Gut microbiota and obesity: studies in gnotobiotic mice

描述（由申请人提供）：发展非认知方法治疗肥胖势在必行。我们一直在用益生小鼠来研究肠道中人-细菌共生的意义，并发现肠道微生物群对脂肪储存有显著的影响。将成年无菌（GF） C57B1/6 （B6）小鼠与从常规饲养小鼠中获取的盲肠微生物群定殖（“常规化”）可使总体脂肪含量和白色脂肪组织（WAT）重量增加60%。尽管食物消耗减少，代谢率增加，但这种快速增加是持续的，并伴随着瘦素水平的增加，胰岛素抵抗和肝脏脂肪生成的增加。脂肪生成反应与碳水化合物反应元件结合蛋白（ChREBP）的核输入增加、胰岛素反应性SREBP-1的适度增加以及ChREBP/SREBPl脂肪生成基因靶点的反式激活有关。WAT肥大与LPL活性增加和Fiaf（编码分泌的LPL抑制剂）的肠道特异性转录抑制有关。此外，GF Fiaf敲除小鼠具有更高的WAT LPL活性和与“常规”（Fiaf抑制）野生型（wt）幼崽相同的体脂含量：它们的脂肪储存不会随着常规而进一步增加。这些结果提出了以下可验证的假设：(a)微生物处理难以消化的膳食多糖（PS）提供单糖，导致ChREBP，并可能是SREBP-1-刺激肝脏脂肪生成；(b)肠道Fiaf的微生物抑制，结合脂肪生成反应，促进了脂蛋白介导的脂肪细胞脂肪储存的增加；(c)增加Fiaf的表达和/或活性应促进瘦。目的1-使用wt B6小鼠来确定饮食碳水化合物和微生物生态对微生物诱导的脂肪储存的作用。GF和常规小鼠将被喂食等热量高PS/低脂肪。高脂肪/低PS或高糖/低脂肪饮食，以及对体脂肪含量、VO2、胃泌素/葡萄糖/胰岛素、WAT LPL和肠/肝/肌肉能量代谢的影响。微生物需求将通过用一个简化的8成员微生物群（改变的Schaedler菌群）的全部或部分成分定植来评估。目的2：通过常规GF小鼠ChREBP敲除、srebp - 1c敲除、ChREBP和srebp - 1c联合缺失或肝细胞特异性Fasl敲除，确定微生物群相关的肝脏脂肪生成反应的贡献。目的3-将小肠上皮组成性表达的转基因引入Fiaf-/-小鼠。我们预测这些小鼠将表现出更瘦的GF wt小鼠，无论它们是否被定植[增加Fiaf表达（或活性）的治疗操作是否会促进瘦的概念证明基因测试]。

DESCRIPTION (provided by applicant): Developing non-cognitive approaches for treating obesity is imperative. We have been using gnotobiotic mice to examine the significance of human-bacterial symbioses in the gut, and discovered that the intestinal microbiota has a remarkable effect on fat storage. Colonization ('conventionalization') of adult germ-free (GF) C57B1/6 (B6) mice with a cecal microbiota harvested from conventionally-raised mice produces a 60% increases in total body fat content and white adipose tissue (WAT) weight. This rapid increase occurs despite decreased chow consumption and increased metabolic rate, is sustained, and accompanied by increased leptin levels, insulin resistance, and increased hepatic lipogenesis. The lipogenic response is associated with increased nuclear import of carbohydrate response element binding protein (ChREBP), modest increases in insulin-responsive SREBP-1, and trans-activation of ChREBP/SREBPl lipogenic gene targets. WAT hypertrophy is associated with increased LPL activity and intestine-specific transcriptional suppression of Fiaf (encodes a secreted LPL inhibitor). Moreover, GF Fiaf knockout mice have higher WAT LPL activity and the same body fat content as 'conventionalized' (Fiaf-suppressed) wild-type (wt) littermates: their fat stores are not increased further with conventionalization. These results suggest the following testable hypothesis: (a) microbial processing of otherwise indigestible dietary polysaccharides (PS) provides monosaccharides that lead to ChREBP, and possibly, SREBP-1- stimulation of hepatic lipogenesis: (b) microbial suppression of intestinal Fiaf, combined with the lipogenic response, promotes LPL-mediated increases in adipocyte fat storage; (c) increasing Fiaf expression and/or activity should promote leanness. Aim 1- Use wt B6 mice to determine the role of dietary carbohydrates and microbial ecology on microbiota-induced fat storage. GF and conventionalized mice will be fed isocaloric high PS/low fat, .high fat/low PS, or high sugar/low fat diets, and the effects on body fat content, VO2, feptin/glucose/insulin, WAT LPL, and intestine/liver/muscle energy metabolism assayed. Microbial requirements will be assessed by colonization with all or some components of a simplified 8-member microbiota (Altered Schaedler Flora). Aim 2- Determine the contribution of the microbiota-associated hepatic lipogenic response by conventionalizing GF mice with a ChREBP knockout, a SREBP-1 c knockout, combined ChREBP and SREBP-1 c deficiencies, or a hepatocyte-specific Fasl knockout. Aim 3- Introduce a transgene constitutively expressed in the small intestinal epithelium into Fiaf-/- mice. We predict that these mice will phenocopy leaner GF wt mice, whether or not they have been colonized [proof-of-concept genetic test of whether therapeutic manipulations that increase Fiaf expression (or activity) will promote leanness].