days. Liver lipid reserves were utilized at the onset of fasting but were thereafter resynthesized. Muscle proteins were also metabolized as the fish were visibly thinner. However, no apparent change in protein content expressed as per gram wet weight was detected as the tissue hydration state was maintained constant. The increased degradation of liver and muscle reserves was coupled to an increase in the activities of key gluconeogenic enzymes in the liver (G6Pase, FDPase, PEPCK, MDH and PC). The increase in glucose synthesis was possibly necessary to counteract hypoglycemia brought about by starvation in B. boddaerti.     

Changes in glucose,glycogen, thyroid activity and hypothalamic catecholamines in tench by starvation and refeeding

The effects of short-term food deprivation (7 days) and refeeding (2 days) on different biochemical and neuroendocrine parameters were studied in tench. A 7-days fast resulted in a significant reduction of plasma glucose and glycogen hepatic content, supporting the key role of liver glycogen as energy depot for being consumed during fasting. The rapid recovery of normal values of blood glucose and glycogen stores by refeeding indicates a rapid replenishment of liver glycogen stores. The short-term starvation decreased circulating thyroid hormones (both T3 and T4) and T4 release from thyroid, supporting an interaction between nutritional state and thyroid function in tench. All these metabolic and hormonal changes were partial or totally reversed under refeeding conditions. An increase in hypothalamic content of norepinephrine and dopamine was found in fasted fish. This result might be a consequence of stress induced by starvation.     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

Energy Budget,Behavior and Leptin in Striped Hamsters Subjected to Food Restriction and Refeeding

Food restriction induces a loss of body mass that is often followed by rapid regaining of the lost weight when the restriction ends, consequently increasing a risk of development of obesity. To determine the physiological and behavioral mechanisms underlining the regaining, striped hamsters were restricted to 85% of initial food intake for 4 weeks and refed ad libitum for another 4 weeks. Changes in body mass, energy budget, activity, body composition and serum leptin level were measured. Body mass, body fat mass and serum leptin level significantly decreased in food-restricted hamsters, and increased when the restriction ended, showing a short “compensatory growth” rather than over-weight or obesity compared with ad libitum controls. During restriction, the time spent on activity increased significantly, which was opposite to the changes in serum leptin level. Food intake increased shortly during refeeding, which perhaps contributed to the rapid regaining of body mass. No correlation was observed between serum leptin and energy intake, while negative correlations were found in hamsters that were refed for 7 and 28 days. Exogenous leptin significantly decreased the time spent on activity during food restriction and attenuated the increase in food intake during refeeding. This suggests that low leptin in restricted animals may function as a starvation signal to induce an increase in activity behavior, and high leptin likely serves as a satiety signal to prevent activity during refeeding. Leptin may play a crucial role in controlling food intake when the restriction ends, and consequently preventing overweight.     

Changes in plasma thyroxine,triiodothyronine and cortisol associated with starvation in rainbow trout (Salmo gairdneri)

Synopsis Chronically starved rainbow trout (Salmo gairdneri) showed a significant fall in liver size, total liver glycogen, liver glycogen concentration and plasma glucose levels. Liver lipid concentration did not differ significantly from controls although total liver lipid reserves fell during the first 40 days of starvation but had partly recovered after 65 days of starvation. Plasma cortisol and T3 levels did not show consistent changes concomitant with food deprivation over the 65 day period of the experiment. However, plasma T4 levels in fish starved for 40 or 65 days were significantly lower than comparably fed animals. The involvement of T4 in intermediate metabolic processes in salmonids is discussed.     

Age-related augmentation of phosphorylase b kinase in hepatic tissue from the glycogen-storage-disease (gsd/gsd) rat.

The effects of food deprivation on body weight, liver weight, hepatic glycogen content, glycogenolytic enzymes and blood metabolites were compared in young and old phosphorylase b kinase-deficient (gsd/gsd) rats. Although the concentration of glycogen in liver from 9-week-old female gsd/gsd rats (730 mumol of glucose equivalents/g wet wt.) was increased by 7-8% during starvation, total hepatic glycogen was decreased by 12% after 24 h without food. In 12-month-old male gsd/gsd rats the concentration of liver glycogen (585 mumol of glucose equiv./g wet wt.) was decreased by 16% and total hepatic glycogen by nearly 40% after food deprivation for 24 h. Phosphorylase b kinase and phosphorylase a were present at approx. 10% of the control activities in 9-week-old gsd/gsd rats, but both enzyme activities were increased more than 3-fold in 12-month-old affected rodents. It is concluded that the age-related ability to mobilize hepatic glycogen appears to result from the augmentation of phosphorylase b kinase during maturation of the gsd/gsd rat.     

Temporal changes in body mass, body composition and metabolism of gibel carp Carassius auratus gibelio during food deprivation

To investigate the response to starvation, gibel carp Carassius auratus gibelio [12.5 ± 0.03 g (mean ± SE, n = 24)] were deprived of food at 25.8 ± 0.2°C (mean ± SE, n = 56) for 56 days. Body mass, proximate composition in whole body and muscle, and respiration were measured at 7‐day intervals. Body mass decreased with prolongation of deprivation, with a significant decline recorded after 7 days deprivation. Fish lost 22% of their fresh mass and 34% of dry mass after 56 days. Fish lost 38% of the body lipid over the first 7 days, and lost body lipid at a rate of 0–11% per week over the remaining 49 days. Body protein was lost at 1–5% per week throughout deprivation. Compared with the initial composition, body lipid concentration was lower and ash concentration higher on day 7. Water as a percentage of body mass was higher after 28 days, and protein concentration lower after 42 days, than at the start of deprivation. Muscle lipid and protein concentration was lower, and % water higher, after 7 days than at the start of deprivation, whereas muscle ash concentration was relatively constant during deprivation. After 56 days, fish lost body water by 18%, body lipid by 84%, body protein by 30%, and body energy by 45%. Oxygen consumption rate dropped from day 1 to day 3, increased from day 4 to day 14, gradually decreased from day 15 to day 35, and maintained a relatively constant level from day 36 to day 56. Results of the present experiment reveal that gibel carp utilize body lipid as a major energy source in the first 7 days of food deprivation, then turn to body protein as an energy fuel when lipid reserves are heavily depleted. Oxygen consumption is maintained at a relatively low and constant level when most lipid reserves are exhausted.     

Effects of intermittent starvation on mature Biomphalaria glabrata (Gastropoda: Planorbidae)

Mature Biomphalaria glabrata, submitted to four weeks of varied starvation times (0, 1, 3, 5, 6 & 7 d.week^–1, were thereafter refed during four weeks. The different intermittent starvation times had no significant effect on snails survival. As weekly starvation increased, the rate of change in body weight and fecundity decreased. In snails fed one or two d.week^–1, the rate of change in body weight was negative, while fecundity remained at a low level. Continuous hunger stopped oviposition.Starvation had no further effect on body growth after the first week of refeeding; however, its effect on fecundity remained significant over the two first weeks.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

Respiration and ammonia excretion of the shrimpCrangon crangon L.: Metabolic response to prolonged starvation

Summary Oxygen consumption and ammonia excretion were measured simultaneously in 25 individual shrimps (C. crangon) every two days during a 30-day imposed starvation.The first response to starvation was a 10% decrease in O₂ consumption and a 25% decrease in ammonia excretion. Following this, O₂ consumption decreased sharply while ammonia excretion tended to rise. From the 14th day until the end of the experiment, the rates of each remained steady, the respiratory rate being about 60% below and rate of excretion about 30% above control values.From the O:N ratio it appeared that carbohydrate reserves were quickly exhausted (3–4 days) and that lipids and proteins were the main substrates oxidized to meet the energetic requirements ofC. crangon. After 2 weeks of starvation the O:N ratio remained constant near a value of 8, indicating that only proteins were being utilised; a 50% loss of body protein after 30 days suggested that structural proteins were heavily catabolized. The meatabolic response ofCrangon to starvation (although seasonal variations must be considered) appears to depend mainly on high nitrogen requirements.     

Refeeding-Induced Brown Adipose Tissue Glycogen Hyper-Accumulation in Mice Is Mediated by Insulin and Catecholamines

Brown adipose tissue (BAT) generates heat during adaptive thermogenesis through a combination of oxidative metabolism and uncoupling protein 1-mediated electron transport chain uncoupling, using both free-fatty acids and glucose as substrate. Previous rat-based work in 1942 showed that prolonged partial fasting followed by refeeding led to a dramatic, transient increase in glycogen stores in multiple fat depots. In the present study, the protocol was replicated in male CD1 mice, resulting in a 2000-fold increase in interscapular BAT (IBAT) glycogen levels within 4–12 hours (hr) of refeeding, with IBAT glycogen stores reaching levels comparable to fed liver glycogen. Lesser effects occurred in white adipose tissues (WAT). Over the next 36 hr, glycogen levels dissipated and histological analysis revealed an over-accumulation of lipid droplets, suggesting a potential metabolic connection between glycogenolysis and lipid synthesis. 24 hr of total starvation followed by refeeding induced a robust and consistent glycogen over-accumulation similar in magnitude and time course to the prolonged partial fast. Experimentation demonstrated that hyperglycemia was not sufficient to drive glycogen accumulation in IBAT, but that elevated circulating insulin was sufficient. Additionally, pharmacological inhibition of catecholamine production reduced refeeding-induced IBAT glycogen storage, providing evidence of a contribution from the central nervous system. These findings highlight IBAT as a tissue that integrates both canonically-anabolic and catabolic stimulation for the promotion of glycogen storage during recovery from caloric deficit. The preservation of this robust response through many generations of animals not subjected to food deprivation suggests that the over-accumulation phenomenon plays a critical role in IBAT physiology.     

Effect of Starvation,Refeeding and Hydrocortisone Administration on Turnover of Myofibrillar Proteins Estimated by Urinary Excretion of Nτ-Methylhistidine in the Rat

Quantitative changes in fractional catabolic and synthetic rates of the myosin-actin pool in rat muscle under starvation and refeeding, during growth or after treatment with hydrocortisone were studied by estimating urinary excretion of N^τ-methylhistidine (3-methyl- histidine; Me-His).

Following deprivation of food, urinary Me-His output increased from 0.35 mg/day to 0.45 mg/day during first 2 day in spite of decreasing body Me-His pool. This high rate of Me-His excretion was maintained for the following 4 days of starvation and then decreased. When rats were refed a 20% casein diet after 10 days of starvation, Me-His excretion continued to decrease even after 3 days of refeeding. On the fifth day of refeeding, it began to rise progressively. During starvation, fractional catabolic rate of myosin-actin was about 3.7 %/day in comparison with 2.6 %/day of fed rats. After refeeding, the fractional catabolic rate decreased rapidly to a minimum value of 1.7 %/day on the third day. After that, it reached to a value of 2.6 %/day of fed rats. On the other hand, fractional synthetic rate of myosin-actin dropped immediately after fasting and the low rate of about 0.4 %/day was maintained during starvation period. Fractional synthetic rate recovered quickly after refeeding.

Urinary output of nitrogen and creatinine rose quickly on the first day after administration of hydrocortisone and on the second day it fell to their normal value. While Me-His excretion increased after injection of hydrocortisone up to 0.52 mg/day on the second day and this high excretion rate remained until the following day. From these results, it was shown that administration of hydrocortisone to rats enhances catabolism and reduces synthesis of myosin-actin. The results also show that the effect of this hormone on myofibrillar protein catabolism appears to last longer than its effect on nitrogen metabolism in the whole body judged from urinary nitrogen output.

Fractional rates of catabolism and synthesis of rat myosin-actin were 3.3 %/day (half- life of 21 days) and 7.2%/day, respectively, at the growth stage of 129 g body weight. These rates were 2.3 %/day (half-life of 30 days) and 2.8 %/day, respectively, at the mature stage of 363 g body weight.

Under the dietary conditions in this experiment, fractional synthetic rate changed far more dramatically than catabolic rate. This suggests that mass of muscle protein is primarily regulated by the rate of synthesis, although the rate of catabolism should not be neglected.     

Rapid activation and inactivation of fatty acid synthesis from glucose in vivo

The flux of glucose carbon to total body fatty acids was measured in unanesthetized mice either after fasting or 50-80 min after they nibbled a small test meal containing 120 mg of glucose (fasted-refed). Flux was calculated from plasma [(14)C]glucose specific activity curves and from total body (14)C-labeled fatty acid 30 min after intravenous injection of tracer [(14)C]glucose. Mobilization of liver glycogen, changes in the body glucose pool size, and total flux of carbon through the glucose pool during periods of fasting and refeeding were defined. Liver glycogen was almost completely depleted 8 hr after food removal. Body glucose pool size fell during fasting and increased after refeeding the test meal. Irreversible disposal rate of glucose C varied directly with body glucose pool size; but flux of glucose C into fatty acids increased exponentially as body glucose concentration increased. Within an hour after nibbling a small test meal, the flux of glucose C into total body fatty acids increased 700% in mice previously starved for 24 hr. However, flux of glucose C into fatty acids in postabsorptive mice (food removed for 2 hr; livers rich in glycogen) was only about 2% of the value calculated from published studies in which the incorporation of an intubated [(14)C]glucose load into total body fatty acid was measured in mice. A possible explanation for this phenomenon is presented.     

Carbohydrate metabolism in starved fifth instar larvae of Manduca sexta

The influence of starvation on carbohydrate metabolism in fifth instar larvae of Manduca sexta was studied. The percentage of active fat body glycogen phosphorylase increased from 10% to approximately 50% within 3 h of starvation; afterward the enzyme was slowly inactivated. The increase of phosphorylase activity might have been caused by a peptide(s) from the CC. The amount of fat body glycogen in starved animals decreased over 24 h by approximately 20 mg. The released glucose molecules seem to be converted mainly to trehalose because the hemolymph trehalose concentration in starved animals was always slightly higher than in the fed controls, and the glucose concentration decreased even when phosphorylase was activated. The chitosan content in starved larvae increased during the first 9 h of treatment to the same extent as in fed controls. It is suggested that fat body glycogen phosphorylase was activated during starvation to provide substrates for chitin synthesis and energy metabolism.     

Long‐term starvation in cave salamander effects on liver ultrastructure and energy reserve mobilization

The morphological alterations of hepatocytes of cave‐dwelling salamander Proteus anguinus anguinus after food deprivation periods of one and 18 months were investigated and the concentrations of glycogen, lipids, and proteins in the liver were determined. Quantitative analyses of the hepatocyte size, the lipid droplets, the number of mitochondria, and volume densities of M and P in the hepatocytes were completed. After one month of food deprivation, the cytological changes in the hepatocytes are mainly related to the distribution and amount of glycogen, which was dispersed in the cytoplasm and failed to form clumps typical of normal liver tissue. After 18 months of food deprivation hepatocytes were reduced in size, lipid droplets were less numerous, peroxisomes formed clusters with small, spherical mitochondria, and specific mitochondria increased in size and lost cristae. Lysosomes, autophagic vacuoles, and clear vacuoles were numerous. The liver integrity was apparently maintained, no significant loss of cytoplasmic constituents have been observed. Biochemical analysis revealed the utilization of stored metabolic reserves in the liver during food deprivation. Glycogen is rapidly utilized at the beginning of the starvation period, whereas lipids and proteins are utilized subsequently, during prolonged food deprivation. In the Proteus liver carbohydrates are maintained in appreciable amounts and this constitutes a very important energy depot, invaluable in the subterranean environment. J. Morphol. 274:887–900, 2013. © 2013 Wiley Periodicals, Inc.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Effects in vivo of food deprivation and 3-mercaptopicolinate in the glycogen-storage-disease (gsd/gsd) rat.

Intraperitoneal injection of 3-mercaptopicolinate into 24 h-food-deprived 27-week-old female control (GSD/GSD) rats lowered the concentration of circulating glucose by 66%, but glycerol and lactate concentrations were increased up to 3- and 4-fold respectively. In phosphorylase b kinase-deficient (gsd/gsd) rats the corresponding changes for blood glucose, lactate and glycerol were half those observed in the controls. Although the concentration of liver glycogen (approx. 12%, w/w) in the gsd/gsd rats was not altered during food deprivation, total hepatic glycogen was decreased by 17%. It is suggested that the gradual breakdown of the extensive hepatic glycogen stores during starvation assists in the maintenance of normoglycaemia in the gsd/gsd rat.