Project: Turning up the Heat on Lipedema

Principal Investigator: Atefeh Rabiee, PhD
University of the Pacific
Thomas J. Long School of Pharmacy & Health Sciences
Stockton, CA

Summary

1. To investigate the differences in the proteome and secretome profiles between Lipedema, obese non-lipidemic, and lean control in response to the browning and thermogenic stimuli.

2. To determine if the mitochondrial dynamic and function in Lipedema fat are affected.

3. To activate the thermogenic potential of Lipedema ASCs using pharmacological and non-pharmacological interventions.

Background

External stimuli such as cold temperature, exercise, and certain diets increase the activity of highly plastic inducible beige adipose tissue (AT) in subcutaneous areas of hips, legs, and tights. As a result, energy expenditure (EE) increases countering weight gain and metabolic consequences. Lipedema is a chronic lipid disease with symmetrical deposition of an extended subcutaneous fat. Although Lipedema fat represents some characteristics of the beige fat including location and a relative reduction in obesity-related metabolic dysfunction (especially at early stages), the fat is often resistant to dietary or exercise-based interventions.

Potential mechanisms to play role in the pathogenesis of Lipedema include the effect of sex hormones, increased vascular permeability and damage (microangiopathy), excessive lipid peroxidation, and disturbances in adipocyte metabolism and cytokine production. We further speculate that the unique proteome and secretome profiles, as well as the mitochondrial dysfunction, are potential contributors to the pathogenesis of Lipedema fat resistance.

Methodology

We will use adipose tissue-derived stem cells (ASCs) isolated from the subcutaneous adipose tissue of the thigh (SVF-T) obtained from Lipedema, obese non-lipedemic, and lean control groups. The browning and thermogenic function will be induced in vitro in cell culture using the established strategies of treating the cells with norepinephrine.

The protein extracts from the whole cell lysates, as well as the adipocytes secretome (the cell culture media), and the protein extract from the isolated mitochondria will be collected prior to and after the thermogenic function has been achieved in lean ASCs. We will then apply a shotgun mass spectrometry (MS) to profile and compare the cellular and mitochondrial proteome and phosphoproteome as well as the secretome of three ACSs groups. Validation of several candidates identified by our MS analysis will be performed using western blotting.

We will also quantify the mitochondrial DNA content and measure the mitochondrial respiration rate in response to thermogenic stimuli. To quantify the mitochondrial DNA content, cells will be harvested, and total DNA will be extracted using the standard phenol-chloroform separation method. The relative mitochondrial DNA content will be assessed by measuring the various mitochondrial genes. To measure the mitochondrial respiration rate, we will use Lucid Scientific’s RESIPHER that utilizes proprietary high-resolution optical oxygen sensors to characterize oxygen consumption and the oxygen environment in cell culture.

Lastly, we will evaluate the thermogenic capacity of Lipedema ASCs in vitro in cell culture by screening for the various known and novel thermogenic stimuli. Upon interventions, live-cell monitoring by CellCyte will be performed to evaluate the cell trajectories, death, or any changes in cell morphology caused by interventions. The thermogenic efficiency of cells will be evaluated and compared using qPCR and western blotting quantifying the expression of well-identified thermogenic markers.

Expected outcomes

Taking advantage of the unbiased high-throughput mass spectrometry-based proteomic and phosphoproteomic techniques, this project aims to investigate the underlying mechanisms of Lipedema thermogenic resistance by identifying the cellular proteins, cellular secretome, and signaling properties that are disturbed/affected in Lipedema. We will also disclose in detail if and how the mitochondrial morphology and function may contribute to the pathogenesis of Lipedema. Investigating these mechanisms, we aim to identify the potential pharmacological and non-pharmacological compounds to convert the Lipedema fat to thermogenic fat, thereby paving the way for future Lipedema therapies.

Practical implications of results

Lipedema is a common but not well recognized subcutaneous fat storage disorder. While Lipedema is likely inherited in 60% of women, the pathophysiologic mechanism responsible for Lipedema is not fully known. On one hand, the excessive amount of scWAT as a source of beige adipocytes in Lipedema patients suggests the therapies to be more effective. On the other hand, Lipedema fat seems to be resilient to exercise and diet. Revealing the underlying mechanisms of Lipedema thermogenic resistance, we hope to provide a better understanding of Lipedema pathophysiology thereby enabling clinicians to diagnose and treat affected patients at an earlier stage.

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