UNDERSTANDING THE MECHANISMS OF ADIPOSE TISSUE EXPANSION IN MULTIPLE SYMMETRIC LIPOMATOSIS AND OBESITY

ABSTRACT Background: Our Center for The Study and The Integrated Treatment of Obesity (Ce.S.I.T.O) manages patients affected by obesity and, thanks to our Endocrine-Metabolic Laboratory, we focus on translational projects in metabolic diseases. Recently, given our scientific interest on the mechanisms and consequences of adipose tissue (AT) expansion, we also investigate another AT disorders, named Multiple Symmetric Lipomatosis (MSL), characterized by enlarging, symmetric and unencapsulated lipomas. The mechanisms involved in lipomatous tissue (LT) formation are still unknown. As the distribution of LT resembles the anatomic location of brown fat depots, the main hypothesis believes that brown cells fail to completely differentiate, accumulating lipids due to mitochondrial dysfunctions, defective noradrenergic regulation or both. Recently, the PtenMyf5cKO mouse model was found to resemble MSL phenotype. PTEN is involved in insulin cascade as an inhibitor factor, whose activity is negatively regulated by protein kinase CK2, thus suggesting that an impairment of insulin pathway could have a role in AT expansion. CK2 is a protein kinase implicated in several essential cellular processes, such as the regulation of insulin signalling cascade. Recently, CK2 has been described to negatively regulate thermogenesis and to inhibit insulin release. Nevertheless, the role of CK2 in adipose tissue (AT) and its involvement in human obesity development and therapy has been poorly investigated. Our work provides a fine characterization of LT and its precursors from a morphological, molecular and functional point of view. Moreover, we investigated the role of CK2 in adipocyte glucose homeostasis and CK2 expression and activity CK2 in a wide variety of AT alterations: mouse models of obesity, obese patients before and after weight loss and lipomas from MSL patients. Material and Methods: We collected paired samples of LT and SAT in non-affected area of 5 Type I MSL patients, during lipectomy. H/E staining for adipocyte sizing evaluation and UCP1 IHC were performed in tissue sections. Gene expression profile of white/brown/beige markers was performed in tissues and cellular fractions. Stromal vascular fractions (SVF) was characterized by ex vivo cytofluorimetric analysis, proliferation assay and adipogenic and clonogenic potential limiting dilution assay. Moreover, to explore CK2 role in AT biology, glucose uptake assay, western blot, GLUT4 immunofluorescence were performed on in vitro differentiated 3T3L1 and human adipocytes in presence of CK2 inhibitors. We analyzed CK2 expression and activity in both visceral (VAT) and subcutaneous AT (SAT) of animal models of obesity (ob/ob) and obesity/diabetes (db/db), of 27 obese patients with different degree of insulin resistance or overt diabetes, in LT, SAT and SFV extracted from the enrolled MSL patients. Finally, CK2 was evaluated also in VAT and/or SAT of 12 patients who had obtained a relevant weight loss after diet or bariatric surgery. Results: We demonstrated that LT contains slightly smaller univacuolated UCP1 negative adipocytes, expressing similar level of white-specific genes than SAT and negligible level of brown marker UCP1. In LT-derived ASCs, defined as CD34+CD31-CD45- fraction, were significantly higher than in SAT. LT-precursors showed a slightly shorter doubling time and a higher clonogenic and adipogenic potential, giving rise to a higher number of clones, able to differentiate to mature adipocytes more efficiently than SAT derived cells. LT-derived cells also exhibited a greater adipogenic potential than SAT at the clonal level, resulted enriched of adipogenic precursors. We demonstrated that in both human and 3T3L1 adipocytes CK2-inhibition decreased insulin-induced glucose uptake by counteracting Akt-signaling and GLUT4-translocation to the plasma membrane. We showed that CK2 promoted insulin-signaling in mouse AT, liver and skeletal muscle and that in vivo acute treatment with CX-4945 impaired tissue insulin signaling activation. Studies in tissues of ob/ob and db/db mice highlighted an up-regulation of CK2 expression and activity only in WAT. CK2 hyper-activation was strongly evident also in SAT and VAT of obese patients, independently from metabolic complications and relevant weight loss completely normalized CK2 level. Interestingly, CK2 has been also found upregulated in LT in comparison with SAT of MSL patients and in LT-derived SVF. Conclusions: LT exhibits a typical white AT signature and morphology and is enriched of adipogenic precursors, with a higher proliferating and clonogenic potential compared with paired SAT. This feature could contribute to explain LT benign expansion. We clearly show that CK2 supports insulin signaling in adipocytes, skeletal muscle and liver. Moreover, we demonstrated in different pathological conditions, such as mouse and human obesity and MSL, that CK2 upregulation is a hallmark of expanding AT, independent from metabolic complications and tightly regulated by AT reduction and remodeling. In conclusion, the present work gives molecular insights in the pathogenesis of obesity and MSL, suggesting a new therapeutic target for both rare and common AT disorders.