**5. Regulation of leptin, adiponectin and osteocalcin**

Hormones such as leptin, adiponectin and osteocalcin play an important role in lipid metabolism. Obese population was characterized by significant lower levels of osteocalcin and adiponectin as well as high leptin level (leptin-resistant) which have been reported in literature. It is now increasingly accepted that leptin can regulate food intake and energy expenditure through hypothalamus and adiponectin can enhance tissue fat oxidation to downstream fatty acids levels and tissue triglyceride content associated with insulin sensitivity [52]. As for osteocalcin, leptin assumed to modulate osteocalcin bioactivity and osteocalcin could stimulate the adiponectin synthesis [53] [54].

#### **5.1. Leptin**

446 Lipid Metabolism

**4. The whole microflora view** 

the metabolism of lipid-regulating drugs in gut.

**5. Regulation of leptin, adiponectin and osteocalcin** 

osteocalcin could stimulate the adiponectin synthesis [53] [54].

Intestinal microbes not only *Lactobacillus* could also exhibit a bile salt deconjugating effect [40], suggesting that other microbes had lipid-reducing potential. Thus, overall intestinal microflora was taken into account for lipid metabolism evaluation. In the past few years, research has focused on new areas of microflora and lipid metabolism with the development

The human gut is consisting of a microbial community of 1014 bacteria with at least 1000 species and the whole microbiome is more than 100-fold the human genome [42].These researches highlight the significance of the whole gut microbiome contribute to energy harvest and the relationship between obesity and changes of gut microbiome [43]. More detailed, obese is mainly characterized by elevated *Firmicutes/Bacteroidetes* ratio in gut [44]. Probiotics serve as one of effective agents for regulation of gut microflora, they can exext benefits on lipid metabolism through downregulating the ratio of *Firmicutes/Bacteroidetes*. Other bacteria such as *Methanobrevibacter smithii* are also at low level in obese people [45]. Interestingly, atherosclerotic disease, which caused by accumulation of cholesterol and inflammation, was recently found its atherosclerotic plaque microbiota was associated with oral and gut microbiota through high throughput 454 pyrosequencing of 16S rRNA genes [46]. Besides, such a huge microflora provide a large reservoir of LPS molecules to circulation through colonizing of Gram-negative bacteria in the gut [47]. A recent study showed *Bifidobacteria* with genes encoding an ATP-binding-cassette-type carbohydrate transporter could protect against Gram-negative *E. coli* O157:H7 colonization in gut due to acetate production [48]. Thus, probiotic can restrict LPS-related microbial communities in the gut.

The whole gut microflora is also known as a target for drug metabolism because of diverse microbial transformations [49]. Manipulation of commensal microbial composition through antibiotics, probiotics or prebiotics was thought to enhance the metabolic activity and production of effective metabolites [50]. Simvastatin, which is an inhibitor of HMG-CoA and widely used for regulating hepatic cholesterol production, was proposed to possess altered pharmacological properties by microflora degradation via changing its capacity to bind to the corresponding receptors [51]. It is indicated that probiotics have potential to influence

Hormones such as leptin, adiponectin and osteocalcin play an important role in lipid metabolism. Obese population was characterized by significant lower levels of osteocalcin and adiponectin as well as high leptin level (leptin-resistant) which have been reported in literature. It is now increasingly accepted that leptin can regulate food intake and energy expenditure through hypothalamus and adiponectin can enhance tissue fat oxidation to downstream fatty acids levels and tissue triglyceride content associated with insulin sensitivity [52]. As for osteocalcin, leptin assumed to modulate osteocalcin bioactivity and

of culture-independent methods for understanding the total microbial diversity [41].

Leptin, an antiobesity hormone produced by adipose tissue, has been reported to regulate body weight by controlling food intake and energy expenditure [55]. However, obesity tend to display markedly higher serum leptin level with a leptin-resistant symptom. Several studies reported a decrease of leptin by probiotic administration. In high-fat fed mice, Lee et al confirmed that *Lactobacillus rhamnosus* PL60 exhibited a reduction in leptin level and antiobesity effect due to production of conjugated linoleic acid [56]. Moreover, serum leptin concentration was reduced by *Lactobacillus gasseri* SBT205 in lean Zucker rats linked with lowered adipocyte size [57]. Another study also report leptin level was reduced by a combined bifidobacteria (*B. pseudocatenulatum* SPM 1204, *B. longum* SPM 1205, and *B. longum*  SPM 1207) in obese rats [58]. Interestingly and controversially, direct injection of *Lactobacillus acidophilus* supernatants (germ free) into the brains of rats lead to weight loss with an increase in leptin expression in neurons and adipose tissue [59].

Leptin-lowering effect of probiotics was also observed in human. Similarly, Naruszewicz et al investigated whether oral administration of *L. plantarum* 299v exert beneficial effect on smokers by detection of cardiovascular risk factors [60]. In this study, smokers showed a great decrease in plasma leptin concentrations and anti-inflammatory properties when supplement of probiotic. Discouragingly, two months of *Lactobacillus acidophilus* and *Bifidobacterium longum* consumption failed to lower plasma leptin levels in male equol excretors [61].

### **5.2. Adiponectin**

As an adipocyte-derived serum protein, adiponectin play an important role in glucose and lipid metabolism since adiponectin deficiency are associated with insulin resistance, inflammation, dyslipidemia and risk of atherogenic vascular disease [62]. In parallel, adiponectin has also been shown to suppress macrophage foam cell formation in atherosclerosis [63]. Several studies showed that probiotic therapy improved adiponectin level or adiponectin gene expression. One comparative research performed in normal microflora (NMF) and germ-free (GF) mice revealed that adiponectin gene expression (Adipoq) was up-regulated in the groups of *Lactobacillus*-treated germ free mice [64]. Moreover, Higurashi et al reported a probiotic cheese could prevent abdominal adipose accumulation and maintained serum adiponectin concentrations in high-calorie fed rats [65]. However, *Lactobacillus plantarum* strain No. 14 exert a white adipose-reducing effect in highfat fed mice with no change of adiponectin [66].

Kadooka et al used a probiotic *L. gasseri* SBT2055 to regulate abdominal adiposity in obese adults, where the probiotic treatment involved a significant reduction in abdominal visceral and subcutaneous fat areas from baseline and significantly increased high-molecular weight adiponectin in their serum [67]. Furthermore, a recent large scale clinical study conducted by Luoto et al confirmed that pregnant women with a consumption of combined *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* probiotics possessed higher colostrum adiponectin concentration compared to placebo which was correlated inversely with maternal weight gain during pregnancy [68].

#### **5.3. Osteocalcin**

In recent years, osteocalcin secreted by osteoblasts has aroused great interest linked to β cell function, adiponectin production, energy expenditure and adiposity [69]. In humans, fat individuals kept a low level of serum osteocalcin [70]. The only study by Naughton et al showed that osteocalcin levels was slightly increased in middle aged rats by consumption of inulin-rich milk fermented by *Lactobacillus* GG and *Bifidobacterium lactis* [71]. It is interesting that osteocalcin is an vitamin K-dependent protein and two main types including vitamin K1 and vitamin K2 are respectively produced from dietary vegetable and microflora [72]. As an effective way to alter microflora, probiotics have potential to enhance vitamin K2 production and related osteocalcin level through changing the microflora.

The Effect of Probiotics on Lipid Metabolism 449

probiotics could modify the expression of PPAR-α, PPAR-β, VDR-α, RAR-γand GR in a marine fish, suggesting extensive crosstalk among NRs activated by probiotic [83]. Concerning about NRs and lipid metabolism linked with probiotic, Aronsson et al observed that *L. paracasei* F19 could reduce the fat storage associated with the drastic changes of PPARs [84]. One most recent study by Zhao et al have also demonstrated probiotic *Pediococcus pentosaceus* LP28 could also acted as a PPAR-γ agonist concomitantly with the

As important pattern recognition receptors, TLRs participate in distinguishing and recognizing a range of microbial components such as peptidoglycan (TLR2) and LPS (TLR4) to activiate immune responses [86]. Up to date, the relationship between TLRs and lipid metabolism is mainly from two aspects. On one hand,TLRs signaling can directly contact and interfere with cholesterol metabolism in macrophages [87]. On the other hand, TLRs signaling (mainly TLR4) are involved in interaction LPS with fatty acid, lipoprotein and organ injury(especially liver and intestine). There is evidence that low dose of LPS can boost *de novo* fatty acid synthesis and lipolysis and lipoprotein production in liver which leading to hepatic hypertriglyceridemia [88]. In mice, moderately higher LPS level could be increased by a fat-enriched diet and contributed to low grade inflammation [34]. In rabbits, high cholesterol intake plus with low dose LPS accelerated the development of atherosclerosis [89]. These two studies are considered as the result of crosstalk between LPS and TLRs leads to intestinal mucosal injury associated with inflammatory response. Besides, foam cell formation in atherosclerosis has been shown to be mediated by TLR2 and 4 and

other TLRs such as TLR3, 7, and 9 may also participate in atherosclerosis [90] [91].

intestinal epithelial cells and macrophages [94].

pro-inammatory cytokines in human subjects [97].

TLR4 appears to be tightly linked to high-fat intake, LPS and inflammation. Probiotics are known to reduced LPS-containing gram-negative organisms (such as *E. coli*) in the gut and influx of LPS into circulation [92] [93]. A great number of probiotics are also able to specically modulate the NF-κB pathway (one of most important inflammatory pathways)in

Due to TLR4 deficiency with anti-obesigenic effects and susceptible to colitis, little information about influence of probiotic on lipid metabolism is obtained in TLR4 knockout model whereas protective effect of probiotic VSL#3 from inflammation was observed in TLR4 knockout mice [95] [96]. With regard to the role of TLR4 in the development of metabolic disorders, Andreasen et al have considered that *L. acidophilus* NCFM may reduce overow of LPS from the gut to the circulation and downregulate the TLR4 signalling and

Immunity homeostasis also have important effect on lipid metabolism. In general, it is well accepted that probiotic bacteria are able to maintain the Th1 and Th2 banlance of immunity through regulating pro-inflammatory and anti-inflammatory cytokines [98]. In addition, Agrawal et al documented that TLR2-derived signaling mainly enhance Th2-cytokine release, while TLR4 triggered by LPS stimulates Th1-type responses [99]. Interestingly,

great reduction of triglyceride and cholesterol in obese mice [85].

**6.2. Toll-like receptors** 
