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Complete study here; http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042529
Discussion
Due to the evident clinical therapeutic effects of berberine for diabetes and dyslipidemia [15], [18], the mechanisms involved in its beneficial effects against metabolic disorders have attracted much attention in recent years. In this study, we demonstrated that berberine co-administration at a dose of 100 mg/kg body weight effectively prevented the weight gain and development of insulin resistance induced by long-term HFD feeding. No obvious side effects were observed in the present study. As extrapolated using the body surface area normalization method [37], such dose is equivalent to about 1.0 g/day/person in humans. In a previous human trial, we also confirmed that oral administration of berberine at such a dose of 1.0 g/day/person for 3 months is effective and safe in the treatment of type 2 diabetes and dyslipidemia [15].
Improvement of insulin sensitivity has been widely reported to be involved in the mechanisms of action of berberine in both animal and human studies. Lee et al. [19] reported that berberine alleviated insulin resistance in both db/db mice and HFD-fed rats, along with downregulating lipogenic genes and upregulating genes involved in energy expenditure. Kong et al. [38] also suggested that berberine restored the impaired insulin sensitivity in rats with type 2 diabetes via a mechanism of protein kinase C-dependent elevation of insulin receptor gene expression. Clinical research further confirmed that the mean percentage of peripheral blood lymphocytes that express insulin receptor on their surface was significantly elevated by 3.6-fold (P<0.01) after 2 months of berberine therapy [16]. A randomized, double-blinded, placebo-controlled, multicenter clinical trial performed by us also revealed that, along with a significant reduction of serum interleukin (IL)-6 levels, berberine treatment significantly improved insulin sensitivity as revealed using hyperinsulinemic euglycemic clamps [15]. However, the primary target of berberine regarding the improvement of insulin sensitivity remains to be elucidated.
Alleviation of inflammation has been identified as an important mechanism in the insulin-sensitizing effects of berberine because of the increasingly evident causative relationship between inflammation and insulin resistance [39]. Pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), can enhance the serine phosphorylation of insulin receptor substrate-1, a crucial event in the induction of insulin resistance [40]. The anti-inflammatory activities of berberine have been widely reported. Jeong et al. [41] reported that berberine significantly suppressed the expression of pro-inflammatory genes, including TNF-α, IL-1β, IL-6, MCP-1, inducible nitric oxide synthase, and cyclooxygenase-2 , in the white adipose tissue of db/db mice. Another study utilized a LPS-injured rat model to demonstrate that berberine significantly reduced LPS-induced intestinal damage and decreased serum levels of downstream inflammatory cytokines [42]. In this study, we also observed that berberine co-administration significantly prevented HFD-induced systemic inflammation. Together with previous reports, we suggest that the alleviation of inflammation may serve as an important mechanism in insulin sensitization in berberine-treated rats.
Accumulating evidence indicates that the gut microbiota plays a pivotal role in modulating host immune systems [7]. Structural imbalances of the gut microbiota, particularly reductions in the abundance of gut barrier-protecting bacteria such as Bifidobacterium spp. and increases in the abundance of Gram-negative endotoxin-producing bacteria such as Desulfovibrio spp., may lead to increases in intestinal permeability and circulating gut-originated antigens, primarily LPS [7], [43]. Upon binding to the complex of CD14 and toll-like receptor 4 on the surface of innate immune cells, LPS can induce systemic inflammation, which eventually impairs insulin sensitivity and induces insulin resistance-related metabolic disorders [7]. Further studies demonstrated that selective increases in the levels of Bifidobacterium spp. via the administration of prebiotics completely abolished the metabolic disorders induced by HFD, possibly by alleviating low-grade inflammation and insulin resistance [11]. In the present study, we measured the serum concentration of LBP, a biomarker of circulating LPS [33], to investigate whether there is a possible role of berberine-mediated modulation of the gut microbiota in the alleviation of host inflammation and amelioration of insulin resistance-related metabolic abnormalities. In accordance with the results previously reported by Cani et al. [7], HFD induced a significant increase in serum LBP levels in our rats, which was essentially prevented by berberine co-administration, suggesting a potential role of antigens derived from the gut microbiota in this pharmacological process.
To study the detailed structural modulation effects of berberine on the gut microbiota and its possible role in alleviating HFD-induced metabolic deteriorations, we performed a MiWAS based on bar-coded 454 pyrosequencing of the V3 region of 16S rRNA genes and multivariate statistics. Significant reductions in bacterial diversity and the total bacterial population were observed in berberine-treated rats. RDA identified 268 key OTUs that were modulated in response to berberine treatment, most of which were eliminated or inhibited by berberine. Berberine has already been reported to have a wide antibacterial spectrum including some opportunistic pathogens, such as Staphylococcus, Streptococcus, Salmonella, Klebsiella, and Pseudomonas [44]. The inhibition of a wide range of intestinal bacteria by berberine might result in a decrease of the free antigen load in the host, as confirmed by the decreased serum LBP levels in berberine-treated HFD-fed rats in this study.
Among the 268 key OTUs identified by RDA, approximately one-third were significantly enriched by berberine treatment. Most significantly, key OTUs in the SCFA-producing genera of Blautia [45] and Allobaculum [46] were enriched by approximately 10-fold compared to their levels in the untreated HFD-fed rats. Determination of fecal SCFA levels by using gas chromatography also indicated that berberine administration significantly increased SCFA concentrations in HFD-fed rats. These results suggest that oral administration of berberine enriches the abundance of SCFA producers to promote colonic fermentation and SCFA production in the intestines of HFD-fed rats. Turnbaugh et al. reported that the obese mice with increased Firmucutes and decreased Bacteriodetes in their guts had an elevated colonic fermentation and SCFA production, which might contribute to obesity by increasing the host's capacity for energy harvesting from foods [5]. However, later studies showed that the relationship between the gut microbial composition, energy harvesting capacity and fecal SCFA levels is more complicated. For example, Murphy et al. showed that the compositional changes of the major phyla Firmicutes, Bacteroidetes and Actinobacteria were unrelated to markers of energy harvest, and the fecal SCFA levels and fecal energy contents in HFD-fed mice were not correlated [47].
Accumulating reports have focused on the alleviating effects of SCFAs on inflammation and their protective effects on gut barrier function. As an important energy source for intestinal epithelial cells, SCFAs improve gut barrier function by either promoting cell differentiation, facilitating tight junction assembly, or upregulating proglucagon gene expression in intestinal L cells [48], [49]. Indeed, the gut barrier-protecting effects of berberine have been reported in animal models challenged with pro-inflammatory cytokines or LPS [42], [50], the mechanisms of which have been suggested to be the promotion of proglucagon mRNA expression and L cell proliferation in the intestine [27], [28]. Our findings suggest that this gut barrier-protecting function of berberine is mediated by elevated levels of SCFAs produced by selectively enriched SCFA producers in the gut.
Anti-inflammation is another well-characterized function of SCFAs. Increased intake of SCFAs has been reported to be clinically beneficial in the treatment of colitis [51]. Follow-up studies suggested that the G-protein coupled receptor 43, a receptor of SCFAs, mediated the effects of SCFAs in regulating inflammatory responses [52]. Another study by Fukuda et al. revealed that SCFAs (namely acetate) produced by certain Bifidobacterium strains promoted the defense functions of host epithelial cells and thereby protected the host against lethal infection with enterohemorrhagic Escherichia coli O157:H7 [53]. A comparative study in children from Europe and rural Africa also suggested that enrichment of SCFA-producing bacteria such as Prevotella and Xylanibacter and increased fecal SCFA concentrations in the intestines of rural Africa children help to inhibit opportunistic pathogens such as Shigella and Escherichia and protect children against inflammation and noninfectious colonic diseases [54]. In this study, significant enrichment of SCFA-producing bacteria and decreased systemic inflammation in berberine-treated HFD-fed rats were also observed. Taken together, the beneficial effects of SCFAs, namely improving gut barrier functions, ameliorating systemic inflammation, or creating a non-permissive environment for pathogens, may mediate the pharmacological effects of berberine against obesity and insulin resistance-related metabolic abnormalities.
Accumulating evidence suggested that diet composition and calorie intake might play an important role in shaping the gut microbiota and modulating host phenotypes [55], [56], [57]. Ravussin et al. demonstrated that, apart from effectively decreasing body weight and fat mass, calorie restriction significantly increased the gut microbial diversity and the relative abundance of Allobaculum in HFD-fed animals, but not in those of NCD-fed [56]. In the current study, berberine significantly reduced the food intake of HFD-fed rats, which is in accordance with some previous reports [23], [58]. Similar to the calorie-restricted HFD-fed animals, selective enrichment of the genus Allobaculum was also observed in berberine-treated HFD-fed rats. However, selective increase of the genus Blautia was unique to berberine, and dramatic decrease of the microbial diversity and significant improvement of insulin sensitivity in berberine-treated HFD-fed rats was also observed. This indicates that although the reduction of food intake may contribute in part to the gut microbiota changes or host metabolic phenotype improvements, the direct modulating effects of berberine on gut microbiota may be more critical for the observed effects of berberine on the host health.
Modulation of gut microbiota with diet or drugs has been indicated to improve host metabolic phenotypes [4], [27]; however, whether the response of the gut microbiota to these environmental perturbations happens at the phylum or specific phylotype level remains controversial [59]. Ley et al. revealed that obese people had fewer Bacteroidetes and more Firmicutes than lean controls [4]. Upon dietary intervention, the ratio of Firmicutes to Bacteroidetes decreased over time as body weight decreased [4]. However, in another similar human study, no association was observed between obesity and the phylum-level changes of the gut microbiota [59]. In a previous report concerning the effects of berberine on the gut microbiota [27], RT-PCR was used to quantify the proportions of Firmicutes and Bacteroidetes to total bacteria, again indicating no significant association between these phylum-level changes with diet types or obesity in mice, although berberine significantly reduced the proportions of both Firmicutes and Bacteroidetes to total bacteria. Accumulating evidence indicates that variations in the species composition of the gut microbiota were related to human obesity [54], [59]. In a previous report [43], we also revealed that the development of metabolic syndromes was relevant to phylotype-specific changes of the gut microbiota. In the present study, we did not find a significant difference in the ratio of Firmicutes to Bacteroidetes between NCD- and HFD-fed rats. Berberine also displayed no significant influence on the proportions of Firmicutes and Bacteroidetes in rats under both feeding conditions. By using the MiWAS strategy, which combines the high-throughput pyrosequencing of 16S rRNA genes with multivariate statistics, we identified 268 key OTUs responding to berberine treatment from a total of 6720 OTUs observed in the samples. PLS regression modeling performed well in predicting the host phenotypes with the abundance data of those identified key OTUs, suggesting a possible close association between those phylotypes and host health phenotypes. In total, 143 and 87 of the 268 OTUs belong to the phyla Firmicutes and Bacteroidetes, respectively. Contrasting responses of the OTUs in the same phylum or even in the same genus were observed. Hence, the present study suggests that phylotype-level profiling of the variation of the gut microbiota by using the MiWAS strategy will serve as a reliable approach for demonstrating the relationship between the gut microbiota and host metabolic phenotypes under diet or drug perturbations.
In conclusion, our findings suggest that marked modulation of gut microbiota by berberine, namely inhibition of a wide range of intestinal microbes and enrichment of some SCFA producers, helps to alleviate systemic inflammation, at least in part, by reducing the antigen load to the host and elevating SCFA levels in the intestine and contributes to the beneficial effects of berberine against insulin resistance, obesity, diabetes, and other metabolic disorders. Functional metagenomic studies and molecular dissection of the host-microbiome cross-talk are needed to further elucidate the mechanisms of action of berberine [3]. This study also suggests that pharmacological or nutritional modulation of gut microbiota is an effective approach for preventive healthcare.
Discussion
Due to the evident clinical therapeutic effects of berberine for diabetes and dyslipidemia [15], [18], the mechanisms involved in its beneficial effects against metabolic disorders have attracted much attention in recent years. In this study, we demonstrated that berberine co-administration at a dose of 100 mg/kg body weight effectively prevented the weight gain and development of insulin resistance induced by long-term HFD feeding. No obvious side effects were observed in the present study. As extrapolated using the body surface area normalization method [37], such dose is equivalent to about 1.0 g/day/person in humans. In a previous human trial, we also confirmed that oral administration of berberine at such a dose of 1.0 g/day/person for 3 months is effective and safe in the treatment of type 2 diabetes and dyslipidemia [15].
Improvement of insulin sensitivity has been widely reported to be involved in the mechanisms of action of berberine in both animal and human studies. Lee et al. [19] reported that berberine alleviated insulin resistance in both db/db mice and HFD-fed rats, along with downregulating lipogenic genes and upregulating genes involved in energy expenditure. Kong et al. [38] also suggested that berberine restored the impaired insulin sensitivity in rats with type 2 diabetes via a mechanism of protein kinase C-dependent elevation of insulin receptor gene expression. Clinical research further confirmed that the mean percentage of peripheral blood lymphocytes that express insulin receptor on their surface was significantly elevated by 3.6-fold (P<0.01) after 2 months of berberine therapy [16]. A randomized, double-blinded, placebo-controlled, multicenter clinical trial performed by us also revealed that, along with a significant reduction of serum interleukin (IL)-6 levels, berberine treatment significantly improved insulin sensitivity as revealed using hyperinsulinemic euglycemic clamps [15]. However, the primary target of berberine regarding the improvement of insulin sensitivity remains to be elucidated.
Alleviation of inflammation has been identified as an important mechanism in the insulin-sensitizing effects of berberine because of the increasingly evident causative relationship between inflammation and insulin resistance [39]. Pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), can enhance the serine phosphorylation of insulin receptor substrate-1, a crucial event in the induction of insulin resistance [40]. The anti-inflammatory activities of berberine have been widely reported. Jeong et al. [41] reported that berberine significantly suppressed the expression of pro-inflammatory genes, including TNF-α, IL-1β, IL-6, MCP-1, inducible nitric oxide synthase, and cyclooxygenase-2 , in the white adipose tissue of db/db mice. Another study utilized a LPS-injured rat model to demonstrate that berberine significantly reduced LPS-induced intestinal damage and decreased serum levels of downstream inflammatory cytokines [42]. In this study, we also observed that berberine co-administration significantly prevented HFD-induced systemic inflammation. Together with previous reports, we suggest that the alleviation of inflammation may serve as an important mechanism in insulin sensitization in berberine-treated rats.
Accumulating evidence indicates that the gut microbiota plays a pivotal role in modulating host immune systems [7]. Structural imbalances of the gut microbiota, particularly reductions in the abundance of gut barrier-protecting bacteria such as Bifidobacterium spp. and increases in the abundance of Gram-negative endotoxin-producing bacteria such as Desulfovibrio spp., may lead to increases in intestinal permeability and circulating gut-originated antigens, primarily LPS [7], [43]. Upon binding to the complex of CD14 and toll-like receptor 4 on the surface of innate immune cells, LPS can induce systemic inflammation, which eventually impairs insulin sensitivity and induces insulin resistance-related metabolic disorders [7]. Further studies demonstrated that selective increases in the levels of Bifidobacterium spp. via the administration of prebiotics completely abolished the metabolic disorders induced by HFD, possibly by alleviating low-grade inflammation and insulin resistance [11]. In the present study, we measured the serum concentration of LBP, a biomarker of circulating LPS [33], to investigate whether there is a possible role of berberine-mediated modulation of the gut microbiota in the alleviation of host inflammation and amelioration of insulin resistance-related metabolic abnormalities. In accordance with the results previously reported by Cani et al. [7], HFD induced a significant increase in serum LBP levels in our rats, which was essentially prevented by berberine co-administration, suggesting a potential role of antigens derived from the gut microbiota in this pharmacological process.
To study the detailed structural modulation effects of berberine on the gut microbiota and its possible role in alleviating HFD-induced metabolic deteriorations, we performed a MiWAS based on bar-coded 454 pyrosequencing of the V3 region of 16S rRNA genes and multivariate statistics. Significant reductions in bacterial diversity and the total bacterial population were observed in berberine-treated rats. RDA identified 268 key OTUs that were modulated in response to berberine treatment, most of which were eliminated or inhibited by berberine. Berberine has already been reported to have a wide antibacterial spectrum including some opportunistic pathogens, such as Staphylococcus, Streptococcus, Salmonella, Klebsiella, and Pseudomonas [44]. The inhibition of a wide range of intestinal bacteria by berberine might result in a decrease of the free antigen load in the host, as confirmed by the decreased serum LBP levels in berberine-treated HFD-fed rats in this study.
Among the 268 key OTUs identified by RDA, approximately one-third were significantly enriched by berberine treatment. Most significantly, key OTUs in the SCFA-producing genera of Blautia [45] and Allobaculum [46] were enriched by approximately 10-fold compared to their levels in the untreated HFD-fed rats. Determination of fecal SCFA levels by using gas chromatography also indicated that berberine administration significantly increased SCFA concentrations in HFD-fed rats. These results suggest that oral administration of berberine enriches the abundance of SCFA producers to promote colonic fermentation and SCFA production in the intestines of HFD-fed rats. Turnbaugh et al. reported that the obese mice with increased Firmucutes and decreased Bacteriodetes in their guts had an elevated colonic fermentation and SCFA production, which might contribute to obesity by increasing the host's capacity for energy harvesting from foods [5]. However, later studies showed that the relationship between the gut microbial composition, energy harvesting capacity and fecal SCFA levels is more complicated. For example, Murphy et al. showed that the compositional changes of the major phyla Firmicutes, Bacteroidetes and Actinobacteria were unrelated to markers of energy harvest, and the fecal SCFA levels and fecal energy contents in HFD-fed mice were not correlated [47].
Accumulating reports have focused on the alleviating effects of SCFAs on inflammation and their protective effects on gut barrier function. As an important energy source for intestinal epithelial cells, SCFAs improve gut barrier function by either promoting cell differentiation, facilitating tight junction assembly, or upregulating proglucagon gene expression in intestinal L cells [48], [49]. Indeed, the gut barrier-protecting effects of berberine have been reported in animal models challenged with pro-inflammatory cytokines or LPS [42], [50], the mechanisms of which have been suggested to be the promotion of proglucagon mRNA expression and L cell proliferation in the intestine [27], [28]. Our findings suggest that this gut barrier-protecting function of berberine is mediated by elevated levels of SCFAs produced by selectively enriched SCFA producers in the gut.
Anti-inflammation is another well-characterized function of SCFAs. Increased intake of SCFAs has been reported to be clinically beneficial in the treatment of colitis [51]. Follow-up studies suggested that the G-protein coupled receptor 43, a receptor of SCFAs, mediated the effects of SCFAs in regulating inflammatory responses [52]. Another study by Fukuda et al. revealed that SCFAs (namely acetate) produced by certain Bifidobacterium strains promoted the defense functions of host epithelial cells and thereby protected the host against lethal infection with enterohemorrhagic Escherichia coli O157:H7 [53]. A comparative study in children from Europe and rural Africa also suggested that enrichment of SCFA-producing bacteria such as Prevotella and Xylanibacter and increased fecal SCFA concentrations in the intestines of rural Africa children help to inhibit opportunistic pathogens such as Shigella and Escherichia and protect children against inflammation and noninfectious colonic diseases [54]. In this study, significant enrichment of SCFA-producing bacteria and decreased systemic inflammation in berberine-treated HFD-fed rats were also observed. Taken together, the beneficial effects of SCFAs, namely improving gut barrier functions, ameliorating systemic inflammation, or creating a non-permissive environment for pathogens, may mediate the pharmacological effects of berberine against obesity and insulin resistance-related metabolic abnormalities.
Accumulating evidence suggested that diet composition and calorie intake might play an important role in shaping the gut microbiota and modulating host phenotypes [55], [56], [57]. Ravussin et al. demonstrated that, apart from effectively decreasing body weight and fat mass, calorie restriction significantly increased the gut microbial diversity and the relative abundance of Allobaculum in HFD-fed animals, but not in those of NCD-fed [56]. In the current study, berberine significantly reduced the food intake of HFD-fed rats, which is in accordance with some previous reports [23], [58]. Similar to the calorie-restricted HFD-fed animals, selective enrichment of the genus Allobaculum was also observed in berberine-treated HFD-fed rats. However, selective increase of the genus Blautia was unique to berberine, and dramatic decrease of the microbial diversity and significant improvement of insulin sensitivity in berberine-treated HFD-fed rats was also observed. This indicates that although the reduction of food intake may contribute in part to the gut microbiota changes or host metabolic phenotype improvements, the direct modulating effects of berberine on gut microbiota may be more critical for the observed effects of berberine on the host health.
Modulation of gut microbiota with diet or drugs has been indicated to improve host metabolic phenotypes [4], [27]; however, whether the response of the gut microbiota to these environmental perturbations happens at the phylum or specific phylotype level remains controversial [59]. Ley et al. revealed that obese people had fewer Bacteroidetes and more Firmicutes than lean controls [4]. Upon dietary intervention, the ratio of Firmicutes to Bacteroidetes decreased over time as body weight decreased [4]. However, in another similar human study, no association was observed between obesity and the phylum-level changes of the gut microbiota [59]. In a previous report concerning the effects of berberine on the gut microbiota [27], RT-PCR was used to quantify the proportions of Firmicutes and Bacteroidetes to total bacteria, again indicating no significant association between these phylum-level changes with diet types or obesity in mice, although berberine significantly reduced the proportions of both Firmicutes and Bacteroidetes to total bacteria. Accumulating evidence indicates that variations in the species composition of the gut microbiota were related to human obesity [54], [59]. In a previous report [43], we also revealed that the development of metabolic syndromes was relevant to phylotype-specific changes of the gut microbiota. In the present study, we did not find a significant difference in the ratio of Firmicutes to Bacteroidetes between NCD- and HFD-fed rats. Berberine also displayed no significant influence on the proportions of Firmicutes and Bacteroidetes in rats under both feeding conditions. By using the MiWAS strategy, which combines the high-throughput pyrosequencing of 16S rRNA genes with multivariate statistics, we identified 268 key OTUs responding to berberine treatment from a total of 6720 OTUs observed in the samples. PLS regression modeling performed well in predicting the host phenotypes with the abundance data of those identified key OTUs, suggesting a possible close association between those phylotypes and host health phenotypes. In total, 143 and 87 of the 268 OTUs belong to the phyla Firmicutes and Bacteroidetes, respectively. Contrasting responses of the OTUs in the same phylum or even in the same genus were observed. Hence, the present study suggests that phylotype-level profiling of the variation of the gut microbiota by using the MiWAS strategy will serve as a reliable approach for demonstrating the relationship between the gut microbiota and host metabolic phenotypes under diet or drug perturbations.
In conclusion, our findings suggest that marked modulation of gut microbiota by berberine, namely inhibition of a wide range of intestinal microbes and enrichment of some SCFA producers, helps to alleviate systemic inflammation, at least in part, by reducing the antigen load to the host and elevating SCFA levels in the intestine and contributes to the beneficial effects of berberine against insulin resistance, obesity, diabetes, and other metabolic disorders. Functional metagenomic studies and molecular dissection of the host-microbiome cross-talk are needed to further elucidate the mechanisms of action of berberine [3]. This study also suggests that pharmacological or nutritional modulation of gut microbiota is an effective approach for preventive healthcare.
