Carboxypeptidase-mediated metabolism of calcitonin gene-related peptide in human gingival crevicular fluid - a role in periodontal inflammation?

Background: Metabolism by peptidases plays an important role in modulating the levels of biologically-active neuropeptides. The metabolism of the anti-inflammatory neuropeptide calcitonin gene-related peptide (GCRP), but not the pro-inflammatory neuropeptides substance P (SP) and neurokinin A (NKA) by components of the gingival crevicular fluid (GCF), could potentiate the inflammatory process in periodontitis.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Metabolism of very low- and low-density lipoproteins isolated from normolipidaemic type 2 (non-insulin-dependent) diabetic patients by human monocyte-derived macrophages

The very low- and low-density lipoprotein fractions were isolated from 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients in good to fair glycaemic control and from corresponding age-, sex-, and race-matched, non-diabetic control subjects. Rates of cholesteryl ester synthesis averaged 268 +/- 31 vs 289 +/- 40 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for very low- and 506 +/- 34 vs 556 +/- 51 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for low-density lipoproteins isolated from the Type 2 diabetic patients and control subjects, respectively, when they were incubated with human macrophages. A group of approximately one-third of the patients was selected for separate analyses because very low-density lipoproteins isolated from these patients did stimulate more cholesteryl ester synthesis when incubated with macrophages. There were no significant differences in the lipid composition of the lipoproteins isolated from the three groups of subjects. The relative proportion of apoprotein C to apoprotein E was significantly decreased (p less than 0.002) in the very low-density lipoproteins from diabetic patients and was further decreased in samples from these selected diabetic patients. The apoprotein C-I content of very low-density lipoproteins isolated from diabetic patients was increased compared to control subjects and was further increased in samples from the selected diabetic patients (p less than 0.02). There were no significant differences in the proportions of apoproteins C-III-0, C-III-1, or C-III-2 among the three groups. These studies suggest that in normolipidaemic Type 2 diabetic patients, the apoprotein composition of VLDL is abnormal and this may alter VLDL macrophage interactions and thus contribute to the increased prevalence of atherosclerosis in diabetic patients.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Untargeted Metabolomic Analysis of Human Plasma Indicates Differentially Affected Polyamine and L-Arginine Metabolism in Mild Cognitive Impairment Subjects Converting to Alzheimer’s Disease

This study combined high resolution mass spectrometry (HRMS), advanced chemometrics and pathway enrichment analysis to analyse the blood metabolome of patients attending the memory clinic: cases of mild cognitive impairment (MCI; n = 16), cases of MCI who upon subsequent follow-up developed Alzheimer's disease (MCI_AD; n = 19), and healthy age-matched controls (Ctrl; n = 37). Plasma was extracted in acetonitrile and applied to an Acquity UPLC HILIC (1.7μm x 2.1 x 100 mm) column coupled to a Xevo G2 QTof mass spectrometer using a previously optimised method. Data comprising 6751 spectral features were used to build an OPLS-DA statistical model capable of accurately distinguishing Ctrl, MCI and MCI_AD. The model accurately distinguished (R2 = 99.1%; Q2 = 97%) those MCI patients who later went on to develop AD. S-plots were used to shortlist ions of interest which were responsible for explaining the maximum amount of variation between patient groups. Metabolite database searching and pathway enrichment analysis indicated disturbances in 22 biochemical pathways, and excitingly it discovered two interlinked areas of metabolism (polyamine metabolism and L-Arginine metabolism) were differentially disrupted in this well-defined clinical cohort. The optimised untargeted HRMS methods described herein not only demonstrate that it is possible to distinguish these pathologies in human blood but also that MCI patients 'at risk' from AD could be predicted up to 2 years earlier than conventional clinical diagnosis. Blood-based metabolite profiling of plasma from memory clinic patients is a novel and feasible approach in improving MCI and AD diagnosis and, refining clinical trials through better patient stratification.

Characterization of the 5' upstream regulatory region of the human myostatin gene: regulation of myostatin gene transcription by dexamethasone in vitro

We cloned and characterized a 3.3-kb fragment containing the 5'-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-kappaB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C(2)C(12) and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay.

Aims/hypothesis: Glycation of insulin, resulting in impaired bioactivity, has been shown within pancreatic beta cells. We have used a novel and specific radioimmunoassay to detect glycated insulin in plasma of Type 2 diabetic subjects.

Methods: Blood samples were collected from 102 Type 2 diabetic patients in three main categories: those with good glycaemic control with a HbA1c less than 7%, moderate glycaemic control (HbA1c 7–9%) and poor glycaemic control (HBA1c greater than 9%). We used 75 age- and sex-matched non-diabetic subjects as controls. Samples were analysed for HbA1c, glucose and plasma concentrations of glycated insulin and insulin.

Results: Glycated insulin was readily detected in control and Type 2 diabetic subjects. The mean circulating concentration of glycated insulin in control subjects was 12.6±0.9 pmol/l (n=75). Glycated insulin in the good, moderate and poorly controlled diabetic groups was increased 2.4-fold (p<0.001, n=44), 2.2- fold (p<0.001, n=41) and 1.1-fold (n=17) corresponding to 29.8±5.4, 27.3±5.7 and 13.5±2.9 pmol/l, respectively.

Conclusion/interpretation: Glycated insulin circulates at noticeably increased concentrations in Type 2 diabetic subjects. [Diabetologia (2003) 46:475–478]

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1e) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M-r of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M-r 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to -9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 mug (.) kg(-1) (.) min(-1), followed by 2 h at 83.0 mug (.) kg(-1) (.) min(-1); corresponding to 0.4 and 2.0 mU (.) kg(-1) (.) min(-1)). At the lower dose, the exogenons glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) d-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-a and significantly increased TNF-a-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-a-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-d reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE2, as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A

AIMS/HYPOTHESIS: Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-beta(II) signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-betaR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-betaR level and chemotaxis.

METHODS: Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted.

RESULTS: At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-betaR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-beta(II). At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-beta(II) inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-betaR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB.

CONCLUSION/INTERPRETATION: The biphasic response in PDGF-betaR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation.