Tae-Hwa Chun

Lipodystrophy (LD) syndromes are characterized by loss of adipose tissue (AT), leading to insulin resistance and the development of metabolic syndrome. We identified a heterozygous nonsense variant in early B cell factor 2 ( EBF2 ) (Chr8:26033143C>A, NM_022659.4: c.493G>T, p.E165X) in a patient with atypical partial LD (PLD). The EBF family is crucial for the differentiation and function of various mesenchymal tissues. Through in vitro and in vivo disease models, we discovered that this variant limited adipocyte differentiation and hampered AT remodeling. Heterozygous-knockin ( Ebf2 ^E165X/+ ) mice showed restricted adipogenesis and defective extracellular matrix remodeling during the post-weaning period and high-fat diet–induced (HFD-induced) AT expansion. A HFD caused abnormal adipocyte hypertrophy, decreased the expression of adiponectin and leptin, and led to glucose intolerance in Ebf2 ^E165X/+ mice. Furthermore, key mitochondrial genes involved in fatty acid metabolism and oxidation were downregulated specifically in Ebf2 ^E165X/+ AT. Our results suggest that EBF2 dysfunction caused by this nonsense variant drives disease pathology, establishing a connection between EBF2 disruption and an atypical form of LD.

Obesity‐associated beta cell dysfunction is a crucial factor in the pathogenesis of Type 2 Diabetes (T2D), driven by a dysfunctional crosstalk between adipose tissues and pancreatic beta cells. Traditional culture systems cannot capture this crosstalk in a dynamic and controlled manner. A flow‐enabled microfluidic is developed with an embedded micro‐Tesla (µTesla) pump to assess the adipocyte‐beta cell crosstalk. This recirculating system allows them to study the transport of soluble‐factor‐based between cultured 3T3 L1 adipocytes and INS1 beta cells. It is found that flow‐enabled incubation with elevated glucose and insulin increased the levels of adipocyte‐derived secretions of IL‐6, TNF‐α, and adiponectin in the media. In turn, adipocyte‐derived IL‐6 enhanced beta‐cell insulin secretions in the same media, establishing a feed‐forward loop. This mechanism can contribute to the hyperinsulinemia and pro‐inflammatory conditions characteristic of obesity‐related T2D. The findings highlight the advantages of flow‐enabled microfluidics in modeling adipocyte‐beta cell crosstalk in obesity, providing novel insights into obesity‐associated beta cell dysfunction.

Pulmonary disorders affect 40%–80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragm muscle weakness. Increased intradiaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs) — mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing the effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 -knockout ( Thbs1 ^–/– ) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGF-β–related expression signatures and augmentation of a Thy1 -expressing subpopulation previously linked to type 2 diabetes. Despite similar weight gain, Thbs1 ^–/– mice were protected from these transcriptomic changes and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1 ^–/– diaphragms maintained normal contractile force and motion after DIO challenge. THBS1 is therefore a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition and a potential therapeutic target in obesity-associated respiratory dysfunction.

Migration and scratch assays are helpful tools to investigate wound healing and tissue regeneration processes, especially under disease conditions such as diabetes. However, traditional migration (injury-free) assays and scratch (with injury) assays are limited in their control over cellular environments and provide only simplified read-outs of their results. On the other hand, microfluidic-based cell assays offer a distinct advantage in their integration and scalability for multiple modalities and concentrations in a single device. Additionally, in situ stimulation and detection helps to avoid variabilities between individual bioassays. To realize an enhanced, smarter migration assay, we leveraged our multilayered oxygen gradient (1-16%) to study HaCaT migrations in diabetic conditions with spatial and metabolic read-outs. An analysis of the spatial migration over time observed a new dynamic between hypoxia (at 4.16-9.14% O2) and hyperglycemia. Furthermore, in situ adenosine triphosphate (ATP) and reactive oxygen species (ROS) responses suggest that this dynamic represents a switch between stationary versus motile modes of metabolism. Thus, elevated glucose and hypoxia are synergistic triggers of this switch under disease conditions. These findings illustrate the benefits of spatial microfluidics for modeling complex diseases such as hypoxia and diabetes, where multimodal measurements provide a more deterministic view of the underlying processes.

Disclosure: M.C. Foss de Freitas: Advisory Board Member; Self; PTC Therapeutics. N. Wys: None. M. Udler: None. L. Pais: None. A. Monteiro da Rocha: None. O.A. MacDougald: None. E.A. Oral: Consulting Fee; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Novo Nordisk, Fractyl Laboratories, Rhythm pharmaceuticals, GID Dynamics. Other; Self; Aegerion Pharmaceuticals. T. Chun: None. Objective/Goals: Partial Lipodystrophy is characterized by the gradual loss of adipose tissues, which progresses after birth. The genetic and molecular underpinning of many cases of partial lipodystrophy have not been fully understood. Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy, we initiated deep sequencing for our patients with partial lipodystrophy syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. Methods: We performed loss-of-function and gene rescue experiments using 3T3-L1 and human primary subcutaneous preadipocytes. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen). After lentivirus transfection (Full-length EBF2 or truncated variant EBF2), cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone, insulin and isobutyl methylxanthine. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. Results: The patient was found to carry a heterozygous nonsense mutation in exon 6 of 16 (NM_022659.4) of EBF2, causing the premature termination of the protein at amino acid position 165 (EBF2 8:26033143 C>A (hg38), c.493G>T, p.Glu165Ter). Adipogenesis was significantly suppressed in 3T3-L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed a marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 p.Glu165Ter did not. Of note, 3T3-L1 cells transfected with the EBF2 variant impaired adipogenesis, suggesting an inhibitory effect of the EBF2 variant on adipogenesis. We next confirmed the inhibition in adipogenesis with the EBF2 variant in primary human preadipocytes from subcutaneous adipose tissues. Using lentiviral gene transfer in human preadipocytes, we observed that the full-length EBF2 increased the gene expression of PPARg and FABP4 while the truncated EBF2 variant did not. Conclusion: Our data suggest that EBF2 is indispensable for adipogenesis of mouse and human preadipocytes. The truncating variant of EBF2 p.Glu165Ter likely plays a pathogenic role in the partial lipodystrophy presentation of this patient. Whole genome (or exome) sequencing undertaken in patients with partial lipodystrophy and subsequent biological functional analysis may lead to identification of novel mechanisms driving novel discoveries. Presentation: Friday, June 16, 2023

Pulmonary disorders impact 40-80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragmatic weakness. Increased intra-diaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs)—mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 knockout ( Thbs1 ^-/- ) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGFβ-related expression signatures, and augmentation of a Thy1 -expressing sub-population previously linked to type 2 diabetes. Despite similar weight gain, Thbs1 ^-/- mice were protected from these transcriptomic changes, and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1 ^-/- diaphragms maintained normal contractile force and motion after DIO challenge. These findings establish THBS1 as a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition, and potential therapeutic target in obesity-associated respiratory dysfunction.

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. METHODS/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). Total RNA was extracted using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. RESULTS/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.

Microfluidics has earned a reputation for providing numerous transformative but disconnected devices and techniques. Active research seeks to address this challenge by integrating microfluidic components, including embedded miniature pumps. However, a significant portion of existing microfluidic integration relies on the time-consuming manual fabrication that introduces device variations. We put forward a framework for solving this disconnect by combining new pumping mechanics and 3D printing to demonstrate several novel, integrated and wirelessly driven microfluidics. First, we characterized the simplicity and performance of printed microfluidics with a minimum feature size of 100 µm. Next, we integrated a microtesla (µTesla) pump to provide non-pulsatile flow with reduced shear stress on beta cells cultured on-chip. Lastly, the integration of radio frequency (RF) device and a hobby-grade brushless motor completed a self-enclosed platform that can be remotely controlled without wires. Our study shows how new physics and 3D printing approaches not only provide better integration but also enable novel cell-based studies to advance microfluidic research.

Thyroid-associated orbitopathy (TAO) is a disfiguring periocular connective tissue disease associated with autoimmune thyroid disorders. TAO is a potentially blinding eye condition, for which no effective pharmacological treatment has been established. Despite a suggested role played by autoimmune thyrotropin receptor activation in the pathogenesis of TAO, the cellular and molecular events contributing to the fibrotic and inflammatory disease process of TAO is not fully defined. By developing a three-dimensional (3D) organoid culture of human orbital fibroblasts (hOFs), we sought to determine the molecular mechanism underlying fibrotic disease process of TAO. In a 3D organoid model, we have demonstrated that hypoxia inducible factor (HIF) 2 alpha (HIF2A), but not its paralog HIF1A, accelerates extracellular matrix (ECM) deposition by inducing a collagen-crosslinking enzyme, lysyl oxidase (LOX). Inhibiting HIF2A and LOX with shRNA or small molecular antagonists effectively ameliorated fibrotic disease process within TAO organoids. Conversely, the overexpression of a constitutively active HIF2A in mouse OFs (mOFs) was sufficient to initiate LOX-dependent fibrotic tissue remodeling in OF organoids. Consistent with these findings, HIF2A and LOX were highly expressed in human TAO tissues paralleling excess ECM deposition. We propose that HIF2A-LOX pathway can be a potential therapeutic target for the prevention and treatment of TAO.

Respiratory dysfunction is a common complication of obesity, conferring cardiovascular morbidity, increased mortality and often necessitating mechanical ventilatory support. While impaired lung expansion in the setting of increased adipose mass and reduced central response to hypercapnia have been implicated as pathophysiological drivers, the impact of obesity on respiratory muscles—in particular, the diaphragm—has not been investigated in detail. Here, we demonstrate that chronic high-fat diet (HFD)-feeding impairs diaphragm muscle function, as assessed in vivo by ultrasonography and ex vivo by measurement of contractile force. During HFD time course, progressive adipose tissue expansion and collagen deposition within the diaphragm parallel contractile deficits. Moreover, intra-diaphragmatic fibro-adipogenic progenitors (FAPs) proliferate with long-term HFD-feeding while giving rise to adipocytes and type I collagen-depositing fibroblasts. Thrombospondin-1 (THBS1), a circulating adipokine, increases with obesity and induces FAP proliferation. These findings suggest a novel role for FAP-mediated fibro-adipogenic diaphragm remodeling in obesity-associated respiratory dysfunction.