Acidosis and glucocorticoids induce branched-chain amino acid catabolism

Chronic renal failure (CRF) is frequently complicated by malnutrition and wasting. The loss of lean body mass in CRF is the result of accelerated protein and amino acid degradation. Both appear to occur via acidosis-induced, glucocorticoid-dependent processes. In skeletal muscle, acidosis stimulates the activity of the rate-limiting enzyme in branched-chain amino acid metabolism, branched-brain ketoacid dehydrogenase (BCKAD). The activation of BCKAD in acidosis is likely to be glucocorticoid-dependent.     

Purification and cloning of the mitochondrial branched-chain amino acid aminotransferase from sheep placenta

This paper presents the first purification of the mitochondrial branched-chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82-85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched-chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched-chain amino acid catabolism is specific to each species.     

The influence of bicarbonate supplementation on plasma levels of branched-chain amino acids in haemodialysis patients with metabolic acidosis

BACKGROUND: It has been hypothesized that correction of metabolic acidosis might improve the nutritional state of acidotic haemodialysis (HD) patients partly because of a reduced oxidation of branched-chain amino acids (BCAA). AIM: We investigated whether bicarbonate (Bic) supplementation in acidotic HD patients results in increased plasma levels of BCAA. METHODS: In a longitudinal study (run-in period, 2 months; study period, 6 months), the effect of Bic supplementation on plasma levels of BCAA was studied in 12 acidotic HD patients (7 men, 5 women, mean age 54 +/- 18 years) with a predialysis bicarbonate (Bic) concentration smaller or equal to 22 mmol/l. Bic was supplemented by increasing Bic concentration of the dialysate and by oral Bic supplementation. RESULTS: Predialysis Bic increased significantly during the study period (18.7 +/- 2.7 vs. 23.1 +/- 11.5 mmol/l). There was no change in nutritional parameters. However, plasma levels of the BCAA valine, leucine, and isoleucine increased significantly. CONCLUSIONS: In haemodialysis patients with metabolic acidosis, Bic supplementation over a 6-months period resulted in an increase in plasma levels of BCAA. Further study is needed to elucidate the mechanisms behind, and the clinical importance of the observed changes in plasma BCAA levels.     

Endomyocardial biopsies for early detection of mitochondrial disorders in hypertrophic cardiomyopathies

An early response to metabolic acidosis is an increase in the degradation of muscle protein to provide the nitrogen needed to increase glutamine production so the kidney can excrete acid. In patients with renal insufficiency, this process may represent an example of a trade-off adaptation to uremia. It requires a hormone (glucocorticoids) and the metabolic response is maladaptive because the inability of the damaged kidney to maintain acid-base balance results in loss of muscle protein. Studies of cultured cells and rats and humans with normal kidneys demonstrate that acidosis stimulates the degradation of both amino acids and protein, which would block the normal adaptive responses to a low-protein diet (ie, to reduce the degradation of essential amino acids and protein). Evidence from studies in rats and humans with chronic uremia show that acidosis is a major stimulus for catabolism. The mechanism includes stimulation of specific pathways for the degradation of protein and amino acids. Since other catabolic conditions (eg, starvation) appear to stimulate the same pathways, understanding the mechanism in acidosis could be applicable to other conditions. Thus, the loss of lean body mass in uremia appears to be a consequence of a normal metabolic response that persists until acidosis is corrected.     

Evidence that elevated plasma corticosterone levels are the cause of reduced hypothalamic corticotrophin-releasing hormone gene expression in diabetes

Uncontrolled diabetes mellitus causes both a sustained activation of the hypothalamic-pituitary-adrenal (HPA) axis and reduced expression of corticotrophin-releasing hormone (CRH) mRNA in the hypothalamic paraventricular nucleus (PVN). To investigate the role of glucocorticoids in the regulation of CRH mRNA expression in the PVN of diabetic rats, we studied surgically adrenalectomized (ADX) and sham-operated male Sprague-Dawley rats 4 days after i.v. injection of streptozotocin (STZ; 65 mg/kg i.v.) or vehicle. Among sham-operated animals, AM plasma corticosterone levels were significantly increased in diabetic as compared to nondiabetic animals (1.46+/-0.54 vs. 0.22+/-0.05 microg/dl; P <0.05), and were positively correlated to both plasma ACTH levels (r = 0.74; P = 0.015) and adrenal gland weight (r = 0.70; P = 0.025). In contrast, CRH mRNA levels measured in the PVN by in situ hybridization were inversely related to the plasma corticosterone level (r = -0.68; P = 0.045). In a second experiment, both diabetic and nondiabetic ADX rats received a continuous subcutaneous infusion of either corticosterone at one of two doses or its vehicle for 4 days. Among vehicle-treated ADX animals, STZ diabetes raised hypothalamic CRH mRNA levels, in contrast to the tendency for diabetes to lower CRH mRNA in intact rats in the first experiment. Corticosterone administration lowered CRH mRNA comparably in both diabetic and nondiabetic ADX rats. In contrast, diabetes reduced arginine vasopressin (AVP) mRNA levels in the PVN of ADX rats and blunted the inhibitory effect of glucocorticoids on AVP mRNA levels in this setting. We conclude (1) glucocorticoids are necessary for the effect of diabetes to reduce hypothalamic CRH gene expression, since diabetes causes a paradoxical increase in CRH mRNA levels in adrenalectomized animals; (2) glucocorticoid inhibition of hypothalamic CRH gene expression is intact in diabetic rats; and (3) the activation of the HPA axis by diabetes is associated with a proportionate decrease in PVN CRH gene expression. These findings support a model in which hypothalamic factors additional to CRH activate the HPA axis in uncontrolled diabetes, and inhibit CRH gene expression indirectly by negative glucocorticoid feedback.     

Leucine metabolism during chronic ethanol consumption

Chronic ethanol consumption is known to increase plasma concentrations of branched-chain amino acids (BCAA) in rats and man, but the mechanisms of this effect are not known. Chronic ethanol consumption may increase levels of BCAA by altering protein turnover and/or by affecting the oxidation of BCAA. These possibilities were investigated in rats pair-fed liquid diets containing either 0% or 36% of total calories as ethanol for 21 days. In the fed state, ethanol-treated rats had a plasma ethanol level of 20 +/- 5 mmol/L and twofold increases in BCAA concentrations in plasma. There were also significant increases (37% to 63%) in muscle, liver, and jejunal mucosa BCAA concentrations. Chronic ethanol consumption significantly increased whole-body rates (mumol/100 g/h) of leucine turnover (73.8 +/- 7.5 v 104 +/- 5.6, P < .01) and oxidation (12.0 +/- 1.7 v 17.7 +/- 1.1, P < .05). In contrast, it significantly decreased leucine incorporation (nmol/mg protein/240 min) into both muscle (0.61 +/- 0.07 v 0.35 +/- 0.05, P < .01) and liver (13.25 +/- 1.40 v 6.78 +/- 0.98, P < .01) proteins. Incorporation of leucine into the mucosal proteins of jejunum (17.42 +/- 1.42 v 15.85 +/- 1.90, P = NS) was not significantly altered by ethanol. These results suggest that reduced protein synthesis and/or increased protein breakdown may account for the elevated tissue BCAA concentrations in chronic ethanol consumption. The consequences of these increased tissue concentrations are increases in tissue oxidation and plasma concentrations of BCAA.     

Regulation of messenger ribonucleic acid for corticotropin releasing hormone receptor in the pituitary during stress

The mechanism regulating pituitary CRH receptors during stress was studied by analysis of the changes in CRH receptor messenger RNA (mRNA) and CRH binding after acute and repeated stress and CRH and vasopressin (VP) administration in intact and adrenalectomized rats. Acute stress caused time- and stress type-dependent changes in pituitary CRH receptor expression. In situ hybridization studies showed biphasic changes in CRH receptor mRNA after immobilization stress for 1 h and decreases by 2 h (P < 0.01). Increases (P < 0.01) were seen 4 and 8 h after the initiation of the stress, and a return to near basal levels by 12 and 18 h. A different pattern, with a decrease by 4 h (P < 0.01) and levels similar to controls after 12 and 18 h, was observed after a single ip injection of hypertonic saline (1.5 M NaCl). Binding autoradiography showed significant increases in pituitary CRH binding 4, 10, and 12 h after immobilization stress, but significant decreases 4, 12, and 18 h after ip hypertonic saline. In contrast, repeated immobilization or ip hypertonic saline for 8 or 14 days increased pituitary CRH receptor mRNA, and CRH binding was decreased. To determine the role of hypothalamic CRH and VP on these stress-induced changes, rats were injected for 14 days with CRH, VP, or their combination at doses mimicking stress levels in pituitary portal circulation (1 microgram/day sc). Repeated injection of CRH or VP increased CRH receptor mRNA and CRH binding (P < 0.05). CRH receptor mRNA levels further increased after combined administration of CRH and VP (P < 0.01), but CRH binding showed a tendency to decrease. The role of glucocorticoids on CRH receptor regulation was studied by analysis of the effects of stress on CRH receptor mRNA and CRH binding in adrenalectomized (ADX) rats with and without corticosterone replacement in the drinking water. Although in 6-day ADX rats pituitary CRH receptor mRNA levels were markedly reduced after acute immobilization, glucocorticoid replacement restored the stimulatory effect of stress to levels observed in intact rats. Similarly, a single sc injection of CRH (1 microgram) decreased CRH receptor mRNA in ADX rats but not in glucocorticoid-replaced ADX rats. CRH binding showed the expected decrease after ADX and was unchanged after stress or CRH injection. The increased pituitary CRH receptor mRNA after stress suggests that stress-induced CRH receptor down-regulation is due to increased receptor occupancy and internalization rather than to a decrease in receptor synthesis. The data suggest that increased hypothalamic secretion of CRH and VP mediates the delayed up-regulatory effect of stress on CRH receptor mRNA, and that resting levels of glucocorticoids are required for this effect. In addition, increased VP levels are permissive for the down-regulation of CRH binding induced by chronic pituitary exposure to stress levels of CRH.     

Stimulation of albumin synthesis by keto analogues of amino acids

(1) The effect of ketoanalogues of branched-chain amino acids on albumin synthesis was examined in two biological systems using the [14C]carbonate technique. (2)alpha-Ketocaproic acid, the ketoanalogue of leucine, was able to reverse the reduced synthesis rate observed when isolated livers, from well-nourished animals were perfused with blood from rats deprived of dietary protein for 48 h. (3) A mixture of ketoanalogues of the three branched-chain amino acids, leucine, isoleucine and valine, was able to increase albumin synthesis per unit dry liver weight to above normal levels when administered intragastrically to rats 16 h after partial hepatectomy.     

Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.     

Reduced kidney branched chain aminotransferase expression in puromycin aminonucleoside-induced nephrotic syndrome

Injection of puromycin aminonucleoside to rats induces nephrotic syndrome characterized by hypoalbuminemia, proteinuria and hypercholesterolemia. In these rats, a low protein diet (6% casein diet) increased serum albumin by 26.3%, decreased proteinuria by 39% and reduced total cholesterol by 32%. Branched chain aminotransferase activity in kidney mitochondria of nephrotic rats fed 20 or 6% casein diet decreased by 30 and 24% with respect to their pair-fed groups and it was not modified by the protein content of the diet. Mitochondrial branched chain aminotransferase mRNA expression decreased by 67.3 and 72.5% in nephrotic rats fed 20 and 6% casein diet in comparison to their pair-fed groups. Total serum branched chain amino acids concentration (leucine, isoleucine, valine) in nephrotic rats was 30% higher than their pair-fed groups and it was associated with a decrease in the branched chain aminotransferase activity and mRNA expression suggesting that the catabolism of branched chain amino acid is reduced to conserve body nitrogen.     

Metabolic acidosis stimulates protein metabolism in uremia

It is well established that chronic renal failure is associated with loss of lean body mass. Possible explanations for protein losses include a limited ability to reduce essential amino acid oxidation and protein degradation when dietary protein is low. Alternatively, uremia could directly stimulate protein catabolism. In rats, we have uncovered evidence that metabolic acidosis not only blunts the responses to a low-protein diet but also stimulates the degradation of muscle protein. We find that the ATP-ubiquitin-proteasome-dependent pathway causing muscle protein degradation is activated by acidosis. Glucocorticoids are required but are not sufficient to elicit this catabolic response.     

Changes in renal metabolite profile and ammoniagenesis during acute and chronic metabolic acidosis in dog and rat

Acute metabolic acidosis was induced by an i.v. administration of hydrochloric acid to dogs and rats to decrease the plasma bicarbonate concentration from 22 to 12 mM in dogs and from 26 to 10 mM in rats. Chronic metabolic acidosis was also induced in dogs by ammonium chloride feeding for 5 days. Rats also were given ammonium chloride for 24 hours. The renal metabolite profile was determined on the freeze-clamped renal tissue before and after 100 min (dogs) or 30 to 240 min (rats) of acsute acidosis. Measurements on chronically acidotic dogs and rats with 24-hour acidosis were obtained also for comparison with acute acidosis. In both species, kidney glutamine, glutamate, and alpha-ketokglutarate concentrations decreased drastically following induction of acute or chronic acidosis, In the dog, or in the rat during the first 2 hours of acidosis, malate concentration was unchanged. Malate concentration fell significantly in the rat kidney only after 2 hours of acidosis without change in phosphoenolpyruvate (PEP) concentration. In chronically acidotic dogs, malate and oxaloacetate rose fivefold with no change in PEP concentration. Phosphoenolpyruvate carboxykinase (PEPCK) activity was not stimulated by chronic metabolic acidosis in the dog in contrast to the rat. Acute acidosis by hydrochloric acid increased net renal glutamine extraction in the rat but not in the dog. These data suggest that an increased metabolic flux occurs between alpha-ketoglutarate and malate in both rat and dog kidney during acute metabolic acidosis. In the rat, however, after 2 hours, PEPCK activation modifies the kidney metabolite profile. Intrarenal glutamine transport seems to be a rate-limiting factor for adaptation to acute acidosis in the dog but not in the rat kidney.     

Effects of insulin-like growth factor I combined with growth hormone on glucocorticoid-induced whole-body protein catabolism in man

Treatment with insulin-like growth factor I (IGF-I) alone failed to affect glucocorticoid-induced protein catabolism in a previous study from our laboratory. To assess the effects of the combination of IGF-I and GH in a similar protocol, 24 normal subjects received (in a double-blind, randomized, placebo-controlled manner) s.c. injections of either GH alone (0.3 IU/kg.day), the combination of IGF-I (80 micrograms/kg.day) and GH (0.3 IU/kg.day), or placebo for a period of 6 days during which they were treated with methylprednisolone (0.5 mg/kg.day). Whole-body protein kinetics measured, using the [1-13C]-leucine infusion technique, demonstrated that leucine flux (a parameter of protein breakdown) increased during administration of glucocorticoids alone (placebo group) and during GH-treatment, whereas the glucocorticoid-induced increase was abolished during IGF-I plus GH (P < 0.03 vs. GH). Leucine oxidation (a parameter of irreversible protein catabolism) increased in the placebo group (+60 +/- 14.5%, P < 0.005, day 7 vs. day 1), remained unchanged in the GH group (+2.5 +/- 10%), and decreased in the combination group (-17.7 +/- 3.3%, P < 0.002, day 7 vs. day 1). Glucose MCR decreased in the group receiving placebo (P < 0.05) and remained unchanged during combined treatment with IGF-I plus GH. It is concluded that glucocorticoid-induced protein, catabolism (leucine oxidation) is abolished during coadministration of GH (anticatabolic effect), whereas treatment with IGF-I and GH results in a net anabolic effect without adverse effects on peripheral glucose clearance.     

Corticosterone binding to tissues of adrenalectomized lean and obese Zucker rats

The binding of corticosterone, dexamethasone and aldosterone was investigated in plasma and in homogenates of liver, kidney, brain, brown adipose tissue and visceral (periovaric) and subcutaneous white adipose tissues of Zucker lean and obese rats: intact controls, adrenalectomized and sham-operated. Corticosterone-binding globulin (CBG) accounted for most of the binding, whereas that of glucocorticoid and mineralocorticoid receptors was much lower. Plasma corticosterone levels increased in sham-operated and obviously decreased in the adrenalectomized animals. Sham-operated and adrenalectomized lean rats showed decreased plasma CBG; in the obese, CBG levels were lower than in controls and were not affected by either surgery. No variation with obesity or surgery was observed either in dexamethasone or aldosterone binding, the latter being practically zero in most samples. When expressed per unit of tissue protein, CBG activity was maximal in adipose tissues, with lowest values in brain and liver. In lean rats, tissue CBG activity decreased with either surgical treatment; no changes were observed in the obese, which also had lower CBG tissue levels. The relative lack of changes in CBG of obese rats suggests that they have lost -- at least in part -- the ability to counter-modulate the changes in glucocorticoid levels through CBG modulation, thus relying only on the control of corticosterone levels. This interpretation agrees with the postulated role of CBG modulating the availability of glucocorticoids to target cells.     

Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic beta cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the alpha subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased ( approximately 55 percent) in the presence of palmitic acid, but increased ( approximately 180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic beta cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.     

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.     

Diurnal changes in plasma amino acids in maple syrup urine disease

Protein turnover is a cyclic process with a net loss of protein in the (catabolic) fasted state and a net gain in the (anabolic) fed state. In maple syrup urine disease (MSUD) the early block of degradation of the branched-chain amino acids (BCAA) brings about the opportunity for evaluation of the diurnal variation in net protein anabolism and catabolism by studying cyclic changes in the plasma concentrations of BCAA. The alterations in plasma BCAA in a 3-y-old boy with classical MSUD were studied in the fed and fasted state over a period of 19 months. For each amino acid a total of 34 data pairs was calculated. The plasma concentrations of the BCAA leucine, valine and isoleucine were constantly higher in the fasted than in the fed state. Plasma concentrations of alloisoleucine, being a non-protein amino acid, did not participate in cyclic changes. In contrast, the essential amino acid pair tyrosine and phenylalanine increased after meals. The fasting concentration of alanine increased after feeding, while glycine did not change significantly. Healthy subjects show a decrease in all amino acids in the fasted (mild catabolic) state and an increase in the fed state. These findings in MSUD suggest a net decrease in non-BCAA as result of a greater rate of amino acid oxidation rate than of protein breakdown and a net entry of BCAA into plasma in the fasted state due to the specific metabolic block. Such changes in amino acid plasma pools have to be taken into account during monitoring of treatment and especially when in vivo leucine oxidation is assessed.     

Oral branched-chain amino acids do not improve exercise capacity in McArdle disease

To determine whether oral branched-chain amino acids (BCAAs) improve exercise capacity, six fasting patients with McArdle's disease were given a solution of BCAA (77 mg/kg) or a control noncaloric beverage 30 minutes before exercise. The BCAA meal tripled plasma BCAA levels, increased BCAA catabolism as indicated by greater exercise increases in plasma glutamine and alanine, but lowered mean peak free fatty acid levels and reduced exercise capacity in five of six patients. Lower work capacity may be attributed to a net reduction in muscle fuel availability after BCAA administration.