Propionic acid counteracts the inflammation of human subcutaneous adipose tissue: a new avenue for drug development
Adipose tissue is a primary site of obesity-induced inflammation, which has been emerging as an important contributor to obesity associated disorders. The factors influencing adipose tissue-induced inflammation and the resulting pathophysiological events remain poorly understood. However, dietary fiber consumptions appear to be protective. Short-chain fatty acids such as propionic acid (PA) are the principal products of the dietary fiber fermentation by microbiota. Therefore, we aim to investigate the influence of PA on inflammation, lipogenesis and glucose uptake markers from human subcutaneous adipose tissue (SAT). We showed that the treatment of SAT with PA resulted in a significant downregulation of inflammatory parameters (e.g. TNF-α and IP-10) and macrophage markers (e.g. CD163 and MMP-9). The expression levels of PA receptors (i.e. G protein coupled receptor-41 and -43) in human primary adipocytes were very low in comparison with SAT and macrophages. Upon PA treatment, no anti-inflammatory effect was observed in human adipocytes. PA significantly upregulated the expression of lipoprotein lipase (LPL), sterol regulatory-element-binding protein-1c (SREBP-1c) and glucose transporter 4 (GLUT-4), which are associated with lipogenesis and glucose uptake. We also showed that the observed anti-inflammatory effects of PA on SAT were partly mediated by Gi/o protein coupled receptor. Our data suggests that PA anti-inflammatory effects on SAT are mediated partly via Gi/o proteins, leading to the improved expression of factors associated with lipogenesis and glucose uptake. These responses appeared to be not mediated by adipocytes; but most probably by macrophages. The current study provides new knowledge, which can be used as a potential new avenue for drug development in preventing obesity-related inflammation and metabolic disorders in future.
KeywordsPropionic acid Subcutaneous adipose tissue Inflammation Gi/o proteins coupled receptors IP-10 and TNF-alpha, macrophage
Obesity has reached epidemic proportions and is still escalating at an alarming rate worldwide. World Health Organization reported that population living on earth is approximately 7.77.7 billion of which more than 1.9 billion adults (~39%) with overweight and over 600 million with obese (~13%) . In Palestine the prevalence of obesity has been shown to be approximately 4 times among women (49%) and 2 times among men (30%) higher than the worldwide observed prevalence .
Obesity is associated with chronic activation of low-grade inflammation , which is implicated in the pathogenesis of obesity-associated diseases including insulin resistance, type-2 diabetes (T2D) [4, 5] and cardiovascular disease [6, 7]. The etiology of obesity and low-grade inflammation is complex and involves intrinsic and extrinsic factors. Recently, it has been shown that specific members of microbiota in humans, in particular Firmicutes, are associated with obesity and its associated afflictions [8, 9, 10, 11].
Furthermore, the colonization of germ-free mice with microbiota derived from obese mice results in significantly greater adiposity than colonization with microbiota from lean mice . Conversely, prebiotic diets such as fructans  are associated with general better health, including the decrease in body weight, fat mass and the severity of T2D [14, 15, 16]. The factors that influence the composition and metabolism of intestinal microbiota and obesity and its related inflammation and pathophysiology remain, however, obscure at best.
Fermentation of dietary fiber/ resistant starch by colonic microbiota is a primary source for the production of short-chain fatty acids (SCFAs), in particular acetic, butyric and propionic acids. It has been demonstrated that SCFAs influence the physiology of humans such as inhibition of inflammation, protection from cancer and promotion of satiety [17, 18, 19, 20]. Recently it has been shown that mice deficient in G protein–coupled receptor 43 (GPR43) have exacerbated and poorly resolving inflammation in animal models of arthritis, allergic airway inflammation and colitis . Germ-free mice model of arthritis has also showed increased inflammation and a much slower resolution of inflammation when compared to conventionally raised mice. GPCR43 is a receptor of SCFAs and consequently providing acetate in the drinking water has reduced inflammation in these mice. Therefore, one could envisage that SCFAs, including PA, may constitute the elusive link between the host and microbiota.
Obesity triggers inflammation in Adipose tissue (AT), which in turn implicated in pathophysiological events such as T2D. AT is also primary organ involved inobesity [20, 22, 23, 24, 25]. In the current study, we determined the anti-inflammatory effect of PA on SAT and adipocytes .
Gentamycin, glucose and PA were purchased from Sigma. M199 media was purchased from Invitrogen. Preadipocytes and their media were purchased from PromoCell. Cluster of differentiation 16A (CD16A), Cluster of differentiation 31 (CD31), matrix metalloproteinase 9 (MMP-9), GPCR41 and GPCR43 primers were purchased from Applied Biosystems.
Human adipose tissue (AT) and cell culture
AT explants were obtained from human subjects who underwent surgery for disorders. None of the human subjects had diabetes and the age average was 48, and Body Mass Index average was 28 kg/m2. This study was approved by the An-Najah National University, Institutional Review Board (IRB) (approves, monitors, and research involving humans) Committee and informed written consent was obtained from all subjects. AT culture was performed as described previously [26, 27] with slight modifications. After the last washing step, tissue explants were incubated for 24 h with or without 3 mM PA. AT explants were pre-incubated with pertussis toxin (PTX) (100 ng/mL) for 2 h. Subsequently, AT explants were treated with PA (3 mM) for 24 h. All tissues were snap frozen in liquid nitrogen and stored at −80 °C until RNA isolation. Secretome (Media fraction collected from cultured tissues) was stored at −80 °C meant for ELISA and multi ELISA measurements. With respect to human preadipocytes, they were cultured and differentiated into adipocytes according to PromoCell instructions.
The sequences of the primers
Primer sequence (5′➔3′)
GGT GAA GGT CGG AGT CAA CG
ACC ATG TAG TTG AGG TCA ATG AAG G
CGC CTG GTC ACC AGG GCT GC
GCT GTG GCT GGT TTC TCC AA
CCC ATA GCC TCC GCA ACA TA
CGA GCA ACT TCA TCA TTG GCA TGG GTT
TGG AGA TGT GGA CCA GCT AGT G
CAG AGA GTC GAT GAA GAG ATG AAT G
CTC CCA CGA GCG CT
GGA TTG CAC TTT CGA AGA CAT G
AGC ATA GGG TGG GTC AAA TAG G
CAG CTT ATC AAC AAC CAA GAC AGT GAC TTC CC
TGC AGA AAA CCC CAC AAA AAG
CAA GGA TCC CGA CTG CAA TAA
AAC AGG TCG CTC ATG CCG TCA GTC A
The analysis of chemokines and cytokines proteins in secreted fraction
Secreted chemokines and cytokines were measured in culture media by multiplex-ELISA assay according to the manufacturer’s instructions (Bio-Rad).
Comparison between two groups was performed by two-sided paired Student’s t test, while the rest were analyzed via either one- or two-ways ANOVA. Results were considered to be statistically significant at P < 0.05.
Propionic Acid (PA) effect on inflammation in human subcutaneous adipose tissue (SAT)
Propionic Acid (PA) inhibited the expression of adipose tissue macrophage (ATM) markers
The comparison of ATM markers expression levels between omental adipose tissue (OAT), subcutaneous adipose tissue (SAT) and adipocytes
Comparison of GPCR 41 and 43 expression levels between SAT, adipocytes and macrophages
Propionic acid positively affects key metabolic genes in human adipose tissue explants
Gi/o-protein coupled receptors role in the anti-inflammatory effect of PA
SCFAs are mainly metabolites of gut microbiota fermentation of escaped undigested food. A numerous of studies has been shown that SCFAs inhibit inflammation with focus on butyrate and to a lesser extent on acetate and PA, . In our previous study we have shown that PA has anti-inflammatory properties in OAT [26, 27]. The anti-inflammatory properties of PA were confirmed by showing that the mice remain lean even when put on a high-fat diet in mice overexpressing PA receptor (GPR43) specifically in OAT. Moreover, these mice exhibit a decrease of macrophages and hallmarks of inflammatory . However, PA effect on human SAT has not (to our knowledge) been investigated. Furthermore, it is known that OAT and SAT are different  with respect to inflammation. To this end, these studies showed that OAT releases more pro-inflammatory and less anti-inflammatory components than SAT [30, 31]. These all promoted us to investigate the effect of PA on inflammation in human SAT, lipogenesis and glucose uptake.
Accumulating evidence shows that AT is considered as one of the major sites of low grade inflammation in obese subjects [23, 24, 25], which contributes to obesity associated energy metabolic disorders such as type 2 diabetes (T2D) and cardiovascular diseases (CVDs) [4, 5, 6, 7]. In the present study we have shown that PA inhibits the secretion of certain pro-inflammatory markers in human SAT. This is in agreement to PA effect on OAT , but to a lesser extent. Notably, this is in accordance with our earlier finding that PA inhibits resistin (a pro-inflammatory parameter) protein release and mRNA expression in both OAT and SAT . Although It is well suggested that obesity is associated with increased infiltration of macrophages into human AT , which are identified via specific ATM markers, AT also contains itself macrophages. In the current study, it has been shown that PA has a great impact on ATM markers supporting the fact that PA has anti-inflammatory properties.
Most of the examined pro-inflammatory markers were not detected in the secretome of adipocytes, while the detected ones were not decreased in response to PA treatment. This may be due to a very low expression level of PA receptors GPCR-41 and -43 in adipocytes as compared to macrophages and SAT. Taking these all together, it implies that non-adipocyte cells, most likely macrophages, respond to the anti-inflammatory effect of PA.
The anti-inflammatory properties of PA are associated with other major metabolic pathways in AT, namely lipogenesis and glucose metabolism. We have found that both LPL and GLUT4 expression was up-regulated by PA. Expression of LPL and GLUT4 is known to be regulated by SREBP1c [33, 34]. Indeed, we have found an increased SREBP1c expression upon PA stimulation, suggesting that SREBP1c is responsible for the increased expression of LPL and GLUT4. These data also suggest that PA has not only an anti-inflammatory effect but also PA could have an anabolic effect similar to insulin, inducing two important metabolic pathways that are also stimulated by insulin. This is in accordance with the response of OAT to PA in our previous studies [16, 26, 35] and to the other studies where SCFAs have been found to increase insulin sensitivity .
The expression of the two receptors, GPRC41 and GPRC43, of PA in human AT (36), suggesting that the effects of PA on the AT might be mediated by these receptors. It has been shown that there is a unique Gi/o coupling for GPCR41, but a dual coupling exists through Gi/o and Gq proteins for GPCR43, . In this study we have found that TNF-α response to PA is via Gi/o protein, suggesting that anti-inflammatory effects of PA mediated by G protein coupled receptors, while IP-10, CD163 and MMP-9 response was not mediated via Gi/o pathway. These results are in line with our earlier study , suggesting that both SAT and OAT employ a similar pathway (i.e. Gi/o protein coupled receptor pathway). However, our findings do not exclude the role of other pathways, such as Gq proteins and PPARγ. Therefore, further investigations are needed to dissect the underlying molecular pathway(s).
Notably, PA exhibits anti-inflammatory effects on human SAT, which is accompanied by improved expression of parameters associated with lipogenesis and glucose uptake. We also demonstrate that anti-inflammatory effects are parttially mediated by Gi/o proteins and most probably via macrophages. This finding is similar to our results what we have observed in OAT in our previous study [26, 37, 38]. The present study provides a new paradigm to understand the relationship between the microbiota and AT’s physiology and its potential power in preventing obesity-related inflammation and energy metabolic disorders. The PA anti-inflammatory properties on macrophages, the role of GPCR41 and GPCR43 receptors and other potential underlying mechanisms such as PPARγ remains to be elucidated.
These studies help us to understand better pathways involved in inflammation in ATs and macrophages for drug research and development.
Compliance with ethical standards
Conflict of interest
The authors have declared that no conflict of interest exists.
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