(M1430-03-19) Exploring Mitochondrial Involvement in Persistent Fat Accumulation of Lipedema

Purpose: Obesity is a condition caused by calorific imbalance and is primarily attributed to the expansion of adipose tissue. What is generally misunderstood though, is that the function of adipose tissue is limited to storing energy in the form of lipid droplets. Although this is true for visceral/white adipose tissue located in the abdominal area, it is not the sole form of fat present. Brown and beige fat are the other two types of fat distinct in their appearance as well as function in terms of their metabolic capacity to burn calories and promote energy expenditure. Brown fat is named so, because of its dense iron-containing mitochondrial content and is present in a small proportion in the neck and shoulder region of human adults. Although metabolically active, this type of fat due to its sparseness is not an ideal target to combat fat accumulation. The third type of fat that forms the core of our research interest is an inducible type of white fat called beige fat which can burn calories upon exposure to external stimuli such as cold temperature, chronic exercise, and certain diets and can burn calories. As this type of fat resides in the abundant subcutaneous depot along the leg and hip region, it is considered an attractive target in diseases such as obesity. Lipedema is a chronic, progressive adipofascial disorder predominantly affecting females, characterized by bilateral and symmetrical deposition of subcutaneous adipose tissue, primarily localized to the lower limbs, gluteal region, and hips. Clinically, patients present with persistent pain, heightened tissue sensitivity, easy bruising, and gradual impairment in mobility. Distinct from generalized obesity, lipedema-associated adipose tissue exhibits pronounced resistance to conventional weight reduction strategies, including caloric restriction, physical activity, bariatric procedures, and pharmacologic anti-obesity therapies. The objective of our project is to elucidate the underlying biological and molecular mechanisms responsible for the treatment resistance of lipedema-associated adipose tissue and to identify targeted therapeutic strategies to overcome these challenges.

Methods: Cell Culture, differentiation, and thermogenic treatment: Adipose-derived stem cells (ADSCs) from subcutaneous adipose tissue were obtained from Lipedema, lean, and obese donors. These ADSCs were expanded in DMEM/F12 growth media supplemented with 10% FBS and 1% Anti-anti solution at 37^◦C. At a confluency of about 80%, these cells were trypsinized and seeded. On achieving 100% confluency the cells were treated with adipogenic induction media for 14-21 days. Verification of differentiation via an increase in the size/number of lipid droplets was confirmed using a confocal microscope. These differentiated cells were fasted overnight (media devoid of FBS) and then treated with either a control DMSO or thermogenic stimuli Forskolin (20 µM). Samples were collected at different time points and used for preliminary gene expression studies and unbiased high throughput mass-spectrometry studies focusing on the role of mitochondria and secreted proteins in fat resistance of Lipedema (mitochondrial proteomics and secretome). Oxygen Consumption rate (OCR) is a direct measure of energy expenditure and mitochondrial health. To measure OCR, we used Resipher, in a 96-well format to compare lipedema cells v/s lean and obese controls.

Results: Validation of differentiation and browning: As part of the preliminary studies, qPCR was for three biological replicates (3 cell lines each from lean, obese, and lipedema donors) to validate differentiation. Adipogenic genes: Fabp4, AdipoQ, Plin1, and Pparg were found to be expressed highly in Lean cells followed by Lipedema and finally obese cells (Day 14 v/s Day 0). As expected, we observed that obese cells differentiated towards a more white fat-like lineage as was confirmed by the upregulated expression of genes such as TLE3, ZFP423 and Resistin. On treatment with forskolin, the lean and lipedema cells showed a relatively high fold change in the expression of classical browning markers, UCP-1 and PGC1a. These initial gene expression studies seemed to indicate lean and lipedema cells respond to forskolin albeit in a slightly different manner. Resipher appeared to indicate a similar trend in which lean, and lipedema cells treated with forskolin exhibited an increase in oxygen consumption. Employing liquid chromatography-mass spectrometry (LC-MS) techniques, we conducted a comprehensive multi-omics analysis of the proteome, secretome, and mitochondrial protein profiles of Lipedema as well as lean and obese controls. Proteomics yielded a list of 4297 proteins of which 795 were confirmed to be mitochondrial proteins. KEGG analysis and GO annotation were performed for regulated proteins in the Lipedema group (mitochondrial proteins) which returned enrichment for terms such as “thermogenesis”, “metabolic pathways”, and “intracellular lipid transport” to name a few among others

Conclusion: Mitochondrial proteomics show how subsets of proteins are regulated across various timepoints differently in lean, obese and lipedema groups. Various proteins which are involved in metabolic processes namely lipid synthesis, beta oxidation, oxidative phosphorylation and precursors of TCA cycle seem to be regulated in the Lipedema group.

Acknowledgements: Project was funded by Lipedema Foundation: LF34-21