NMR-based metabolic profiling in healthy individuals overfed different types of fat: links to changes in liver fat accumulation and lean tissue mass

Background:

Overeating different dietary fatty acids influence the amount of liver fat stored during weight gain, however, the mechanisms responsible are unclear. We aimed to identify non-lipid metabolites that may differentiate between saturated (SFA) and polyunsaturated fatty acid (PUFA) overfeeding using a non-targeted metabolomic approach. We also investigated the possible relationships between plasma metabolites and body fat accumulation.

Methods:

In a randomized study (LIPOGAIN study), n=39 healthy individuals were overfed with muffins containing SFA or PUFA. Plasma samples were precipitated with cold acetonitrile and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition techniques were used to overview the data, identify variables contributing to group classification and to correlate metabolites with fat accumulation.

Results:

We previously reported that SFA causes a greater accumulation of liver fat, visceral fat and total body fat, whereas lean tissue levels increases less compared with PUFA, despite comparable weight gain. In this study, lactate and acetate were identified as important contributors to group classification between SFA and PUFA (P<0.05). Furthermore, the fat depots (total body fat, visceral adipose tissue and liver fat) and lean tissue correlated (P(corr)>0.5) all with two or more metabolites (for example, branched amino acids, alanine, acetate and lactate). The metabolite composition differed in a manner that may indicate higher insulin sensitivity after a diet with PUFA compared with SFA, but this needs to be confirmed in future studies.

Conclusion:

A non-lipid metabolic profiling approach only identified a few metabolites that differentiated between SFA and PUFA overfeeding. Whether these metabolite changes are involved in depot-specific fat storage and increased lean tissue mass during overeating needs further investigation.     

The gut microbiota keeps enteric glial cells on the move; prospective roles of the gut epithelium and immune system

The enteric nervous system (ENS) coordinates the major functions of the gastrointestinal tract. Its development takes place within a constantly changing environment which, after birth, culminates in the establishment of a complex gut microbiota. How such changes affect ENS development and its subsequent function throughout life is an emerging field of study that holds great interest but which is inadequately explored thus far. In this addendum, we discuss our recent findings showing that a component of the ENS, the enteric glial cell network that resides in the gut lamina propria, develops after birth and parallels the evolution of the gut microbiota. Importantly, this network was found to be malleable throughout life by incorporating new cells that arrive from the area of the gut wall in a process of directional movement which was controlled by the lumen gut microbiota. Finally, we postulate on the roles of the intestinal epithelium and the immune system as potential intermediaries between gut microbiota and ENS responses.     

Quantitative assessment of oral and pharyngeal function in Parkinson's disease

Oral and pharyngeal dysfunction is common in Parkinson's disease. To reveal the frequency of swallowing dysfunction and correlate swallowing dysfunction with locomotor disturbances, we studied 75 patients with Parkinson's disease staged I–IV according to the Hoehn and Yahr score. We assessed oral and pharyngeal swallow during optimal medication by a quantitative test of swallowing (the ROSS test) measuring the suction pressure, bolus volume, swallowing capacity, and time for important events in the swallowing cycle. We found abnormal results in 7/12 patients (58%) in stage 1 of the Hoehn and Yahr score, in 13/14 patients (93%) in stage 2, in 29/32 patients (91%) in stage 3, and in 16/17 patients (94%) in stage 4. Abnormal test results in stages, 1, 2, and 3 were seldom related to swallowing difficulties noticed by the patients. In advanced disease (Hoehn and Yahr stage 4), the abnormal results were often considerable, with swallowing difficulties obvious to the patient. Two of 17 patients coughed during or immediately after the test and 3/17 patients were unable to complete the test. The degree of swallowing disturbance increased during stress (forced, repetitive swallow). The Hoehn and Yahr score and the results in the ROSS test did not correlate, indicating that swallowing disturbances are due to nondopaminergic degeneration. Silent swallowing impairment may interfere with the nutrition and quality of life in Parkinson's disease, thus it is of interest to monitor this in clinical practice.     

Thyroxine transport and distribution in Nagase analbuminemic rats.

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin-bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550). To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values. Evaluation of the plasma disappearance curves of simultaneously injected 125I-T4 and 131I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51 +/- 21 ng/min, mean +/- SD) and in the control rats (54 +/- 15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13 +/- 0.53 vs. 1.22 +/- 0.36 micrograms). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9 +/- 3.3%, n = 3, vs. 11.4 +/- 3.4%). When greater than 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3 +/- 2.1%, n = 5, vs. 15.2 +/- 2.5%). These data provide the first valid experimental test of the enhanced dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.     

Masticatory ability in experimentally induced xerostomia

The masticatory ability of 15 nondysphagic volunteers with complete natural dentition was tested using different chewing parameters including preparation of a two-color plastic chewing gum (bolus shape, and color mixture), particle reduction of a piece of silicone, and number of strokes before swallow of almonds. The tests were performed under conditions of normal salivation and experimental oral dryness caused by intramuscular injection of methylscopolamine. The chewing gum tests as well as the silicone particle reduction tests were not influenced by lack of salivation. The number of chewing strokes prior to the initiation of swallowing of almonds was significantly increased. Oral dryness seems to cause accumulation of particles in the oral cavity from friable food and the particulate material is not transported posteriorly into a “readyto-swallow” positioning. The absorption of saliva by dry oral content such as an almond further impaired oral manipulation of food.     

Dietary Fiber,Kidney Function,Inflammation, and Mortality Risk

Background and objectives

In the United States population, high dietary fiber intake has been associated with a lower risk of inflammation and mortality in individuals with kidney dysfunction. This study aimed to expand such findings to a Northern European population.

Design, setting, participants, & measurements

Dietary fiber intake was calculated from 7-day dietary records in 1110 participants aged 70–71 years from the Uppsala Longitudinal Study of Adult Men (examinations performed during 1991–1995). Dietary fiber was adjusted for total energy intake by the residual method. Renal function was estimated from the concentration of serum cystatin C, and deaths were registered prospectively during a median follow-up of 10.0 years.

Results

Dietary fiber independently and directly associated with eGFR (adjusted difference, 2.6 ml/min per 1.73 m² per 10 g/d higher; 95% confidence interval [95% CI], 0.3 to 4.9). The odds of C-reactive protein >3 mg/L were lower (linear trend, P=0.002) with higher fiber quartiles. During follow-up, 300 participants died (incidence rate of 2.87 per 100 person-years at risk). Multiplicative interactions were observed between dietary fiber intake and kidney dysfunction in the prediction of mortality. Higher dietary fiber was associated with lower mortality in unadjusted analysis. These associations were stronger in participants with kidney dysfunction (eGFR<60 ml/min per 1.73 m²) (hazard ratio [HR], 0.58; 95% CI, 0.35 to 0.98) than in those without (HR, 1.30; 95% CI, 0.76 to 2.22; P value for interaction, P=0.04), and were mainly explained by a lower incidence of cancer-related deaths (0.25; 95% CI, 0.10 to 0.65) in individuals with kidney dysfunction versus individuals with an eGFR≥60 ml/min per 1.73 m² (1.61; 95% CI, 0.69 to 3.74; P value for interaction, P=0.01).

Conclusions

High dietary fiber was associated with better kidney function and lower inflammation in community-dwelling elderly men from Sweden. High dietary fiber was also associated with lower (cancer) mortality risk, especially in individuals with kidney dysfunction.     

Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A₄ hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB₄. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA₄ hydrolase. Here, we determined the crystal structure of LTA₄ hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA₄ hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB₄ synthesis in human neutrophils (IC₅₀ of ∼0.5 μM) and conversion of LTA₄ into LTB₄ by purified LTA4H with a K_i of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA₄ hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA₄, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA₄ into LTB₄, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA₄ hydrolase inhibitor that holds promise for improved anti-inflammatory properties.Leukotriene (LT) A₄ hydrolase/aminopeptidase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the formation of the potent chemotactic agent LTB₄, a key lipid mediator in the innate immune response (1, 2). Previous work has shown that LTA₄ hydrolase (LTA4H) is an aminopeptidase with high affinity for N-terminal arginines of various synthetic tripeptides (3, 4). The two enzyme activities of LTA4H are exerted via distinct but overlapping active sites and depend on the catalytic zinc, bound within the signature HEXXH-(X)₁₈-E, typical of M1 metallopeptidases (5–7). In LTA4H, His295, His299, and Glu318 are the zinc-binding ligands, whereas Glu296 is the general base catalyst for peptide hydrolysis (8, 9).LTA4H’s crystal structure has been determined (10). The enzyme folds into an N-terminal domain, a catalytic domain, and a C-terminal domain, each with ∼200 amino acids. The interface of the domains forms a cavity, where the active site is located (Fig. 1). The cavity narrows at the zinc-binding site, forming a tunnel into the catalytic domain. The opening and wider parts of the cavity are highly polar; the tunnel is more hydrophobic. The cavity is mostly defined by the catalytic and C-terminal domains; part of the tunnel is defined by the N-terminal domain. Bound substrate is in contact with all three domains.Open in a separate windowFig. 1.Position and extension of the active center in LTA4H. Cartoon representation of the structure of LTA4H with a tunnel for LTA₄ (red mesh) and peptide substrates (blue mesh). The catalytic zinc (yellow sphere) is located in a wide section of the active site from which a narrow, L-shaped, hydrophobic tunnel protrudes ∼15 Å deeper into the protein. LTA₄ is believed to bind with its ω-end at the end of the hydrophobic tunnel. The volume of the active center was calculated in CAVER (31).Recently, it was discovered that LTA4H cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro, thus identifying a previously unrecognized endogenous, physiologically significant aminopeptidase substrate (11). Inasmuch as Pro-Gly-Pro attracts neutrophils and promotes inflammation, these data also suggest that LTA4H plays dual and opposite roles during an inflammatory response (i.e., production of chemotactic LTB₄, as well as inactivation of chemotactic Pro-Gly-Pro). Previous efforts to develop inhibitors of LTA4H have used the aminopeptidase activity for screening purposes, and these molecules therefore block both catalytic activities of LTA4H (12).Here, we used crystallography, MS, and a stable peptide analog to determine the binding mode of Pro-Gly-Pro at the active site of LTA4H, as well as the mechanism of peptide cleavage. Based on the structure, we also designed a lead compound that selectively blocks the conversion of LTA₄ into LTB₄, although sparing the hydrolysis of Pro-Gly-Pro.     

Biomarkers of liver fibrosis: prospective comparison of multimodal magnetic resonance,serum algorithms and transient elastography

Abstract

Background and aims

Accurate biomarkers for quantifying liver fibrosis are important for clinical practice and trial end-points. We compared the diagnostic performance of magnetic resonance imaging (MRI), including gadoxetate-enhanced MRI and ³¹P-MR spectroscopy, with fibrosis stage and serum fibrosis algorithms in a clinical setting. Also, in a subset of patients, MR- and transient elastography (MRE and TE) was evaluated when available.