The tricarboxylic acid (TCA) cycle: a malleable metabolic network to counter cellular stress.

The tricarboxylic acid (TCA) cycle is a primordial metabolic pathway that is conserved from bacteria to humans. Although this network is often viewed primarily as an energy producing engine fueling ATP synthesis via oxidative phosphorylation, mounting evidence reveals that this metabolic hub orchestrates a wide variety of pivotal biological processes. It plays an important part in combatting cellular stress by modulating NADH/NADPH homeostasis, scavenging ROS (reactive oxygen species), producing ATP by substrate-level phosphorylation, signaling and supplying metabolites to quell a range of cellular disruptions. This review elaborates on how the reprogramming of this network prompted by such abiotic stress as metal toxicity, oxidative tension, nutrient challenge and antibiotic insult is critical for countering these conditions in mostly microbial systems. The cross-talk between the stressors and the participants of TCA cycle that results in changes in metabolite and nucleotide concentrations aimed at combatting the abiotic challenge is presented. The fine-tuning of metabolites mediated by disparate enzymes associated with this metabolic hub is discussed. The modulation of enzymatic activities aimed at generating metabolic moieties dedicated to respond to the cellular perturbation is explained. This ancient metabolic network has to be recognized for its ability to execute a plethora of physiological functions beyond its well-established traditional roles.

Natural and synthetic 2-oxoglutarate derivatives are substrates for oncogenic variants of human isocitrate dehydrogenase 1 and 2.

Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.

Genomic and biochemical analysis of repeatedly observed variants in DBT in individuals with maple syrup urine disease of Central American ancestry.

Maple syrup urine disease (MSUD) is an intoxication-type inherited metabolic disorder in which hyperleucinemia leads to brain swelling and death without treatment. MSUD is caused by branched-chain alpha-ketoacid dehydrogenase deficiency due to biallelic loss of the protein products from the genes BCKDHA, BCKDHB, or DBT, while a distinct but related condition is caused by loss of DLD. In this case series, eleven individuals with MSUD caused by two pathogenic variants in DBT are presented. All eleven individuals have a deletion of exon 2 (delEx2, NM_001918.3:c.48_171del); six individuals are homozygous and five individuals are compound heterozygous with a novel missense variant (NM_001918.5:c.916 T > C [p.Ser306Pro]) confirmed to be in trans. Western Blot indicates decreased amount of protein product in delEx2;c.916 T > C liver cells and absence of protein product in delEx2 homozygous hepatocytes. Ultrahigh performance liquid chromatography-tandem mass spectrometry demonstrates an accumulation of branched-chain amino acids and alpha-ketoacids in explanted hepatocytes. Individuals with these variants have a neonatal-onset, non-thiamine-responsive, classical form of MSUD. Strikingly, the entire cohort is derived from families who immigrated to the Washington, DC, metro area from Honduras or El Salvador suggesting the possibility of a founder effect.

Characterization of acetolactate decarboxylase of Streptococcus thermophilus and its stereoselectivity in decarboxylation of α-hydroxy-ß-ketoacids.

Acetolactate decarboxylase (ALDC) is a well-characterized catabolic enzyme catalyzes the decarboxylation of (±)-acetolactate to produce a single product, (R)-acetoin. It can also convert other racemic α-hydroxy-ß-ketoacids to corresponding α -hydroxyketones in R-configuration. In this work, we prepared ALDC of Streptococcus thermophilus (StALDC) and explored its stereoselectivity on different substrates. The enzyme displays no enantioselectivity on substrate (±)-acetolactate, but R-selectivity on product acetoin, which are identical with the data reported for various ALDCs. When compound (±)-2-propionyl-2-hydroxybutyrate is used as a substrate, however, the enzyme exhibits S-selectivity on both substrate and product, namely it can only decarboxylate (S)-2-propionyl-2-hydroxybutyrate to generate (S)-4-hydroxy-3-hexanone rather than its R-isomer, which is totally discriminate from the data published for the ALDC of Bacillus subtilis. As far as we know, this is the first time that substrate dependent enantioselectivity of ALDC is reported and the feature of StALDC is also discussed on the basis of homology modeling and molecular docking experiments.

PDC1, a pyruvate/α-ketoacid decarboxylase, is involved in acetaldehyde, propanal and pentanal biosynthesis in melon (Cucumis melo L.) fruit.

Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.

A Hydroperoxide-Mediated Decarboxylation of α-Ketoacids Enables the Chemoselective Acylation of Amines.

Strategies for the formation of amide bonds, that is, one of the most basic and important transformations in organic synthesis, have so far focused predominantly on dehydration reactions. Herein, we report and demonstrate the practical utility of a novel decarboxylative amidation of α-ketoacids by using inexpensive tert-butyl hydroperoxide (TBHP), which is characterized by high yields, a broad substrate scope, mild reaction conditions, and a unique chemoselectivity. These features enable the synthesis of peptides from amino acid derived α-ketoacids under preservation of the stereochemical information.

Low-protein diet supplemented with ketoacids delays the progression of diabetic nephropathy by inhibiting oxidative stress in the KKAy mice model.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. We aimed to investigate the effect of the low-protein diets (LPD) supplemented with ketoacids (LPD+KA) in KKAy mice, an early type 2 DN model. KKAy mice were treated with normal protein diet (NPD), LPD or LPD+KA from 12 to 24 weeks of age. A period of 12-week treatment with LPD significantly reduced albuminuria as compared with that observed after NPD treatment. Treatment with LPD+KA further reduced albuminuria as compared with that observed with LPD treatment alone. Moreover, LPD treatment reduced mesangial expansion, thickness of glomerular basement membrane and the severity of the podocyte foot process effacement in KKAy mice; these effects were more pronounced in KKAy mice treated with LPD+KA. Both LPD and LPD+KA treatments slightly reduced total body weight, but had no significant effect on kidney weight and blood glucose concentrations when compared with NPD-treated KKAy mice. LPD treatment slightly attenuated oxidative stress in kidneys as compared with that observed in NPD-treated KKAy mice; however, LPD+KA treatment remarkably ameliorated oxidative stress in diabetic kidneys as shown by decreased malondialdehyde concentrations, protein carbonylation, nitrotyrosine expression and increased superoxide dismutase expression. Nutritional therapy using LPD+KA confers additional renal benefits as compared with those of LPD treatment alone in early type 2 DN through inhibition of oxidative stress.

The contribution of muscle, kidney, and splanchnic tissues to leucine transamination in humans.

The first steps of leucine utilization are reversible deamination to α-ketoisocaproic acid (α-KIC) and irreversible oxidation. Recently, the regulatory role of leucine deamination over oxidation was underlined in rodents. Our aim was to measure leucine deamination and reamination in the whole body, in respect to previously determined rates across individual organs, in humans. By leucine and KIC isotope kinetics, we determined whole-body leucine deamination and reamination, and we compared these rates with those already reported across the sampled organs. As an in vivo counterpart of the "metabolon" concept, we analysed ratios between oxidation and either deamination or reamination. Leucine deamination to KIC was greater than KIC reamination to leucine in the whole body (p = 0.005), muscles (p = 0.005), and the splanchnic area (p = 0.025). These rates were not significantly different in the kidneys. Muscle accounted for ≈60% and ≈78%, the splanchnic bed for ≈15% and ≈15%, and the kidney for ≈12% and ≈18%, of whole-body leucine deamination and reamination rates, respectively. In the kidney, percent leucine oxidation over either deamination or reamination was >3-fold greater than muscle and the splanchnic bed. Skeletal muscle contributes by the largest fraction of leucine deamination, reamination, and oxidation. However, in relative terms, the kidney plays a key role in leucine oxidation.

Safety and effectiveness of low-protein diet supplemented with ketoacids in diabetic patients with chronic kidney disease.

BACKGROUND: The impact of the low-protein diet on nutrition in CKD diabetics is uncertain. METHODS: The metabolic and nutritional effects of a low-protein (0.5-0.6 g/kg/d), normal-high energy (30-35 kcal/kg/d) diet supplemented with ketoacids (LPD-KA) were prospectively evaluated in CKD patients with (DM) and without (non-DM) diabetes mellitus. RESULTS: 197 patients on CKD stages 3-5 were enrolled. DM (n = 81) and non-DM (n = 116) were comparable for gender (Male 58 vs 55%), age (66 ± 9 vs 63 ± 18 years), renal function (eGFR 23 ± 13 vs 24 ± 13 mL/min). After 6-month, serum urea (DM: 131 ± 58 to 105 ± 49 mg/dl, p < 0.05; non-DM: 115 ± 52 to 88 ± 36, p < 0.05) and phosphate (DM: 4.5 ± 1.3 to 4.1 ± 1.2 mg/dl, p = 0.06; non-DM: 4.3 ± 1.0 to 3.7 ± 0.8, p < 0.05) declined. Fasting glucose decreased in DM (126 ± 52 to 103 ± 29 mg/dl, p < 0.05) without insulin dose increase. These effects were preserved after 3-year. Serum albumin did not change after 6 months (DM: 3.7 ± 0.6 to 3.8 ± 0.4 mg/dl; non-DM: 4.0 ± 0.6 to 4.0 ± 0.4) and in the long-term. Body weight (BW) declined after the diet start (DM: 68.9 ± 14.3 to 65.1 ± 12.1 kg, p < 0.05; non-DM: 66.6 ± 15.1 to 64.1 ± 15.1, p < 0.05) and was stable at 6 months and 3 years. Muscle strength at baseline was reduced in all patients and remained stable during the diet period. Changes of nutritional markers during the study were similar among groups and diabetes was not associated to any nutritional change at the multivariate analysis. As attain wasting, lower BMI (< 23 kg/m2) and albumin (< 3.8 g/dl) levels were present in 1/3 patients at start and along 3 years, cholesterol never dropped below the lower threshold (< 100 mg/dl) and poorer FM (< 10%) was less than 10% during the study in both groups. CONCLUSIONS: In diabetic CKD patients a low-protein diet supplemented with ketoacids improves uremia and diabetes, causes sudden decline of body weight which remains stable over time and has not a negative effect on wasting and muscle mass and fitness. In diabetic CKD patients the LPD-KA is safe and the nutritional impact is the same as in non-diabetics CKD.

Reactive nitrogen species (RNS)-resistant microbes: adaptation and medical implications.

Nitrosative stress results from an increase in reactive nitrogen species (RNS) within the cell. Though the RNS - nitric oxide (·NO) and peroxynitrite (ONOO-) - play pivotal physiological roles, at elevated concentrations, these moieties can be poisonous to both prokaryotic and eukaryotic cells alike due to their capacity to disrupt a variety of essential biological processes. Numerous microbes are known to adapt to nitrosative stress by elaborating intricate strategies aimed at neutralizing RNS. In this review, we will discuss both the enzymatic systems dedicated to the elimination of RNS as well as the metabolic networks that are tailored to generate RNS-detoxifying metabolites - α-keto-acids. The latter has been demonstrated to nullify RNS via non-enzymatic decarboxylation resulting in the production of a carboxylic acid, many of which are potent signaling molecules. Furthermore, as aerobic energy production is severely impeded during nitrosative stress, alternative ATP-generating modules will be explored. To that end, a holistic understanding of the molecular adaptation to nitrosative stress, reinforces the notion that neutralization of toxicants necessitates significant metabolic reconfiguration to facilitate cell survival. As the alarming rise in antimicrobial resistant pathogens continues unabated, this review will also discuss the potential for developing therapies that target the alternative ATP-generating machinery of bacteria.

Metabolic defence against oxidative stress: the road less travelled so far.

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly-reactive chalcogen-oxygen (O2 ). They are known to evoke intricate strategies to defend themselves from the reactive by-products of oxygen-reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)-cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH-the reductive engine of many ROS-combating enzymes-is maintained by metabolic enzymes including, but not exclusively, glucose-6 phosphate dehydrogenase (G6PDH) and NADP-dependent isocitrate dehydrogenase (ICDH-NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro-oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids-moieties capable of non-enzymatic decarboxylation in the presence of oxidants-as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid-mediated detoxification of the ROS will be commented on within the context of oxidative stress.

Stereoselective Synthesis of Functionalized Bicyclic Scaffolds by Passerini 3-Center-2-Component Reactions of Cyclic Ketoacids.

We report the use of bifunctional starting materials (ketoacids) in a diastereoselective Passerini three-center-two-component reaction. Study of the reaction scope revealed the required structural features for stereoselectivity in the isocyanide addition. In this system, an interesting isomerization of the primary Passerini product - the α-carboxamido lactone - into an atypical product, an α-hydroxy imide, was found to occur under acidic conditions. Furthermore, enantioenriched Passerini products can be generated from an enantioenriched ketoacid obtained by chemoenzymatic synthesis.

Low-protein diet supplemented with ketoacids ameliorates proteinuria in 3/4 nephrectomised rats by directly inhibiting the intrarenal renin-angiotensin system.

Low-protein diet plus ketoacids (LPD+KA) has been reported to decrease proteinuria in patients with chronic kidney diseases (CKD). However, the mechanisms have not been clarified. As over-activation of intrarenal renin-angiotensin system (RAS) has been shown to play a key role in the progression of CKD, the current study was performed to investigate the direct effects of LPD+KA on intrarenal RAS, independently of renal haemodynamics. In this study, 3/4 subtotal renal ablated rats were fed 18 % normal-protein diet (Nx-NPD), 6 % low-protein diet (Nx-LPD) or 5 % low-protein diet plus 1 % ketoacids (Nx-LPD+KA) for 12 weeks. Sham-operated rats fed NPD served as controls. The level of proteinuria and expression of renin, angiotensin II (AngII) and its type 1 receptors (AT1R) in the renal cortex were markedly higher in Nx-NPD group than in the sham group. LPD+KA significantly decreased the proteinuria and inhibited intrarenal RAS activation. To exclude renal haemodynamic impact on intrarenal RAS, the serum samples derived from the different groups were added to the culture medium of mesangial cells. It showed that the serum from Nx-NPD directly induced higher expression of AngII, AT1R, fibronectin and transforming growth factor-ß1 in the mesangial cells than in the control group. Nx-LPD+KA serum significantly inhibited these abnormalities. Then, proteomics and biochemical detection suggested that the mechanisms underlying these beneficial effects of LPD+KA might be amelioration of the nutritional metabolic disorders and oxidative stress. In conclusion, LPD+KA could directly inhibit the intrarenal RAS activation, independently of renal haemodynamics, thus attenuating the proteinuria in CKD rats.

The role of formate in combatting oxidative stress.

The interaction of keto-acids with reactive oxygen species (ROS) is known to produce the corresponding carboxylic acid with the concomitant formation of CO2. Formate is liberated when the keto-acid glyoxylate neutralizes ROS. Here we report on how formate is involved in combating oxidative stress in the nutritionally-versatile Pseudomonas fluorescens. When the microbe was subjected to hydrogen peroxide (H2O2), the levels of formate were 8 and two-fold higher in the spent fluid and the soluble cell-free extracts obtained in the stressed cultures compared to the controls respectively. Formate was subsequently utilized as a reducing force to generate NADPH and succinate. The former is mediated by formate dehydrogenase (FDH-NADP), whose activity was enhanced in the stressed cells. Fumarate reductase that catalyzes the conversion of fumarate into succinate was also markedly increased in the stressed cells. These enzymes were modulated by H2O2. While the stressed whole cells produced copious amounts of formate in the presence of glycine, the cell-free extracts synthesized ATP and succinate from formate. Although the exact role of formate in anti-oxidative defence has to await further investigation, the data in this report suggest that this carboxylic acid may be a potent reductive force against oxidative stress.

Very low-protein diet plus ketoacids in chronic kidney disease and risk of death during end-stage renal disease: a historical cohort controlled study.

BACKGROUND: Very low-protein intake during chronic kidney disease (CKD) improves metabolic disorders and may delay dialysis start without compromising nutritional status, but concerns have been raised on a possible negative effect on survival during dialysis. This study aimed at evaluating whether a very low-protein diet during CKD is associated with a greater risk of death while on dialysis treatment. METHODS: This is an historical, cohort, controlled study, enrolling patients at dialysis start previously treated in a tertiary nephrology clinic with a very low-protein diet supplemented with amino acids and ketoacids (s-VLPD group, n = 184) or without s-VLPD [tertiary nephrology care (TNC) group, n = 334] and unselected patients [control (CON) group, n = 9.092]. The major outcome was survival rate during end-stage renal disease associated to s-VLPD treatment during CKD. The propensity score methods and Cox regression model were used to match groups at the start of dialysis to perform survival analysis and estimate adjusted hazard ratio (HR). RESULTS: In s-VLPD, TNC and CON groups, average age was 67.5, 66.0 and 66.3 years, respectively (P = 0.521) and male prevalence was 55, 55 and 62%, respectively (P = 0.004). Diabetes prevalence differed in the three groups (P < 0.001), being 18, 17 and 31% in s-VLPD, CON and TNC, respectively. A different prevalence of cardiovascular (CV) disease was found (P < 0.001), being similar in TNC and CON (31 and 25%) and higher in s-VLPD (41%). Median follow-up during renal replacement therapy (RRT) was 36, 32 and 36 months in the three groups. Adjusted HR estimated on matched propensity patients was 0.59 (0.45-0.78) for s-VLPD versus CON. Subgroup analysis showed a lower mortality risk in s-VLPD versus matched-CON in younger patients (<70 years) and those without CV disease. No significant difference in HRs was found between s-VLPD and TNC. CONCLUSION: s-VLPD during CKD does not increase mortality in the subsequent RRT period.

Copper-catalyzed one-pot denitrogenative-dehydrogenative-decarboxylative coupling of ß-ketoacids with trifluorodiazoethane: facile access to trifluoromethylated aldol products.

A novel copper-catalyzed one-pot cross-coupling of ß-ketoacids with in situ generated trifluorodiazoethane has been developed. This reaction provides a direct and efficient method, in which one C-C bond and one C-O bond were formed in a carbenoid center with concomitant denitrogenation-dehydrogenation-decarboxylation, to afford trifluoromethylated aldol products. In several preliminary experiments, good to high enantioselectivities were also obtained.

Abomasal infusion of branched-chain amino acids or branched-chain keto-acids alter lactation performance and liver triglycerides in fresh cows.

BACKGROUND: Dairy cows are at high risk of fatty liver disease in early lactation, but current preventative measures are not always effective. Cows with fatty liver have lower circulating branched-chain amino acid (BCAA) concentrations whereas cows with high circulating BCAA levels have low liver triglyceride (TG). Our objective was to determine the impact of BCAA and their corresponding ketoacids (branched-chain ketoacids, BCKA) on production performance and liver TG accumulation in Holstein cows in the first 3 weeks postpartum. METHODS: Thirty-six multiparous Holstein cows were used in a randomized block design experiment. Cows were abomasally infused for the first 21 d postpartum with solutions of 1) saline (CON, n = 12); 2) BCA (67 g valine, 50 g leucine, and 34 g isoleucine, n = 12); and 3) BCK (77 g 2-ketovaline calcium salt, 57 g 2-ketoleucine calcium salt, and 39 g 2-ketoisoleucine calcium salt, n = 12). All cows received the same diet. Treatment effects were determined using PROC GLIMMIX in SAS. RESULTS: No differences were detected for body weight, body condition score, or dry matter intake averaged over the first 21 d postpartum. Cows receiving BCK had significantly lower liver TG concentrations compared to CON (6.60% vs. 4.77%, standard error of the mean (SEM) 0.49) during the first 3 weeks of lactation. Infusion of BCA increased milk yield (39.5 vs. 35.3 kg/d, SEM 1.8), milk fat yield (2.10 vs. 1.69 kg/d, SEM 0.08), and lactose yield (2.11 vs. 1.67 kg/d, SEM 0.07) compared with CON. Compared to CON, cows receiving BCA had lower plasma glucose (55.0 vs. 59.2 mg/dL, SEM 0.86) but higher ß-hydroxybutyrate (9.17 vs. 6.00 mg/dL, SEM 0.80). CONCLUSIONS: Overall, BCAA supplementation in this study improved milk production, whereas BCKA supplementation reduced TG accumulation in the liver of fresh cows.

Transcriptomics and UHPLC-QQQ-MS analyses reveal the dysregulation of branched chain amino acids metabolism in renal fibrotic rats.

The dysregulated levels of branched chain amino acids (BCAA) contribute to renal fibrosis in chronic kidney disease (CKD), yet specific analysis of BCAA contents and how they are regulated still remain unclear. It is therefore of great scientific interest to understand BCAA catabolism in CKD and develop a sensitive method for simultaneous determination of individual BCAA and their metabolites branched chain α-ketoacids (BCKA). In this work, the important role of BCAA metabolism that drives renal fibrosis in the process of CKD was first revealed by using transcriptomics. The key target genes controlling BCAA metabolism were then validated, that is, mRNA levels of BCKDHA and BCKDHB, the regulating rate-limiting enzymes during BCAA metabolism were abnormally reduced by quantitative PCR (qPCR), and a similar drop-off trend of protein expression of BCKDH, HIBCH and MCCC2 that are closely related to BCAA metabolism was also confirmed by western blotting. Furthermore, we established a novel strategy that simultaneously determines 6 individual BCAA and BCKA in serum and tissue. The method based on dansylhydrazine derivatization and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS) achieved to simultaneously determine the contents of BCAA and BCKA, which is efficient and stable. Compared with normal rats, levels of BCAA including leucine, isoleucine and valine in serum and kidney of CKD rats was decreased, while BCKA including α-ketoisocaproic acid, α-ketomethylvaleric acid and α-ketoisovaleric acid was increased. Together, these findings revealed the abnormality of BCAA metabolism in driving the course of kidney fibrosis and CKD. Our current study sheds new light on changes in BCAA metabolism during CKD, and may facilitate development of drugs to treat CKD and renal fibrosis.

Optimizing Nutrition in Renal Patients: Effects of a Low-Protein Diet Supplemented With Ketoacids.

Background Chronic kidney disease (CKD) is a non-communicable disease; it is a major cause of morbidity and mortality in Nigeria as the incidence has been increasing in Nigeria over the last few years. A low-protein diet supplemented with ketoacids has been duly documented to reduce the malnutrition associated with CKD as well as improve estimated glomeruli filtration rate while delaying the onset of dialysis in predialysis CKD patients. Objective The aim of this study was to determine the effects of a low-protein diet supplemented with ketoacids compared to a conventional low protein on nutritional indices in predialysis CKD patients. Methods and materials A randomized controlled trial with a total of 60 participants was conducted at Delta State University Teaching Hospital (DELSUTH), Oghara, Nigeria. Participants were patients older than 18 years with CKD stage 3-5 who were not on dialysis. They were recruited and randomized into the intervention group (low-protein diet supplemented with ketoacids) with 30 participants and the non-intervention group (low protein with placebo) with 30 participants. The mean outcome was changed in the nutritional indices from baseline till the end of the study. Results A total of 60 patients were randomly allocated to receive a low-protein diet supplemented with ketoacids (n=30) or control (n=30). All participants were included in the analysis of all outcomes. The mean change score in serum total protein, albumin, and triglycerides between the intervention and non-intervention groups were 1.1±1.1 g/dL vs 0.1±1.1 g/dL (p<0.001), 0.2±0.9 g/dL vs -0.3±0.8 g/dL (p<0.001), and 3.0±3.5 g/dL vs 1.8±3.7 g/dL, respectively. Conclusion and recommendation The use of low-protein diet supplemented with ketoacids improved the anthropometric and nutritional indices in patients with stage 3-5 CKD.

Metabolic Role of GABA in the Secretory Function of Pancreatic ß-Cells: Its Hypothetical Implication in ß-Cell Degradation in Type 2 Diabetes.

The stimulus-secretion coupling of a glucose-induced release is generally attributed to the metabolism of the hexose in the ß-cells in the glycolytic pathway and the citric acid cycle. Glucose metabolism generates an increased cytosolic concentration of ATP and of the ATP/ADP ratio that closes the ATP-dependent K+-channel at the plasma membrane. The resultant depolarization of the ß-cells opens voltage-dependent Ca2+-channels at the plasma membrane that triggers the exocytosis of insulin secretory granules. The secretory response is biphasic with a first and transient peak followed by a sustained phase. The first phase is reproduced by a depolarization of the ß-cells with high extracellular KCl maintaining the KATP-channels open with diazoxide (triggering phase); the sustained phase (amplifying phase) depends on the participation of metabolic signals that remain to be determined. Our group has been investigating for several years the participation of the ß-cell GABA metabolism in the stimulation of insulin secretion by three different secretagogues (glucose, a mixture of L-leucine plus L-glutamine, and some branched chain alpha-ketoacids, BCKAs). They stimulate a biphasic secretion of insulin accompanied by a strong suppression of the intracellular islet content of gamma-aminobutyric acid (GABA). As the islet GABA release simultaneously decreased, it was concluded that this resulted from an increased GABA shunt metabolism. The entrance of GABA into the shunt is catalyzed by GABA transaminase (GABAT) that transfers an amino group between GABA and alpha-ketoglutarate, resulting in succinic acid semialdehyde (SSA) and L-glutamate. SSA is oxidized to succinic acid that is further oxidized in the citric acid cycle. Inhibitors of GABAT (gamma-vinyl GABA, gabaculine) or glutamic acid decarboxylating activity (GAD), allylglycine, partially suppress the secretory response as well as GABA metabolism and islet ATP content and the ATP/ADP ratio. It is concluded that the GABA shunt metabolism contributes together with the own metabolism of metabolic secretagogues to increase islet mitochondrial oxidative phosphorylation. These experimental findings emphasize that the GABA shunt metabolism is a previously unrecognized anaplerotic mitochondrial pathway feeding the citric acid cycle with a ß-cell endogenous substrate. It is therefore a postulated alternative to the proposed mitochondrial cataplerotic pathway(s) responsible for the amplification phase of insulin secretion. It is concluded the new postulated alternative suggests a possible new mechanism of ß-cell degradation in type 2 (perhaps also in type 1) diabetes.