Butyrate oxidation attenuates the butyrate-induced improvement of insulin sensitivity in myotubes

Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short -chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mito-chondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate -induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism.Diabetes mellitus: pathophysiological changes and therap

The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner

All cells and organisms exhibit stress-coping mechanisms to ensure survival. Cytoplasmic protein-RNA assemblies termed stress granules are increasingly recognized to promote cellular survival under stress. Thus, they might represent tumor vulnerabilities that are currently poorly explored. The translation-inhibitory eIF2α kinases are established as main drivers of stress granule assembly. Using a systems approach, we identify the translation enhancers PI3K and MAPK/p38 as pro-stress-granule-kinases. They act through the metabolic master regulator mammalian target of rapamycin complex 1 (mTORC1) to promote stress granule assembly. When highly active, PI3K is the main driver of stress granules; however, the impact of p38 becomes apparent as PI3K activity declines. PI3K and p38 thus act in a hierarchical manner to drive mTORC1 activity and stress granule assembly. Of note, this signaling hierarchy is also present in human breast cancer tissue. Importantly, only the recognition of the PI3K-p38 hierarchy under stress enabled the discovery of p38’s role in stress granule formation. In summary, we assign a new pro-survival function to the key oncogenic kinases PI3K and p38, as they hierarchically promote stress granule formation

T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice

Atherosclerosis is a chronic inflammatory disease driven by hypercholesterolemia. During aging, T cells accumulate cholesterol, potentially affecting inflammation. However, the effect of cholesterol efflux pathways mediated by ATP-binding cassette A1 and G1 (ABCA1/ABCG1) on T cell-dependent age-related inflammation and atherosclerosis remains poorly understood. In this study, we generate mice with T cell-specific Abca1/Abcg1-deficiency on the low-density-lipoprotein-receptor deficient (Ldlr-/-) background. T cell Abca1/Abcg1-deficiency decreases blood, lymph node, and splenic T cells, and increases T cell activation and apoptosis. T cell Abca1/Abcg1-deficiency induces a premature T cell aging phenotype in middle-aged (12-13 months) Ldlr-/- mice, reflected by upregulation of senescence markers. Despite T cell senescence and enhanced T cell activation, T cell Abca1/Abcg1-deficiency decreases atherosclerosis and aortic inflammation in middle-aged Ldlr-/- mice, accompanied by decreased T cells in atherosclerotic plaques. We attribute these effects to T cell apoptosis downstream of T cell activation, compromising T cell functionality. Collectively, we show that T cell cholesterol efflux pathways suppress T cell apoptosis and senescence, and induce atherosclerosis in middle-aged Ldlr-/- mice

Rebalancing of mitochondrial homeostasis through an NAD+-SIRT1 pathway preserves intestinal barrier function in severe malnutrition.

BACKGROUND: The intestine of children with severe malnutrition (SM) shows structural and functional changes that are linked to increased infection and mortality. SM dysregulates the tryptophan-kynurenine pathway, which may impact processes such as SIRT1- and mTORC1-mediated autophagy and mitochondrial homeostasis. Using a mouse and organoid model of SM, we studied the repercussions of these dysregulations on malnutrition enteropathy and the protective capacity of maintaining autophagy activity and mitochondrial health. METHODS: SM was induced through feeding male weanling C57BL/6 mice a low protein diet (LPD) for 14-days. Mice were either treated with the NAD +-precursor, nicotinamide; an mTORC1-inhibitor, rapamycin; a SIRT1-activator, resveratrol; or SIRT1-inhibitor, EX-527. Malnutrition enteropathy was induced in enteric organoids through amino-acid deprivation. Features of and pathways to malnutrition enteropathy were examined, including paracellular permeability, nutrient absorption, and autophagic, mitochondrial, and reactive-oxygen-species (ROS) abnormalities. FINDINGS: LPD-feeding and ensuing low-tryptophan availability led to villus atrophy, nutrient malabsorption, and intestinal barrier dysfunction. In LPD-fed mice, nicotinamide-supplementation was linked to SIRT1-mediated activation of mitophagy, which reduced damaged mitochondria, and improved intestinal barrier function. Inhibition of mTORC1 reduced intestinal barrier dysfunction and nutrient malabsorption. Findings were validated and extended using an organoid model, demonstrating that resolution of mitochondrial ROS resolved barrier dysfunction. INTERPRETATION: Malnutrition enteropathy arises from a dysregulation of the SIRT1 and mTORC1 pathways, leading to disrupted autophagy, mitochondrial homeostasis, and ROS. Whether nicotinamide-supplementation in children with SM could ameliorate malnutrition enteropathy should be explored in clinical trials. FUNDING: This work was supported by the Bill and Melinda Gates Foundation, the Sickkids Research Institute, the Canadian Institutes of Health Research, and the University Medical Center Groningen

Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites

The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the present study, precision-cut intestinal slices (PCIS) prepared from the jejunum of 18 human donors were used as an ex vivo model to investigate whether DCF intestinal metabolites are responsible for its intestinal toxicity in man. PCIS were incubated with a concentration range of DCF (0-600 A mu M) up to 24 h. DCF (a parts per thousand yen400 A mu M) caused direct toxicity to the intestine as demonstrated by ATP depletion, morphological damage, caspase 3 activation, and lactate dehydrogenase leakage. Three main metabolites produced by PCIS (4'-hydroxy DCF, 5-hydroxy DCF, and DCF acyl glucuronide) were detected by HPLC. Protein adducts were detected by immunohistochemical staining and showed correlation with the intestinal metabolites. DCF induced similar toxicity to each of the samples regardless of the variation in metabolism among them. Less metabolites were produced by slices incubated with 400 A mu M DCF than with 100 A mu M DCF. The addition of the metabolic inhibitors such as ketoconazole, cimetidine, or borneol decreased the metabolite formation but increased the toxicity. The results suggest that DCF can induce intestinal toxicity in human PCIS directly at therapeutically relevant concentrations, independent of the reactive metabolites 4'-OH DCF, 5-OH DCF, or diclofenac acylglucuronide produced by the liver or formed in the intestine

Consequences of Mrp2 deficiency for diclofenac toxicity in the rat intestine ex vivo

The non-steroidal anti-inflammatory drug diclofenac (DCF) has a high prevalence of intestinal side effects in humans and rats. It has been reported that Mrp2 transporter deficient rats (Mrp2) are more resistant to DCF induced intestinal toxicity. This was explained in vivo by impaired Mrp2-dependent biliary transport of DCF-acylglucuronide (DAG), leading to decreased intestinal exposure to DAG and DCF. However, it is not known to what extent adaptive changes in the Mrp2 intestine itself influence its sensitivity to DCF toxicity without the influence of liver metabolites. To investigate this, DCF toxicity and disposition were studied ex vivo by precision-cut intestinal slices and Ussing chamber using intestines from wild type(WT) and Mrp2 rats. The results show that adaptive changes due to Mrp2 deficiency concerning Mrp2, Mrp3 and BCRP gene expression, GSH content and DAG formation were different between liver and intestine. Furthermore, Mrp2 intestine was intrinsically more resistant to DCF toxicity than its WT counterpart ex vivo. This can at least partly be explained by a reduced DCF uptake by the Mrp2 intestine, but isnot related to the other adaptive changes in the intestine. The extrapolation of this data to humans with MRP2 deficiency is uncertain due to species differences in activity and regulation of transporters

Organoids as a model to study intestinal and liver dysfunction in severe malnutrition

Hospitalized children with severe malnutrition face high mortality rates and often suffer from hepatic and intestinal dysfunction, with negative impacts on their survival. New treatments cannot be developed without understanding the underlying pathophysiology. We have established and characterized translational organoid models of severe malnutrition of the liver and the intestine. In these models, amino acid starvation recapitulates the expected organ-specific functional changes (e.g., hepatic steatosis, barrier dysfunction) accompanied by reduced mitochondrial and peroxisomal proteins, and altered intestinal tight junction proteins. Re-supplementation of amino acids or pharmacological interventions with rapamycin or fenofibrate lead to partial recovery. Restoration of protein levels aligned with signs of improved peroxisomal function in both organoids, and increased mitochondrial proteins and tight junction protein claudin-3 in intestinal organoids. We present two organoid models as novel tools to gain mechanistic insights and to act as a testing platform for potential treatments for intestinal and hepatic dysfunction in severe malnutrition