Indole-3-Propionic Acid, a Gut-Derived Tryptophan Metabolite, Associates with Hepatic Fibrosis.

BACKGROUND AND AIMS: Gut microbiota-derived metabolites play a vital role in maintenance of human health and progression of disorders, including obesity and type 2 diabetes (T2D). Indole-3-propionic acid (IPA), a gut-derived tryptophan metabolite, has been recently shown to be lower in individuals with obesity and T2D. IPA's beneficial effect on liver health has been also explored in rodent and cell models. In this study, we investigated the association of IPA with human liver histology and transcriptomics, and the potential of IPA to reduce hepatic stellate cell activation in vitro. METHODS: A total of 233 subjects (72% women; age 48.3 ± 9.3 years; BMI 43.1 ± 5.4 kg/m2) undergoing bariatric surgery with detailed liver histology were included. Circulating IPA levels were measured using LC-MS and liver transcriptomics with total RNA-sequencing. LX-2 cells were used to study hepatoprotective effect of IPA in cells activated by TGF-ß1. RESULTS: Circulating IPA levels were found to be lower in individuals with liver fibrosis compared to those without fibrosis (p = 0.039 for all participants; p = 0.013 for 153 individuals without T2D). Accordingly, levels of circulating IPA associated with expression of 278 liver transcripts (p < 0.01) that were enriched for the genes regulating hepatic stellate cells (HSCs) activation and hepatic fibrosis signaling. Our results suggest that IPA may have hepatoprotective potential because it is able to reduce cell adhesion, cell migration and mRNA gene expression of classical markers of HSCs activation in LX-2 cells (all p < 0.05). CONCLUSION: The association of circulating IPA with liver fibrosis and the ability of IPA to reduce activation of LX-2 cells suggests that IPA may have a therapeutic potential. Further molecular studies are needed to investigate the mechanisms how IPA can ameliorate hepatic fibrosis.

Quantitative assessment of betainized compounds and associations with dietary and metabolic biomarkers in the randomized study of the healthy Nordic diet (SYSDIET).

BACKGROUND: Recently, a group of betainized compounds have been suggested to play a role in health effects in relation to a whole-grain-rich diet. OBJECTIVES: The aims of this study were to develop a quantitative mass spectrometric method for selected betainized compounds in human plasma, and to investigate their association with nutrient intake and measures of metabolic health in participants of the SYSDIET study. METHODS: The SYSDIET study was a controlled randomized intervention including individuals with metabolic syndrome, where the healthy Nordic diet (HND) group increased intakes of whole grains, canola oil, berries, and fish, whereas the control diet (CD) group consumed low-fiber cereal products, milk fat, and restricted amounts of fish and berries. A quantitative LC combined with triple quadrupole MS method for betainized compounds was developed and applied to fasting plasma samples from baseline (week 0) and the end of the intervention (week 18 or 24). Concentrations of betainized compounds were correlated with intakes of selected nutrients and fiber and measures of metabolic health. RESULTS: Pipecolic acid betaine (PAB) concentrations were significantly higher in the HND group than in the CD group (P = 0.00032) at the end of the intervention and correlated directly (P < 0.0001) with intakes of dietary fiber (r = 0.376) and a biomarker related to whole-grain rye intake, namely the ratio of alkylresorcinol C17:0 to C21:0 (r = 0.442). PAB was associated inversely with fasting plasma insulin consistently at the beginning and at the end of the intervention (P < 0.001, r = -0.300; P < 0.01, r = -0.250, respectively), as well as IL-1 receptor antagonist (P < 0.01, r = -0.232 at the beginning; P < 0.01, r = -0.236 at the end) and serum LDL/HDL cholesterol (P < 0.01, r = -0.239 at the beginning; P < 0.01, r = -0.241 at the end). CONCLUSIONS: Among adults with the metabolic syndrome, PAB plasma concentrations were associated with fasting insulin, inflammation, and lipids and were significantly increased with adoption of the HND. Further studies are needed to clarify the biological functions of betainized compounds. This trial was registered at clinicaltrials.gov as NCT00992641.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes.

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3-22 µM and 38-78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to ß-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of ß-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

Differentially cleaving peptides as a strategy for controlled drug release in human retinal pigment epithelial cells.

Currently, drug delivery to the posterior eye segment relies on intravitreal injections of therapeutics. This approach requires frequent injections and does not guarantee drug delivery to intracellular targets. Controlled release systems and nanoparticles are being investigated to mitigate these challenges but most of these approaches lack translational success to the clinics. In our present study, we report a peptide-based delivery system that utilizes enzyme assisted cleavable linkers to release conjugated cargo within the retinal pigment epithelial (RPE) cells. Peptide linkers with differential cleavage rates were developed and tested in the vitreous humor, RPE cell homogenates and intact RPE cells. Selected peptide linkers were conjugated to cell penetrating peptides and d-peptide cargoes. The peptide-based delivery systems were non-toxic to the RPE cells, chemically stable in porcine vitreous and delivered cargo prototypes (hydrophobic & hydrophilic) to the RPE cells. Importantly, we show quantitatively with LC/MS analytics that the intracellular cargo release is controlled by the sequence of the peptide linker. The controlled cleavage of the peptide linkers is not only a useful strategy for intracellular drug delivery to the RPE targets but might also be useful in utilizing the RPE cells as mediators of drug delivery to intracellular targets and surrounding tissues (such as neural retina and choroid).

Needle metabolome, freezing tolerance and gas exchange in Norway spruce seedlings exposed to elevated temperature and ozone concentration.

Northern forests are currently experiencing increasing mean temperatures, especially during autumn and spring. Consequently, alterations in carbon sequestration, leaf biochemical quality and freezing tolerance (FT) are likely to occur. The interactive effects of elevated temperature and ozone (O(3)), the most harmful phytotoxic air pollutant, on Norway spruce (Picea abies (L.) Karst.) seedlings were studied by analysing phenology, metabolite concentrations in the needles, FT and gas exchange. Sampling was performed in September and May. The seedlings were exposed to a year-round elevated temperature (+1.3 °C), and to 1.4× ambient O(3) concentration during the growing season in the field. Elevated temperature increased the concentrations of amino acids, organic acids of the citric acid cycle and some carbohydrates, and reduced the concentrations of phenolic compounds, some organic acids of the shikimic acid pathway, sucrose, cyclitols and steroids, depending on the timing of the sampling. Although growth onset occurred earlier at elevated temperature, the temperature of 50% lethality (LT(50)) was similar in the treatments. Photosynthesis and the ratio of photosynthesis to dark respiration were reduced by elevated temperature. Elevated concentrations of O(3) reduced the total concentration of soluble sugars, and tended to reduce LT(50) of the needles in September. These results show that alterations in needle chemical quality can be expected at elevated temperatures, but the seedlings' sensitivity to autumn and spring frosts is not altered. Elevated O(3) has the potential to disturb cold hardening of Norway spruce seedlings in autumn, and to alter the water balance of the seedling through changes in stomatal conductance (g(s)), while elevated temperature is likely to reduce g(s) and consequently reduce the O(3)-flux inside the leaves.

Characterization of the glyoxalase 1 gene TcGLX1 in the metal hyperaccumulator plant Thlaspi caerulescens.

Stress tolerance is currently one of the major research topics in plant biology because of the challenges posed by changing climate and increasing demand to grow crop plants in marginal soils. Increased Zn tolerance and accumulation has been reported in tobacco expressing the glyoxalase 1-encoding gene from Brassica juncea. Previous studies in our laboratory showed some Zn tolerance-correlated differences in the levels of glyoxalase 1-like protein among accessions of Zn hyperaccumulator Thlaspi caerulescens. We have now isolated the corresponding gene (named here TcGLX1), including ca. 570 bp of core and proximal promoter region. The predicted protein contains three glyoxalase 1 motifs and several putative sites for post-translational modification. In silico analysis predicted a number of cis-acting elements related to stress. The expression of TcGLX1 was not responsive to Zn. There was no correlation between the levels of TcGLX1 expression and the degrees of Zn tolerance or accumulation among T. caerulescens accessions nor was there co-segregation of TcGLX1 expression with Zn tolerance or Zn accumulation among F3 lines derived from crosses between plants from accessions with contrasting phenotypes for these properties. No phenotype was observed in an A. thaliana T-DNA insertion line for the closest A. thaliana homolog of TcGLX1, ATGLX1. These results suggest that glyoxalase 1 or at least the particular isoform studied here is not a major determinant of Zn tolerance in the Zn hyperaccumulator plant T. caerulescens. In addition, ATGLX1 is not essential for normal Zn tolerance in the non-tolerant, non-accumulator plant A. thaliana. Possible explanations for the apparent discrepancy between this and previous studies are discussed.

Leaf proteome responses of Arabidopsis thaliana exposed to mild cadmium stress.

The leaf proteome of 3-week-old Arabidopsis thaliana seedlings exposed for 1 week to low, environmentally realistic Cd concentrations was investigated. The data indicated that at 1muMCd, A. thaliana plants adapted their metabolism to cope with the Cd exposure. As a result, only moderate protein changes were observed. However, at 10muMCd, severe stress was indicated by growth reduction and chlorosis of rosette leaves at the macroscopic level and by lipid peroxidation and enhanced peroxidase activity at the cellular level. Of the 730 reproducible proteins among all gels, 21 were statistically upregulated in response to Cd. These proteins can be functionally grouped into 5 classes: proteins involved in (1) oxidative stress response, (2) photosynthesis and energy production, (3) protein metabolism, (4) gene expression and finally, (5) proteins with various or unknown function. In order to provide greater insight into the mechanisms induced on Cd exposure, a working model is proposed.