Gene therapy restores adipose tissue and metabolic health in a pre-clinical mouse model of lipodystrophy

The authors are extremely grateful to Dr Donna MacCallum (University of Aberdeen) for assistance with AAV vector i.v. tail vein injections and Pat Bain (University of Aberdeen) for design and generation of the graphical abstract. The authors would also like to thank the staff at the University of Aberdeen’s Microscopy and Histology Core Facility and the Medical Research Facility for support with animal breeding and maintenance. This research was supported by funding from the EFSD/Lilly Young Investigator Research Award Programme, Wellcome Trust ISSF Fellowship Support Fund, and Diabetes UK RD Lawrence Fellowship (21/0006280) awarded to G.D.M. and Diabetes UK (18/0005884) awarded to J.J.R.Peer reviewedPublisher PD

Bscl2 Deficiency Does Not Directly Impair the Innate Immune Response in a Murine Model of Generalized Lipodystrophy

Funding: Work was supported by Diabetes UK (JJR;18/0005884, MD;17/0005621) the Medical Research Council (JJR; MR/L002620/1, MC/PC/15077), the British Heart Foundation (MD; PG/14/43/30889), The Agency for Science, Technology and Research, Singapore (A*STAR) (WH), The Wellcome Trust (ISSF Funding to GDM) and the European Union’s Horizon 2020 ERC consolidator award (MB:2016-726152-TYPHI).Peer reviewedPublisher PD

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

Abstract: 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

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities

Adipose Loss and Metabolic Disease Due to Seipin Deficiency: Systemic Versus Tissue Specific Effects

This thesis aimed to understand the molecular mechanisms via which BSCL2 disruption causes metabolic disease in congenital generalised lipodystrophy type 2. The results presented provide evidence that seipin has significant roles in adipose tissue, by regulating lipolysis, but also in pancreatic ß-cells where it influences insulin secretion. This work also reveals an interaction between seipin and glucagon-like peptide 1 receptor. This thesis demonstrates the therapeutic potential of liraglutide to treat metabolic disease in CGL2 patients

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

Additional file 3: of Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells

Figure S3. (related to Fig. 2). A. WM115 H2B-mCherry cell counting following 48 h of incubation with either apoptotic or CICD conditioned media. B. Cell counting for assessing the potential effect of doxycycline treatment on WM115 H2B-mCherry cells. C. Cell proliferation of WM115 H2B-mCherry cells in response to doxycycline treatment (1 μg/ml) was assessed by quantifying the optical density of methylene blue staining. D. Same as in C, representative methylene blue-stained cells are shown. E-F. Incucyte analysis for the proliferation of WM239A H2B-mCherry cells grown in the presence of APO (E) or CICD (F) conditioned media. n = 3 independent experiments; mean values +/− s.e.m. G-H. Cell proliferation of WM239A H2B-mCherry cells grown for 48 h in APO or CICD conditioned media was measured by quantifying the optical density (O.D.) of methylene blue staining (G), while in H same approach was done to exclude a potential effect of DOX treatment on cell proliferation. I. Incucyte analysis for the cell death induction (SYTOX Green exclusion) triggered by CICD conditioned media in WM239A H2B-mCherry cells. Actinomycin D treatment (1 μM) is used as positive control for cell death induction. A representative experiment is shown. J Immunofluorescence representative images for the nuclear translocation of p65 in WM115 H2B-mCherry cells grown in presence of apoptotic or CICD conditioned media. Treatment for 3 h with TNFα (20 ng/ml) was used as positive control for p65 nuclear translocation. (PDF 706 kb

Additional file 4: of Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells

Figure S4. (related to Fig. 2). A. SYTOX Green analysis for the cell death kinetics allowing to identify the 24 and 40 h time frame where apoptosis and CICD, respectively, reach the same extent of execution. A representative experiment is shown. B-C. WM115 H2B-mCherry cells were grown in either apoptotic (obtained at 24 h following doxycycline treatment) (B) or CICD conditioned media (collected 40 h after triggering CICD) (C) and the number of H2B-mCherry positive cells was automatically counted using an Incucyte imager. n = 3 independent experiments; mean values +/− s.e.m. D-E. Representative images of methylene blue staining (D) and the optical density quantification (E) for assessing the proliferation of WM115 H2B-mCherry cells grown as described in B-C. F-I. Same as in B-E, the proliferation of WM239A H2B-mCherry cells is determined by either Incucyte imager or methylene blue staining. For the Incucyte analysis, n = 4–5 independent experiments; mean values +/− s.e.m. (PDF 329 kb

Additional file 2: of Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells

Figure S2. (related to Fig. 2). A-B. Detection of PGE2 by ELISA in apoptotic (A) or CICD (B) conditioned media obtained in the presence or absence of celecoxib (5 μM). C. Incucyte analysis for the proliferation of WM115 H2B-mCherry cells grown in apoptotic conditioned media obtained in the presence or absence of celecoxib (5 μM). n = 4 independent experiments; mean values +/− s.e.m. (PDF 142 kb

Additional file 1: of Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells

Figure S1. (related to Fig. 1). A. Immunoblotting validation of CRISPR/Cas9-mediated APAF-1 KO in 501Mel cells. Actin is used as loading control. B. Cell death kinetics for apoptosis and CICD triggered by doxycycline treatment in tetON BAX expressing 501Mel. A representative experiment is shown. C. Representative SYTOX Green positive staining for either apoptotic or cells undergoing CICD at 24 h in tetON BAX expressing 501Mel cells. D. Cytokine antibody array immunoblotting for CICD conditioned media. tetON BAX-expressing APAF-1 KO WM115 cells were treated with DOX (1 μg/ml) for 24 h then the conditioned media was processed for the cytokine antibody microarray. E. Relative quantification of selected cytokines in CICD conditioned media. For D and E, data is presented from one representative experiment (out of two). (PDF 461 kb