NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis.

Adipose tissue is central to regulation of energy homeostasis. Adaptive thermogenesis, which relies on mitochondrial oxidative phosphorylation (Ox-Phos), dissipates energy to counteract obesity. On the other hand, chronic inflammation in adipose tissue is linked to type 2 diabetes and obesity. Here, we show that nuclear factor I-A (NFIA), a transcriptional regulator of brown and beige adipocytes, improves glucose homeostasis by upregulation of Ox-Phos and reciprocal downregulation of inflammation. Mice with transgenic expression of NFIA in adipocytes exhibited improved glucose tolerance and limited weight gain. NFIA up-regulates Ox-Phos and brown-fat-specific genes by enhancer activation that involves facilitated genomic binding of PPARγ. In contrast, NFIA in adipocytes, but not in macrophages, down-regulates proinflammatory cytokine genes to ameliorate adipose tissue inflammation. NFIA binds to regulatory region of the Ccl2 gene, which encodes proinflammatory cytokine MCP-1 (monocyte chemoattractant protein-1), to down-regulate its transcription. CCL2 expression was negatively correlated with NFIA expression in human adipose tissue. These results reveal the beneficial effect of NFIA on glucose and body weight homeostasis and also highlight previously unappreciated role of NFIA in suppressing adipose tissue inflammation.

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes.

Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.

NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation.

The transcription factor nuclear factor I-A (NFIA) is a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA (which we call pro#3 domain) are required for the transcriptional activity of NFIA. Full-length NFIA-but not deletion mutant lacking pro#3 domain-rescued impaired expression of PPARγ, the master transcriptional regulator of adipogenesis and impaired adipocyte differentiation in NFIA-knockout cells. Mechanistically, the ability of NFIA to penetrate chromatin and bind to the crucial Pparg enhancer is mediated through pro#3 domain. However, the deletion mutant still binds to Myod1 enhancer to repress expression of MyoD, the master transcriptional regulator of myogenesis as well as proximally transcribed non-coding RNA called DRReRNA, via competition with KLF5 in terms of enhancer binding, leading to suppression of myogenic gene program. Therefore, the negative effect of NFIA on the myogenic gene program is, at least partly, independent of the positive effect on PPARγ expression and its downstream adipogenic gene program. These results uncover multiple ways of action of NFIA to ensure optimal regulation of brown and beige adipocyte differentiation.

Epidemiological Study of Fusarium Species Causing Invasive and Superficial Fusariosis in Japan.

In Japan, Fusarium species are known etiological agents of human fungal infection; however, there has been no report of a large-scale epidemiological study on the etiological agents of fusariosis. A total of 73 Fusarium isolates from patients with invasive fusariosis (IF, n= 36) or superficial fusariosis (SF, n= 37), which were obtained at hospitals located in 28 prefectures in Japan between 1998 and 2015, were used for this study. Fusarium isolates were identified using Fusarium- and Fusarium solani species complex (FSSC) -specific real-time PCR and partial DNA sequences of the elongation factor-1 alpha (EF-1α) gene and the nuclear ribosomal internal transcribed spacer (ITS) region. FSSC was predominately isolated from both patients with IF and SF (IF, 77.8% and SF, 67.6%). Distribution of the phylogenetic species of FSSC isolates from patients with IF and SF exhibited different spectra; specifically, F. keratoplasticum (FSSC 2) (25.0%) was the most frequent isolate from patients with IF, whereas F. falciforme (FSSC 3+4) (32.4%) was the most frequent isolate from patients with SF. Fusarium sp. (FSSC 5) was the second most frequent isolate from both patients with IF and SF (IF, 22.2% and SF, 24.3%). Notably, F. petroliphilum (FSSC 1) was isolated only from patients with IF. Each species was isolated from a broad geographic area, and an epidemic was not observed. This is the first epidemiological study of Fusarium species causing IF and SF in Japan.