Detailed Analysis of Prebiotic Fructo- and Galacto-Oligosaccharides in the Human Small Intestine.

Galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) are food ingredients that improve human health, but their degradation throughout the human small intestine is not well understood. We studied the breakdown kinetics of FOS and GOS in the intestines of seven healthy Dutch adults. Subjects were equipped with a catheter in the distal ileum or proximal colon and consumed 5 g of chicory-derived FOS (degree of polymerization (DP) DP2-10), and 5 g of GOS (DP2-6). Postprandially, intestinal content was frequently collected until 350 min and analyzed for mono-, di-, and oligosaccharides. FOS and GOS had recoveries of 96 ± 25% and 76 ± 28%, respectively. FOS DP ≥ 2 and GOS DP ≥ 3 abundances in the distal small intestine or proximal colon matched the consumed doses, while GOS dimers (DP2) had lower recoveries, namely 22.8 ± 11.1% for ß-D-gal-(1↔1)-α-D-glc+ß-D-gal-(1↔1)-ß-D-glc, 19.3 ± 19.1% for ß-D-gal-(1 → 2)-D-glc+ß-D-gal-(1 → 3)-D-glc, 43.7 ± 24.6% for ß-D-gal-(1 → 6)-D-gal, and 68.0 ± 38.5% for ß-D-gal-(1 → 4)-D-gal. Lactose was still present in the distal small intestine of all of the participants. To conclude, FOS DP ≥ 2 and GOS DP ≥ 3 were not degraded in the small intestine of healthy adults, while most prebiotic GOS DP2 was hydrolyzed in a structure-dependent manner. We provide evidence on the resistances of GOS with specific ß-linkages in the human intestine, supporting the development of GOS prebiotics that resist small intestine digestion.

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.

Intraintestinal fermentation of fructo- and galacto-oligosaccharides and the fate of short-chain fatty acids in humans.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

Age and Diet Modulate the Insulin-Sensitizing Effects of Exercise: A Tracer-Based Oral Glucose Tolerance Test.

Diet modulates the development of insulin resistance during aging. This includes tissue-specific alterations in insulin signaling and mitochondrial function, which ultimately affect glucose homeostasis. Exercise stimulates glucose clearance and mitochondrial lipid oxidation and also enhances insulin sensitivity (IS). It is not well known how exercise interacts with age and diet in the development of insulin resistance. To investigate this, oral glucose tolerance tests with tracers were conducted in mice ranging from 4 to 21 months of age, fed a low-fat diet (LFD) or high-fat diet (HFD) with or without life-long voluntary access to a running wheel (RW). We developed a computational model to derive glucose fluxes, which were commensurate with independent values from steady-state tracer infusions. Values for an IS index derived for peripheral tissues (IS-P) and one for the liver (IS-L) were steeply decreased by aging and an HFD. This preceded the age-dependent decline in the mitochondrial capacity to oxidize lipids. In young animals fed an LFD, RW access enhanced the IS-P concomitantly with the muscle ß-oxidation capacity. Surprisingly, RW access completely prevented the age-dependent IS-L decrease; however this only occurred in animals fed an LFD. Therefore, this study indicates that endurance exercise can improve the age-dependent decline in organ-specific IS if paired with a healthy diet. ARTICLE HIGHLIGHTS: Exercise is a known strategy to improve insulin sensitivity (IS), whereas aging and a lipid-rich diet decrease IS. Using a tracer-based oral glucose tolerance test, we investigated how exercise, age, and diet interact in the development of tissue-specific insulin resistance. Exercise (voluntary access to a running wheel) mainly improved IS in animals fed a low-fat diet. In these animals, exercise improved peripheral IS only at young age but fully prevented the age-dependent decline of hepatic IS. The prevention of age-dependent decline in IS by exercise is tissue-specific and blunted by a lipid-rich diet.

Age and diet modulate the insulin-sensitizing effects of exercise: a tracer-based oral glucose tolerance test.

Diet modulates the development of insulin resistance during aging. This includes tissue-specific alterations in insulin signaling and mitochondrial function, which ultimately affect glucose homeostasis. Exercise stimulates glucose clearance, mitochondrial lipid oxidation and enhances insulin sensitivity. It is not well known how exercise interacts with age and diet in the development of insulin resistance. To investigate this, oral glucose tolerance tests (OGTT) with a tracer were conducted in mice ranging from 4 to 21 months of age, fed a low- (LFD) or high-fat diet (HFD), with or without life-long voluntary access to a running wheel (RW). We developed a computational model to derive glucose fluxes, which were commensurate with independent values from steady-state tracer infusions. Both insulin sensitivity indices derived for peripheral tissues and liver (IS-P and IS-L, respectively) were steeply decreased by aging and a HFD. This preceded the age-dependent decline in the mitochondrial capacity to oxidize lipids. In LFD young animals, RW access enhanced the IS-P concomitantly with the muscle ß- oxidation capacity. Surprisingly, RW access completely prevented the age-dependent IS-L decrease, but only in LFD animals. This study indicates, therefore, that endurance exercise can improve the age-dependent decline in organ-specific IS mostly in the context of a healthy diet.

Differential effects of oleate on vascular endothelial and liver sinusoidal endothelial cells reveal its toxic features in vitro.

Several fatty acids, in particular saturated fatty acids like palmitic acid, cause lipotoxicity in the context of non-alcoholic fatty liver disease . Unsaturated fatty acids (e.g. oleic acid) protect against lipotoxicity in hepatocytes. However, the effect of oleic acid on other liver cell types, in particular liver sinusoidal endothelial cells (LSECs), is unknown. Human umbilical vein endothelial cells (HUVECs) are often used as a substitute for LSECs, however, because of the unique phenotype of LSECs, HUVECs cannot represent the same biological features as LSECs. In this study, we investigate the effects of oleate and palmitate (the sodium salts of oleic acid and palmitic acid) on primary rat LSECs in comparison to their effects on HUVECs. Oleate induces necrotic cell death in LSECs, but not in HUVECs. Necrotic cell death of LSECs can be prevented by supplementation of 2-stearoylglycerol, which promotes cellular triglyceride (TG) synthesis. Repressing TG synthesis, by knocking down DGAT1 renders HUVECs sensitive to oleate-induced necrotic death. Mechanistically, oleate causes a sharp drop of intracellular ATP level and impairs mitochondrial respiration in LSECs. The combination of oleate and palmitate reverses the toxic effect of oleate in both LSECs and HUVECs. These results indicate that oleate is toxic and its toxicity can be attenuated by stimulating TG synthesis. The toxicity of oleate is characterized by mitochondrial dysfunction and necrotic cell death. Moreover, HUVECs are not suitable as a substitute model for LSECs.

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 mitochondrial 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.

Dynamic Methods for Childhood Hypoglycemia Phenotyping: A Narrative Review.

Hypoglycemia results from an imbalance between glucose entering the blood compartment and glucose demand, caused by a defect in the mechanisms regulating postprandial glucose homeostasis. Hypoglycemia represents one of the most common metabolic emergencies in childhood, potentially leading to serious neurologic sequelae, including death. Therefore, appropriate investigation of its specific etiology is paramount to provide adequate diagnosis, specific therapy and prevent its recurrence. In the absence of critical samples for biochemical studies, etiological assessment of children with hypoglycemia may include dynamic methods, such as in vivo functional tests, and continuous glucose monitoring. By providing detailed information on actual glucose fluxes in vivo, proof-of-concept studies have illustrated the potential (clinical) application of dynamic stable isotope techniques to define biochemical and clinical phenotypes of inherited metabolic diseases associated with hypoglycemia. According to the textbooks, individuals with glycogen storage disease type I (GSD I) display the most severe hypoglycemia/fasting intolerance. In this review, three dynamic methods are discussed which may be considered during both diagnostic work-up and monitoring of children with hypoglycemia: 1) functional in vivo tests; 2) in vivo metabolic profiling by continuous glucose monitoring (CGM); 3) stable isotope techniques. Future applications and benefits of dynamic methods in children with hypoglycemia are also discussed.

The Effects of Butyrate on Induced Metabolic-Associated Fatty Liver Disease in Precision-Cut Liver Slices.

Metabolic-associated fatty liver disease (MAFLD) starts with hepatic triglyceride accumulation (steatosis) and can progress to more severe stages such as non-alcoholic steatohepatitis (NASH) and even cirrhosis. Butyrate, and butyrate-producing bacteria, have been suggested to reduce liver steatosis directly and systemically by increasing liver ß-oxidation. This study aimed to examine the influence of butyrate directly on the liver in an ex vivo induced MAFLD model. To maintain essential intercellular interactions, precision-cut liver slices (PCLSs) were used. These PCLSs were prepared from male C57BL/6J mice and cultured in varying concentrations of fructose, insulin, palmitic acid and oleic acid, to mimic metabolic syndrome. Dose-dependent triglyceride accumulation was measured after 24 and 48 h of incubation with the different medium compositions. PCLSs viability, as indicated by ATP content, was not affected by medium composition or the butyrate concentration used. Under induced steatotic conditions, butyrate did not prevent triglyceride accumulation. Moreover, it lowered the expression of genes encoding for fatty acid oxidation and only increased C4 related carnitines, which indicate butyrate oxidation. Nevertheless, butyrate lowered the fibrotic response of PCLSs, as shown by reduced gene expression of fibronectin, alpha-smooth muscle actin and osteopontin, and protein levels of type I collagen. These results suggest that in the liver, butyrate alone does not increase lipid ß-oxidation directly but might aid in the prevention of MAFLD progression to NASH and cirrhosis.

Bistability in fatty-acid oxidation resulting from substrate inhibition.

In this study we demonstrated through analytic considerations and numerical studies that the mitochondrial fatty-acid ß-oxidation can exhibit bistable-hysteresis behavior. In an experimentally validated computational model we identified a specific region in the parameter space in which two distinct stable and one unstable steady state could be attained with different fluxes. The two stable states were referred to as low-flux (disease) and high-flux (healthy) state. By a modular kinetic approach we traced the origin and causes of the bistability back to the distributive kinetics and the conservation of CoA, in particular in the last rounds of the ß-oxidation. We then extended the model to investigate various interventions that may confer health benefits by activating the pathway, including (i) activation of the last enzyme MCKAT via its endogenous regulator p46-SHC protein, (ii) addition of a thioesterase (an acyl-CoA hydrolysing enzyme) as a safety valve, and (iii) concomitant activation of a number of upstream and downstream enzymes by short-chain fatty-acids (SCFA), metabolites that are produced from nutritional fibers in the gut. A high concentration of SCFAs, thioesterase activity, and inhibition of the p46Shc protein led to a disappearance of the bistability, leaving only the high-flux state. A better understanding of the switch behavior of the mitochondrial fatty-acid oxidation process between a low- and a high-flux state may lead to dietary and pharmacological intervention in the treatment or prevention of obesity and or non-alcoholic fatty-liver disease.

Age-related susceptibility to insulin resistance arises from a combination of CPT1B decline and lipid overload.

BACKGROUND: The skeletal muscle plays a central role in glucose homeostasis through the uptake of glucose from the extracellular medium in response to insulin. A number of factors are known to disrupt the normal response to insulin leading to the emergence of insulin resistance (IR). Advanced age and a high-fat diet are factors that increase the susceptibility to IR, with lipid accumulation in the skeletal muscle being a key driver of this phenomenon. It is debated, however, whether lipid accumulation arises due to dietary lipid overload or from a decline of mitochondrial function. To gain insights into the interplay of diet and age in the flexibility of muscle lipid and glucose handling, we combined lipidomics, proteomics, mitochondrial function analysis and computational modelling to investigate young and aged mice on a low- or high-fat diet (HFD). RESULTS: As expected, aged mice were more susceptible to IR when given a HFD than young mice. The HFD induced intramuscular lipid accumulation specifically in aged mice, including C18:0-containing ceramides and diacylglycerols. This was reflected by the mitochondrial ß-oxidation capacity, which was upregulated by the HFD in young, but not in old mice. Conspicuously, most ß-oxidation proteins were upregulated by the HFD in both groups, but carnitine palmitoyltransferase 1B (CPT1B) declined in aged animals. Computational modelling traced the flux control mostly to CPT1B, suggesting a CPT1B-driven loss of flexibility to the HFD with age. Finally, in old animals, glycolytic protein levels were reduced and less flexible to the diet. CONCLUSION: We conclude that intramuscular lipid accumulation and decreased insulin sensitivity are not due to age-related mitochondrial dysfunction or nutritional overload alone, but rather to their combined effects. Moreover, we identify CPT1B as a potential target to counteract age-dependent intramuscular lipid accumulation and thereby IR.

Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry.

13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography-mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.

A toolbox for the comprehensive analysis of small volume human intestinal samples that can be used with gastrointestinal sampling capsules.

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.

Impaired Very-Low-Density Lipoprotein catabolism links hypoglycemia to hypertriglyceridemia in Glycogen Storage Disease type Ia.

Prevention of hypertriglyceridemia is one of the biomedical targets in Glycogen Storage Disease type Ia (GSD Ia) patients, yet it is unclear how hypoglycemia links to plasma triglyceride (TG) levels. We analyzed whole-body TG metabolism in normoglycemic (fed) and hypoglycemic (fasted) hepatocyte-specific glucose-6-phosphatase deficient (L-G6pc-/- ) mice. De novo fatty acid synthesis contributed substantially to hepatic TG accumulation in normoglycemic L-G6pc-/- mice. In hypoglycemic conditions, enhanced adipose tissue lipolysis was the main driver of liver steatosis, supported by elevated free fatty acid concentrations in GSD Ia mice and GSD Ia patients. Plasma very-low-density lipoprotein (VLDL) levels were increased in GSD Ia patients and in normoglycemic L-G6pc-/- mice, and further elevated in hypoglycemic L-G6pc-/- mice. VLDL-TG secretion rates were doubled in normo- and hypoglycemic L-G6pc-/- mice, while VLDL-TG catabolism was selectively inhibited in hypoglycemic L-G6pc-/- mice. In conclusion, fasting-induced hypoglycemia in L-G6pc-/- mice promotes adipose tissue lipolysis and arrests VLDL catabolism. This mechanism likely contributes to aggravated liver steatosis and dyslipidemia in GSD Ia patients with poor glycemic control and may explain clinical heterogeneity in hypertriglyceridemia between GSD Ia patients.

Enantiomer-specific pharmacokinetics of D,L-3-hydroxybutyrate: Implications for the treatment of multiple acyl-CoA dehydrogenase deficiency.

D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased Cmax and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for Cmax : 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (Cmax : 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB.

Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet.

Mouse models are frequently used to study mechanisms of human diseases. Recently, we observed a spontaneous bimodal variation in liver weight in C57BL/6JOlaHsd mice fed a semisynthetic diet. We now characterized the spontaneous variation in liver weight and its relationship with parameters of hepatic lipid and bile acid (BA) metabolism. In male C57BL/6JOlaHsd mice fed AIN-93G from birth to postnatal day (PN)70, we measured plasma BA, lipids, Very low-density lipoprotein (VLDL)-triglyceride (TG) secretion, and hepatic mRNA expression patterns. Mice were sacrificed at PN21, PN42, PN63 and PN70. Liver weight distribution was bimodal at PN70. Mice could be subdivided into two nonoverlapping groups based on liver weight: 0.6 SD 0.1 g (approximately one-third of mice, small liver; SL), and 1.0 SD 0.1 g (normal liver; NL; p<0.05). Liver histology showed a higher steatosis grade, inflammation score, more mitotic figures and more fibrosis in the SL versus the NL group. Plasma BA concentration was 14-fold higher in SL (p<0.001). VLDL-TG secretion rate was lower in SL mice, both absolutely (-66%, p<0.001) and upon correction for liver weight (-44%, p<0.001). Mice that would later have the SL-phenotype showed lower food efficiency ratios during PN21-28, suggesting the cause of the SL phenotype is present at weaning (PN21). Our data show that approximately one-third of C57BL/6JOlaHsd mice fed semisynthetic diet develop spontaneous liver disease with aberrant histology and parameters of hepatic lipid, bile acid and lipoprotein metabolism. Study designs involving this mouse strain on semisynthetic diets need to take the SL phenotype into account. Plasma lipids may serve as markers for the identification of the SL phenotype.

Transcriptome analysis suggests a compensatory role of the cofactors coenzyme A and NAD+ in medium-chain acyl-CoA dehydrogenase knockout mice.

During fasting, mitochondrial fatty-acid ß-oxidation (mFAO) is essential for the generation of glucose by the liver. Children with a loss-of-function deficiency in the mFAO enzyme medium-chain acyl-Coenzyme A dehydrogenase (MCAD) are at serious risk of life-threatening low blood glucose levels during fasting in combination with intercurrent disease. However, a subset of these children remains asymptomatic throughout life. In MCAD-deficient (MCAD-KO) mice, glucose levels are similar to those of wild-type (WT) mice, even during fasting. We investigated if metabolic adaptations in the liver may underlie the robustness of this KO mouse. WT and KO mice were given a high- or low-fat diet and subsequently fasted. We analyzed histology, mitochondrial function, targeted mitochondrial proteomics, and transcriptome in liver tissue. Loss of MCAD led to a decreased capacity to oxidize octanoyl-CoA. This was not compensated for by altered protein levels of the short- and long-chain isoenzymes SCAD and LCAD. In the transcriptome, we identified subtle adaptations in the expression of genes encoding enzymes catalyzing CoA- and NAD(P)(H)-involving reactions and of genes involved in detoxification mechanisms. We discuss how these processes may contribute to robustness in MCAD-KO mice and potentially also in asymptomatic human subjects with a complete loss of MCAD activity.

Running wheel access fails to resolve impaired sustainable health in mice feeding a high fat sucrose diet.

Diet and physical activity are thought to affect sustainable metabolic health and survival. To improve understanding, we studied survival of mice feeding a low-fat (LF) or high-saturated fat/high sugar (HFS) diet, each with or without free running wheel (RW) access. Additionally several endocrine and metabolic health indices were assessed at 6, 12, 18 and 24 months of age. As expected, HFS feeding left-shifted survival curve of mice compared to LF feeding, and this was associated with increased energy intake and increased (visceral/total) adiposity, liver triglycerides, and increased plasma cholesterol, corticosterone, HOMA-IR, and lowered adiponectin levels. Several of these health parameters improved (transiently) by RW access in HFS and LF fed mice (i.e., HOMA-IR, plasma corticosterone), others however deteriorated (transiently) by RW access only in HFS-fed mice (i.e., body adiposity, plasma resistin, and free cholesterol levels). Apart from these multiple and sometimes diverging health effects of RW access, RW access did not affect survival curves. Important to note, voluntary RW activity declined with age, but this effect was most pronounced in the HFS fed mice. These results thus challenge the hypothesis that voluntary wheel running can counteract HFS-induced deterioration of survival and metabolic health.

A large pooled analysis refines gene expression-based molecular subclasses in cutaneous melanoma.

This study aimed to establish the number of expression-based molecular subclasses in cutaneous melanoma, identify their dominant biological pathways and evaluate their clinical relevance. To this end, consensus clustering was performed separately on two independent datasets (n = 405 and n = 473) composed of publicly available cutaneous melanoma expression profiles from previous studies. Four expression-based molecular subclasses were identified and labelled 'Oxidative phosphorylation', 'Oestrogen response/p53-pathway', 'Immune' and 'Cell cycle', based on their dominantly expressed biological pathways determined by gene set enrichment analysis. Multivariate survival analysis revealed shorter overall survival in the 'Oxidative phosphorylation' subclass compared to the other subclasses. This was validated in a third independent dataset (n = 214). Finally, in a pooled cohort of 76 patients treated with anti-PD-1 therapy a trend towards a difference in response rates between subclasses was observed ('Immune' subclass: 65% responders, 'Oxidative Phosphorylation' subclass: 60% responders, other subclasses: <50% responders). These findings support the stratification of cutaneous melanoma in four expression-based molecular subclasses.

One- vs two-phase extraction: re-evaluation of sample preparation procedures for untargeted lipidomics in plasma samples.

Lipidomics is a rapidly developing field in modern biomedical research. While LC-MS systems are able to detect most of the known lipid classes in a biological matrix, there is no single technique able to extract all of them simultaneously. In comparison with two-phase extractions, one-phase extraction systems are of particular interest, since they decrease the complexity of the experimental procedure. By using an untargeted lipidomics approach, we explored the differences/similarities between the most commonly used two-phase extraction systems (Folch, Bligh and Dyer, and MTBE) and one of the more recently introduced one-phase extraction systems for lipid analysis based on the MMC solvent mixture (MeOH/MTBE/CHCl3). The four extraction methods were evaluated and thoroughly compared against a pooled extract that qualitatively and quantitatively represents the average of the combined extractions. Our results show that the lipid profile obtained with the MMC system displayed the highest similarity to the pooled extract, indicating that it was most representative of the lipidome in the original sample. Furthermore, it showed better extraction efficiencies for moderate and highly apolar lipid species in comparison with the Folch, Bligh and Dyer, and MTBE extraction systems. Finally, the technical simplicity of the MMC procedure makes this solvent system highly suitable for automated, untargeted lipidomics analysis.