Evert M. van Schothorst

Meat consumption, cigarette smoking, and genetic susceptibility in the etiology of colorectal cancer: results from a Dutch prospective study

Objective: We evaluated the effect of meat consumption and cigarette smoking in combination with N-acetyltransferases 1 and 2 (NAT1 and NAT2), and glutathione S-transferase M1 (GSTM1) genotypes on colorectal cancer. Methods: From a Dutch prospective study, after 8.5 years of follow-up, data of 102 incident colorectal cancer cases and a random sample of 537 controls frequency-matched for gender and age were analyzed. Baseline information on dietary and smoking habits, as well as blood samples for DNA isolation and genotyping, were available. Results: Red meat intake increased colorectal cancer risk among men (OR 2.7; 95% CI 1.1–6.7 highest vs. lowest intake), whereas poultry and fish decreased risk among women (OR 0.5; 95% CI 0.2–1.07). Cigarette smoking for at least 16 years increased colorectal cancer risk among former smokers only (OR 2.7; 95% CI 1.0–7.4), compared to those having smoked for 15 years or less. NAT1 and NAT2 polymorphisms did not significantly modify these associations. High consumption of poultry and fish was inversely associated with colorectal cancer only in the presence of GSTM1. Conclusions: In this study meat consumption and former long-term smoking were associated with colorectal cancer. Associations of colorectal cancer with different types of meat were modified by gender and GSTM1 genotype.

Skeletal muscle mitochondrial uncoupling drives endocrine cross-talk through the induction of FGF21 as a myokine

UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in approximately fivefold elevated circulating FGF21 in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF21, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The cofactor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

Four selenoproteins, protein biosynthesis, and Wnt signalling are particularly sensitive to limited selenium intake in mouse colon

Selenium is an essential micronutrient. Its recommended daily allowance is not attained by a significant proportion of the population in many countries and its intake has been suggested to affect colorectal carcinogenesis. Therefore, microarrays were used to determine how both selenoprotein and global gene expression patterns in the mouse colon were affected by marginal selenium deficiency comparable to variations in human dietary intakes. Two groups of 12 mice each were fed a selenium-deficient (0.086 mg Se/kg) or a selenium-adequate (0.15 mg Se/kg) diet. After 6 wk, plasma selenium level, liver, and colon glutathione peroxidase (GPx) activity in the deficient group was 12, 34, and 50%, respectively, of that of the adequate group. Differential gene expression was analysed with mouse 44K whole genome microarrays. Pathway analysis by GenMAPP identified the protein biosynthesis pathway as most significantly affected, followed by inflammation, Delta-Notch and Wnt pathways. Selected gene expression changes were confirmed by quantitative real-time PCR. GPx1 and the selenoproteins W, H, and M, responded significantly to selenium intake making them candidates as biomarkers for selenium status. Thus, feeding a marginal selenium-deficient diet resulted in distinct changes in global gene expression in the mouse colon. Modulation of cancer-related pathways may contribute to the higher susceptibility to colon carcinogenesis in low selenium status.

Alterations in hepatic one-carbon metabolism and related pathways following a high-fat dietary intervention

Obesity frequently leads to insulin resistance and the development of hepatic steatosis. To characterize the molecular changes that promote hepatic steatosis, transcriptomics, proteomics, and metabolomics technologies were applied to liver samples from C57BL/6J mice obtained from two independent intervention trials. After 12 wk of high-fat feeding the animals became obese, hyperglycemic, and insulin resistant, had elevated levels of blood cholesterol and VLDL, and developed hepatic steatosis. Nutrigenomic analysis revealed alterations of key metabolites and enzyme transcript levels of hepatic one-carbon metabolism and related pathways. The hepatic oxidative capacity and the lipid milieu were significantly altered, which may play a key role in the development of insulin resistance. Additionally, high choline levels were observed after the high-fat diet. Previous studies have linked choline levels with insulin resistance and hepatic steatosis in conjunction with changes of certain metabolites and enzyme levels of one-carbon metabolism. The present results suggest that the coupling of high levels of choline and low levels of methionine plays an important role in the development of insulin resistance and liver steatosis. In conclusion, the complexities of the alterations induced by high-fat feeding are multifactorial, indicating that the interplay between several metabolic pathways is responsible for the pathological consequences.

Quercetin induces hepatic lipid omega-oxidation and lowers serum lipid levels in mice.

Elevated circulating lipid levels are known risk factors for cardiovascular diseases (CVD). In order to examine the effects of quercetin on lipid metabolism, mice received a mild-high-fat diet without (control) or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H nuclear magnetic resonance were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify possible mechanisms underlying altered circulating lipid levels. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, triglycerides (TG) were decreased with 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) were increased with 13% (p<0.01). Palmitic acid, oleic acid, and linoleic acid were all decreased by 9–15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega (ω)-oxidation. Gene expression profiling showed that quercetin increased hepatic lipid metabolism, especially ω-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450s) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up-regulated in the quercetin-fed mice. We conclude that quercetin intake increased hepatic lipid ω-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects on CVD.

Induction of lipid oxidation by polyunsaturated fatty acids of marine origin in small intestine of mice fed a high-fat diet

BackgroundDietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process.ResultsThe main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -in a second animal experiment- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon.ConclusionWe show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.

Preservation of Metabolic Flexibility in Skeletal Muscle by a Combined Use of n-3 PUFA and Rosiglitazone in Dietary Obese Mice

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil–based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Effects of a high-fat, low- versus high-glycemic index diet: retardation of insulin resistance involves adipose tissue modulation

Beneficial effects of low glycemic index (GI) diets in rodents have been studied using healthy low‐fat diets, while the effects might be different on high‐fat diets inducing progression of insulin resistance. We fed C57BL/6J male mice high‐fat low/ high‐GI (LGI/HGI) diets for 13 wk. Glucose and insulin tolerance and serum substrates, including adipokines, were measured longitudinally. The LGI group showed a significantly higher glucose tolerance from wk 2 onwards, which was supported by lower serum insulin and free fatty acids levels at 8 wk, and a tendency for lower leptin levels, while resistin levels remained similar. At 11 wk, when differences in serum resistin started to increase, differences in serum insulin were diminished. Although food intake was similar throughout the study, body weights and epididymal adipose tissue mass became significantly lower in the LGI group at necropsy. Several serum substrates and adipose tissue leptin mRNA levels, as analyzed by Q‐PCR, were, again, significantly lower, whereas adiponectin mRNA levels were higher. Taken together, an LGI high‐fat diet maintains higher glucose tolerance and insulin sensitivity via adipose tissue modulation solely because of a difference in the type of carbohydrate, supporting a nutritional approach in the fight against insulin resistance.—Van Schothorst, E. M., Bunschoten, A., Schrauwen, P., Mensink, R. P., Keijer, J. Effects of a high‐fat, low versus high glycemic index diet: retardation of insulin resistance involves adipose tissue modulation. FASEB J. 23, 1092–1101 (2009)

Supplemental Calcium Attenuates the Colitis-Related Increase in Diarrhea, Intestinal Permeability, and Extracellular Matrix Breakdown in HLA-B27 Transgenic Rats

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

Short-term, high fat feeding-induced changes in white adipose tissue gene expression are highly predictive for long-term changes.

SCOPE We aimed to evaluate the predictability of short-term (5 days) changes in epididymal white adipose tissue (eWAT) gene expression for long-term (12 weeks) changes induced by high-fat diet (HFD) feeding. METHODS AND RESULTS Mice were fed semisynthetic diets containing 10 (low-fat diet) or 40 (HFD) energy% of fat. Global gene expression in eWAT was analyzed using microarrays and confirmed by quantitative PCR. As expected, HFD feeding resulted in increased body fat accumulation and reduced glucose tolerance after 12 weeks. A total of 4678 transcripts were significantly changed by HFD after 12 weeks and 973 after 5 days, with an overlap of 764 transcripts encoding 549 genes. Of these, 79% were downregulated and 21% were upregulated by HFD, all in the same direction and highly correlated (r(2) = 0.90) between the time points. Pathway analysis showed downregulation of the main identified processes: lipid metabolism, carbohydrate metabolism, and oxidative phosphorylation. Mest (mesoderm-specific transcript) was highly upregulated, confirming its role as an early marker of fat cell expansion. CONCLUSION The high predictive value of short-term gene expression changes for long-term effects of high fat feeding is a promising step to establish robust early biomarkers that could shorten animal trials to assess health-promoting food compounds.