Functional and molecular profiling of fasted piglets reveals decreased energy metabolic function and cell proliferation in the small intestine.

The small intestine requires energy to exert its important role in nutrient uptake and barrier function. Pigs are an important source of food and a model for humans. Young piglets and infants can suffer from periods of insufficient food intake. Whether this functionally affects the small intestinal epithelial cell (IEC) metabolic capacity and how this may be associated with an increased vulnerability to intestinal disease is unknown. We therefore performed a 48-h fasting intervention in young piglets. After feeding a standard weaning diet for 2 wk, 6-wk-old piglets (n = 16/group) were fasted for 48 h, and midjejunal IECs were collected upon euthanasia. Functional metabolism of isolated IECs was analyzed with the Seahorse XF analyzer and gene expression was assessed using RNA-sequencing. Fasting decreased the mitochondrial and glycolytic function of the IECs by 50% and 45%, respectively (P < 0.0001), signifying that overall metabolic function was decreased. The RNA-sequencing results corroborated our functional metabolic measurements, showing that particularly pathways related to mitochondrial energy production were decreased. Besides oxidative metabolic pathways, decreased cell-cycle progression pathways were most regulated in the fasted piglets, which were confirmed by 43% reduction of Ki67-stained cells (P < 0.05). Finally, the expression of barrier function genes was reduced upon fasting. In conclusion, we found that the decreased IEC energy metabolic function in response to fasting is supported by a decreased gene expression of mitochondrial pathways and is likely linked to the observed decreased intestinal cell proliferation and barrier function, providing insight into the vulnerability of piglets, and infants, to decreased food intake.NEW & NOTEWORTHY Fasting is identified as one of the underlying causes potentiating diarrhea development, both in piglets and humans. With this study, we demonstrate that fasting decreases the metabolism of intestinal epithelial cells, on a functional and transcriptional level. Transcriptional and histological data also show decreased intestinal cell proliferation. As such, fasting-induced intestinal energy shortage could contribute to intestinal dysfunction upon fasting.

Butyrate Alters Pyruvate Flux and Induces Lipid Accumulation in Cultured Colonocytes.

Butyrate is considered the primary energy source of colonocytes and has received wide attention due to its unique health benefits. Insight into the mechanistic effects of butyrate on cellular and metabolic function relies mainly on research in in-vitro-cultured cells. However, cells in culture differ from those in vivo in terms of metabolic phenotype and nutrient availability. For translation, it is therefore important to understand the impact of different nutrients on the effects of butyrate. We investigated the metabolic consequences of butyrate exposure under various culturing conditions, with a focus on the interaction between butyrate and glucose. To investigate whether the effects of butyrate were different between cells with high and low mitochondrial capacity, we cultured HT29 cells under either low- (0.5 mM) or high- (25 mM) glucose conditions. Low-glucose culturing increased the mitochondrial capacity of HT29 cells compared to high-glucose (25 mM) cultured HT29 cells. Long-term exposure to butyrate did not alter mitochondrial bioenergetics, but it decreased glycolytic function, regardless of glucose availability. In addition, both high- and low-glucose-grown HT29 cells showed increased lipid droplet accumulation following long-term butyrate exposure. Acute exposure of cultured cells (HT29 and Caco-2) to butyrate increased their oxygen consumption rate (OCR). A simultaneous decrease in extracellular acidification rate (ECAR) was observed. Furthermore, in the absence of glucose, OCR did not increase in response to butyrate. These results lead us to believe that butyrate itself was not responsible for the observed increase in OCR, but, instead, butyrate stimulated pyruvate flux into mitochondria. Indeed, blocking of the mitochondrial pyruvate carrier prevented a butyrate-induced increase in oxygen consumption. Taken together, our results indicate that butyrate itself is not oxidized in cultured cells but instead alters pyruvate flux and induces lipid accumulation.

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.

The Molecular and Physiological Effects of Protein-Derived Polyamines in the Intestine.

Consumption of a high-protein diet increases protein entry into the colon. Colonic microbiota can ferment proteins, which results in the production of protein fermentation end-products, like polyamines. This review describes the effects of polyamines on biochemical, cellular and physiological processes, with a focus on the colon. Polyamines (mainly spermine, spermidine, putrescine and cadaverine) are involved in the regulation of protein translation and gene transcription. In this, the spermidine-derived hypusination modification of EIF5A plays an important role. In addition, polyamines regulate metabolic functions. Through hypusination of EIF5A, polyamines also regulate translation of mitochondrial proteins, thereby increasing their expression. They can also induce mitophagy through various pathways, which helps to remove damaged organelles and improves cell survival. In addition, polyamines increase mitochondrial substrate oxidation by increasing mitochondrial Ca2+-levels. Putrescine can even serve as an energy source for enterocytes in the small intestine. By regulating the formation of the mitochondrial permeability transition pore, polyamines help maintain mitochondrial membrane integrity. However, their catabolism may also reduce metabolic functions by depleting intracellular acetyl-CoA levels, or through production of toxic by-products. Lastly, polyamines support gut physiology, by supporting barrier function, inducing gut maturation and increasing longevity. Polyamines thus play many roles, and their impact is strongly tissue- and dose-dependent. However, whether diet-derived increases in colonic luminal polyamine levels also impact intestinal physiology has not been resolved yet.