HOXC10 Suppresses Browning to Maintain White Adipocyte Identity.

Promoting beige adipocyte development within white adipose tissue (WAT) is a potential therapeutic approach to staunch the current obesity epidemic. Previously, we identified homeobox-containing transcription factor HOXC10 as a suppressor of browning in subcutaneous WAT. Here, we provide evidence for the physiological role of HOXC10 in regulating WAT thermogenesis. Analysis of an adipose-specific HOXC10 knockout mouse line with no detectable HOXC10 in mature adipocytes revealed spontaneous subcutaneous WAT browning, increased expression of genes involved in browning, increased basal rectal temperature, enhanced cold tolerance, and improved glucose homeostasis. These phenotypes were further exacerbated by exposure to cold or a ß-adrenergic stimulant. Mechanistically, cold and ß-adrenergic exposure led to reduced HOXC10 protein level without affecting its mRNA level. Cold exposure induced cAMP-dependent protein kinase-dependent proteasome-mediated degradation of HOXC10 in cultured adipocytes, and shotgun proteomics approach identified KCTD2, 5, and 17 as potential E3 ligases regulating HOXC10 proteasomal degradation. Collectively, these data demonstrate that HOXC10 is a gatekeeper of WAT identity, and targeting HOXC10 could be a plausible therapeutic strategy to unlock WAT thermogenic potentials.

Oligomers of the lipodystrophy protein seipin may co-ordinate GPAT3 and AGPAT2 enzymes to facilitate adipocyte differentiation.

Seipin deficiency causes severe congenital generalized lipodystrophy (CGL) and metabolic disease. However, how seipin regulates adipocyte development and function remains incompletely understood. We previously showed that seipin acts as a scaffold protein for AGPAT2, whose disruption also causes CGL. More recently, seipin has been reported to promote adipogenesis by directly inhibiting GPAT3, leading to the suggestion that GPAT inhibitors could offer novel treatments for CGL. Here we investigated the interactions between seipin, GPAT3 and AGPAT2. We reveal that seipin and GPAT3 associate via direct interaction and that seipin can simultaneously bind GPAT3 and AGPAT2. Inhibiting the expression of seipin, AGPAT2 or GPAT3 led to impaired induction of early markers of adipocyte differentiation in cultured cells. However, consistent with normal adipose mass in GPAT3-null mice, GPAT3 inhibition did not prevent the formation of mature adipocytes. Nonetheless, loss of GPAT3 in seipin-deficient preadipocytes exacerbated the failure of adipogenesis in these cells. Thus, our data indicate that GPAT3 plays a modest positive role in adipogenesis and argue against the potential of GPAT inhibitors to rescue white adipose tissue mass in CGL2. Overall, our study reveals novel mechanistic insights regarding the molecular pathogenesis of severe lipodystrophy caused by mutations in either seipin or AGPAT2.

Discovering metabolic disease gene interactions by correlated effects on cellular morphology.

OBJECTIVE: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation. METHODS: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. RESULTS: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions. CONCLUSIONS: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.

Obesity-associated gene TMEM18 has a role in the central control of appetite and body weight regulation.

An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is TMEM18, although the molecular mechanisms mediating these effects remain entirely unknown. Tmem18 expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of Tmem18 in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of Tmem18 in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that TMEM18 itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.

Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis.

OBJECTIVE: Disruption of the genes encoding either seipin or 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) causes severe congenital generalized lipodystrophy (CGL) in humans. However, the function of seipin in adipogenesis remains poorly defined. We demonstrated recently that seipin can bind the key adipogenic phosphatidic acid (PA) phosphatase lipin 1 and that seipin forms stable dodecamers. As AGPAT2 generates PA, the substrate for lipin 1, we investigated whether seipin might bind both enzymes of this lipid biosynthetic pathway, which is required for adipogenesis to occur. METHODS: We employed co-immunoprecipitation and immunofluorescence methods to determine whether seipin can interact with AGPAT2 and the consequences of this in developing adipocytes. Atomic force microscopy was used to determine whether these interactions involved direct association of the proteins and to define the molecular architecture of these complexes. RESULTS: Our data reveal that seipin can bind AGPAT2 during adipogenesis and that stabilizing this interaction during adipogenesis can increase the nuclear accumulation of PPARγ. Both AGPAT2 and lipin 1 can directly associate with seipin dodecamers, and a single seipin complex can simultaneously bind both AGPAT2 and lipin with a defined orientation. CONCLUSIONS: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL. Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis. Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

Analyzing the functions and structure of the human lipodystrophy protein seipin.

Disruption of the gene BSCL2, which encodes the protein seipin, causes severe generalized lipodystrophy in humans with a near complete absence of adipose tissue. Moreover, cell culture studies have demonstrated that seipin plays a critical cell-autonomous role in adipocyte differentiation. These observations reveal seipin as a critical regulator of human adipose tissue development; however, until recently very little has been known about the potential molecular functions of this intriguing protein. Despite significant recent interest in the function of seipin, our understanding of its molecular role(s) remains limited. The topology of seipin and lack of evidence for any enzymatic domains or activity indicate that it may act principally as a scaffold for other proteins or play a structural role in altering membrane curvature and/or budding. Work in this area has been hampered by several factors, including the lack of homology that might imply testable functions, the poor availability of antibodies to the endogenous protein and the observation that this hydrophobic ER membrane-resident protein is difficult to analyze by standard Western blotting techniques. Here we summarize some of the techniques we have applied to investigate the association of seipin with a recently identified binding partner, lipin 1. In addition, we describe the use of atomic force microscopy (AFM) to image oligomers of the seipin protein. We believe that AFM will offer a valuable tool to examine the association of candidate binding proteins with the seipin oligomer.

Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms.

AIMS/HYPOTHESIS: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL. METHODS: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations. RESULTS: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not. CONCLUSIONS/INTERPRETATION: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1.

Disruption of the gene BSCL2 causes a severe, generalised lipodystrophy, demonstrating the critical role of its protein product, seipin, in human adipose tissue development. Seipin is essential for adipocyte differentiation, whilst the study of seipin in non-adipose cells has suggested a role in lipid droplet formation. However, its precise molecular function remains poorly understood. Here we demonstrate that seipin can inducibly bind lipin 1, a phosphatidic acid (PA) phosphatase important for lipid synthesis and adipogenesis. Knockdown of seipin during early adipogenesis decreases the association of lipin 1 with membranes and increases the accumulation of its substrate PA. Conversely, PA levels are reduced in differentiating cells by overexpression of wild-type seipin but not by expression of a mutated seipin that is unable to bind lipin 1. Together our data identify lipin as the first example of a seipin-interacting protein and reveals a novel molecular function for seipin in developing adipocytes.