Hyperglycemia Changes Expression of Key Adipogenesis Markers (C/EBPα and PPARᵞ)and Morphology of Differentiating Human Visceral Adipocytes

Abstract

Disturbances in adipose tissue significantly contribute to the development of metabolic disorders, which are connected with hyperglycemia (HG) and underlain by epigenetics-based mechanisms. Therefore, we aimed to evaluate the effect of hyperglycemia on proliferating, differentiating and maturating human visceral pre/adipocytes (HPA-v). Three stages of cell culture were conducted under constant or variable glycemic conditions. Adipogenesis progress was assessed using BODIPY 505/515 staining. Lipid content typical for normal and hyperglycemic conditions of adipocytes was analyzed using Raman spectroscopy and imaging. Expression of adipogenic markers, PPARγ and C/EBPα, was determined at the mRNA and protein levels. We also examined expression of miRNAs proven to target PPARγ (miR-34a-5p) and C/EBPα (miR-137-3p), employing TaqMan Low-Density Arrays (TLDA) cards. Hyperglycemia altered morphology of differentiating HPA-v in relation to normoglycemia by accelerating the formation of lipid droplets and making their numbers and volume increase. Raman results confirmed that the qualitative and quantitative lipid composition under normal and hyperglycemic conditions were different, and that the number of lipid droplets increased in (HG)-treated cells. Expression profiles of both examined genes markedly changed either during adipogenesis under physiological and hyperglycemic conditions, orat particular stages of adipogenesis upon chronic and/or variable glycemia. Expression levels of PPARγ seemed to correspond to some expression changes of miR-34a-5p. miR-137-3p, whose expression was rather stable throughout the culture, did not seem to affect C/EBPα. Our observations revealed that chronic and intermittent hyperglycemia change the morphology of visceral pre/adipocytes during adipogenesis. Moreover, hyperglycemia may utilize miR-34a-5p to induce some expression changes in PPARγ.

Chronic and Transient Hyperglycemia Induces Changes in the Expression Patterns of IL6 and ADIPOQ Genes and Their Associated Epigenetic Modifications in Differentiating Human Visceral Adipocytes

“…Therefore, the inhibition of IL-6 transcription could be protective for C/EBPα expression and Ads commitment. Consistently, transcription of CEBPA was opposed to the IL-6 mRNA levels in this model, whereas chronic HG accelerated adipogenesis, as we reported previously [18]. On the other hand, Chinese researchers described a gradual increase in IL-6 production during adipogenic differentiation in human bone marrow mesenchymal stem cells (BM-MSCs), while we observed increased IL-6 production in mature Ads [19].…”

“…The most important stage of differentiation requiring the glucose pulse was also assessed using a design reflecting our transient HG exposure, but with contrasting results [32]. Indeed, the authors observed around 75% lower lipid accumulation in 4 mM of glucose, and, previously, we had consistently observed that HG accelerated adipogenesis [18]. Thus, more research is needed to explore the concentration of glucose dysregulating adipokines and Ads metabolism.…”

“…PAds were differentiated to mature Ads in three stages, proliferation (5 days), differentiation (12 days), and maturation (6 days), with culture in preadipocyte medium (PAM), preadipocyte differentiation medium (PADM), and adipocyte medium (AdM) media in subsequent stages, respectively. Completion of every stage of cell culture was confirmed with BODIPY 505/515 staining (Life Technologies, Eugene, OR, USA) as described previously [18]. To create hyperglycemic conditions, full medium was supplemented with glucose to obtain a concentration of 30 mM (d-(+)-Glucose, Sigma-Aldrich, Saint Louis, MO, USA).…”

Visceral Adipose Tissue of Prediabetic and Diabetic Females Shares a Set of Similarly Upregulated microRNAs Functionally Annotated to Inflammation, Oxidative Stress and Insulin Signaling

“…The complete list of 78 miRNAs and 4 small nuclear RNAs (snRNAs) is provided in Table S1 . Selection of the set of molecules was based on: (i) preliminary data obtained via previously used TLDA cards on 1 healthy and 1 diabetic subject, (ii) literature search for miRNAs associated with T2DM, IR, obesity in all insulin-responsive tissues, (iii) providing a broad panel of candidate reference genes (literature search), (iv) results of our previous research on hyperglycemia-exposed visceral pre/adipocytes [ 18 , 19 ].…”

Chronic and Intermittent Hyperglycemia Modulates Expression of Key Molecules of PI3K/AKT Pathway in Differentiating Human Visceral Adipocytes

“…IRS binds to the kinase regulatory subunit of PI3K (PI3K-R), the central molecule of the pathway. This, in turn, causes activation of the catalytic PI3K (PI3K-C) subunit that converts phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3) [8]. The main insulin pathway regulator, PTEN, (phosphatase and tensin homolog), inhibits further signal transduction by PIP3 dephosphorylation [10].…”