Roel J. Vonk

Biological effects of propionic acid in humans; metabolism, potential applications and underlying mechanisms

Undigested food is fermented in the colon by the microbiota and gives rise to various microbial metabolites. Short-chain fatty acids (SCFA), including acetic, propionic and butyric acid, are the principal metabolites produced. However, most of the literature focuses on butyrate and to a lesser extent on acetate; consequently, potential effects of propionic acid (PA) on physiology and pathology have long been underestimated. It has been demonstrated that PA lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus increased production of PA by the microbiota might be considered beneficial in the context of prevention of obesity and diabetes type 2. The molecular mechanisms by which PA may exert this plethora of physiological effects are slowly being elucidated and include intestinal cyclooxygenase enzyme, the G-protein coupled receptors 41 and 43 and activation of the peroxisome proliferator-activated receptor γ, in turn inhibiting the sentinel transcription factor NF-κB and thus increasing the threshold for inflammatory responses in general. Taken together, PA emerges as a major mediator in the link between nutrition, gut microbiota and physiology.

Characterization of the Human Visceral Adipose Tissue Secretome

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48–114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing l-[13C6,15N2]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with ≥99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.

Human Primary Adipocytes Exhibit Immune Cell Function: Adipocytes Prime Inflammation Independent of Macrophages

Background Obesity promotes inflammation in adipose tissue (AT) and this is implicated in pathophysiological complications such as insulin resistance, type 2 diabetes and cardiovascular disease. Although based on the classical hypothesis, necrotic AT adipocytes (ATA) in obese state activate AT macrophages (ATM) that then lead to a sustained chronic inflammation in AT, the link between human adipocytes and the source of inflammation in AT has not been in-depth and systematically studied. So we decided as a new hypothesis to investigate human primary adipocytes alone to see whether they are able to prime inflammation in AT. Methods and Results Using mRNA expression, human preadipocytes and adipocytes express the cytokines/chemokines and their receptors, MHC II molecule genes and 14 acute phase reactants including C-reactive protein. Using multiplex ELISA revealed the expression of 50 cytokine/chemokine proteins by human adipocytes. Upon lipopolysaccharide stimulation, most of these adipocyte-associated cytokines/chemokines and immune cell modulating receptors were up-regulated and a few down-regulated such as (ICAM-1, VCAM-1, MCP-1, IP-10, IL-6, IL-8, TNF-α and TNF-β highly up-regulated and IL-2, IL-7, IL-10, IL-13 and VEGF down-regulated. In migration assay, human adipocyte-derived chemokines attracted significantly more CD4+ T cells than controls and the number of migrated CD4+ cells was doubled after treating the adipocytes with LPS. Neutralizing MCP-1 effect produced by adipocytes reduced CD4+ migration by approximately 30%. Conclusion Human adipocytes express many cytokines/chemokines that are biologically functional. They are able to induce inflammation and activate CD4+ cells independent of macrophages. This suggests that the primary event in the sequence leading to chronic inflammation in AT is metabolic dysfunction in adipocytes, followed by production of immunological mediators by these adipocytes, which is then exacerbated by activated ATM, activation and recruitment of immune cells. This study provides novel knowledge about the prime of inflammation in human obese adipose tissue, opening a new avenue of investigations towards obesity-associated type 2 diabetes.

EFFECTS OF SUPPLEMENTAL DIETARY CALCIUM ON THE INTESTINAL ASSOCIATION OF CALCIUM, PHOSPHATE, AND BILE-ACIDS

It has been suggested that supplemental dietary calcium decreases hyperproliferation of colonic epithelial cells because calcium precipitates and thus inactivates luminal bile acids. Therefore, 12 healthy men were studied before and after dietary calcium supplementation (35.5 mmol/day) to quantify intestinal associations of calcium, phosphate, and bile acids. The supplemental dietary calcium was almost completely (95%) recovered, mainly in feces. Calcium increased the fecal excretion of both phosphate (31%) and bile acids (53%) and decreased the ratio of dihydroxy to trihydroxy bile acids in duodenal bile almost twofold. In vitro studies showed that precipitation of glycodeoxycholic acid was caused by the formation of insoluble calcium phosphate. Water-soluble and calcium-associated amounts of phosphate and bile acids in feces were measured by resolubilization studies, using the calcium chelator ethylenediaminetetraacetate. In both the control and calcium periods, significant amounts of phosphate (80% and 90%) and bile acids (33% and 50%) were calcium-associated. Moreover, the calcium-induced increments in fecal phosphate and bile acids were completely calcium-associated. Calcium decreased the amount of water-soluble phosphate but not of bile acids. These results indicate that supplemental calcium stimulates formation of insoluble calcium phosphate in the intestinal lumen and thus increases binding of luminal bile acids.

Regulation of adipokine production in human adipose tissue by propionic acid

Eur J Clin Invest 2010; 40 (5): 401–407

Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose-intolerant subjects

Aims:  Colonic metabolism of lactose may play a role in lactose intolerance. We investigated whether a 2‐week supplementation of Bifidobacterium longum (in capsules) and a yogurt enriched with Bifidobacterium animalis could modify the composition and metabolic activities of the colonic microbiota in 11 Chinese lactose‐intolerant subjects.

Localization of the Wilson's disease protein in human liver.

BACKGROUND & AIMS Wilsons disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilsons disease protein in hepatic copper handling, its localization in human liver was investigated. METHODS By use of a specific antibody, localization of the Wilsons disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. RESULTS The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. CONCLUSIONS These results show that in human liver the Wilsons disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

DIFFERENCES IN PROPIONATE-INDUCED INHIBITION OF CHOLESTEROL AND TRIACYLGLYCEROL SYNTHESIS BETWEEN HUMAN AND RAT HEPATOCYTES IN PRIMARY CULTURE

Propionate is a short-chain fatty acid formed in the colon and supposedly involved in the cholesterol-lowering effect of soluble fibre. To explore the underlying mechanism(s) of this fibre action, we have used human hepatocytes in primary culture to study the effects of propionate on hepatic lipid synthesis. Initial experiments with mevalonate and mevinolin, a competitive inhibitor of hydroxymethylglutaryl (HMG)-CoA reductase (EC 1.1.1.88) were performed to evaluate basic regulatory mechanisms in these cells; results were compared with those obtained with rat hepatocytes. Incubation for 24 h with mevalonate caused a similar, concentration-dependent inhibition of [14C]acetate incorporation into cholesterol in human and rat hepatocytes. Likewise, mevinolin (100 mumol/l) inhibited the formation of cholesterol from radiolabelled acetate by about 80% in cells from both species. Propionate inhibited cholesterol as well as triacylglycerol synthesis from [14C]acetate with a similar concentration-dependency in rat hepatocytes. Fifty percent inhibition was obtained at a propionate concentration of only 0.1 mmol/l. This propionate-induced inhibition was not affected by a 100-fold excess of unlabelled acetate. Human hepatocytes were much less susceptible in this respect: propionate concentrations of 10-20 mmol/l were required to obtain similar inhibitory effects in these cells, i.e. values greatly exceeding reported portal propionate concentrations in humans. The results suggest the existence of differences in the regulation of hepatic cholesterol (and triacylglycerol) synthesis between human and rat liver cells. These results do not support the hypothesis that the fibre-induced decrease in plasma cholesterol concentration in man is mediated by a direct effect of propionate on hepatic cholesterol synthesis.

Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects

Eur J Clin Invest 2012; 42 (4): 357–364

Factors related to colonic fermentation of nondigestible carbohydrates of a previous evening meal increase tissue glucose uptake and moderate glucose-associated inflammation

BACKGROUND Evening meals that are rich in nondigestible carbohydrates have been shown to lower postprandial glucose concentrations after ingestion of high-glycemic-index breakfasts. This phenomenon is linked to colonic fermentation of nondigestible carbohydrates, but the underlying mechanism is not fully elucidated. OBJECTIVE We examined the way in which glucose kinetics and related factors change after breakfast as a result of colonic fermentation. DESIGN In a crossover design, 10 healthy men ingested as an evening meal white wheat bread (WB) or cooked barley kernels (BA) that were rich in nondigestible carbohydrates. In the morning after intake of 50 g (13)C-enriched glucose, the dual-isotope technique was applied to determine glucose kinetics. Plasma insulin, free fatty acid, interleukin-6, tumor necrosis factor-alpha, and short-chain fatty acid concentrations and breath-hydrogen excretion were measured. RESULTS The plasma glucose response after the glucose drink was 29% lower after the BA evening meal (P = 0.019). The insulin response was the same, whereas mean (+/-SEM) tissue glucose uptake was 30% higher (20.2 +/- 1.9 compared with 15.5 +/- 1.8 mL/2 h; P = 0.016) after the BA evening meal, which indicated higher peripheral insulin sensitivity (P = 0.001). The 4-h mean postprandial interleukin-6 (19.7 +/- 5.1 compared with 5.1 +/- 0.7 pg/mL; P = 0.024) and tumor necrosis factor-alpha (7.8 +/- 2.1 compared with 5.3 +/- 1.6 pg/mL; P = 0.008) concentrations after the glucose drink were higher after the WB evening meal. Butyrate concentrations (P = 0.041) and hydrogen excretion (P = 0.005) were higher in the morning after the BA evening meal. CONCLUSION In healthy subjects, factors related to colonic fermentation of nondigestible carbohydrates increase peripheral insulin sensitivity and moderate glucose-associated inflammation.