Deletion of the Mammalian INDY Homologue in Mice Mimics Aspects of Dietary Restriction and Protects Against Diet and Age-Induced Adiposity and Insulin Resistance

Reduction in expression of the INDY gene in Drosophila melanogaster and Caenorhabditis elegans prolongs life span, and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which INDY does this is unknown. In order to examine the effect that INDY might have on energy metabolism and insulin sensitivity in mammals we created the first knock out mouse model of the mammalian INDY homologue SLC13A5. Here, we show that deletion of the mammalian homologue of INDY, SLC13A5 (mINDY) in mice (mINDY-/- mice) have increased mitochondrial biogenesis, hepatic lipid oxidation, and energy expenditure, but decreased hepatic de novo lipogenesis. Loss of mIndy activates hepatic AMPK, which induces PGC-1?, inhibits ACC-2, and reduces SREBP-1c levels. In contrast to wild- type mice, mINDY-/- mice have reduced body weight and are protected from insulin resistance that evolves with high-fat feeding and aging. These studies demonstrate that mIndy functions as a novel regulator of mammalian energy metabolism and suggest that mIndy might be a novel therapeutic target for the treatment of obesity and type 2 diabetes. An SLC13A5 knockout mouse model (mINDY-/-) was generated. The litters produced when heterozygous (KO/+) mINDY mice (mINDY+/-) were bred, contained the expected ratio of Illumina Mouse-ref8Indy wild-type (mINDY+/+), mINDY+/- mice, and homozygous mIndy (mINDY-/-) pups, indicating no substantial embryonic lethality. In wild-type mice, Indy mRNA expression was highest in liver, and very low in white adipose tissue, brown adipose tissue, skeletal muscle, small and large intestines and the pancreas confirming previous mINDY expression levels in rodents, and humans. In the liver, mINDY mRNA expression was completely abolished in the INDY-/- mice and ~50% reduced in heterozygous mINDY+/- mice. Total RNA from the Liver tissue of 5 replicate Wt and 5 replicate Knockout mice were labeled and hybridized to Illumina Mouse-ref8 v2 bead arrays. Analysis of gene expression was carried out by Hierarchy clustering/K-means clustering and Principal Components Analysis (PCA), as well as The Parameterized Analysis of Gene Enrichment (PAGE) and Ingenuity Pathways Analysis IPA (Ingenuity Systems, CA, USA).在黑腹果蝇（Drosophila melanogaster）与秀丽隐杆线虫（Caenorhabditis elegans）中，INDY基因（INDY gene）的表达下调可延长生物体寿命；而在黑腹果蝇中，该基因表达下调还能以类似热量限制（caloric restriction）的方式增强线粒体生物发生（mitochondrial biogenesis）。不过，INDY介导此类效应的细胞机制至今仍不明晰。为探究INDY对哺乳动物能量代谢与胰岛素敏感性的潜在影响，我们构建了首个靶向哺乳动物INDY同源物SLC13A5的基因敲除（knockout）小鼠模型。本研究证实，敲除小鼠体内的INDY哺乳动物同源基因SLC13A5（下称mINDY）后（mINDY-/-小鼠），其线粒体生物发生、肝脏脂质氧化及能量消耗均显著升高，而肝脏的从头脂肪生成（de novo lipogenesis）则受到抑制。mIndy的缺失会激活肝脏AMP活化蛋白激酶（AMPK），该激酶可诱导PGC-1α的表达、抑制ACC-2的活性并降低SREBP-1c的蛋白水平。与野生型（wild-type）小鼠相比，mINDY-/-小鼠体重更低，且可免受高脂喂养与衰老过程中诱发的胰岛素抵抗（insulin resistance）。本研究表明，mIndy是哺乳动物能量代谢的新型调控因子，提示其有望成为治疗肥胖与2型糖尿病（type 2 diabetes）的全新治疗靶点。 我们成功构建了SLC13A5基因敲除小鼠模型（mINDY-/-）。将杂合子（KO/+）mINDY小鼠（mINDY+/-）繁育所得的子代中，Illumina Mouse-ref8Indy野生型（mINDY+/+）、mINDY+/-及纯合子mIndy（mINDY-/-）幼崽的比例符合孟德尔遗传预期，说明该基因敲除未引发显著的胚胎致死现象。在野生型小鼠体内，Indy mRNA在肝脏中表达水平最高，而在白色脂肪组织、棕色脂肪组织、骨骼肌、大小肠及胰腺中表达量极低，该结果验证了此前针对啮齿类与人类体内mINDY表达模式的研究结论。在肝脏组织中，mINDY-/-小鼠的mINDY mRNA表达完全被敲除，杂合子mINDY+/-小鼠的该基因表达量则降低约50%。 我们对5只野生型小鼠与5只基因敲除小鼠的肝脏组织提取总RNA（total RNA）进行标记，并将标记产物与Illumina Mouse-ref8 v2微珠芯片（Illumina Mouse-ref8 v2 bead arrays）进行杂交。基因表达分析采用层次聚类（Hierarchy clustering）/K均值聚类（K-means clustering）、主成分分析（Principal Components Analysis, PCA）、基因富集参数化分析（Parameterized Analysis of Gene Enrichment, PAGE）以及Ingenuity通路分析（Ingenuity Pathways Analysis, IPA，美国加利福尼亚州Ingenuity Systems公司）完成。