Antiobesity Effect of Lacticaseibacillus paracasei LM-141 on High-Fat Diet-Induced Rats through Alleviation of Inflammation and Insulin Resistance

In this study, we set out to evaluate the antiobesity activities of our newly isolated Lacticaseibacillus paracasei LM-141 (LPLM141) using a high-fat diet (HFD)-fed rat model. Male Sprague–Dawley rats were fed with a HFD with or without low-dosage (2 × 107 CFU/day per rat) or high-dosage (2 × 109 CFU/day per rat) LPLM141 for 14 weeks. The results showed that administration of LPLM141 significantly decreased body weight gain, liver weight, adipose tissue weight, and epididymal white adipocyte size increased by HFD feeding. The abnormal serum lipid profile induced by HFD feeding was normalized by administration of LPLM141. The enhanced chronic low-grade inflammation in HFD-fed rats was reduced by LPLM141 supplementation, as reflected by decreased serum lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) levels, reduced macrophage infiltration in adipose tissue, and increased serum adiponectin concentration. In addition, the elevations of proinflammatory cytokine genes and suppression of PPAR-γ mRNA in adipose tissues of rats fed with a HFD were markedly reversed by LPLM141 administration. Oral administration of LPLM141 induced browning of epididymal white adipose tissue (eWAT) and activation of interscapular brown adipose tissue (iBAT) in rats fed with HFD. Consumption of LPLM141 exhibited a significant amelioration in insulin resistance, which were mechanistically caused by downregulation of the serum leptin level and upregulation of hepatic IRS-1 and p-Akt protein expressions, in HFD treated rats. LPLM141 consumption significantly decreased hepatic lipogenic gene expressions and preserved liver function stimulated by HFD treatment. Administration of LPLM141 obviously mitigated hepatic steatosis observed in HFD feeding rats. Our current findings shed light on LPLM141 supplementation that exhibited an antiobesity effect in HFD-fed rats by alleviating inflammation and insulin resistance, which further highlighted the potential of utilizing LPLM141 as a preventive/therapeutic probiotic agent for obesity.


Introduction
Metabolic syndrome is a cluster of metabolic disorders including hyperglycemia, hyperinsulinemia, and hyperlipidemia, resulting from metabolic dysregulation [1]. Tese metabolic disorders are considered to be critical contributors to obesity-related diseases, such as type 2 diabetes, insulin resistance, and fatty liver disease [2]. Obesity is a complicated disease characterized by accumulation of lipids in metabolic tissues, mainly adipose tissue and the liver, and is a serious public health issue in the whole world in view of its morbidity and mortality [3]. According to the report from the World Health Organization [4], worldwide obesity has nearly tripled since 1975 and approximately 39% of adults are now overweight and 13% are obese in 2016. Terefore, it is imperative to study the available strategy that can assist with preventing and treating obesity.
Over several decades, the habitual consumption toward high fat and/or high glucose of western-type diets signifcantly increases the prevalence of obesity [5]. More specifcally, accumulating evidences demonstrate that the excess accumulation of body fat frequently causes the increase of chronic low-grade infammation in obesity [6]. In addition, previous studies also suggested that the chronic low-grade infammation in obesity can increase macrophage infltration and proinfammatory adipokine secretion, including interleukin (IL)-1, -6 and tumor necrosis factor (TNF)-α by adipose tissues, which interfere with the insulinsignaling pathway in related tissues and ultimately lead to the development of insulin resistance and the subsequent progression to type 2 diabetes [7,8]. Tus, the persistent chronic low-grade infammation might be one of the key points for the development of obesity and insulin resistance, which can be regarded as the target for the screening of antiobesity supplementations.
More and more studies indicate that the intestinal microbiota play a crucial role in the development of obesity [9,10]. Gut microbiota have been reported to regulate energy metabolic balance and infammatory status by acting on intestinal integrity [11,12]. Te impairment of gut integrity causes upregulation of the systemic endotoxin level and further enhances chronic low-grade infammation, thereby promoting the development of obesity. Tese results raise the possibility that the intentional manipulation of the community structure of gut microbiota may be a potential strategy to treat obesity. As an essential component of gut microbiota, probiotics gradually emerge as benefcial microbes to human health through various mechanisms including modulation of the community structure of gut microbiota, modifcation of microenvironment with the gut, increase of the gut epithelial barrier function, balancing of energy homeostasis, and regulation of the host immune response [13]. A large body of evidences has demonstrated that the best way for controlling the fora balance in intestine is achieved by intake of probiotics, which is thought to be efective in treating obesity [14,15]. Probiotics are defned as viable nonpathogenic microorganisms that confer health benefts to the host when ingested in adequate amount as food ingredients. Among probiotics, Lacticaseibacillus spp. and Bifdobacterium spp. are the most relevant in ameliorating obesity and improving metabolic parameters [16]. Nevertheless, not all discovered probiotics possess healthpromoting or antiobesogenic efects, some results indicate no health benefts or even obesogenic efects associated with probiotics [17][18][19]. Tis leads to the existence of speciesspecifc efects with diferent mechanistic actions of probiotics on obesity or other metabolic disorders' improvement. Terefore, it is necessary and imperative to carefully screen out the probiotic strains with higher efcacy in obesity management.
Recently, we isolated several strains of Lactobacillus spp., including Lacticaseibacillus rhamnosus, Lacticaseibacillus plantarum, Lacticaseibacillus paracasei, Lacticaseibacillus casei, and Lactobacillus delbrueckii (subsp. bulgaricus) from Taiwanese kimchi, breast milk, newborn feces, and Tempeh (a kind of Indonesian fermented soy food). Tese isolated Lactobacillus strains were then identifed by 16S rRNA gene sequencing, followed by the ribotyping technique [20]. As aforementioned, obesity is characterized by a chronic lowgrade infammation that may afect the insulin activity in its metabolically sensitive tissues. Terefore, the antiinfammatory ability is considered to be the most important criterion for strain selection. After coculture of murine macrophages with our isolated Lactobacillus strains, Lacticaseibacillus paracasei LM-141 (LPLM141) exhibited the best anti-infammatory efect, as shown by the highest interleukin 10 (IL-10) versus IL-6 (IL-10/IL-6; anti-/proinfammatory) secretion ratio. Te present study aimed to explore whether the selected probiotic in terms of LPLM141 may reduce obesity, infammation, and insulin resistance in rats fed with a high-fat diet (HFD).

Preparation of Lacticaseibacillus paracasei LM-141
(LPLM141). LPLM141 cells were prepared according to previous protocols [21]. After centrifugation, LPLM141 cells were washed with sterile 0.85% NaCl solution followed by lyophilizing and then kept at −80°C until use. Te amount of LPLM141 cells in lyophilized powder was higher than 10 11 cells/gram. Te lyophilized powder containing LPLM141 cells was dissolved with sterile RO water and orally administered to the rats.

Animal Model.
Te animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (approval no. 108019) and conducted according to the guidelines laid down by the IACUC. Five-week-old pathogen-free male Sprague-Dawley (SD) rats were purchased from BioLASCO (BioLASCO Taiwan Co., Ltd, Yi-Lan, Taiwan) and housed at 24 ± 1°C, 50% humidity, and 12 h light-dark cycles with free access to water and food.

Experimental Design.
After one week of acclimatization, a total of thirty-two male rats were randomly divided into four groups (n � 8/group) as follows: (1) the control group (control) consumed a standard diet; (2) the high-fat diet (HFD) group consumed a high-fat diet (a diet containing 59.3% energy from fat); (3) the HFD + low-dosage group that consumed a high-fat diet was fed with low-dosage LPLM141 (2 × 10 7 CFU/day per rat); and (4) the HFD + high-dosage group that consumed a high-fat diet was fed with high-dosage LPLM141 (2 × 10 9 CFU/day per rat). Five weeks after feeding with standard diet or high-fat diet, the rats were administered with extra RO water (for control and HFD groups) or diferent dosages of LPLM141 (for HFD + low-or high-dosage groups) till the end of experiments. Te oral gavage was performed at fxed time every day. Te composition of the standard diet and the high-fat diet is listed in Table 1. Body weight was recorded twice a week, and diet intake was recorded once a week. At the end of the experiments (14 weeks), the rats were fasted overnight before sacrifcing.

Blood and Tissue
Collection. Blood and tissue samples were collected from animals sacrifced by CO 2 anesthesia at the end of the experiments. Te blood samples were collected in a sterile tube by cardiac puncture. After having been centrifuged at 4, 000g for 10 min at 4°C, the serum was collected and stored at −80°C for further experiments. Tissue samples including the liver, kidney, and diferent adipose tissues (including subcutaneous white adipose tissue (sWAT), epididymal white adipose tissue (eWAT), and interscapular brown adipose tissue (iBAT)) were weighed and washed with saline and then stored at −80°C until use.

Oral Glucose Tolerance Test (OGTT).
Tirteen weeks after treatment, the oral glucose tolerance test was performed. After fasting for 12 h, an oral glucose load (2 g/body weight (kg)) was given to each rat. Blood samples collected from the tail vein at 0, 30, 60, 90, and 120 min after glucose administration were subjected to glucose level determinations using a glucometer Rightest GM550 (Bionime, Taichung, Taiwan).

Serum Biochemical Parameter Evaluation.
Serum biochemical parameters including TG, LDL-C, HDL-C, cholesterol, AST, and ALT were analyzed using an automatic clinical analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan). Serum glucose concentrations were determined using a glucometer Rightest GM550 (Bionime). Serum levels of insulin, adiponectin, leptin, LPS, and MCP-1 were measured using commercial rat ELISA kits (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Histological
Studies. Both the rat hepatic and eWAT were fxed in 10% neutral -bufered formalin at 4°C for two days and then were frozen in Tissue-Tek. Te liver sections (8 μm thick) were rehydrated and stained with oil red O (Sigma-Aldrich, St. Louis, MO, USA) with Mayer's hematoxylin as counterstaining [22]. For hematoxylin and eosin (H&E) staining, the liver and eWAT isolated from rats were fxed in 10% formalin and embedded in parafn. Tree micrometer thick sections were deparafnized in xylene and stained with hematoxylin-eosin. Te images of staining were

Statistical Analysis.
Te data were presented as the mean ± SEM. Statistical analyses were performed by Student's t-test when two groups were compared and by oneway ANOVA followed by Tukey's test procedures using SPSS 20.0 (SPSS Inc., Chicago, IL, USA) when more than two groups were compared. All the assays were obtained from at least three independent experiments. A p value < 0.05 was considered statistically signifcant.

Administration of Lacticaseibacillus paracasei LM-141 (LPLM141) Mitigates Obesity and Lipid Accumulation in
High-Fat Diet-Fed Rats. At the beginning of the experiment, the initial body weight among diferent groups showed no signifcant diference. Te body weight and body weight gain in the HFD group was signifcantly increased as compared to the control group at the 14th week, while this increase was downregulated by the administration of low-and highdosage LPLM141. Of note, the food intake showed no signifcant diference among all groups, indicating that the efects of LPLM141 on body weight were not due to the changes in appetite (Table 3). In addition, compared to the control group, the liver and the subcutaneous and eWAT weights, which were signifcantly increased in the HFD group, were markedly decreased after oral low-or highdosage LPLM141 treatment. However, the iBAT weight was not signifcantly diferent among diferent groups. Tere were no signifcant diferences in kidney and spleen weights among diferent groups (Table 3). In addition to decrease body weights and some tissue weights' (liver and adipose tissues) increase by the high-fat diet after the administration of LPLM141, the average epididymal adipocyte size was also signifcantly reduced by LPLM141 supplementation as compared to the HFD group (Figures 1(a) and 1(b)). Te anatomical images of abdominal fat accumulation shown in Figure 1(c) further corroborated that LPLM141 intervention efectively improved obesity and lipid accumulation in HFDfed rats.

Lacticaseibacillus paracasei LM-141 Improves Serum Lipid Profles in High-Fat Diet-Fed Rats.
It is well known that the excessive fat accumulation often accompanies metabolic disorders, so the levels of serum TC, TG, LDL-C, and HDL-C were detected. Te results demonstrated in Figure 2 revealed that an increase in the serum TC, TG, and LDL-C levels and a decrease in HDL-C levels were detected in the HFD group when compared with the control group. However, both low-and high-dosage LPLM141 supplementation signifcantly reduced serum TC, TG, and LDL-C levels and increased serum HDL-C levels as compared to the HFD group.

Lacticaseibacillus paracasei LM-141 Suppresses Infammation in High-Fat Diet-Fed Rats.
Chronic low-grade infammation is an important character associated with obesity. It was evidenced that LPS derived from Gramnegative bacteria in the gut played a critical role in the development of tissue infammation in obesity [23]. Te result shown in Figure 3(a) demonstrated that a signifcant higher serum LPS level was observed in the HFD group compared with the control group. LPLM141 administration could efectively decrease LPS concentration which was increased in the HFD group. Furthermore, the serum level of anti-infammatory adipokine, namely, adiponectin was decreased in the HFD group as compared to the control group.
Evidence-Based Complementary and Alternative Medicine However, both low-and high-dosage LPLM141 interventions signifcantly upregulated serum adiponectin concentrations reduced by high-fat diet treatment (Figure 3(b)). MCP-1 is a proinfammatory chemokine that recruits and activates macrophages in adipose tissues of obese animals [24]. Our present results indicated that the serum level of MCP-1 in the HFD group was signifcantly elevated compared with the control group. However, both low-and high-dosage LPLM141 supplementation reduced the serum MCP-1 level in rats fed with a high-fat diet (Figure 3(c)). We further examined the infltration of macrophages in adipose tissue by immunohistochemical analysis with an anti-F4/80 antibody to evaluate the involvement of adipose tissue infammation. Te results shown in Figure 3(d) demonstrated that a signifcant percentage of macrophage infltration in adipocytes was  Evidence-Based Complementary and Alternative Medicine observed in the HFD group (42.3 ± 3.2%) when compared with the control group (19.7 ± 1.4%). However, the HFDinduced infltration of macrophages in the adipocytes was signifcantly inhibited by both low-and high-dosage of LPLM141 treatment (30.0 ± 2.5% and 11.1 ± 1.2%, respectively) (Figure 3(d)). It is generally recognized that the infammatory response emerging in the presence of obesity is predominantly attributed to the infammation in adipose tissue, although other metabolically critical sites may also be involved [25]. Terefore, we next monitored mRNA expression levels of proinfammatory cytokines in epididymal and subcutaneous adipose tissues after high-fat diet feeding with or without LPLM141 administration. As shown in Figure 3(e), TNF-α, IL-6, and IL-1β mRNA expression levels in eWAT were higher in the HFD group compared with the control group. Both low-and high-dosage LPLM141 supplementation signifcantly reduced cytokine expression levels compared with the HFD group. Similar to the results demonstrated in eWAT (Figure 3(e)), the upregulation of TNF-α and IL-1β mRNA expressions in sWAT after HFD feeding were downregulated by both low-and high-dosage LPLM141 treatments (Figure 3(f )). Although not statistically signifcant, the increased IL-6 mRNA expression induced by HFD in subcutaneous adipose tissue was decreased by both low-and high-dosage LPLM141 administration (Figure 3(f )). PPAR-c, highly expressed in adipose tissue, is known to decrease adipose tissue infammation by decreasing infammatory gene expression [26,27]. As demonstrated in Figures 3(e) and 3(f ), high-fat diet treatment signifcantly suppressed PPAR-c mRNA expression in epididymal and subcutaneous adipose tissue compared with the control group. Both low-and high-dosage LPLM141 administration increased PPAR-c mRNA expression which was decreased in the HFD group. Based on these results, we concluded that LPLM141 administration efectively alleviated infammation by decreasing the serum LPS level, TNFα, IL-6, and IL-1β mRNA expression and infltrated macrophages in adipose tissues, increasing serum adiponectin concentration and PPAR-c mRNA in adipose tissues.

Lacticaseibacillus paracasei LM-141 Administration Induces Browning in eWAT and Activation in iBAT in High-Fat
Diet-Fed Rats. In addition to its role in regulating tissue infammation, PPAR-c was also considered the master regulator as well as a critical determinant of adipocyte diferentiation and activation [28]. Since both low-and high-dosage LPLM141 treatments markedly increased PPAR-c gene expression decreased by HFD feeding in white adipose tissues (Figure 3), we next set out to assess mRNA expression levels of PPAR-c downstream genes related to browning in eWAT and activation in iBAT. Te results shown in Figure 4(a) indicated that the levels of brown adipocyte marker genes including UCP1, Prdm16, and Pgc1α in eWAT were signifcantly reduced in the HFD group as compared to the control group. Tese reducing mRNA expression levels of brown adipocyte markers were recovered after LPLM141 administration. Besides, the mRNA expressions of two beige adipocyte markers, namely, Tmem26 and CD137 decreased by HFD feeding, were increased by LPLM141 treatment. We also evaluated whether the increased PPAR-c mRNA expression by LPLM141 intervention in WAT could infuence the activation of iBAT. Te results demonstrated that HFD   Evidence-Based Complementary and Alternative Medicine signifcantly suppressed the mRNA expression levels of known BAT markers including Ucp1, Prdm16, Pgc1α, and Cidea in iBAT. However, the administration of LPLM141 mitigated the decrease of the mRNA expression levels (Figure 4(b)). Tese results suggested that the upregulation of PPAR-c after LPLM141 intervention may promote WAT browning and increase BAT activity.

Lacticaseibacillus paracasei LM-141 Administration Attenuates Insulin Resistance and Improves Glucose Tolerance in Rats Fed with High-Fat Diet.
It is evidenced that infammation is a triggering factor in the development of insulin resistance [27]. Terefore, it is reasonable to explore the efects of LPLM141 administration on insulin resistance induced by high-fat diet feeding since our  Evidence-Based Complementary and Alternative Medicine aforementioned results validated the anti-infammatory efects of LPLM141 intervention. We next conducted OGTT and HOMA-IR to verify the insulin sensitivity by LPLM141. As shown in Figures 5(a) and 5(b), it was apparent that impaired glucose tolerance in the HFD group compared with the control group was noticed, which was clearly verifed by a higher AUC, while both low-and high-dosage LPLM141 supplementation significantly improved this impairment ( Figure 5(b)). HOMA-IR is calculated by fasting glucose and insulin levels, and the results shown in Figures 5(c) and 5(d) revealed that both fasting blood glucose and fasting insulin levels were signifcantly higher in the HFD group compared to the control group. Tis enhancing efect was abrogated by the intervention of both low-and highdosage LPLM141. Furthermore, the results showed that the value of the HOMA-IR index in the HFD group was approximately 2.5-fold higher than the control group ( Figure 5(e)), while the value of HOMA-IR in the HFD + low-dosage LPLM141 group and the HFD + highdosage LPLM141 group both exhibited an apparent reduction compared to the HFD group ( Figure 5(e)). As a member of adipokines, leptin can act as a proinfammatory cytokine and play some role in the development of obesity and insulin resistance [29,30]. Our present fndings indicated that the serum level of leptin in the HFD group was signifcantly upregulated when compared to the control group. However, the elevated serum leptin concentration induced by high-fat diet feeding was markedly reduced by administration of both low-and high-dosage LPLM141 ( Figure 5(f )).
In order to clarify the possible mechanisms underlying the development of insulin resistance induced by high-fat diet treatment, we determined the relevant molecule expression on the insulin signaling pathway using Western blotting analysis. Te results shown in Figure 6(a) indicated that both IRS-1 and phosphorylated Akt at Ser473 (p-Akt Ser473 ) protein expressions in hepatic tissues were reduced in the HFD group compared with the control group. Notably, both low-and high-dosage LPLM141 supplementation obviously reversed this inhibitory efect afected by high-fat diet feeding. Te densitometry analysis of IRS-1 and p-Akt Ser473 derived from the blotting plot is shown in Figures 6(b) and 6(c), respectively. Tese results clearly demonstrated that LPLM141 intervention mitigated insulin resistance in obese rats.

Lacticaseibacillus paracasei LM-141 Ameliorates Hepatic Lipid Accumulation and Liver Damage in Rats Fed with High-Fat Diet.
Obesity is a well-known risk factor for the development of nonalcoholic fatty liver disease (NAFLD) characterized by steatosis, infammation, hepatocellular ballooning, and fbrosis [31]. Te fndings revealed in Figure 7(a) demonstrated that the mRNA expressions of hepatic lipogenic genes, namely, Elovl6, FAS, and SREBP-1c, in hepatic tissues were signifcantly higher in the HFD group than those in the control group, while the expression of these genes were reduced via administration of both low-and high-dosage LPLM141. In addition, two important liver function markers, namely, ALT and AST, were signifcantly elevated in the serum of the HFD group rats as compared to those of the control group, while these alterations were reversed by the intervention of both lowand high-dosage LPLM141 (Figure 7(b)). We next examined the accumulation of fat in hepatic tissues by histological studies. Te H&E (Figure 7(c)) and oil red O (Figure 7(d)) staining of the liver in the control group exhibited a normal histological architecture characterized by the negligible appearance of large vacuoles and minimal lipid accumulation (Figures 7(c) and 7(d), respectively, control group). Compared to the control group, high-fat diet treatment caused prominent difuse macrovesicular and microvesicular steatosis (Figure 7(c), HFD group) and obvious oil red O staining in the liver (Figure 7(d), HFD group). However, both low-and high-

Discussion
Our current fndings clearly showed that administration of both low-and high-dosage LPLM141 reduced body weight gain, liver weight, and adipose tissue weight without Evidence-Based Complementary and Alternative Medicine afecting the total amount of diet intake in high-fat diet treated rats (Table 3), indicating that this lowering efect was not explained by decreased food intake or loss of appetite but was associated with reduced food efciency or increased energy expenditure evidenced by our current fndings demonstrating that LPLM141 intervention facilitated energy expenditure and thermogenesis (Figure 4).
In this research, exposure of both low-and high-dosage LPLM141 decreased lipid accumulation in adipose tissues (Figure 1(c)) and the size of epididymal white adipocyte (Figures 1(a) and 1(b)) and suppressed serum total cholesterol, triglyceride, and LDL-C levels induced by high-fat diet treatment, whereas the downregulation of serum HDL-C concentration in the HFD group was reversed by both low-and high-dosage LPLM141 intervention (Figure 2). Tese results suggested that LPLM141 administration attenuated lipid accumulation in adipose tissue and relieved the disorder of lipid metabolism in high-fat diet-fed rats.
A systemic low-grade infammation characterized by elevation of proinfammatory cytokines is usually observed in obesity. Te levels of circulating LPS, adiponectin, and MCP-1 play a critical role in the development of infammation and obesity [32][33][34]. In our present study, signifcantly increased serum levels of LPS and MCP-1 and a decreased serum level of adiponectin were noticed in the rats fed with a HFD. Administration of both low-and highdosage LPLM141 obviously reversed these alterations (Figures 3(a)-3(c), respectively). Furthermore, the results from immunohistochemical staining in eWAT demonstrated that the increased macrophage infltration in HFDfed rats was dramatically decreased by both low-and highdosage LPLM141 treatments (Figure 3(d)). It is now admitted that the expression of proinfammatory cytokines, namely, TNF-α, IL-6, and IL-1β, by infltrated macrophages in adipose tissues could enhance and perpetuate the infammation of adipose tissues often seen in obese humans and animals [35][36][37]. As demonstrated in the Figures 3(e) and 3(f ) that correlated well with the macrophage infltration in eWAT shown in the present results (Figure 3(d)), the mRNA expressions of TNF-α, IL-6, and IL-1β in epididymal and subcutaneous white adipose tissues in HFD-fed rats were signifcantly increased, whereas both low-and high-dosage LPLM141 intervention markedly suppressed this enhancing efect. In addition to proinfammatory cytokines, adipose tissue macrophages also produce a certain amount of PPAR-c, a member of the nuclear hormone receptor family of transcription factors, to negatively regulate a large set of infammatory pathway genes [26,38]. Te previous study demonstrated that addition of TNF-α downregulated PPAR-c expression and ultimately contributed to infammation in adipose tissues [39]. Tese results suggested that PPAR-c is strongly correlated with the anti-infammatory response in adipose tissue. We found that both low-and high-dosage LPLM141 intervention signifcantly increased the PPAR-c mRNA expression in adipose tissue which was decreased in high-fat diet-fed rats (Figure 3(e)). Taken together, our results revealed that LPLM141 exerted its anti-infammatory efects by decreasing serum LPS, MCP-1, proinfammatory cytokines, and infltrated macrophages in eWAT and simultaneously increasing serum adiponectin and PPAR-c expression in adipose tissues.
Obesity and its associated metabolic complications are always accompanied with insulin resistance. Our fndings demonstrated that administration of both low-and highdosage LPLM141 signifcantly prevented the development of hyperglycemia and ameliorated insulin resistance in HFDinduced obese rats (Figures 5(a)-5(e)). Leptin is an important hormone secreted by adipose tissue which regulates appetite and energy balance [40]. A large body of results from humans and animal studies indicated that elevated leptin levels were observed in obese hosts, especially in dietinduced obesity [40,41]. Paradoxically, in our present fnding shown in Figure 5(f ), the increased serum leptin concentration in HFD-fed rats was not accompanied with the expected appetite reducing and body weight lowering efects (see Table 3). However, administration of both lowand high-dosage LPLM141 signifcantly downregulated the serum leptin levels upregulated by high-fat diet feeding ( Figure 5(f )). Tis phenomenon can be explained by the occurrence of leptin resistance, which is defned by the reduced ability of leptin to suppress appetite and weight gains [42,43]. More importantly, accumulating evidences demonstrated that the obese animals and humans who displayed leptin resistance may directly contribute to the suppression of lipid oxidation in insulin-sensitive organs and ultimately lead to accumulation of lipids and insulin resistance [40,41,44,45]. Insulin resistance induced by obesity is characterized by a dysfunction of insulin to activate the IRS/phosphoinositide 3-kinase/AKT pathway, leading to suppression of the insulin-induced glucose uptake in the insulin-sensitive organs, such as the liver [46,47]. In our present studies (Figure 6), high-fat diet treatment signifcantly downregulated IRS-1 and p-Akt Ser473 protein expressions in rat hepatic tissues, whereas administration of both low-and high-dosage LPLM141 efectively reversed the high-fat diet-induced decline of IRS-1 and p-Akt Ser473 (Figures 6(b) and 6(c), respectively). Tese results implied that LPLM141 intervention signifcantly mitigated insulin resistance by enhancing the IRS-1/p-AktSer473 insulin signaling pathway and lowering the serum leptin level in high-fat diet-fed rats and subsequently improving glucose intolerance and normalizing the HOMA-IR index. Nonalcoholic fatty liver disease (NAFLD), a chronic liver disease characterized by excess fat deposition in the liver, is strongly associated with obesity and the metabolic syndrome [48]. Srebp-1c is an important transcription factor reported to be involved in the transcriptional activation of genes encoding rate-limiting enzymes in lipogenesis and is also associated with increased de novo lipogenesis in NAFLD [49]. Upon insulin stimulation, SREBP-1c regulates hepatic fatty acid and TG biosynthesis by upregulating the expression of key genes, such as FAS [50]. In addition, the previous study indicated that inhibition of Elovl6, a key lipogenic enzyme elongating longchain saturated and unsaturated fatty acids, can ameliorate insulin resistance in fatty livers and suppress fatty acid synthesis [51]. Moreover, Elovl6 is regulated directly and primarily by SREBP-1c [52]. Our present results revealed that administration of both low-and high-dosage LPLM141 signifcantly counteracted the increase in liver lipogenic genes, namely, Elvol6, FAS, and Srebp-1c expressions in rats fed with HFD (Figure 7(a)). Administration with lowand high-dosage LPLM141 obviously attenuated steatosis and oil red O staining which were enhanced in liver tissue of HFD-fed rats (Figures 7(c) and 7(d)) and further corroborated their inhibitory efect on lipid accumulation. Several lines of evidence indicate that the animals fed with high-fat diet induce obesity accompanied by liver damage, similar to the phenotype observed in humans sufering from NAFLD [53]. In line with the previous studies, our present fndings exhibited that a signifcant increase in serum AST and ALT was noticed in HFD-fed rats, whereas administration of both low-and high-dosage LPLM141 markedly alleviated these two liver damage markers induced by HFD treatment (Figure 7(b)). Tese results suggested that LPLM141 intervention exerted hepatoprotective activity via suppression of lipogenesis and fat accumulation.
Although our preliminary results supported LPLM141 efectively alleviating infammation and insulin resistance in HDF-induced obese rats, some limitations are still needed to be mentioned here. First, it has been well documented that probiotics supplementation exerted its antiobesity efect by restoring the gut microbiota and mitigating metabolic endotoxemia by decreasing intestinal permeability [54][55][56]. Terefore, the impact on the composition of gut microbial populations and intestinal barrier integrity in HFD-induced obese rats after LPLM141 intervention will be explored. Second, for future long-term studies, the actual energy expenditure should be measured to consolidate our present fndings indicating increased expression of genes encoding beige adipocyte activation and thermogenesis in eWAT and iBAT, respectively, in HFD-induced obese rats after LPLM141 treatment. Upon fnishing these tasks, we can elucidate the mechanism of the antiobesity efects of LPLM141 more comprehensively.

Conclusion
In summary, we preliminarily proved that LPLM141 possessed an excellent antiobesity efect in a HFD-induced obese rat model. Te potential mechanisms of this efect might be related to signifcantly attenuated body weight gain, reduced fat accumulation in adipose tissues, enhanced eWAT browning and iBAT activation, protected rats from glucose intolerance, and insulin resistance induced by HFD. Tese aforementioned efects were at least in part due to the anti-infammatory actions of LPLM141 intervention in adipose tissues. Moreover, upregulation of serum adiponectin and downregulation of serum leptin concentrations after LPLM141 administration in HFD-fed rats might make contributions to alleviation of systemic infammation and improvement of insulin resistance, respectively. We also further clarifed that the underlying mechanism on improving insulin resistance after LPLM141 intervention was an attribute to the recoveries of IPS-1 and p-Akt expressions which were decreased in HFD-fed rats. Finally, our fndings indicated that administration of LPLM141 remarkably ameliorated hepatic steatosis and exerted hepatoprotective efect in rats fed with a high-fat diet. Our current studies could ofer LPLM141 as a promising probiotic for the prevention or therapy of obesity and its associated metabolic disorders.

Data Availability
Te datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Hsieh FC is the President and Director of R&D of Larmood Company Limited. LPLM141 was obtained from Larmood Company Limited. Te other authors declare that they have no conficts of interest.