Gut microbiota and obesity: studies in gnotobiotic mice

描述（由申请人提供）：开发用于治疗肥胖的非认知方法势在必行。我们一直在使用gnotobiotic小鼠来研究肠道中人类细菌共生的重要性，并发现肠道微生物群对脂肪储存有显着影响。成年无菌（GF）C57 B1/6（B6）小鼠与从常规饲养的小鼠收获的盲肠微生物群的定殖（“常规化”）产生总身体脂肪含量和白色脂肪组织（WAT）重量的60%增加。尽管食物消耗减少和代谢率增加，但这种快速增加是持续的，并伴随着瘦素水平增加、胰岛素抵抗和肝脏脂肪生成增加。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、胰岛素响应性SREBP-I的适度增加以及ChREBP/SREBPl脂肪生成基因靶标的反式激活相关。WAT肥大与增加的LPL活性和Fiaf（编码分泌的LPL抑制剂）的丝氨酸特异性转录抑制相关。此外，GF Fiaf敲除小鼠具有更高的WAT LPL活性和与“常规化”（Fiaf抑制）野生型（wt）同窝出生小鼠相同的体脂含量：它们的脂肪储存不会随着常规化而进一步增加。这些结果提示了以下可检验的假设：（a）微生物处理其他难消化的膳食多糖（PS）提供单糖，其导致ChREBP，并且可能导致SREBP-1-刺激肝脂肪生成：（B）微生物抑制肠FAF，与脂肪生成反应结合，促进LPL介导的脂肪细胞脂肪储存的增加;（c）增加Fiaf表达和/或活性应促进瘦肉。目的1-使用野生型B6小鼠来确定膳食碳水化合物和微生物生态学对微生物群诱导的脂肪储存的作用。将GF和常规小鼠喂食等热量的高PS/低脂、高脂/低PS或高糖/低脂饮食，并测定对体脂含量、VO 2、feptin/葡萄糖/胰岛素、WAT LPL和肠/肝/肌肉能量代谢的影响。将通过简化的8成员微生物群（改变的Schaedler植物群）的全部或部分组分定殖来评估微生物要求。目的2-通过常规化具有ChREBP敲除、SREBP-Ic敲除、ChREBP和SREBP-Ic组合缺陷或肝细胞特异性FasL敲除的GF小鼠来确定微生物群相关的肝脂肪生成应答的贡献。目的3-将在小肠上皮中组成型表达的转基因导入Fiaf-/-小鼠。我们预测这些小鼠将表型模仿更瘦的GF wt小鼠，无论它们是否已经被定殖[增加Fiaf表达（或活性）的治疗操作是否会促进瘦的概念验证遗传测试]。

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].