Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle.

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m2 (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

Early-onset preeclampsia, plasma microRNAs, and endothelial cell function.

BACKGROUND: Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. OBJECTIVE: We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro. STUDY DESIGN: We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age-matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether overexpression of the microRNAs in endothelial cells affected endothelial cell function in vitro. Functional assays were the wound-healing assay (which measures cell migration and proliferation), the proliferation assay, and the tube-formation assay (which assesses formation of endothelial cell tubes during the angiogenic process). To determine whether the miRNAs are able to decrease gene expression of certain genes, RNA was isolated from transfected endothelial cells and gene expression (by measuring RNA expression) was evaluated by gene expression microarray (Genechip Human Gene 2.1 ST arrays; Life Technologies). For the microarray, we used pooled samples, but the differently expressed genes in the microarray were validated by real-time quantitative polymerase chain reaction in individual samples. RESULTS: No significant differences (fold change <-1.2 or >1.2 with a false-discovery rate <0.05) were found in miRNA plasma concentrations between healthy pregnant and nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy were miR-574-5p, miR-1972, and miR-4793-3p. Transfection of endothelial cells with these miRNAs in showed that miR-574-5p decreased (P<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.1) to decrease both the wound-healing capacity and tube formation in vitro. Gene expression analysis of transfected endothelial cells revealed that miR-574-5p tended (P<.1) to decrease the expression of the proliferation marker MKI67. CONCLUSION: We conclude that in the early-onset preeclampsia group in our study different concentrations of plasma miRNAs are present as compared with healthy pregnancy. Our results suggest that miR-574-5p and miR-1972 decrease the proliferation (probably via decreasing MKI67) and/or migration as well as the tube-formation capacity of endothelial cells. Therefore, these miRNAs may be antiangiogenic factors affecting endothelial cells in preeclampsia.

No effect of 25-hydroxyvitamin D supplementation on the skeletal muscle transcriptome in vitamin D deficient frail older adults.

OBJECTIVE: Vitamin D deficiency is common among older adults and has been linked to muscle weakness. Vitamin D supplementation has been proposed as a strategy to improve muscle function in older adults. The aim of this study was to investigate the effect of calcifediol (25-hydroxycholecalciferol) on whole genome gene expression in skeletal muscle of vitamin D deficient frail older adults. METHODS: A double-blind placebo-controlled trial was conducted in vitamin D deficient frail older adults (aged above 65), characterized by blood 25-hydroxycholecalciferol concentrations between 20 and 50 nmol/L. Subjects were randomized across the placebo group and the calcifediol group (10 µg per day). Muscle biopsies were obtained before and after 6 months of calcifediol (n = 10) or placebo (n = 12) supplementation and subjected to whole genome gene expression profiling using Affymetrix HuGene 2.1ST arrays. RESULTS: Expression of the vitamin D receptor gene was virtually undetectable in human skeletal muscle biopsies, with Ct values exceeding 30. Blood 25-hydroxycholecalciferol levels were significantly higher after calcifediol supplementation (87.3 ± 20.6 nmol/L) than after placebo (43.8 ± 14.1 nmol/L). No significant difference between treatment groups was observed on strength outcomes. The whole transcriptome effects of calcifediol and placebo were very weak, as indicated by the fact that correcting for multiple testing using false discovery rate did not yield any differentially expressed genes using any reasonable cut-offs (all q-values ~ 1). P-values were uniformly distributed across all genes, suggesting that low p-values are likely to be false positives. Partial least squares-discriminant analysis and principle component analysis was unable to separate treatment groups. CONCLUSION: Calcifediol supplementation did not significantly affect the skeletal muscle transcriptome in frail older adults. Our findings indicate that vitamin D supplementation has no effects on skeletal muscle gene expression, suggesting that skeletal muscle may not be a direct target of vitamin D in older adults. TRIAL REGISTRATION: This study was registered at clinicaltrials.gov as NCT02349282 on January 28, 2015.

Identification of a mammalian silicon transporter.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

Combined Activities of JNK1 and JNK2 in Hepatocytes Protect Against Toxic Liver Injury.

BACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1(Δhepa)) or combination of Jnk1 and Jnk2 (Jnk(Δhepa)), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk(Δhepa) mice produced a greater level of liver injury than that observed in Jnk1(Δhepa) or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in Jnk(Δhepa) mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1(Δhepa) or control mice. Hepatocytes from Jnk(Δhepa) mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk(Δhepa) and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.

Detection of prokaryotic mRNA signifies microbial viability and promotes immunity.

Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

The Muscle Metabolome Differs between Healthy and Frail Older Adults.

Populations around the world are aging rapidly. Age-related loss of physiological functions negatively affects quality of life. A major contributor to the frailty syndrome of aging is loss of skeletal muscle. In this study we assessed the skeletal muscle biopsy metabolome of healthy young, healthy older and frail older subjects to determine the effect of age and frailty on the metabolic signature of skeletal muscle tissue. In addition, the effects of prolonged whole-body resistance-type exercise training on the muscle metabolome of older subjects were examined. The baseline metabolome was measured in muscle biopsies collected from 30 young, 66 healthy older subjects, and 43 frail older subjects. Follow-up samples from frail older (24 samples) and healthy older subjects (38 samples) were collected after 6 months of prolonged resistance-type exercise training. Young subjects were included as a reference group. Primary differences in skeletal muscle metabolite levels between young and healthy older subjects were related to mitochondrial function, muscle fiber type, and tissue turnover. Similar differences were observed when comparing frail older subjects with healthy older subjects at baseline. Prolonged resistance-type exercise training resulted in an adaptive response of amino acid metabolism, especially reflected in branched chain amino acids and genes related to tissue remodeling. The effect of exercise training on branched-chain amino acid-derived acylcarnitines in older subjects points to a downward shift in branched-chain amino acid catabolism upon training. We observed only modest correlations between muscle and plasma metabolite levels, which pleads against the use of plasma metabolites as a direct read-out of muscle metabolism and stresses the need for direct assessment of metabolites in muscle tissue biopsies.

Haematopoietic cell-derived Jnk1 is crucial for chronic inflammation and carcinogenesis in an experimental model of liver injury.

BACKGROUND & AIMS: Chronic liver injury triggers complications such as liver fibrosis and hepatocellular carcinoma (HCC), which are associated with alterations in distinct signalling pathways. Of particular interest is the interaction between mechanisms controlled by IKKγ/NEMO, the regulatory IKK subunit, and Jnk activation for directing cell death and survival. In the present study, we aimed to define the relevance of Jnk in hepatocyte-specific NEMO knockout mice (NEMO(Δhepa)), a genetic model of chronic inflammatory liver injury. METHODS: We generated Jnk1(-/-)/NEMO(Δhepa) and Jnk2(-/-)/NEMO(Δhepa) mice by crossing NEMO(Δhepa) mice with Jnk1 and Jnk2 global deficient animals, respectively, and examined the progression of chronic liver disease. Moreover, we investigated the expression of Jnk during acute liver injury, evaluated the role of Jnk1 in bone marrow-derived cells, and analysed the expression of NEMO and p-JNK in human diseased-livers. RESULTS: Deletion of Jnk1 significantly aggravated the progression of liver disease, exacerbating apoptosis, compensatory proliferation and carcinogenesis in NEMO(Δhepa) mice. Conversely, Jnk2(-/-)/NEMO(Δhepa) displayed hepatic inflammation. By using bone marrow transfer, we observed that Jnk1 in haematopoietic cells had an impact on the progression of chronic liver disease in NEMO(Δhepa) livers. These findings are of clinical relevance since NEMO expression was downregulated in hepatocytes of patients with HCC whereas NEMO and p-JNK were expressed in a large amount of infiltrating cells. CONCLUSIONS: A synergistic function of Jnk1 in haematopoietic cells and hepatocytes might be relevant for the development of chronic liver injury. These results elucidate the complex function of Jnk in chronic inflammatory liver disease.

An acute intake of plant stanol esters alters immune-related pathways in the jejunum of healthy volunteers.

Plant sterols and stanols inhibit intestinal cholesterol absorption and consequently lower serum LDL-cholesterol (LDL-C) concentrations. The underlying mechanisms are not yet known. In vitro and animal studies have suggested that changes in intestinal sterol metabolism are attributed to the LDL-C-lowering effects of plant stanol esters. However, similar studies in human subjects are lacking. Therefore, we examined the effects of an acute intake of plant stanol esters on gene expression profiles of the upper small intestine in healthy volunteers. In a double-blind cross-over design, fourteen healthy subjects (eight female and six male; age 21-55 years), with a BMI ranging from 21 to 29 kg/m², received in random order a shake with or without plant stanol esters (4 g). At 5 h after consumption of the shake, biopsies were taken from the duodenum (around the papilla of Vater) and from the jejunum (20 cm distal from the papilla of Vater). Microarray analysis showed that the expression profiles of genes involved in sterol metabolism were not altered. Surprisingly, the pathways involved in T-cell functions were down-regulated in the jejunum. Furthermore, immunohistochemical analysis showed that the number of CD3 (cluster of differentiation number 3), CD4 (cluster of differentiation number 4) and Foxp3⁺ (forkhead box P3-positive) cells was reduced in the plant stanol ester condition compared with the control condition, which is in line with the microarray data. The physiological and functional consequences of the plant stanol ester-induced reduction of intestinal T-cell-based immune activity in healthy subjects deserve further investigation.

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis.

OBJECTIVE: The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. DESIGN: Jnk1(f/f) wildtype (WT), Jnk1(-/-) and Jnk1(Δhepa) (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. RESULTS: Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1(-/-) compared with WT and Jnk1(Δhepa) mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1(-/-) compared with WT and Jnk1(Δhepa) livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow-derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1(-/-) livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. CONCLUSIONS: Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Hepatocyte specific deletion of c-Met leads to the development of severe non-alcoholic steatohepatitis in mice.

BACKGROUND & AIMS: Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development. METHODS: Hepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet. RESULTS: MCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted. CONCLUSIONS: c-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8.

Differential regulation of pancreatic digestive enzymes during chronic high-fat diet-induced obesity in C57BL/6J mice.

Exocrine pancreatic digestive enzymes are essential for the digestion of dietary components and are regulated by them. Chronic excess dietary high fat (HF) consumption is a contributing factor of diet-induced obesity (DIO) and associated chronic diseases and requires adaptation by the pancreas. The aim of the present study was to investigate the effects of chronic HF diet feeding on exocrine pancreatic digestive enzyme transcript levels in DIO C57BL/6J mice. C57BL/6J mice were fed diets containing either 10 or 45% energy (E%) derived from fat for 12 weeks (n 10 mice per diet group). Pancreatic tissue and blood samples were collected at 0, 4 and 12 weeks. The expression of a panel of exocrine pancreatic digestive enzymes was analysed using quantitative RT-PCR and Western blot analysis. The HF (45 E%) diet-fed C57BL/6J mice developed obesity, hyperleptinaemia, hyperglycaemia and hyperinsulinaemia. The transcript levels of pancreatic lipase (PL), pancreatic lipase-related protein 2 (PLRP2) and pancreatic phospholipase A2 (PLA2) were initially elevated; however, they were down-regulated to basal control levels at week 12. The transcript levels of colipase were significantly affected by diet and time. The protein levels of PL and PLRP2 responded to HF diet feeding. The transcript levels of amylase and proteases were not significantly affected by diet and time. The transcript levels of specific lipases in hyperinsulinaemic, hyperleptinaemic and hyperglycaemic DIO C57BL/6J mice are down-regulated. However, these mice compensate for this by the post-transcriptional regulation of the levels of proteins that respond to dietary fat. This suggests a complex regulatory mechanism involved in the modulation of fat digestion.

Dietary heme induces acute oxidative stress, but delayed cytotoxicity and compensatory hyperproliferation in mouse colon.

Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by generating cytotoxic and oxidative stress. Recently, we found that this surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signaling. It is unknown whether this changed signaling is caused by cytotoxic stress and/or oxidative stress, as these processes were never studied separately. The aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified, humanized, control diet or the diet supplemented with 0.2 µmol heme/g. Oxidative and cytotoxic stress were measured in fecal water. Proliferation was determined by Ki67-immunohistochemistry and mucosal responses by whole-genome transcriptomics. After heme ingestion, there was an acute increase in reactive oxygen species (ROS) leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an acute antioxidant response, but no change in cell turnover. After day 4, cytotoxicity of the colonic contents was increased and this coincided with differential signaling and hyperproliferation, indicating that cytotoxicity was the causal factor. Simultaneously, several oncogenes were activated, whereas the tumor suppressor p53 was inhibited. In conclusion, luminal cytotoxicity, but not ROS, caused differential surface to crypt signaling resulting in mucosal hyperproliferation and the differential expression of oncogenes and tumor suppressor genes.

Palmitic acid follows a different metabolic pathway than oleic acid in human skeletal muscle cells; lower lipolysis rate despite an increased level of adipose triglyceride lipase.

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100µM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid ß-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

JNKs protect from cholestatic liver disease progression by modulating Apelin signalling.

Background & Aims: Cholestatic liver injury is associated with c-Jun N-terminal kinases (JNK) activation in distinct cell types. Its hepatocyte-specific function during cholestasis, however, has not yet been established. Therefore, in our present study, we investigated the role of JNK1/2 during cholestasis and dissected its hepatocyte-specific function. Methods: A cohort of patients with primary biliary cholangitis (n = 29) and primary sclerosing cholangitis (n = 37) was examined. Wild-type, hepatocyte-specific knockout mice for Jnk2 (Jnk2Δhepa) or Jnk1 and Jnk2 (Jnk1Δhepa/2Δhepa) were generated. Mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) treatment. Finally, Apelin signalling was blocked using a specific inhibitor. As an interventional approach, Jnk1/2 were silenced in wild-type mice using lipid nanoparticles for small interfering RNA delivery. Results: JNK activation was increased in liver specimens from patients with chronic cholestasis (primary biliary cholangitis and primary sclerosing cholangitis) and in livers of Mdr2-/- and BDL-treated animals. In Jnk1Δhepa/2Δhepa animals, serum transaminases increased after BDL, and liver histology demonstrated enhanced cell death, compensatory proliferation, hepatic fibrogenesis, and inflammation. Furthermore, microarray analysis revealed that hepatocytic Jnk1/2 ablation induces JNK-target genes involved in oxidative stress and Apelin signalling after BDL. Consequently, blocking Apelin signalling attenuated BDL-induced liver injury and fibrosis in Jnk1Δhepa/2Δhepa mice. Finally, we established an interventional small interfering RNA approach of selective Jnk1/2 targeting in hepatocytes in vivo, further demonstrating the essential protective role of Jnk1/2 during cholestasis. Conclusions: Jnk1 and Jnk2 work together to protect hepatocytes from cholestatic liver disease by controlling Apelin signalling. Dual modification of JNK signalling in hepatocytes is feasible, and enhancing its expression might be an attractive therapeutic approach for cholestatic liver disease. Impact and Implications: The cell-specific function of Jnk genes during cholestasis has not been explicitly explored. In this study, we showed that combined Jnk1/2, but not Jnk2 deficiency, in hepatocytes exacerbates liver damage and fibrosis by enhancing Apelin signalling, which contributes to cholestasis progression. Combined cell-specific Jnk targeting may be a new molecular strategy for treating cholestatic liver disease.

Detailed transcriptomics analysis of the effect of dietary fatty acids on gene expression in the heart.

Fatty acids comprise the primary energy source for the heart and are mainly taken up via hydrolysis of circulating triglyceride-rich lipoproteins. While most of the fatty acids entering the cardiomyocyte are oxidized, a small portion is involved in altering gene transcription to modulate cardiometabolic functions. So far, no in vivo model has been developed enabling study of the transcriptional effects of specific fatty acids in the intact heart. In the present study, mice were given a single oral dose of synthetic triglycerides composed of one single fatty acid. Hearts were collected 6 h thereafter and used for whole genome gene expression profiling. Experiments were conducted in wild-type and peroxisome proliferator-activated receptor (PPAR)α-/- mice to allow exploration of the specific contribution of PPARα. It was found that: 1) C18:3 had the most pronounced effect on cardiac gene expression. 2) The largest similarity in gene regulation was observed between C18:2 and C18:3. Large similarity was also observed between PPARα agonist Wy14643 and C22:6. 3) Many genes were regulated by one particular treatment only. Genes regulated by one particular treatment showed large functional divergence. 4) The majority of genes responding to fatty acid treatment were regulated in a PPARα-dependent manner, emphasizing the importance of PPARα in mediating transcriptional regulation by fatty acids in the heart. 5) Several genes were robustly regulated by all or many of the fatty acids studied, mostly representing well-described targets of PPARs (e.g., Acot1, Angptl4, Ucp3) but also including Zbtb16/PLZF, a transcription factor crucial for natural killer T cell function. 6) Deletion and activation of PPARα had a major effect on expression of numerous genes involved in metabolism and immunity. Our analysis demonstrates the marked impact of dietary fatty acids on gene regulation in the heart via PPARα.

In male rats with concurrent iron and (n-3) fatty acid deficiency, provision of either iron or (n-3) fatty acids alone alters monoamine metabolism and exacerbates the cognitive deficits associated with combined deficiency.

Concurrent deficiencies of iron (Fe) (ID) and (n-3) fatty acids [(n-3)FAD)] in rats can alter brain monoamine pathways and impair learning and memory. We examined whether repletion with Fe and DHA/EPA, alone and in combination, corrects the deficits in brain monoamine activity (by measuring monoamines and related gene expression) and spatial working and reference memory [by Morris water maze (MWM) testing] associated with deficiency. Using a 2 × 2 design, male rats with concurrent ID and (n-3)FAD [ID+(n-3)FAD] were fed an Fe+DHA/EPA, Fe+(n-3)FAD, ID+DHA/EPA, or ID+(n-3)FAD diet for 5 wk [postnatal d 56-91]. Biochemical measures and MWM performance after repletion were compared to age-matched control rats. The provision of Fe in combination with DHA/EPA synergistically increased Fe concentrations in the olfactory bulb (OB) (Fe x DHA/EPA interaction). Similarly, provision of DHA/EPA in combination with Fe resulted in higher brain DHA concentrations than provision of DHA alone in the frontal cortex (FC) and OB (P < 0.05). Dopamine (DA) receptor D1 was upregulated in the hippocampus of Fe+DHA/EPA rats (fold-change = 1.25; P < 0.05) and there were significant Fe x DHA/EPA interactions on serotonin (5-HT) in the OB and on the DA metabolite dihydroxyphenylacetic acid in the FC and striatum. Working memory performance was impaired in ID+DHA/EPA rats compared with controls (P < 0.05). In the reference memory task, Fe+DHA/EPA improved learning behavior, but Fe or DHA/EPA alone did not. These findings suggest that feeding either Fe or DHA/EPA alone to adult rats with both ID and (n-3)FAD affects the DA and 5-HT pathways differently than combined repletion and exacerbates the cognitive deficits associated with combined deficiency.