NMR-Based Metabonomic Analysis of Physiological Responses to Starvation and Refeeding in the Rat.

Starvation is a postabsorptive condition derived from a limitation on food resources by external factors. Energy homeostasis is maintained under this condition by using sources other than glucose via adaptive mechanisms. After refeeding, when food is available, other adaptive processes are linked to energy balance. However, less has been reported about the physiological mechanisms present as a result of these conditions, considering the rat as a supraorganism. Metabolic profiling using (1)H nuclear magnetic resonance spectroscopy was used to characterize the physiological metabolic differences in urine specimens collected under starved, refed, and recovered conditions. In addition, because starvation induced lack of faecal production and not all animals produced faeces during refeeding, 24 h pooled faecal water samples were also analyzed. Urinary metabolites upregulated by starvation included 2-butanamidoacetate, 3-hydroxyisovalerate, ketoleucine, methylmalonate, p-cresyl glucuronide, p-cresyl sulfate, phenylacetylglycine, pseudouridine, creatinine, taurine, and N-acetyl glycoprotein, which were related to renal and skeletal muscle function, ß-oxidation, turnover of proteins and RNA, and host-microbial interactions. Food-derived metabolites, including gut microbial cometabolites, and tricarboxylic acid cycle intermediates were upregulated under refed and recovered conditions, which characterized anabolic urinary metabotypes. The upregulation of creatine and pantothenate indicated an absorptive state after refeeding. Fecal short chain fatty acids, 3-(3-hydroxyphenyl)propionate, lactate, and acetoin provided additional information about the combinatorial metabolism between the host and gut microbiota. This investigation contributes to allow a deeper understanding of physiological responses associated with starvation and refeeding.

Different variations of tissue B-group vitamin concentrations in short- and long-term starved rats.

Prolonged starvation changes energy metabolism; therefore, the metabolic response to starvation is divided into three phases according to changes in glucose, lipid and protein utilisation. B-group vitamins are involved in energy metabolism via metabolism of carbohydrates, fatty acids and amino acids. To determine how changes in energy metabolism alter B-group vitamin concentrations during starvation, we measured the concentration of eight kinds of B-group vitamins daily in rat blood, urine and in nine tissues including cerebrum, heart, lung, stomach, kidney, liver, spleen, testis and skeletal muscle during 8 d of starvation. Vitamin B1, vitamin B6, pantothenic acid, folate and biotin concentrations in the blood reduced after 6 or 8 d of starvation, and other vitamins did not change. Urinary excretion was decreased during starvation for all B-group vitamins except pantothenic acid and biotin. Less variation in B-group vitamin concentrations was found in the cerebrum and spleen. Concentrations of vitamin B1, vitamin B6, nicotinamide and pantothenic acid increased in the liver. The skeletal muscle and stomach showed reduced concentrations of five vitamins including vitamin B1, vitamin B2, vitamin B6, pantothenic acid and folate. Concentrations of two or three vitamins decreased in the kidney, testis and heart, and these changes showed different patterns in each tissue and for each vitamin. The concentration of pantothenic acid rapidly decreased in the heart, stomach, kidney and testis, whereas concentrations of nicotinamide were stable in all tissues except the liver. Different variations in B-group vitamin concentrations in the tissues of starved rats were found. The present findings will lead to a suitable supplementation of vitamins for the prevention of the re-feeding syndrome.

Effect of fasting on the urinary excretion of water-soluble vitamins in humans and rats.

Recent studies showed that the urinary excretion of the water-soluble vitamins can be useful as a nutritional index. To determine how fasting affects urinary excretion of water-soluble vitamins, a human study and an animal experiment were conducted. In the human study, the 24-h urinary excretion of water-soluble vitamins in 12 healthy Japanese adults fasting for a day was measured. One-day fasting drastically decreased urinary thiamin content to 30%, and increased urinary riboflavin content by 3-fold. Other water-soluble vitamin contents did not show significant change by fasting. To further investigate the alterations of water-soluble vitamin status by starvation, rats were starved for 3 d, and water-soluble vitamin contents in the liver, blood and urine were measured during starvation. Urinary excretion of thiamin, riboflavin, vitamin B(6) metabolite 4-pyridoxic acid, nicotinamide metabolites and folate decreased during starvation, but that of vitamin B(12), pantothenic acid and biotin did not. As for blood vitamin levels, only blood vitamin B(1), plasma PLP and plasma folate levels decreased with starvation. All water-soluble vitamin contents in the liver decreased during starvation, whereas vitamin concentrations in the liver did not decrease. Starvation decreased only concentrations of vitamin B(12) and folate in the skeletal muscle. These results suggest that water-soluble vitamins were released from the liver, and supplied to the peripheral tissues to maintain vitamin nutrition. Our human study also suggested that the effect of fasting should be taken into consideration for subjects showing low urinary thiamin and high urinary riboflavin.

Nitrogen excretion in rats on a protein-free diet and during starvation.

Nitrogen balances (six days) were determined in male Wistar rats during feeding a diet with sufficient protein or a nearly protein-free diet (n = 2 x 24), and then during three days of starvation (n = 2 x 12). The objective was to evaluate the effect of protein withdrawal on minimum nitrogen excretion in urine (UN), corresponding to endogenous UN, during feeding and subsequent starvation periods. The rats fed the protein free-diet had almost the same excretion of urinary N during feeding and starvation (165 and 157 mg/kg W(0.75)), while it was 444 mg/kg W(0.75) in rats previously fed with protein, demonstrating a major influence of protein content in a diet on N excretion during starvation. Consequently, the impact of former protein supply on N losses during starvation ought to be considered when evaluating minimum N requirement necessary to sustain life.

Macro- and micronutrient losses and nutritional status resulting from 44 days of total fasting in a non-obese man.

OBJECTIVE: We wanted to establish and understand how the fractional losses of fat, fat-free tissues, and selected nutrients compare with that of body mass during a 44-d voluntary starvation (water only) and measurements of nutrient status. METHODS: We used anthropometry, sequential measurements of urinary substances during the fast, and blood analytes at the end of the fast. RESULTS: At the start of the fast, body weight was 96.0 kg (20% fat) and body mass index was 28.36 kg/m(2). The changes in body mass and arm anthropometry and in the pattern of urinary excretion of creatinine, ammonia, sodium, and ketone bodies during the study were consistent with starvation. At the end of the fast, body mass had decreased by 25.5%, of which a quarter to a third was due to loss of fat and the remainder to fat-free mass, predominantly muscle. There was an estimated loss of 20% of total body protein, 20-25% of fat-free mass, and a greater fractional loss of fat. Total energy expenditure was estimated to be 1638-2155 kcal/d of which 13.0-17.1% was from protein oxidation. Differential losses of minerals in urine ranged from 1.2% of estimated initial body content for manganese to 17.3% for selenium and 40.5% for zinc. At the end of the study, plasma concentrations of zinc and vitamin B12 were increased, those of copper, selenium, and manganese were normal, and there was biochemical evidence of deficiency in thiamine, riboflavin, and vitamin K (prothrombin time). CONCLUSION: The data confirm and extend the available information on prolonged fasting in lean individuals and have relevance to the understanding of the physiologic responses to starvation and the associated homeostatic mechanisms.

Identification of functions of peroxisome proliferator-activated receptor alpha in proximal tubules.

Peroxisome proliferator-activated receptor alpha(PPARalpha) is a member of the steroid/nuclear receptor superfamily that is intensively expressed in the kidney, but its physiologic function is unknown. In this study, PPARalpha-null mice were used to help clarify the function. Starved PPARalpha-null mice were found to secrete significantly more quantities of urine albumin than starved wild-type mice. Furthermore, the appearance of giant lysosomes, marked accumulation of albumin, and an impaired ability concerning albumin digestion were found only in proximal tubules of the starved PPARalpha-null mice. These abnormalities were probably derived from ATP insufficiency as a result of the starvation-induced decline of carbohydrate metabolism and a lack of PPARalpha-dependent fatty acid metabolism. It is interesting that these abnormalities disappeared when glucose was administered. Taken together, these findings demonstrate important functions of PPARalpha in the proximal tubules, the dynamic regulation of the protein-degradation system through maintenance of ATP homeostasis, and emphasize the importance of the fatty acid metabolism in renal physiology.

Plasma N tau-methylhistidine concentration is a sensitive index of myofibrillar protein degradation during starvation in rats.

Urinary excretion of N tau-methylhistidine (MeHis) in rats was linearly elevated by starvation for 2 days. Plasma concentration of MeHis on day 1 and day 2 of starvation were increased 2.4- and 2.6-fold, respectively. The amount of released MeHis from the isolated muscles into medium during a 2-h incubation period was increased with starvation corresponded to the plasma MeHis concentration. The results of this study suggest that plasma MeHis is a sensitive index of myofibrillar protein degradation.

Effects of starvation and of selenium deficiency on the urinary excretion of electrolytes, ketone bodies, creatinine, urea and uric acid.

The aim of this study was to investigate whether urinary excretion of other compounds than ketone bodies are also increased in starved, selenium (Se)-deficient rats. Two groups of male rats were fed an Se-deficient diet with 0.009 mg Se/kg, ("Se-deficient" and "Se-repleted") and one group was fed the same diet with 0.23 mg Se/kg as control for eleven weeks. The urinary excretion of ketone bodies was highly enhanced in Se deficiency, with a 7-fold increase in 3-hydroxybutyrate and an 18-fold increase in acetoacetate. Despite this, the plasma concentration of ketone bodies and the glomerular filtration rate were unaffected in the Se-deficient rats. Starvation resulted in a significant decrease in the urinary content of potassium, magnesium and calcium, in both dietary groups of rats and of urea in the Se-adequate group. No Se-dependent difference was noted for the urinary excretion of these compounds or of sodium, phosphate, creatinine and uric acid in any of the groups. This was unexpected in view of certain previous results and indicates that disturbances in the renal handling of compounds are progressive in Se deficiency, with increased excretion of ketone bodies being an early event while more severe deficiency is required to impair the renal handling of electrolytes and other compounds studied.

Central nervous system demyelinating diseases and increased release of cholesterol into the urinary system of rats.

The question of what happens to cholesterol in the adult central nervous system during its slow turnover has been addressed using rats with brain and spinal cord labeled with [4-14C]cholesterol upon intracerebral injection of labeled cholesterol into rats at 10-12 days of age. At six months after injection, 14C was found only in the brain and spinal cord and was slowly released via the rat's urine. When labeled rats were given demyelinating agents (triethyl tin chloride, hexachlorophene, sodium cyanide) and when experimental allergic encephalomyelitis was induced, a measurable increase in urinary 14C label above control levels was found. It was concluded that there is a direct relationship between the experimental demyelination induced and the increased release of cholesterol metabolites into urine. The study suggests that a clinical method could be developed to determine the rate of central nervous system demyelination by measuring the amount of urinary cholesterol metabolites.

Substrate uptake and utilization by the kidney of fed and starved rats in vivo.

In order to obtain information (1) on the quantitative contribution of various circulating substrates to renal metabolism and (2) on the relative importance of net luminal and basolateral transport for substrate uptake, we have precisely quantified the renal blood flow, the urinary flow, and the rates of substrate handling by the kidney of anesthetized fed and 72-hour-starved rats. For this, the concentration of twelve metabolites were simultaneously measured in arterial and venous whole blood and plasma as well as in urine of each rat thanks to the use of microassays based on enzymatic cycling. In fed rats, the main potential energy sources were glucose and lactate followed by fatty acids, ketone bodies, citrate and glycerol. Starvation caused a large increase in renal uptake and metabolism of fatty acids, ketone bodies, glutamine and glycerol, and a large inhibition of lactate utilization. The net peritubular uptake of acetoacetate, citrate, glycerol and free fatty acids demonstrated in both nutritional states was increased by starvation only for glycerol and free fatty acids; net peritubular efflux of both beta-hydroxybutyrate and ammonium ions was stimulated whereas that of glutamine was converted into net peritubular uptake by starvation.

Increase of urinary ketone body excretion in selenium-deficient rats is a ketone-specific change.

The effects of selenium (Se) deficiency on urinary ketone body excretion in starved rats were examined. Rats were fed a basal diet which was Se-deficient (Se content: 0.011 micrograms/g) or a Se-adequate diet (the basal diet supplemented with 0.1 micrograms Se/g as sodium selenite). On the 11th and 22nd week of the feeding period, Se-deficient status in rats fed the basal diet was verified by the observation that the Se content and glutathione peroxidase activity in their plasma, erythrocytes, and livers were markedly lowered. On the 4th, 6th, 11th, 15th, and 22nd week, the rats were starved for 48 h and the urinary excretion of ketone bodies (acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHBA)), urea, and creatinine were examined. The urinary excretion of AcAc and 3-OHBA during the second 24 h of the 48-h starvation period were markedly higher in the Se-deficient rats than in the Se-adequate rats for all weeks examined, while the urine volume and the excretion of urea and creatinine were similar in the Se-deficient and Se-adequate rats, irrespective of the feeding period and the number of hours of starvation. On the 22nd week, the plasma ketone body levels were also determined and significantly higher plasma 3-OHBA levels were observed in the Se-deficient rats than in the Se-adequate rats 72 h after starvation began. These results indicate that Se deficiency causes an increase of urinary ketone body excretion in starved rats and that the increase is ketone-specific with no changes in major urinary profiles.

Lipid mobilising factors specifically associated with cancer cachexia.

Both urine and plasma from mice and humans with cancer cachexia have been shown to contain higher levels of lipid mobilising activity than normal controls, even after acute starvation. There was no significant increase in the urinary lipid mobilising activity of either mice or humans after acute starvation, suggesting that the material in the cachectic situation was probably not due to an elevation of hormones normally associated with the catabolic state in starvation. Further characterisation of the lipid mobilising activity in the urine of cachectic mice using Sephadex G50 exclusion chromatography showed four distinct peaks of activity of apparent molecular weights of greater than 20, 3, 1.5 and less than 0.7 kDa. No comparable peaks of activity were found in the urine of a non tumour-bearing mouse. The high molecular weight activity was probably formed by aggregation of low molecular weight material, since treatment with 0.5 M NaCl caused dissociation to material with a broad spectrum of molecular weights between 3 and 0.7 kDa. Lipolytic species of similar molecular weights were also found in the urine of cachectic cancer patients, but not in normal urine even after 24 h starvation. The lipid mobilising species may be responsible for catabolism of host adipose tissue in the cachectic state.

Urinary cortisol and urea nitrogen responses in irreversibly undernourished mule deer fawns.

We examined the concentration of urinary cortisol and urea nitrogen of five hand-reared mule deer (Odocoileus hemionus) fawns that failed to recover from winter starvation, and compared them to levels found in fawns that recovered. The fawns wintered in fenced pastures stocked with wild deer, and were put back on supplemental feed after losing 15% of their body mass. The five fawns that died began receiving supplemental feed up to 3 wk before death. All continued to lose weight, and were consequently removed from the pasture and fed ad libitum 4 to 10 days before death. In the animals that died, cortisol levels continued to increase regardless of food availability, and were correlated with those of urea nitrogen. Postmortem cortisol and urea nitrogen measurements were significantly greater than concentrations found in the weeks preceding death. We hypothesize that uncontrolled protein catabolism is promoted by high levels of cortisol. These cortisol levels may reach a point at which irreversible multiple-system organ failure occurs, leading to the animal's death.

Role of the liver in carnitine metabolism: the mechanism of development of carnitine-deficient status in guinea-pigs.

It was shown that carnitine deficiency and an impairment of the conversion of butyrobetaine into carnitine develops not only in ascorbic acid-deficient guinea-pigs but also in partially starved animals. We propose that the same mechanism, an absolute or relative ascorbic acid deficiency, is operating in both nutritional states. An increased urinary excretion greatly contributes to the development of carnitine deficiency in guinea-pigs, both in ascorbic acid deficiency and starvation. With respect to the greatly increased excretion, guinea-pig carnitine deficiency resembles the human disorder and may serve as model for it.

[Hormonal and metabolic reactions in the human body during prolonged starvation]. / Gormonal'nye i metabolicheskie reaktsii organizma cheloveka pri dlitel'nom golodanii.

This paper discusses hormonal and metabolic reactions of healthy volunteers exposed to 14-day starvation. This exposure led to many-fold increase of plasma and urinary epinephrine (E); drastic increase of ACTH and beta-endorphin (BE), morning and integrated concentrations of cortisol and STH, aldosterone, T3, glucagon, cAMP, cGMP, cAMP-cGMP, acetyl choline (AC), free fatty acids (FFA), lactate, metanephrine (MN) excretion; decrease of plasma norepinephrine (NE) and unchanged NE excretion; decrease of plasma concentrations of TTH, T4, T3, prolactin (PL), insulin (morning and integrated concentrations), C-peptide, FSH, LH, testosterone, histamine, prostaglandins (PG) A + E, PG F2, glucose and pH, as well as decrease of excretion of homovanillic acid (HVA), vanillyl mandelic acid (VMA), normetanephrine (NMN) and MN-E, NMN:NE. On recovery day 14 concentrations of E, NE, BE, STH, AC, cAMP, cGMP, FFA as well as E and dopamine excretion remained elevated while concentrations of T3, PL, FT, LT, testosterone PG A + E, PG 2 and excretion of MN, HVA, VMA, MN:E remained decreased, while other parameters returned to the normal.

[The significance of pregnancy-induced ketonuria in sheep and goats]. / Zur Bedeutung der graviditätsbedingten Ketonurie bei Schaf und Ziege.

The last third of pregnancy in sheep and goats is characterized by a considerable reduction in the volume of the rumen. When the animal is carrying more than one fetus there is thus a latent ketosis caused by starvation. A discrete hypoglycemia is in close correlation to this. If other special factors like increasing age of the pregnant animal, chronic liver disease, unbalanced nutrition containing too little carbohydrates are also present, the latent starvation ketosis can give rise to an acute gestation ketosis. The clinical picture is described in detail and contrasted with the gestation hypocalcemia. Furthermore, the parameters relevant to laboratory diagnosis will be discussed.

Urinary excretion of a glucose-containing tetrasaccharide. A parameter for increased degradation of glycogen.

The urinary excretion of a glucose-containing oligosaccharide, Glc alpha[1-6Glc alpha[1-4Glc alpha[1-4Glc, (Glc4) has been measured in various physiological and pathological conditions. The Glc4 content of 24 h samples from the same individual was relatively constant, whereas 2 h samples showed up to 4-fold variations in Glc4 concentration. This variation is associated mainly with increased excretion of Glc4 after meals. A carbohydrate-rich diet, starvation or a protein-rich diet, and intense physical activity all affected the urinary excretion of Glc4. Both oral and intravenous administration of glycogen in a Rhesus monkey resulted in increased excretion of Glc4. When Glc4 itself was injected intravenously in small amounts renal clearance was rapid and complete. In contrast, injection of a larger amount resulted in incomplete (approximately 10%) renal clearance, probably due to uptake and metabolism of the oligosaccharide. In patients with glycogen storage diseases, certain malignancies, and pancreatitis, 24 h urinary Glc4 excretion exceeded the normal range. The diagnostic implications of these observations deserve evaluation. The results presented suggest a need for standardization of nutritional status and physical activity when monitoring urinary Glc4 excretion for diagnostic purposes.

Protein and substrate metabolism during starvation and parenteral refeeding.

1. Healthy male volunteers underwent 10 days of hospitalized protein-calorie starvation and a subsequent 10 day repletion phase with complete intravenous nutritional support (IVF). Non-protein calories were provided as either all D-glucose or as 50% D-glucose/50% lipid. 2. In comparison with starvation, whole-body protein breakdown, as assessed by [15N]glycine, [13C]leucine and urinary excretion of 3-methylhistidine (3-MH), was diminished during IVF. The administration of parenteral nutrition did not specifically suppress peripheral tissue protein breakdown, as measured by extremity 3-MH efflux. 3. Despite the differential insulin response to D-glucose/amino acid (50 +/- 6 m-units/ml) as compared with the D-glucose/lipid/amino acid regimen (25 +/- 4 m-units/ml), there was no difference in nitrogen retention between the regimens. Indirect calorimetric determinations revealed that oxidation of substrate during IVF was related to the proportion of D-glucose and lipid infusion.

Effect of starvation on biochemical indices of renal function in the rat.

The effect of starvation on urinary output and biochemical indices of renal function was investigated in rats. Starvation resulted in a marked fall in water intake. Urinary output paradoxically increased during the first day following starvation, but fell dramatically thereafter. Urinary creatinine excretion and creatinine clearance fell markedly, but plasma creatinine concentration did not alter. Plasma urea concentration and urinary urea excretion fell. Plasma sodium concentration increased, whilst plasma potassium concentration did not alter; urinary sodium and potassium excretion fell. Plasma bicarbonate concentration fell marginally, but the anion gap increased to a greater extent. Following re-feeding, water intake and urine output increased, as did urinary creatinine excretion and creatinine clearance. Plasma urea and urinary urea concentrations, as well as sodium and potassium excretion, increased. Plasma bicarbonate increased and the anion gap decreased. These indices improved within 2 days of re-feeding and were restored to normal in 5 days.