Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria.

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2-16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2-0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.

Expression of tyrosine pathway enzymes in mice demonstrates that homogentisate 1,2-dioxygenase deficiency in the liver is responsible for homogentisic acid-derived ochronotic pigmentation.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase (HGD) deficiency. This study aimed to determine if HGD and other enzymes related to tyrosine metabolism are associated with the location of ochronotic pigment. Liver, kidney, skin, bone, brain, eyes, spleen, intestine, lung, heart, cartilage, and muscle were harvested from 6 AKU BALB/c Hgd -/- (3 females, 3 males) and 4 male C57BL/6 wild type (WT) mice. Hgd, 4-hydroxyphenylpyruvate dioxygenase (4-Hppd), tyrosine hydroxylase (Th), and tyrosinase (Tyr) mRNA expression was investigated using qPCR. Adrenal gland and gonads from AKU Hgd tm1a -/- mice were LacZ stained, followed by qPCR analysis of Hgd mRNA. The liver had the highest expression of Hgd, followed by the kidney, with none detected in cartilage or brain. Low-level Hgd expression was observed within developing male germ cells within the testis and epididymis in Hgd tm1a -/-. 4-Hppd was most abundant in liver, with smaller amounts in kidney and low-level expression in other tissues. Th was expressed mainly in brain and Tyr was found primarily in the eyes. The tissue distribution of both Hgd and 4-Hppd suggest that ochronotic pigment in AKU mice is a consequence of enzymes within the liver, and not from enzymatic activity within ochronotic tissues. Excessive accumulation of HGA as ochronotic pigment in joints and other connective tissues originates from the circulation and therefore the extracellular fluid. The tissue distribution of both Th and Tyr suggests that these enzymes are not involved in the formation of HGA-derived ochronotic pigment.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces.

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl's staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd-/-, Hgd tm1a-/-), using Schmorl's staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 µmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 µmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 µmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 µmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 µmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.

Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

c-Fos induction by gut hormones and extracellular ATP in osteoblastic-like cell lines.

It is widely accepted that the c-Fos gene has a role in proliferation and differentiation of bone cells. ATP-induced c-Fos activation is relevant to bone homeostasis, because nucleotides that are present in the environment of bone cells can contribute to autocrine/paracrine signalling. Gut hormones have previously been shown to have an effect on bone metabolism. In this study, we used the osteoblastic Saos-2 cell line transfected with a c-Fos-driven reporter stimulated with five gut hormones: glucose inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin and obestatin, in the presence or absence of ATP. In addition, TE-85 cells were used to determine the time course of c-Fos transcript induction following stimulation with GLP-1, and GLP-2 with or without ATP, using reverse transcription qPCR. The significant results from the experiments are as follows: higher level of c-Fos induction in presence of GIP, obestatin (p = 0.019 and p = 0.011 respectively), and GIP combined with ATP (p < 0.001) using the luciferase assay; GLP-1 and GLP-2 combined with ATP (p = 0.034 and p = 0.002, respectively) and GLP-2 alone (p < 0.001) using qPCR. In conclusion, three of the gut peptides induced c-Fos, providing a potential mechanism underlying the actions of these hormones in bone which can be directed or enhanced by the presence of ATP.

Differential effects of adiponectin in osteoblast-like cells.

The skeleton should maintain an adequate volume, vigour and strength to carry out the role for which it is designed: to hold the whole soft tissue mass that shapes the body and to protect the vital organs. To fulfil this task a satisfactory food intake is required and regulators that are released in the feeding and fasting states, among other signals indicate how much soft mass needs to be built up. Those signals include the secretion of adipocytokines which could represent a relevant link between soft mass (adipose tissue) and skeleton. We studied the presence of adiponectin receptors (AdipoR1, AdipoR2) and its direct effects in osteosarcoma cell line Saos-2. The results indicated that adiponectin receptors were present in the osteoblastic cells with a higher expression of AdipoR1. Human recombinant globular adiponectin was able to increase viability levels and decrease cytotoxicity rates in cell cultures. Also, adiponectin significantly inhibited alkaline phosphatase activity in supernatants. Osteoprotegerin mRNA expression was significantly reduced after 72 h treatment. The FOS induction was studied and the results exhibited a significant increase caused by adiponectin. In conclusion, all these observations suggest that adiponectin influences bone metabolism decreasing the levels of bone formation. Regulators of adiponectin or its receptors could be circulating to modulate the activities of this peptide.

Adiponectin receptors are present in RANK-L-induced multinucleated osteoclast-like cells.

Adiponectin is an adipokine that has been related to bone metabolism. Data on adiponectin receptors (AdipoR1, -R2) in osteoclasts have shown discrepancies. In this study we carried out observations of AdipoR1, -R2 in peripheral blood mononuclear cells that were induced to differentiate into osteoclasts. AdipoR1, -R2 were screened using reverse transcription and quantitative PCR and immunofluorescence. Acid phosphatase and Cathepsin-K were evaluated as osteoclastic markers. Results showed that acid phosphatase was expressed from day 1 whereas Cathepsin-K started from day 7. AdipoR1 and -R2 showed expression from day 1, with greater expression for AdipoR1 than AdipoR2. The immunofluorescent patterns were observed in the cells cultured under three different conditions: non-supplemented medium, added M-CSF, or medium with M-CSF, and RANK-L. The non-supplemented control did not display specific fluorescence whereas specific and strong signals were detected in cells cultured with combined M-CSF and RANK-L from day 7. The fluorescence patterns were detected mainly at the periphery of the cells, and in the cytoplasm, showing a localized patchy pattern for both receptors. In contrast, a diffuse fluorescent pattern was detected in the cytoplasm of the cells with M-CSF alone. In summary, AdipoR1 and -R2 were detected by quantitative PCR and immunofluorescence. The immunofluorescence patterns suggest that adiponectin receptors are located, or re-located, in the plasma membrane with distribution in the cytoplasm when mononuclear cells are committed to differentiate to osteoclasts. These findings could be a reasonable explanation for the controversy found in the published literature regarding the role of adiponectin in bone metabolism.

Primary human osteoblast cultures.

Osteoblast cultures can be used to investigate the mechanisms of bone formation, to probe the cellular and molecular basis of bone disease, and to screen for potential therapeutic agents that affect bone formation. Here, we describe the methods for establishing and characterising primary human osteoblast cultures.

Receptors and effects of gut hormones in three osteoblastic cell lines.

BACKGROUND: In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. METHODS: mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. RESULTS: The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. CONCLUSIONS: These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.