Acetate, Propionate and Butyrate Reduce Appetite and Fat Accumulation in Mice via Modulating Relevant Genes and Hormones

Acetate, propionate and butyrate, three of the most common short chain fatty acids (SCFAs), can be produced when some non-digestible carbohydrates enter the large intestine and undergo bacterial fermentation. This study was designed to investigate the effects of these three SCFAs on appetite regulation and lipid metabolism, and to what extent appetite contributed to the benecial inuences of SCFAs. In a 35-day study, a total of 48 C57BL/6 male mice were randomly allocated into six groups : (1) control; (2) 5% sodium acetate; (3) 5% sodium propionate; (4) 5% sodium butyrate; (5) pair fed 1; (6) pair fed 2. The results showed that sodium acetate reduced serum triglyceride, free fatty acids, glucose and interleukin (IL) 6 levels (P < 0.05), increased serum glucagon-like peptide 1 and leptin levels (P < 0.05), down-regulated the mRNA expressions of fatty acid synthase, peroxisome proliferator activated receptor and lipoprotein lipase (P < 0.05), and up-regulated the mRNA expressions of fasting induced adipose factor, nuclear respiratory factor 1, mitochondrial transcription factor A, tumor necrosis factor receptor superfamily member 9, cytochromec oxidase IV and free fatty acid receptor 2 (P < 0.05). Sodium propionate also reduced serum IL-1β level (P < 0.05), increased serum peptide YY level (P < 0.05), down-regulated the mRNA expressions of acetyl-CoA carboxylase and sterol regulatory element binding protein 1c (P < 0.05), and up-regulated the mRNA expression of transmembrane protein 26 (P < 0.05). Besides, Sodium butyrate decreased average daily feed intake (P < 0.05), down-regulated the mRNA expression of myosin heavy-chain (MyHc) (cid:0) b (P < 0.05), and up-regulated the mRNA expressions of lipase hormone-sensitive, MyHC (cid:0) a and carnitine palmitoyltransferase-1α (P < 0.05). Moreover, the metabolic benets of SCFAs were partly attributed to the reduction of feed intake. Taken together, SCFAs could reduce appetite and fat accumulation via modulating relevant genes and hormones, which might further illustrate the potential mechanisms that underlay the impacts of SCFAs on lipid homeostasis and body weight control. carboxylase; SREBP-1c sterol regulatory element 1c; peroxisome proliferator LIPE


Introduction
Obesity, one of the most severe health problems that contemporary people are faced with, has inevitably drawn our great attention. Due to the imbalance between energy intake and expenditure, a lot of complex symptoms called metabolic syndrome are likely to occur [1]. It increases the risk of metabolic diseases like type 2 diabetes and cardiovascular disease [1]. Currently, more and more studies have suggested dietary treatment as one of the most e cient strategies to control obesity level. Among them, dietary ber has been associated with suppressed appetite, reduced body weight gain, and improved postprandial glucose response [2,3]. Thus, there has been a refocus on the investigation of dietary ber and its potential mechanisms.
Dietary ber passes through the small intestine without being in uenced by digestive enzymes [4]. However, it can be catabolized by bacteria in the hindgut [5]. And the main products of bacterial intestinal fermentation are short chain fatty acids (SCFAs), with acetate, propionate and butyrate as the most abundant ones, in the approximate ratio of 60:20:20 [6]. It has been suggested that the effects of dietary ber on metabolism, to a certain degree, are mediated by SCFAs and their receptors, namely free fatty acid receptor 2 and free fatty acid receptor 3 [7]. Previous studies demonstrated that acetate could be used for de novo synthesis of lipid and propionate was classically regarded as a gluconeogenic substrate [8]. Moreover, butyrate was a main energy source for both hosts and cells [9]. Despite all of these, recent studies showed that SCFAs could act as signaling molecules to be involved in several physiological process, including reductions in insulin resistance and appetite, thus contributing to glucose homeostasis and body weight control [10,11]. Besides, propionate and butyrate activated intestinal gluconeogenesis, which was necessary for the metabolic bene ts generated by SCFAs or dietary ber [12]. Thus, more studies are needed badly to investigate this controversy and the speci c roles of acetate, propionate and butyrate, respectively.
Therefore, in this study, we determined the effects of different SCFAs on the appetite regulation and lipid metabolism of mice, and more importantly, to what extent appetite contributed to the bene cial in uences of SCFAs.

Methods And Materials
Animal, management and diet Experimental procedure and animal care were accomplished in accordance with the guide for the care and use of laboratory animals provided by the institutional animal care advisory committee for Sichuan Agricultural University. All animal protocols used in this study were approved by the animal care and use committee of Sichuan Agricultural University under permit number DKY-B20131704.
In the present study, a total of 56 C57BL/6 male mice (aged 4-week, purchased from Chengdu Dashuo Experimental Animal Co, Ltd) were randomly allocated to 7 groups (n = 8): (1) control; (2) 5% sodium acetate; (3) 5% sodium propionate; (4) 5% sodium butyrate; (5) pair fed 1; (6) pair fed 2; (7) pair fed 3. All of the mice received a high fat diet (D12492) without (control and pair fed) or with 5% (w/w) sodium acetate, propionate and butyrate, respectively. Soy oil and lard were used as fat sources for the high fat diet. Sodium acetate, propionate and butyrate were purchased from Sigma. As sodium acetate, propionate and butyrate were supposed to reduce feed intake, three pair fed groups received the same amount of high fat diet as those of sodium acetate, propionate and butyrate, respectively. All of the mice were individually caged under a 12 hour light and 12 hour dark cycle, with free access to water. The whole experiment lasted for 5 weeks.

Growth Performance
The body weight of each mice was measured every week. And the feed intake was recorded every day.
The average daily body weight gain (ADG), average daily feed intake (ADFI) and the ratio of feed to gain (F/G) were calculated based on the values mentioned above.

Slaughter And Sample Collection
For the ADFI of sodium acetate and sodium propionate was almost the same, so at the end of the experiment, control group, SCFAs groups, and two pair fed groups were sacri ced. At 8:00 on day 36, after overnight fasting and ether anesthesia, blood samples were collected by heart puncture, centrifuged at 3000 × g, and stored at -20℃. All of the mice were sacri ced according to previously described methods [13]. Epididymal fat, gastrocnemius and liver were obtained and stored at -80℃ for further analyses.

Real-time Quantitative Pcr
After reverse transcription, the mRNA levels of several relevant genes were detected by real-time quantitative PCR with SYBR Premix Ex Taq reagents (TaKaRa Biotechnology, Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) according to previously described methods [14]. All primers presented in Table 1  Resistin. The cycling conditions were: rst pre-denaturation at 95 ℃ for 30 s, then 40 cycles at 95 ℃ for 5 s, next at annealing temperature for 30 s, nally at 72 ℃ for 60 s. A melting curve analysis was also carried out to verify the purity and speci city of reactions. β-actin was chosen as the reference gene to normalize the mRNA expressions of target genes, and the relative gene expression compared to reference gene was calculated based on previously described methods [16]. All sample analyses were run repeatedly in triplicate at the same time, and on the same plate. At last, an average one was utilized to calculate the values mentioned above.

Statistical analysis
Descriptive statistic program was performed to asses whether data was normally distributed by using SPSS 20.0 (Statistical Product and Service Solutions, Inc, USA). Then one-way ANOVA test was performed to compare the difference of normally distributed data among groups, followed by Duncan's multiple-range test. Results were shown as mean and SEM. P < 0.05 was considered statistically signi cant. And, P < 0.1 was considered a tendency.

Growth performance
As shown in Table 2, sodium butyrate signi cantly reduced the ADFI of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding ADG and F/G (P > 0.05).

Serum Metabolites
According to Table 4, sodium acetate and butyrate signi cantly reduced the serum TG level of C57BL/6 mice compared with the control group (P < 0.05). Sodium acetate also signi cantly reduced the serum FFA level of C57BL/6 mice compared with the control group (P < 0.05). Besides, sodium SCFAs signi cantly reduced the serum glucose level of C57BL/6 mice compared with the control group (P < 0.05). In addition, the serum TG level of sodium butyrate group was signi cantly lower than that of pair fed 2 group (P < 0.05). The serum FFA level of sodium acetate group was signi cantly lower that that of pair fed 1 group (P < 0.05). However, no signi cant differences were observed among the six groups regarding TC, HDL-c and LDL-c (P > 0.05). Within a row, means without a common superscript differ (P < 0.05).

Serum Hormones
As shown in Table 5, sodium acetate and propionate signi cantly increased the serum GLP-1 and leptin levels of C57BL/6 mice compared with the control group (P < 0.05). Sodium propionate and butyrate also signi cantly increased the serum PYY level of C57BL/6 mice compared with the control group (P < 0.05).
In addition, the serum GLP-1 and and leptin levels of sodium acetate and propionate groups were signi cantly higher than those of pair fed 1 group (P < 0.05). The serum GLP-1 and PYY levels of sodium butyrate group were signi cantly higher than those of pair fed 2 group (P < 0.05). However, no signi cant differences were observed among the six groups regarding adiponectin, resistin, ghrelin and insulin (P > 0.05).

Serum Cytokines
According to Table 6, sodium propionate and butyrate signi cantly reduced the serum IL-1β level of C57BL/6 mice compared with the control group (P < 0.05). Sodium acetate also signi cantly reduced the serum IL-6 level of C57BL/6 mice compared with the control group (P < 0.05). In addition, the serum IL-6 level of sodium acetate group was signi cantly lower than that of pair fed 1 group (P < 0.05). However, no signi cant differences were observed among the six groups regarding IL-10 and TNF-α (P > 0.05). Within a row, means without a common superscript differ (P < 0.05).

The Mrna Expressions Of Related Genes In Epididymal Fat
As shown in Table 7  Within a row, means without a common superscript differ (P < 0.05).
According to Table 8, sodium SCFAs signi cantly up-regulated the mRNA expressions of PGC-1α, NRF-1 and Tfam in epididymal fat of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding β-F1-ATPase, COX IV and Cyt-c (P > 0.05). Within a row, means without a common superscript differ (P < 0.05).
As shown in Table 9, sodium propionate and butyrate signi cantly up-regulated the mRNA expression of Tmem 26 in epididymal fat of C57BL/6 mice compared with the control group (P < 0.05). Sodium acetate and propionate also signi cantly up-regulated the mRNA expression of CD137 in epididymal fat of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding TBX-1 (P > 0.05). a−c Within a row, means without a common superscript differ (P < 0.05).

The Mrna Expressions Of Related Genes In Gastrocnemius Muscle
According to Table 10, sodium acetate signi cantly down-regulated the mRNA expression of PPAR in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). Sodium butyrate also signi cantly up-regulated the mRNA expression of LIPE in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding FAS, ACC, SREBP-1c, LPL and CPT-1α (P > 0.05). a−b Within a row, means without a common superscript differ (P < 0.05).
As shown in Table 11, sodium SCFAs signi cantly up-regulated the mRNA expressions of PGC-1α and COX IV in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). Sodium propionate and butyrate also signi cantly up-regulated the mRNA expression of NRF-1 in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). Sodium acetate and propionate signi cantly up-regulated the mRNA expression of Tfam in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding β-F1-ATPase and Cyt-c (P > 0.05). a−c Within a row, means without a common superscript differ (P < 0.05).
According to Table 12, sodium butyrate signi cantly up-regulated the mRNA expression of MyHC a, and down-regulated the mRNA expression of MyHC b in gastrocnemius of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding MyHC and MyHC x (P > 0.05). a−b Within a row, means without a common superscript differ (P < 0.05).

The Mrna Expressions Of Related Genes In Liver
According to Table 13, sodium propionate signi cantly down-regulated the mRNA expressions of ACC and SREBP-1c in liver of C57BL/6 mice compared with the control group (P < 0.05). Sodium butyrate also signi cantly up-regulated the mRNA expression of CPT-1α in liver of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding FAS, PPAR, LIPE and LPL (P > 0.05). a−c Within a row, means without a common superscript differ (P < 0.05).
As shown in Table 14, sodium propionate and butyrate signi cantly up-regulated the mRNA expressions of PGC-1α in liver of C57BL/6 mice compared with the control group (P < 0.05). However, no signi cant differences were observed among the six groups regarding NRF-1, Tfam,β-F1-ATPase, COX IV and Cyt-c (P > 0.05). Within a row, means without a common superscript differ (P < 0.05).

The Mrna Expressions Of Ffar2 And Ffar3
According to Table 15, sodium propionate and butyrate signi cantly up-regulated the mRNA expression FFAR3 in epididymal fat of C57BL/6 mice compared with the control group (P < 0.05). Sodium SCFAs also signi cantly up-regulated the mRNA expression of FFAR2 in epididymal fat of C57BL/6 mice compared with the control group (P < 0.05). In addition, the mRNA expression of FFAR 2 of sodium SCFAs groups was signi cantly higher than that of pair fed groups (P < 0.05). However, no signi cant differences were observed among the six groups regarding FFAR2 and FFAR3 in gastrocnemius muscle and liver (P > 0.05). Within a row, means without a common superscript differ (P < 0.05).

Discussion
The prevalence of obesity has increased rapidly over last decades, and the roles of gut microorganisms and SCFAs in metabolic homeostasis have been highlighted. Previous studies showed that SCFAs could reduce appetite via the gut-brain neural circuit [17]. According to our study, sodium butyrate signi cantly reduced the ADFI of C57BL/6 mice. Besides, sodium SCFAs increased the concentrations of GLP-1, PYY and/or leptin in serum. GLP-1 and PYY are secreted by L cells, and respond closely and quickly to feed intake [18,19]. Administrations of these gut hormones resulted in enhanced satiety and reduced energy intake, which were considered as good strategies to combat obesity [20,21]. Leptin is produced primarily by white adipose tissue and involved in many physiological processes like feeding behaviour and metabolic status [22]. Leptin signalling failure was associated with hyperphagia while an infusion of it could reduce feed intake and increase energy expenditure [23,24]. Thus, sodium SCFAs reduced ADFI possibly via regulating GLP-1, PYY and leptin, with sodium acetate having the greatest impacts.
SCFAs contribute greatly to improved glucose homeostasis and insulin sensitivity [25]. They controlled body energy utilization and maintained metabolic status via FFAR2, a sensitive sensor for excessive dietary energy [26]. Previous studies also showed that SCFAs activated intestinal gluconeogenesis, which was crucial for the bene ts generated by SCFAs [12]. According to our results, we found that sodium SCFAs reduced serum TG, FFA and/or glucose levels, which were partly consistent with other studies [8,27]. Besides, reduced postprandial glucose level was often associated with increased GLP-1 and PYY levels [28,29], thus SCFAs might modulate glucose level via gut-derived hormones. More importantly, in the light of our study, SCFAs regulated these metabolic parameters mainly or partly by reduced feed intake.
Chronic low-grade in ammation is one of the key factors that result in obesity and its complications, such as non-alcoholic fatty liver disease [30]. IL-6 concentration was associated with circulating lipopolysaccharides level, which was supposed to initiate in ammation-related insulin resistance [31]. And IL-1β was correlative with G protein-coupled receptor 109A, whose signalling was involved in type 2 diabetes [32]. Our results showed that sodium SCFAs reduced the concentrations of IL-6 and/or IL-1β in serum, which indicated that SCFAs could attenuate chronic low-grade in ammation and contribute to insulin sensitivity.
SCFAs could modulate adipogenesis and lipolysis in adipose tissue, skeletal muscle and liver via several mechanisms [25]. FAS is a core enzyme that catalyzes fatty acid synthesis, and ACC as well as its product, malonyl-CoA, can act as building blocks for de novo fatty acid synthesis [33,34]. SCFAs regulated these gene expressions through activating AMP activated protein kinase (AMPK) [27]. Besides, SREBP-1c enhances the transcription of targeted genes that encode the enzymes of cholesterol biosynthesis and uptake [35]. Consistent with previous studies [36], we found that SCFAs reduced the mRNA expressions of FAS, ACC and/or SREBP-1c, which could attenuate adipogenesis and cholestrol synthesis. Moreover, suppression of Fiaf increased LPL activity in adipocytes, thereby increasing triglyceride storage [37]. Our study demonstrated that sodium SCFAs up-regulated the mRNA expression of Fiaf while down-regulated the mRNA expression of LPL, which indicated a reduction of triglyceride storage in adipose tissue. In addition, LIPE is the chief enzyme responsible for FFA mobilization and CPT-1α participates in fatty acid oxidation and catalyzes the very rst step [38,39]. According to our study, SCFAs enhanced the mRNA expressions of LIPE and/or CPT-1α, thus promoting lipolysis and fatty acid oxidation.
Impairment of mitochondrial function is associated with diabetes due to the fact that reduced ATP synthesis rate is often observed before decreased glucose tolerance [40]. PGC-1α acts as a crucial transcriptional coactivator of both nuclear and non-nuclear receptor transcription factors involved in the energy metabolism of cells, such as PPAR and NRFs [41]. Also, NRFs regulate Tfam, a nuclear factor that activates mitochondria replication and transcription [42]. Moreover, mitochondrial cytochrome c oxidase, known as complex IV, catalyzes nitrite reduction under anaerobiosis [42]. Therefore, our results found that SCFAs improved mitochondrial function by enhancing the mRNA expressions of PGC-1α, NRF-1, Tfam and/or COX IV, which further contributed to glucose regulation and body weight control. Besides, generally, muscles with a high proportion of fast-twitch ber (MyHC-IIb) are relatively poor in mitochondrial activity [43]. Our studies showed that sodium butyrate enhanced MyHC a mRNA level while reduced MyHC-IIb level in gastrocnemius, suggesting an improvement of muscle mitochondrial function.
Typically, adipocytes could be divided into two types, namely white fat cells and brown fat cells. White fat cells store energy while brown fat cells produce heat and combat obesity and diabetes [44]. Specially, brown fat could dissipate chemical energy as heat by utilizing mitochondrial contents [44]. Apart from classical brown fat, some brown-like cells called beige cells are derived from white adipose, and they are also characterized by strong antiobesity properties [45]. When animals are under cold circumstances or given chronic β-adrenergic stimulation, beige adipocytes will experience a kind of phenotypic transdifferentiation, then browning will occur morphologically and histochemically [44]. Our study found that some beige adipocyte markers, such as Tmem26 and/or CD137, were elevated by sodium SCFAs, indicating a promotion in beige adipogenesis.
FFAR 2 and FFAR 3, also called G-protein-coupled receptor 43 and 41, could be bound and activated by SCFAs. They are widely expressed in both small and large intestine [46]. Previous study demonstrated that SCFAs attenuated obesity induced by high fat diet via these receptors [15]. Our results showed that sodium SCFAs only increased the mRNA expressions of FFAR 2 and FFAR 3 in epididymal fat, but not in gastrocnemius muscle or liver, suggesting more direct or indirect in uences of SCFAs on these tissues could be investigated further in the future.

Conclusion
In summary, our study found that sodium SCFAs could suppress appetite and attenuate fat deposition via modulating related genes and hormones involved in adipogenesis, lipolysis, mitochondrial function and beige adipogenesis. More importantly, SCFAs regulated some metabolic processes mainly or partly by reduced feed intake. These all provided some new insights into the roles of SCFAs in obesity and nonalcoholic fatty liver.