Gut microbiota and obesity: studies in gnotobiotic mice

描述(申请人提供)：开发治疗肥胖症的非认知方法势在必行。我们一直在利用诺生菌小鼠来检查肠道中人-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。成年无菌(GF)C57B1/6(B6)小鼠在常规饲养的小鼠的盲肠微生物群中定居(“常规化”)，可使体内总脂肪含量和白色脂肪组织(WAT)重量增加60%。尽管食物消耗量减少，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。成脂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/SREBP1成脂基因靶点的反式激活有关。WAT肥大与LPL活性增加和肠道特异性转录抑制FIAF(编码一种分泌的LPL抑制物)有关。此外，GF Fiaf基因敲除小鼠具有更高的Wat LPL活性和相同的体脂含量，与常规的(Fiaf抑制的)野生型(Wt)小鼠相同：它们的脂肪储存不会随着常规的进一步增加而进一步增加。这些结果提出了以下可检验的假说：(A)微生物对原本无法消化的膳食多糖(PS)进行加工提供单糖，从而导致ChREBP，并可能，SREBP-1-刺激肝脏脂肪生成：(B)微生物抑制肠道FIAF，结合生脂反应，促进LPL介导的脂肪储存的增加；(C)增加FIAF的表达和/或活性应促进瘦肉。目的1-利用wt B6小鼠确定膳食碳水化合物和微生物生态在微生物区系诱导的脂肪储存中的作用。以等热量高PS/低脂、高脂/低PS、高糖/低脂饲料喂养GF和常规小鼠，测定其对体脂含量、VO2、Feptin/葡萄糖/胰岛素、Wat LPL和肠/肝/肌肉能量代谢的影响。微生物需求将通过与简化的8成员微生物区系(改变的舍德勒植物群)的全部或部分成分进行定居来评估。目的2-通过使具有ChREBP基因敲除、SREBP-1c基因敲除、ChREBP和SREBP-1c联合缺陷或肝细胞特异性FasL基因敲除的GF小鼠常规化，来确定微生物区系相关的肝脏生脂反应的贡献。目的3-将一种在小肠上皮细胞中组成性表达的转基因导入Fiaf-/-小鼠。我们预测，这些小鼠将表现为更瘦的GF wt小鼠，无论它们是否已经被殖民[增加Fiaf表达(或活动)的治疗操作是否会促进瘦身的概念验证基因测试]。

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].