Effect of Hyperglycemia on Gene Expression during Early Organogenesis in Mice

BACKGROUND: Cardiovascular and neural malformations are common sequels of diabetic pregnancies, but the underlying molecular mechanisms remain unknown. We hypothesized that maternal hyperglycemia would affect the embryos most shortly after the glucose-sensitive time window at embryonic day (ED) 7.5 in mice. METHODS: Mice were made diabetic with streptozotocin, treated with slow-release insulin implants and mated. Pregnancy aggravated hyperglycemia. Gene expression profiles were determined in ED8.5 and ED9.5 embryos from diabetic and control mice using Serial Analysis of Gene Expression and deep sequencing. RESULTS: Maternal hyperglycemia induced differential regulation of 1,024 and 2,148 unique functional genes on ED8.5 and ED9.5, respectively, mostly in downward direction. Pathway analysis showed that ED8.5 embryos suffered mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylation (all P ≤ E-5). A query of the Mouse Genome Database showed that 20-25% of the differentially expressed genes were caused by cardiovascular and/or neural malformations, if deficient. Despite high glucose levels in embryos with maternal hyperglycemia and a ~150-fold higher rate of ATP production from glycolysis than from oxidative phosphorylation on ED9.5, ATP production from both glycolysis and oxidative phosphorylation was reduced to ~70% of controls, implying a shortage of energy production in hyperglycemic embryos. CONCLUSION: Maternal hyperglycemia suppressed cell proliferation during gastrulation and cytoskeletal remodeling during early organogenesis. 20-25% of the genes that were differentially regulated by hyperglycemia were associated with relevant congenital malformations. Unexpectedly, maternal hyperglycemia also endangered the energy supply of the embryo by suppressing its glycolytic capacity

Towards functional effects of polyphenols : modulation of energy metabolism revealed

A diet rich in fruits and vegetables contains high levels of polyphenols (up to 1 gram per day). Epidemiological studies suggest that a high dietary intake of selected polyphenols can be protective against development of cardiovascular heart diseases in humans. In addition, mechanistic studies demonstrate that polyphenols possess beneficial properties ininvitro and animal model systems. Due to the possible beneficial health effects of polyphenols, they are currently being sold extensively as food supplements. However, the basis for most of the health claims attributed to polyphenols in food supplements is often very small. Our objective was to elucidate relevant mechanisms of action of selected polyphenols. We studied the tissue distribution and in vivo physiological effects of quercetin (a polyphenol abundant in the human diet) after chronic dietary exposure, followed by in vitro elucidation of possible biological mechanisms. We revealed lungs as novel tissue target of quercetin and demonstrated that dietary quercetin alters fatty acid catabolism pathways in rats. In addition, dietary quercetin lowered tumor incidence in the colon of rats in a model of colon carcinogenesis. Furthermore, a major in vivo metabolite of quercetin, quercetin 3-O-glucuronide, opposed the effect of quercetin aglycone on SIRT1 activation in vitro , whereas quercetin 3-O-glucuronide attenuated glucose utilization in cultured adipocytes in a similar fashion as quercetin aglycone. Although we used high dietary dosages of quercetin and further studies should elucidate physiological effects of a normal dietary intake of polyphenols, the experiments described in this thesis point to a possible beneficial effect of dietary polyphenols. However, as long as the molecular mechanisms in humans are unknown and the risk of increasing dietary intakes of polyphenols via food supplements is not thoroughly investigated, there is no scientific justification for supplementing the diet with large amounts of polyphenols. Nevertheless, our approach successfully identified modulation of energy metabolism by polyphenols as an important process involved in mediating the possible health effects associated with dietary polyphenol intake

In vivo assessment of mitochondrial capacity using NIRS in locomotor muscles of young and elderly males with similar physical activity levels

Mitochondrial capacity is pivotal to skeletal muscle function and is suggested to decline with age. However, there is large heterogeneity in current data, possibly due to effect modifiers such as physical activity, sex and muscle group. Yet, few studies have compared multiple muscle groups in different age groups with comparable physical activity levels. Here, we newly used near-infrared spectroscopy (NIRS) to characterise mitochondrial capacity in three different locomotor muscles in young (19–25 year) and older (65–71 year), healthy males with similar physical activity levels. Mitochondrial capacity and reperfusion after arterial occlusion was measured in the vastus lateralis (VL), the gastrocnemius (GA) and the tibialis anterior (TA). Physical activity was verified using accelerometry and was not different between the age groups (404.3 ± 214.9 vs 494.9 ± 187.0 activity kcal per day, p = 0.16). Mitochondrial capacity was significantly lower in older males in the GA and VL, but not in the TA (p = 0.048, p = 0.036 and p = 0.64, respectively). Reperfusion rate was not significantly different for the GA (p = 0.55), but was significantly faster in the TA and VL in the young group compared to the older group (p = 0.0094 and p = 0.039, respectively). In conclusion, we identified distinct modes of mitochondrial ageing in different locomotor muscles in a young and older population with similar physical activity patterns. Furthermore, we show that NIRS is suitable for relatively easy application in ageing research and can reveal novel insights into mitochondrial functioning with age

Functional metabolic capacity of pig colonocytes is differentially modulated by fermentable fibre and poorly digestible protein

The intestine is a highly metabolic organ that relies on energy production within the intestinal cells to sustain its functions. In the colon, intestinal cell metabolic function could be affected positively and negatively by microbiota-derived metabolites. Protein fermentation metabolites are known to negatively impact intestinal metabolic function, while fibre fermentation metabolites are generally thought beneficial. We aimed to investigate whether proteins of different digestibility in the absence and presence of fibres impact the energy metabolism of colonocytes, with potentially adverse health effects. We fed 32, 9-week-old boars one of four experimental diets for 14 days in a 2 × 2 factorial arrangement. Whey and collagen were added as a well and a poorly digestible protein source, respectively, and fibre was either included at 5% or 23%. We examined the effects of the diets on the flux of fermentation metabolites in colon digesta and assessed the impact of the diets on functional metabolic capacity of isolated colonocytes using the Seahorse XF analyzer. Feeding the poorly digestible protein source collagen indeed increased nitrogen flow into the colon by 135% compared to the well-digestible whey-protein source. Feeding high fermentable fibre increased colonic fluxes of both fibre-derived metabolites acetate, propionate, butyrate and caproate, but also increased flux of protein-derived metabolites ammonia, isobutyrate, isovalerate, valerate and isocaproate. To analyse the impact of the diets and the induced differential metabolic composition of the intestinal lumen on functional metabolic capacity of the intestine, we used extracellular flux analysis on freshly isolated pig colonocytes. Colonocytes isolated from high fermentable fibre-fed pigs in the whey-protein diet, but not in the collagen-protein diet, had a reduced mitochondrial capacity, as indicated by a 35% reduction of maximal respiration (interaction P < 0.05) and a 20% reduction of spare respiratory capacity (interaction P < 0.05). Colonocytes from high fermentable fibre-fed pigs had a 37% decreased glycolytic activity compared to the colonocytes isolated from the low fermentable fibre-fed pigs (P < 0.001). This indicated that different diets, and in particular different protein sources and fibre levels, differentially affect colonic epithelial cell metabolism in pigs. Especially, high fermentable fibre lowered both colonocyte mitochondrial and glycolytic metabolism, indicating that high-fibre intake in pigs could lower colonocyte energetic status. Because the metabolic capacity of colonocytes is tightly linked with their functionality, assessment of intestinal cell metabolic capacity may be a valuable tool for future research

SIRT1/PGC1a-dependent increase in oxidative phosphorylation supports chemotherapy resistance of colon cancer

PURPOSE: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms.EXPERIMENTAL DESIGN: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy.RESULTS: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy.CONCLUSIONS: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable

The white adipose tissue transcriptional response to withdrawal of vitamin B3

Distinct markers for early, mild vitamin B3 deficiency are lacking. To identify these, we examined the molecular responses of white adipose tissue to vitamin B3 withdrawal. We performed a dietary intervention in male C57Bl/6JRcc mice. A diet with a low but adequate level of tryptophan without nicotinamide riboside (NR) was compared to the same diet with NR at the recommended vitamin B3 (30 mg NR per kg diet). Physiological and circulating parameters were determined and global transcriptomics, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were done. We observed a decreased insulin sensitivity and a shift from carbohydrate to fatty acid oxidation. This was consistent with molecular changes in eWAT, where we observed an altered MEK/ERK signalling, a lowering of glucose utilization markers and an increase in makers of fatty acid catabolism, which may be related to the consistent reduction of mitochondrial OXPHOS Complex I (mRNAs and protein). The synthesis pathway of tetrahydropteridine (BH4), an essential cofactor for neurotransmitter synthesis, was found to be increased. Based on our results, we propose the technically validated downregulation of Anp32a, Tnk2 and the upregulation of Mapk1, Map2k1, Mthfs, Mthfsl and Qdpr as a WAT transcriptional signature marker for mild vitamin B3 deficienc

The white adipose tissue transcriptional response to withdrawal of vitamin B3

Distinct markers for early, mild vitamin B3 deficiency are lacking. To identify these, we examined the molecular responses of white adipose tissue to vitamin B3 withdrawal. We performed a dietary intervention in male C57Bl/6JRcc mice. A diet with a low but adequate level of tryptophan without nicotinamide riboside (NR) was compared to the same diet with NR at the recommended vitamin B3 (30 mg NR per kg diet). Physiological and circulating parameters were determined and global transcriptomics, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were done. We observed a decreased insulin sensitivity and a shift from carbohydrate to fatty acid oxidation. This was consistent with molecular changes in eWAT, where we observed an altered MEK/ERK signalling, a lowering of glucose utilization markers and an increase in makers of fatty acid catabolism, which may be related to the consistent reduction of mitochondrial OXPHOS Complex I (mRNAs and protein). The synthesis pathway of tetrahydropteridine (BH4), an essential cofactor for neurotransmitter synthesis, was found to be increased. Based on our results, we propose the technically validated downregulation of Anp32a, Tnk2 and the upregulation of Mapk1, Map2k1, Mthfs, Mthfsl and Qdpr as a WAT transcriptional signature marker for mild vitamin B3 deficienc

Tissue Distribution of Quercetin in Pigs after Long-Term Dietary Supplementation

Although the flavonol quercetin is intensively investigated, our knowledge about its bioavailability and possible target organs is far from being complete. The aim of this study was to check the potential of quercetin to accumulate in various tissues after long-term dietary treatment compared with a single treatment with flavonol. Pigs ingested either a single dose of quercetin aglycone (25 mg/kg body weight; Expt. 1) or received the flavonol twice a day at the same dose mixed into their regular meals (i.e 50 mg·kg¿1·d¿1) for 4 wk (Expt. 2). In both experiments, we took plasma and tissue samples 90 min after the final meal and analyzed them using HPLC. Additionally, the specific activity of the enzyme ß-glucuronidase was measured in selected tissues. Higher flavonol concentrations than in plasma were found in only the liver (Expt. 1) or the intestinal wall and kidneys (Expt. 2). All tissues except blood plasma contained a variable amount of deconjugated quercetin in the range of 30¿100% of total flavonols. However, the specific ß-glucuronidase activity was not correlated with the proportions of deconjugated flavonols in the various tissues. Long-term dietary intake of the flavonol did not lead to a greater accumulation in any tissue compared with the single treatment. Flavonol concentrations only exceeded the plasma concentration within organs involved in its metabolism and excretion, including liver, small intestine, and kidneys

The white adipose tissue transcriptional response to withdrawal of vitamin B3

Distinct markers for early, mild vitamin B3 deficiency are lacking. To identify these, we examined the molecular responses of white adipose tissue to vitamin B3 withdrawal. We performed a dietary intervention in male C57Bl/6JRcc mice. A diet with a low but adequate level of tryptophan without nicotinamide riboside (NR) was compared to the same diet with NR at the recommended vitamin B3 (30 mg NR per kg diet). Physiological and circulating parameters were determined and global transcriptomics, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were done. We observed a decreased insulin sensitivity and a shift from carbohydrate to fatty acid oxidation. This was consistent with molecular changes in eWAT, where we observed an altered MEK/ERK signalling, a lowering of glucose utilization markers and an increase in makers of fatty acid catabolism, which may be related to the consistent reduction of mitochondrial OXPHOS Complex I (mRNAs and protein). The synthesis pathway of tetrahydropteridine (BH4), an essential cofactor for neurotransmitter synthesis, was found to be increased. Based on our results, we propose the technically validated downregulation of Anp32a, Tnk2 and the upregulation of Mapk1, Map2k1, Mthfs, Mthfsl and Qdpr as a WAT transcriptional signature marker for mild vitamin B3 deficienc