Glycomacropeptide, a low-phenylalanine protein isolated from cheese whey, supports growth and attenuates metabolic stress in the murine model of phenylketonuria

Phenylketonuria (PKU) is caused by a mutation in the phenylalanine (phe) hydroxylase gene and requires a low-phe diet plus amino acid (AA) formula to prevent cognitive impairment. Glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to AA formula. Our objective was to compare growth, body composition, and energy balance in Pah(enu2) (PKU) and wild-type mice fed low-phe GMP, low-phe AA, or high-phe casein diets from 3-23 wk of age. The 2 × 2 × 3 design included main effects of genotype, sex, and diet. Fat and lean mass were assessed by dual-energy X-ray absorptiometry, and acute energy balance was assessed by indirect calorimetry. PKU mice showed growth and lean mass similar to wild-type littermates fed the GMP or AA diets; however, they exhibited a 3-15% increase in energy expenditure, as reflected in oxygen consumption, and a 3-30% increase in food intake. The GMP diet significantly reduced energy expenditure, food intake, and plasma phe concentration in PKU mice compared with the casein diet. The high-phe casein diet or the low-phe AA diet induced metabolic stress in PKU mice, as reflected in increased energy expenditure and intake of food and water, increased renal and spleen mass, and elevated plasma cytokine concentrations consistent with systemic inflammation. The low-phe GMP diet significantly attenuated these adverse effects. Moreover, total fat mass, %body fat, and the respiratory exchange ratio (CO(2) produced/O(2) consumed) were significantly lower in PKU mice fed GMP compared with AA diets. In summary, GMP provides a physiological source of low-phe dietary protein that promotes growth and attenuates the metabolic stress induced by a high-phe casein or low-phe AA diet in PKU mice.     

Hoe 140 abolishes the blood pressure lowering effect of taurine in high fructose-fed rats

Summary. High fructose feeding induces moderate increases in blood pressure of normal rats, associated with hyperinsulinemia, insulin resistance and impaired glucose tolerance. Increased vascular resistance, and sodium retention have been proposed to contribute to the blood pressure elevation in this model. Taurine, a sulphur-containing amino acid has been reported to have antihypertensive and antinatriuretic actions. In addition, taurine is shown to increase the excretion of nitrite and kinin availability and hence would be expected to improve the vascular tone. In the present study, the involvement of kinins in the blood pressure lowering effect of taurine was investigated by coadministration of Hoe 140, a kinin B₂ receptor antagonist along with taurine. The effects of taurine on plasma and urinary concentrations of sodium and tissue kallikrein activity were studied in high fructose-fed rats. Fructose-fed rats had elevated blood pressure and decreased levels of sodium in urine. Treatment with 2% taurine in drinking water prevented the blood pressure elevation and coadministration of Hoe 140 abolished this effect of taurine in high fructose-fed rats. The findings confirm the antinatriuretic action of taurine and also suggest a role for the kinins in the mechanism of taurine action in diet-induced hypertension.     

Endogenous Control of Photosynthetic Activity during Progressive Drought: Influence of Final Products of Photosynthesis

Photosynthetic activities and the redox states of photosystem I (PSI) and photosystem II (PSII) in intact leaves of cucumber plants (Cucumis sativus L.), as well as the sucrose and starch contents were examined under conditions of ongoing soil water deficit imposed by the cessation of watering. As the soil drought progressed, the maximum rate of photosynthetic CO₂ fixation was shown to decrease. These changes in the maximum photosynthetic rate occurred synchronously with changes in the maximum quantum yield of photosynthesis. Under soil water deficit, the reduced form of PSII primary acceptor Q _A was accumulated only at photon flux densities of about 100 mol/(m² s). At such photon flux densities, the changes in nonphotochemical quenching (qN) induced by soil water deficit were opposite to changes in photochemical quenching parameter (1 – qP). Irrespective of the duration of soil drought, the relationship between steady-state concentrations of photochemically inactive reaction centers of PSI and PSII (the fractions of P700 and Q _A in the oxidized and reduced state, respectively) was almost linear, which provides evidence for the concerted operation of both photosystems. The conditions of soil water deficit promoted sucrose accumulation in the source leaf, which was paralleled by a substantial decrease in the amount of starch in the same leaf. The highest content of sucrose in the leaf after a 7-day drought was correlated with the largest decrease in photosynthetic activity. It is concluded that the progressive drought triggers an endogenous mechanism that regulates photosynthesis through feedback relations, namely, the inhibition of photosynthesis by its end products.     

Inhibitors of polyamine metabolism: Review article

Summary. The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases.The early development of polyamine biosynthetic single enzyme inhibitors such as -difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer.The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N¹-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Low Bone Strength Is a Manifestation of Phenylketonuria in Mice and Is Attenuated by a Glycomacropeptide Diet

Purpose

Phenylketonuria (PKU), caused by phenylalanine (phe) hydroxylase loss of function mutations, requires a low-phe diet plus amino acid (AA) formula to prevent cognitive impairment. Glycomacropeptide (GMP), a low-phe whey protein, provides a palatable alternative to AA formula. Skeletal fragility is a poorly understood chronic complication of PKU. We sought to characterize the impact of the PKU genotype and dietary protein source on bone biomechanics.

Procedures

Wild type (WT; Pah^+/+) and PKU (Pah^enu2/enu2) mice on a C57BL/6J background were fed high-phe casein, low-phe AA, and low-phe GMP diets between 3 to 23 weeks of age. Following euthanasia, femur biomechanics were assessed by 3-point bending and femoral diaphyseal structure was determined. Femoral ex vivo bone mineral density (BMD) was assessed by dual-enengy x-ray absorptiometry. Whole bone parameters were used in prinicipal component analysis. Data were analyzed by 3-way ANCOVA with genotype, sex, and diet as the main factors.

Findings

Regardless of diet and sex, PKU femora were more brittle, as manifested by lower post-yield displacement, weaker, as manifested by lower energy and yield and maximal loads, and showed reduced BMD compared with WT femora. Four principal components accounted for 87% of the variance and all differed significantly by genotype. Regardless of genotype and sex, the AA diet reduced femoral cross-sectional area and consequent maximal load compared with the GMP diet.

Conclusions

Skeletal fragility, as reflected in brittle and weak femora, is an inherent feature of PKU. This PKU bone phenotype is attenuated by a GMP diet compared with an AA diet.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Intakes of whey protein hydrolysate and whole whey proteins are discriminated by LC–MS metabolomics

Whey protein improves fasting lipids and insulin response in overweight and obese individuals. Whey hydrolysate was recently shown to be more active than whole protein but the differences in metabolite profiles after intake remain unknown. This study discriminates plasma profiles after intake of four different whey protein fractions and establishes new hypotheses for the observed effects. Obese, non-diabetic subjects were included in the randomized, blinded, cross-over meal study. Subjects ingested a high-fat meal containing whey isolate (WI), whey concentrate hydrolysate (WH), α-lactalbumin or caseinoglycomacropeptide as the protein source. Plasma samples were collected at five time points and metabolites analysed using LC–Q-TOF–MS. Plasma concentrations of ten amino acids (AAs) were different between the meals. The plasma levels of AAs and AA derivatives were generally directly related to the AA composition of the meals. Highly elevated plasma levels of a number of cyclic dipeptides and other AA metabolites were found following intake of the WH meal and these metabolites are primary candidates to explain the superior insulinotropic effect of WH. The manufacturing process of WH caused oxidization of methionine to methionine sulfoxide which in turn caused in vivo generation of N-phenylacetyl-methionine and N-phenylacetyl-methionine sulfoxide. These two compounds have not previously been described in biological systems.     

Snakes allocate amino acids acquired during vitellogenesis to offspring: are capital and income breeding consequences of variable foraging success?

Reproductive allocation strategies have been historically described as lying on a continuum between capital and income breeding. Capital breeders have been defined as species that allocate stored reserves to reproduction, whereas income breeders have been defined as species that allocate relatively recently‐ingested food resources to reproduction. Snakes are considered capital breeders because they efficiently store large amounts of nutrients and energy, potentially enough to support an entire reproductive bout without feeding. We examined the abilities of five viviparous snake species to allocate income to follicles during vitellogenesis. We fed ¹⁵N‐labelled L‐leucine to experimental females of each species during vitellogenesis, whereas control females were fed unlabelled meals. After ovulation, we measured yolk ¹⁵N p.p.m. using mass spectrometry. Maternal scale samples taken before labelling were used to estimate endogenous ¹⁵N concentrations, which should represent ‘capital’. Scale samples taken at ovulation were used to determine whether snakes assimilated ¹⁵N‐labelled‐leucine from labelled diets. Yolks and post‐ovulatory scales of labelled females were significantly more enriched in ¹⁵N than those of unlabelled females in all species, indicating significant assimilation and allocation of income‐derived amino acids to the yolk during vitellogenesis. The lack of among‐species differences suggests that all species allocated income amino acids to vitellogenesis. The results obtained in the present study suggest that proportional utilization of income or capital depends on the frequency and timing of foraging success during reproductive events. Therefore, capital and income breeding may be consequences of both life‐history and environmental constraints on foraging success, rather than strategies of reproductive allocation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 390–404.     

Seasonal soil‐surface carbon fluxes from the root systems of young Pinus radiata trees growing at ambient and elevated CO2 concentration

Elevated atmospheric CO₂ concentration may result in increased below‐ground carbon allocation by trees, thereby altering soil carbon cycling. Seasonal estimates of soil surface carbon flux were made to determine whether carbon losses from Pinus radiata trees growing at elevated CO₂ concentration were higher than those at ambient CO₂ concentration, and whether this was related to increased fine root growth. Monthly soil surface carbon flux density (f) measurements were made on plots with trees growing at ambient (350) and elevated (650 μmol mol^?1) CO₂ concentration in large open‐top chambers. Prior to planting the soil carbon concentration (0.1%) and f (0.28 μmol m^?2 s^?1 at 15 °C) were low. A function describing the radial pattern of f with distance from tree stems was used to estimate the annual carbon flux from tree plots. Seasonal estimates of fine root production were made from minirhizotrons and the radial distribution of roots compared with radial measurements of f. A one‐dimensional gas diffusion model was used to estimate f from soil CO₂ concentrations at four depths. For the second year of growth, the annual carbon flux from the plots was 1671 g y^?1 and 1895 g y^?1 at ambient and elevated CO₂ concentrations, respectively, although this was not a significant difference. Higher f at elevated CO₂ concentration was largely explained by increased fine root biomass. Fine root biomass and stem production were both positively related to f. Both root length density and f declined exponentially with distance from the stem, and had similar length scales. Diurnal changes in f were largely explained by changes in soil temperature at a depth of 0.05 m. Ignoring the change of f with increasing distance from tree stems when scaling to a unit ground area basis from measurements with individual trees could result in under‐ or overestimates of soil‐surface carbon fluxes, especially in young stands when fine roots are unevenly distributed.     

Taurine protected myocardial mitochondria injury induced by hyperhomocysteinemia in rats

Summary. Taurine can protect against cardiovascular diseases, whereas elevated levels of plasma homocysteine are associated with atherosclerotic and thromboembolic cardiovascular diseases. To illustrate the effects of taurine on hyperhomocysteinemia, we observed the myocardial mitochondria dysfunction in the rats with hyperhomocysteinemia induced by diet methionine loading, and the therapeutic effect of taurine. A methionine diet increased plasma homocysteine concentration (133.51±27.91mol/L vs 12.31±2.58mol/L in control, P<0.01), stimulated the production of reactive oxygen species (ROS) in the myocardial mitochondria, and inhibited the activities of mitochondrial Mn-superoxide dismutase and catalase. The ⁴⁵Ca uptake and Ca²⁺-ATPase activity in the myocardial mitochondria were significantly lowered in rats with hyperhomocysteinemia. Taurine supplements effectively attenuated the hyperhomocysteinemia-induced ROS production and inhibition of Mn-superoxide dismutase and catalase activities in the myocardial mitochondria, and increased its ⁴⁵Ca uptake and Ca²⁺-ATPase activity. Thus, taurine antagonizes the oxidative stress injury in the myocardial mitochondria induced by the hyperhomocysteinemia.     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Polyamines biosynthesis and oxidation in free-living amoebae

Summary. In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae.The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N⁸-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.     

Characteristics of basal taurine release in the rat striatum measured by microdialysis

Summary. Taurine is a sulfur-containing amino acid thought to be an osmoregulator, neurotransmitter or neuromodulator in the brain. Our objective was to establish how much taurine is released in the striatum and examine the mechanisms controlling extracellular taurine concentrations under resting conditions. The experiments were made on rats by microdialysis in vivo. Changes in taurine were compared with those in glutamate, glycine and the non-neuroactive amino acid threonine. Using the zero net flux approach we showed the extracellular concentration of taurine to be 25.2±5.1M. Glutamate was increased by tetrodotoxin and decreased by Ca²⁺ omission, glycine and threonine were not affected and both treatments increased extracellular taurine. The basal taurine release was increased by the taurine transport inhibitor guanidinoethanesulfonate and reduced by the anion channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid.     

Effect of adding dietary L-lysine, L-threonine and L-methionine to a low gluten diet on urea synthesis in rats

Summary. We have shown that urinary urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten+0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10d. The urinary excretion of urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic urea-cycle enzymes was not related to the urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline urea synthesis.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Endocrine changes during pregnancy, parturition and post-partum in guanacos (Lama guanicoe)

Plasma concentrations of progesterone (P4), estradiol-17β (E2), estrone (E1) and estrone sulfate (E1S) were measured during gestation in eight guanacos kept in captivity. Gestational length was 346.1 ± 9.8 days. P4 plasma concentrations increased after ovulation and remained elevated until parturition. However, during the last 4 weeks of gestation, a gradual decrease from 4.17 × 1.17^±1 nmol/L to 2.02 × 1.95^±1 nmol/L on day 5 before parturition was observed, followed by a more abrupt final decline to baseline concentrations which were reached on the day after parturition. Mean E2 plasma concentrations started to increase during the eighth month of gestation, and were significantly elevated up to maximum concentrations of 484.7 × 1.21^±1 pmol/L during the last 2 months of pregnancy. Concentrations returned to baseline during the last 2 days of gestation. An increase of E1S concentrations (p < 0.01) was observed in the eleventh month of gestation. Mean E1S concentrations remained rather constant during the last 3 weeks of gestation between 4 to 8 nmol/L until parturition, when a steep precipitous decline was observed. E1 concentrations were slightly elevated during the last 4 weeks of gestation, however, maximum concentrations did not exceed 1.5 nmol/L. The results show distinct species specific features of gestational steroid hormone profiles in the guanaco in comparison to domestic South American camelids, such as a more pronounced gradual prepartal decrease of P4 concentrations prior to the final decline to baseline, and clearly lesser E1S concentrations during the last 4 weeks of gestation, which lack a continuous prepartal increase.     

Lipoprotein Subpopulation Distributions in Lean,Obese, and Type 2 Diabetic Women: A Comparison of African and White Americans

Objective: Abnormal subpopulation distributions of plasma lipoproteins have been reported in white American (WA) women with obesity and type 2 diabetes that explain part of the elevated rate of cardiovascular disease in these patients. This study examined if these perturbations also occur in obese and diabetic African American (AA) women and compared the lipoprotein profiles with WA counterparts. Research Methods and Procedures: We determined the lipoprotein subpopulation distribution in the plasma of 51 lean women (29 WA, 22 AA, body mass index [BMI] < 30), 50 obese women (27 WA, 23 AA, BMI > 30), and 43 obese women with type 2 diabetes (27 WA, 16 AA), by nuclear magnetic resonance spectroscopy. Results: AA diabetic women, like WA diabetic women, had a larger average very low density lipoprotein (VLDL) size, elevated levels of small low density lipoprotein cholesterol (LDL‐C), and lower levels of small high density lipoprotein cholesterol (HDL‐C), when compared to lean controls (p < 0.05). These differences were accompanied by higher VLDL‐triglycerides (TG) and LDL‐C in WA (p < 0.05), but not in AA. Although the effects of obesity and diabetes on lipoprotein subpopulation were fairly similar for AA and WA, some racial differences, particularly with respect to HDL, were observed. Discussion: The atherogenic perturbations in lipoprotein profiles of obese AA women, particularly those with diabetes, were relatively similar to those found in WA women and may be contributing to the increased rate of cardiovascular disease (CVD) in AA with obesity and diabetes. The parameters of subpopulation distribution may provide better markers for CVD than lipid concentrations alone, particularly in AA women. Furthermore, subtle racial differences in lipoprotein profiles suggest that race‐specific criteria may be needed to screen patients for CVD.