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.     

Effects of 24-hour starvation period on metabolic parameters of 20-day-old rats

The effects of a 24-hour starvation period upon 20-day-old rat pups were studied both in animals kept in the presence of their starved dams and in their absence. The relative loss of body weight was more intense in the rats that received no milk, being however, severe in both groups. There was a significant maintenance of both plasma glucose levels and total amino acids, with differences in blood glucose compartmentation and a remarkable uniformity in the effects of starvation upon individual amino-acid concentrations. A significant increase in plasma urea was observed, higher in the rats kept in the presence of the dam. Ketone bodies increased with starvation but their final levels were lower than in adults. The general pattern of metabolic change observed suggests a situation, after 24-h food deprivation, similar to that of long term starvation in adults; with an active protein amino-acid catabolism but with a remarkable maintenance of circulating foodstuff levels.     

Effect of starvation and protein-feeding on blood amino acid compartmentation of domestic fowl hatchlings

The amino acid concentrations in plasma and blood cells of 5-day old domestic fowl hatchlings that received either standard feeding, protein-feeding or were starved have been determined. The effects of 5-day starvation or protein feeding did not alter significantly the combined amino acid concentration of blood plasma, but decreased blood cell levels. The patterns of individual amino acid changes observed in starvation or protein-feeding were similar in both groups when compared with those of controls. However, starvation-induced effects were actually more marked than those observed in protein-fed animals. The patterns of change with starvation of individual amino acids in the hatchling blood compartments were very different from those observed in mammals subjected to short or medium-term starvation. The mechanisms controlling circulating amino acid concentrations act in both situations studied to maintain the plasma amino acid concentrations despite marked changes in the availability of 2-amino nitrogen energy to the animal; changes in blood amino acid compartmentation buffering plasma amino acid availability.     

The effects of starvation and force-feeding on the metabolism of the Northern pike, Esox lucius L.

The effects of starvation and force-feeding on certain tissue and blood constituents were studied in the Northern pike, Esox lucius L. Starvation resulted in a reduction of liver and muscle glycogen and liver lipid. Blood glucose concentration and haematocrit were reduced, total plasma cholesterol levels were increased, while the levels of plasma free fatty acids (FFA), amio acid nitrogen and protein remained unaltered. No significant changes were observed in either muscle protein, muscle water or the response to amino acid loading during the starvation period.
The force-feeding of pike starved for 3 months resulted in liver lipid and muscle glycogen being increased to levels higher than those observed in freshly-captured fish. Liver glycogen, however, increased to values only slightly higher than those of starved animals. Furthermore, while force-feeding had little effect on plasma FFA or protein concentrations, blood glucose, plasma cholesterol and haematocrit returned to the levels found in freshlycaptured fish and those of amino acid nitrogen were higher.
The results indicate that pike are well adapted for periods of prolonged starvation and that hepatic and extra-hepatic lipid and glycogen stores serve for metabolic needs during food shortage, while body protein is conserved. The endocrine basis for these changes in the tissue and blood constituents is discussed.     

The effects of starvation and refeeding on plasma amino acid levels in carp, Cyprinus carpio L., 1758

Plasma amino acid (AA) levels of carp, Cyprinus carpio L., 1758, were analysed after various periods of starvation as well as after 12 days of refeeding. The levels were compared to control groups, which had been previously fasted for 24 h. A positive correlation between dietary and plasma essential amino acid (EAA) concentrations was observed in all of the control groups.
The effect of starvation on the dynamics of AA concentration was different according to the period of starvation. Fasting already produced a decrease of total α-AA levels at 2 days, and these low levels were maintained until 5 and 8 days. These short periods of fasting affected the levels of EAA (especially branched-chain AA) more than those of non-essential amino acids (NEAA). The only AA that increased was Ala, which rose at 5 days of starvation, surpassing the levels of the control group. These high levels were maintained until 19 days of starvation.
A different situation was observed at 19 days, when an important increase of total α-AA levels was produced, the branched-chain AA being the most notable among EAA and Glu/Gln among NEAA. Later, at 50 days of starvation, total α-AA, EAA (except Leu and Ile) and NEAA decreased.
After 50 days of starvation, 12 days of refeeding did not modify the levels of EAA and NEAA, and their concentrations were lower than those of the control group, which presented an increase of total AA at this time. The differences observed on the changes of individual AA levels are discussed.     

Effect of short term fasting on plasma composition of lactating rats

The effects of a 24-h fast upon plasma and blood components of the lactating rat on day 20 after parturition have been determined. Body weight decrease observed with fasting was more marked in nursing dams than in controls. The changes observed agree with a limited use of lipidic resources, exhaustion of liver glycogen and considerable utilization of amino acids as energetic - as well as gluconeogenetic - substrates. Blood and plasma glucose were maintained in fasted dams at a level comparable with that of controls. The actual variations observed in the plasma aminogram were limited, showing a remarkable maintenance of the dam's own amino-acid homeostasis.     

Plasma amino acids of lean and obese Zucker rats subjected to a cafeteria diet after weaning.

Plasma amino acids of Zucker obese (fa/fa) and lean (Fa/?) rats fed either a reference nonpurified pellet or a cafeteria diet have been studied from 30 to 60 days after birth. Obese rats showed higher plasma branched chain amino acid levels but similar total amino acids, urea and glucose concentrations. The ingestion of a cafeteria diet induced higher levels in many amino acids, as well as in the composite figure in lean rats, but failed to alter total 2-amino nitrogen concentrations in obese rats, despite high levels in several non-essential amino acids and lower values in essential amino acids; urea levels were much lower in rats fed the cafeteria diet. The results are consistent with an impairment of amino acid nitrogen elimination via urea cycle in cafeteria diet-fed rats. This is independent of the hyperinsulinemia-driven plasma accumulation of several essential amino acids induced by genetic obesity. The effects were, then additive.     

Effects of growth hormone administration on wool growth in merino sheep

The effects of daily administration of 10 mg of highly purified ovine growth hormone (GH) for a period of 4 weeks on wool growth have been measured in 12 Merino ewes fed either a calculated maintenance energy intake or 1.6 times this amount (six on each ration). Concentrations of hormones, glucose, urea, alpha-amino N and amino acids in the blood were monitored and faeces and urine collected for measurement of nitrogen balance. Wool growth rate decreased by 20% during the 4 weeks of GH treatment in sheep fed the high energy diet, largely because of reduced wool fibre diameter. This was followed by restoration of normal growth and then an increase of up to 20% above control levels, a response which persisted for 12 weeks following cessation of GH administration, and which was due to increases in both fibre length and diameter. GH administration caused marked increases in plasma concentrations of GH, insulin and somatomedin C, glucose and free fatty acids, all of which returned to basal levels following cessation of GH administration. No consistent changes in plasma concentration of T3, T4, cortisol, prolactin or alpha amino N were detected. Plasma urea and methionine levels decreased during GH treatment and returned to, or were raised above, basal levels after the GH treatment period. GH injection also resulted in a net retention of N during treatment, followed by a transient period of net N loss. The GH-induced changes in wool growth may be caused by a change in the partitioning of amino acids between the muscle mass and the skin. No other contributing factor(s) were identified.     

Amino Acids in Plasma and CSF and Monoamine Metabolites in CSF: Interrelationship in Healthy Subjects

Plasma and cerebrospinal fluid (CSF) concentrations of amino acids were measured in 65 healthy volunteers (50 men and 15 women). The CSF levels of the monoamine metabolites homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), and 5-hydroxyindoleacetic acid (5-HIAA) were also determined. Sex differences were observed in both plasma and CSF amino acid levels as well as in the relationship between these concentrations. No significant correlations were observed between the CSF levels of HVA and 5-HIAA, and the concentrations of their precursor amino acids in either plasma or CSF. The MOPEG level in CSF correlated positively with the plasma concentrations of several amino acids.     

Amino acid compartmentation in chick blood during the peri-hatching period

Individual amino acid levels and compartmentation in chick blood were measured on day 20 of incubation, at hatching (day 0), or after 1 or 5 days of free life, and compared with those of adult chickens. Blood cell amino acid concentrations were almost one order of magnitude higher than those of plasma, with higher values than those found in mammalian erythrocytes. This difference may be due to the capability for protein synthesis of the nucleated cells coupled with a postulated utilization of amino acids as fuel. The most common pattern of individual plasma amino acid levels was a slight rise at hatching followed by a large decrease, with minimal values for adults. The patterns in the cells did not always coincide with those for plasma. Total blood amino acid levels increased steadily during the period studied due to the increase in intracellular amino acids, giving rise to increasing blood-cell/plasma concentration ratios. These changes showed higher availability of plasma amino acids just after hatching, while the cell concentrations increased steadily to the maximum values in adults. The increase in alanine levels in cells with little changes in plasma can be correlated with the role of this amino acid as the main 2-amino nitrogen carrier in the avian bloodstream. The high amino acid levels in the cells suggest that these cells act as inter-organ transporters and reservoirs of amino acids, they have a different role in their handling and metabolism from those of mammals.     

Plasma aldosterone and renal function in runners during a 20-day road race

To evaluate the effects that repeated long-distance running has on plasma aldosterone concentration and urinary excretion of solutes, fifteen male runners were studied during a 20-day, 500-km road race. Venous blood samples were taken on day 1 prior to running, on day 11 after 10 days of running, on day 13 after a 70-h rest, and on day 18 after an additional five days of running. Overnight urine samples were obtained on day 10 before and after running and on days 11, 12, and 13 during the 70-h rest period. Plasma sodium concentrations on days 13 and 18 and plasma potassium concentrations on days 11 and 13 were decreased (P less than 0.05). Plasma aldosterone levels were increased on days 11 and 18 after running and returned to pre-race levels on day 13 after 70 h of rest. Plasma cortisol concentrations were not altered. The urinary excretion rates of sodium were elevated and of aldosterone were decreased after 70 h of rest. Increase in excretion rate of urinary sodium correlated with decrease in concentration of plasma aldosterone. These findings show that plasma aldosterone levels are chronically elevated with repeated long-distance running, resulting in a decrease in urinary excretion rate of sodium.     

Influence of a 36 h fast followed by refeeding with glucose, glycerol or placebo on metabolism and performance during prolonged exercise in man

Five men were studied during exercise to exhaustion on an electrically braked cycle ergometer at 70% of VO2max. The four experimental treatments were as follows: fasted for 36 h (A); fasted (36 h) and refed with glucose (B) or glycerol (C); postabsorptive (overnight fast, D). In B and C the subjects were given a drink containing glucose or glycerol (1g per kg body weight) 45 min before starting exercise. A placebo drink was given 45 min before exercise on treatments A and D. Despite an increased availability of circulating free fatty acids, beta-hydroxybutyrate and glycerol exercise time to exhaustion was significantly lower after fasting (treatment A 77.7 +/- 6.8 min) compared with treatment D (119.5 +/- 5.8 min). Refeeding with glucose or glycerol did not significantly improve performance (92.4 +/- 11.8 min and 80.8 +/- 3.6 min respectively) compared with treatment A and lowered circulating levels of FFA and beta-HB during exercise compared with A. Despite the probability of low liver glycogen levels after fasting, none of the subjects became hypoglycaemic (blood glucose less than 4 mmol.l-1) during exercise and their blood lactate concentrations were not high at exhaustion. Plasma levels of branched chain amino acids (BCAA) decreased progressively during exercise on treatments A, B and C and were considerably lower at exhaustion compared with treatment D. Falling plasma concentrations of BCAA during prolonged exercise may be implicated in the generation of central fatigue.     

Effects of hypo- and hyper-thyroidism on liver composition, blood glucose, ketone bodies and insulin in the male rat

1. Thyroidectomized rats injected daily with 0, 0.1, 2 or 25mug of l-thyroxine/100g body wt. were compared with intact controls. In plasma, the protein-bound iodine was decreased in the rats given the 0 or 0.1mug doses and increased in those given the 25mug dose. 2. Blood glucose decreased in those given 2mug and was augmented in those given 25mug, and ketone bodies were the same in all the groups. 3. Plasma insulin was lowest in the rats given the 0 or 0.1mug doses and was highest in those given the 2 or 25mug doses of thyroxine. 4. After 48h starvation, the decrease in blood glucose and increase in ketone bodies observed in all the groups was greatest in the group not supplemented with thyroxine. 5. Plasma insulin concentrations remained at the value for fed animals in the rats given the 25mug dose of thyroxine but decreased in the other groups. 6. In fed animals, concentrations of hepatic DNA P, citrate, total fatty acids and acetyl-CoA were similar in all the groups, and glycogen was low only in the rats given the 25mug dose of thyroxine. 7. After 48h starvation, liver DNA P, total fatty acids and acetyl-CoA increased in all the groups, except in the rats given the 25mug dose, where both total fatty acids and acetyl-CoA remained at the value for fed animals. Liver citrate did not change in the groups given the 0 or 25mug doses of thyroxine, but decreased in the other groups. 8. The results are discussed in relation to the regulation of intermediary metabolism in hypo- and hyper-thyroidism.     

Diurnal Changes in Blood Metabolites and Their Relation to Plasma Growth Hormone and Time of Feeding in Mithun Heifers (Bos frontalis)

The aim of the present study was to investigate what, if any, diurnal changes occur in blood metabolites in relation to plasma growth hormone (GH) and feeding time among mithun (Bos frontalis), a semi‐wild ruminant. Blood samples were collected at hourly intervals during a 24 h span from 6 mithun heifers (averaging 2.5 yr of age and averaging 230 kg in weight) that were fed twice a day at 11:00 and 16:00 h. Samples were assayed for plasma GH and blood metabolites, non‐esterified fatty acids (NEFA), glucose, and alpha‐amino nitrogen. The total sampling period was divided into a 1) postprandial (after meal) period (period I: 11:00 to 21:00 h) and 2) interprandial period (period II: 22:00 to 10:00 h) and also into night (20:00 to 05:00 h) and day (06:00 to 10:00 h) periods for statistical analysis. Plasma glucose and alpha‐amino nitrogen levels increased (p<0.01), and plasma NEFA and GH decreased (p<0.01) after each meal. No diurnal rhythmicity was detected in plasma glucose or alpha‐amino nitrogen levels. Interestingly, plasma NEFA and GH levels were higher (p<0.01) during the interprandial (period II) and night periods, indicating an energy deficit that occurred progressively during the interprandial period of nocturnal feed deprivation. In twice‐daily‐fed mithuns we conclude that: 1) plasma metabolites and GH exhibited a definite pattern of change with time of feeding; 2) concentrations of plasma NEFA were higher nocturnally due to an energy deficit and that GH levels were higher during the interprandial period after the second meal; 3) the interprandial period after the second feeding may be considered to constitute a short‐term food deprivation; 4) the longer interprandial period of 19 h in this study between the second and subsequent morning meal may be changed into equally divided feedings to minimize the short‐term energy deficit; and 5) blood sampling for blood metabolites in mithuns should be conducted at a fixed time of day with special emphasis on time of feeding.     

Modulation of the amino acid control of hepatic protein degradation by caloric deprivation. Two modes of alanine co-regulation

Intracellular protein degradation in perfused livers of fed rats has been shown to be directly regulated by 7 amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) and co-regulated by alanine. Responses to graded increases of regulatory amino acids (individually or combined) are multiphasic and include (a) an initial inhibition at 0.5 times normal plasma concentrations, (b) a localized, zonal loss of inhibition at normal levels, and (c) suppression to basal rates at 4 times normal concentrations or greater; the zonal loss of inhibition is prevented by 0.5 mM (normal) alanine. In further perfusion studies carried out at the usual time (1100 h), we have occasionally observed a sharp decrease in proteolytic responsiveness at normal amino acid concentrations. The decrease, which occurred spontaneously in normal fed rats, was attributed to a nearly 90% loss in the sensitivity of alanine co-regulation. In all instances, alanine sensitivity was restored after 4 to 24 h of starvation. The cause of the insensitivity and the mechanism of its reversal by caloric deprivation are not presently known. Starvation for 24 h also appeared to alter the individual inhibitory effectiveness of Leu, Tyr, and Gln. On the other hand, inhibition by the full regulatory group at 4 times normal plasma levels was unchanged when compared with the complete plasma mixture except for a concentration shift in the peak zonal loss of proteolytic inhibition from 1.25 to 0.6 times plasma levels. Since the shift paralleled known changes in portal vein regulatory amino acids, it may have been adaptive in nature. As with fed animals, the zonal loss in starvation was abolished by 0.5 mM alanine, but not with high levels of lactate and pyruvate (10 mM), a finding consistent with the view that co-regulation is mediated by the recognition of alanine per se rather than its metabolism.     

Pituitary and extrapituitary effects of somatostatin in normal man.

The effects of synthetic linear somatostatin on basal circulating levels on several pituitary and pancreatic hormones, and of glucose and free fatty acids (FFA) were studied in 6 normal men after an overnight fast. A priming intravenous infusion of 250 mug of somatostatin in 18 sec was followed by a constant infusion of 500 mug over a period of 60 min. A decrease in plasma values of GH, prolactin, TSH, insulin and glucagon and in blood glucose was observed during somatostatin infusion, while FFA levels increased progressively. Plasma IRI and blood glucose increased rapidly when the somatostatin infusion was stopped, while FFA decreased progressively; GH, prolactin, TSH and glucagon remained low as compared to basal levels for one hour after the end of the infusion, i.e. until the end of the experiment. A slight but significant increase of LH and ACTH was observed after the end of the infusion.     

Effect of 24-hour starvation on amino acid pool composition and enzyme activities of rat brown adipose tissue

The effect of 24-hr starvation on the amino acid pool composition and its concentration ratios with respect to blood and plasma as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase have been studied in rat brown adipose tissue. Starvation induced a considerable decrease of pool amino acid concentration. Alanine and taurine were the amino acids in which the decrease was more marked. Small changes were observed in the activities of the enzymes studied, with decreases only in glutamate dehydrogenase and adenylate deaminase. These changes agree with a decrease in amino acid utilization in this tissue induced by starvation.     

Diminished hormonal responses to exercise in trained rats

Male rats (120 g) either were subjected to a 12-wk physical training program (T rats) or were sedentary controls (C rats). Subsequently the rats were killed at rest or after a 45- or 90-min forced swim. At rest, T rats had higher liver and muscle glycogen concentrations but lower plasma insulin. During exercise, blood glucose increased 60% in T rats but decreased 20% in C rats. Plasma glucagon and insulin concentrations did not change in T rats but plasma glucagon increased and insulin decreased markedly in C rats. Plasma epinephrine (90 min: range, 0.78-2.96 ng-ml-1, (T) vs. 4.42-15.67 (C)) and norepinephrine (90 min: 0.70-2.22 (T) vs. 2.50-6.10 (C)) were lower in T than in C rats. Hepatic glycogen decreased substantially and, as with muscle glycogen, the decrease was parallel in T and C rats. The plasma concentrations of free fatty acids were higher but lactate and alanine lower in T than in C rats. In trained rats the hormonal response to exercise is blunted partly due to higher glucose concentrations. In these rats adipose tissue sensitivity to catecholamines is increased, and changes in glucagon and insulin concentrations are not necessary for increased lipolysis and hepatic glycogen depletion during exercise.     

Effect of the Phenylalanine-free Diet on the Nitrogen Balance,Body Weight and Plasma Free Amino Acid Levels in the Domestic Fowl at Various Ages

Effect of a phenylalanine (Phe)-free diet on body weight, nitrogen balance and plasma free Phe levels were studied with male White Leghorn aged 2 weeks, 6, 12 and 15 months. Effects of feeding an isoleucine (Ile)- or amino acid-free diet and fasting on plasma amino acid levels in 15-month-old roosters were also studied.

Two-week-old chicks lost appetite for the Phe-free diet and their body weights decreased gradually. Six-month-old cockerels consumed the Phe-free diet completely but could not maintain normal growth rate and positive nitrogen balances. Twelve- and 15-month-old roosters maintained their body weights and nitrogen equilibrium on the Phe-free diet.

Plasma Phe levels of 2-week- and 6-month-old fowls at the 6th hr after the last feeding of the Phe-free diet did not decrease significantly compared to those of the control groups. However, plasma Phe levels of 12- and 15-month-old roosters decreased rapidly within a day on the Phe-free diet. Fasting resulted in a significant increase in plasma amino acid concentrations whereas feeding an Ile- or amino acid-free diet resulted in concentrations of plasma amino acids which were not lower than those observed in the control roosters.