New therapeutic targets for noncognitive reductions in energy intake, absorption, or storage are crucial given the worldwide epidemic of obesity. The gut microbial community (microbiota) is essential for processing dietary polysaccharides. We found that conventionalization of adult germ-free (GF) C57BL/6 mice with a normal microbiota harvested from the distal intestine (cecum) of conventionally raised animals produces a 60% increase in body fat content and insulin resistance within 14 days despite reduced food intake. Studies of GF and conventionalized mice revealed that the microbiota promotes absorption of monosaccharides from the gut lumen, with resulting induction of de novo hepatic lipogenesis. Fasting-induced adipocyte factor (Fiaf), a member of the angiopoietin-like family of proteins, is selectively suppressed in the intestinal epithelium of normal mice by conventionalization. Analysis of GF and conventionalized, normal and Fiaf knockout mice established that Fiaf is a circulating lipoprotein lipase inhibitor and that its suppression is essential for the microbiota-induced deposition of triglycerides in adipocytes. Studies of Rag1 -/- animals indicate that these host responses do not require mature lymphocytes. Our findings suggest that the gut microbiota is an important environmental factor that affects energy harvest from the diet and energy storage in the host.

THE endothelial cell-specific vascular endothelial growth factor (VEGF)^1–5 and its cellular receptors Flt-1 (refs 6,7) and Flk-1 (refs 8,9) have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis^10,11 and the impaired vessel formation in Flk-1 (ref. 12) and Flt-1 (ref. 13) deficient embryos. However, because Flt-1 also binds placental growth factor^14,15, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF^+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES)^16,17, and even more impaired in homozygous VEGF-deficient (VEGF^-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F₁-VEGF^-/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inacti-vation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.

Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell <==> tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.

Obesity-associated noncoding sequences within FTO are functionally connected with IRX3, and long-range enhancers in this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval is part of IRX3 regulation; Irx3-deficient mice have lower body weight and are resistant to diet-induced obesity, suggesting IRX3 as a novel determinant of body mass and composition. The search for genetic correlates of obesity has highlighted a noncoding region in the FTO gene: variations within this intron are associated with increased risk for obesity and type 2 diabetes. Although the biological actions of FTO have been intensely scrutinized, it is still not clear how these genetic variants influence FTO expression and biology. This paper shows that these noncoding sequences are functionally connected — at megabase distances — with the homeobox gene IRX3. The obesity-associated interval appears to belong to the regulatory functions of IRX3, rather than FTO. In addition, mice lacking Irx3 have reduced body weight and are resistant to diet-induced obesity. Taken together, the data suggest that IRX3 is an important metabolic regulator associated with human obesity and type 2 diabetes. Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D)1,2,3. Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes4,5,6. However, no direct connection between the obesity-associated variants and FTO expression or function has been made7,8,9. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.