Adipose tissue place of origin and obesity influence sphingolipid signaling pathway in the adipocytes differentiated from ADMSCs isolated from morbidly obese women.

Adipose derived mesenchymal stem cells (ADMSCs) are a component of adipose tissue that in recent years has gained on importance. The progenitor cells serve as an essentially unlimited source of new adipocytes and therefore are considered to be an important determinant of the tissue's physiology. In this paper we investigated mature adipocytes differentiated from ADMSCs obtained from subcutaneous/visceral fat of patients with different metabolic status (lean, obese without and with metabolic syndrome). We focused our interests on the sphingolipid signaling pathway, i.e.a signal transduction system indispensable for cells functioning, but also implicated in the development of medical conditions associated with obesity. We observed that the cells derived from visceral tissue had significantly greater levels of almost all the examined sphingolipids (especially Cer, dhCer, SM). Moreover, obesity and metabolic syndrome present in donor patients was associated with an increased level of sphingosine kinase (SPHK) and the product of its reaction sphingosine-1-phosphate (S1P). Moreover, the condition appeared to display a tissue specific pattern. Namely, the adipocytes of subcutaneous provenance had an increased activation of ceramide de novo synthesis pathway when the donors of ADMSCs had metabolic syndrome. The above translated into greater accumulation of ceramide in the cells. To our knowledge this is the first study that demonstrated altered sphingolipid profile in the mature adipocytes differentiated from ADMSCs with respect to the stem cells tissue of origin and the donor patient metabolic status.

Efficacy of adipose-derived mesenchymal stem cell therapy in the treatment of chronic micro- and macrovascular complications of diabetes.

Diabetes mellitus is a highly prevalent disease characterized by hyperglycaemia that damages the vascular system, leading to micro- (retinopathy, neuropathy, nephropathy) and macrovascular diseases (cardiovascular disease). There are also secondary complications of diabetes (cardiomyopathy, erectile dysfunction or diabetic foot ulcers). Stem cell-based therapies have become a promising tool targeting diabetes symptoms and its chronic complications. Among all stem cells, adipose-derived mesenchymal stem cells (ADMSCs) are of great importance because of their abundance, non-invasive isolation and no ethical limitations. Characteristics that make ADMSCs good candidates for cell-based therapy are their wide immunomodulatory properties and paracrine activities through the secretion of an array of growth factors, chemokines, cytokines, angiogenic factors and anti-apoptotic molecules. Besides, after transplantation, ADMSCs show great ex vivo expansion capacity and differentiation to other cell types, including insulin-producing cells, cardiomyocytes, chondrocytes, hepatocyte-like cells, neurons, endothelial cells, photoreceptor-like cells, or astrocytes. Preclinical studies have shown that ADMSC-based therapy effectively improved visual acuity, ameliorated polyneuropathy and foot ulceration, arrested the development and progression of diabetic kidney disease, or alleviated the diabetes-induced cardiomyocyte hypertrophy. However, despite the positive results obtained in animal models, there are still several challenges that need to be overcome before the results of preclinical studies can be translated into clinical applications. To date, there are several clinical trials or ongoing trials using ADMSCs in the treatment of diabetic complications, most of them in the treatment of diabetic foot ulcers. This narrative review summarizes the most recent outcomes on the usage of ADMSCs in the treatment of long-term complications of diabetes in both animal models and clinical trials.

Adipose-derived Mesenchymal Stem Cells Therapy as a new Treatment Option for Diabetes Mellitus.

The worldwide increase in the prevalence of diabetes mellitus has raised the demand for new therapeutic strategies targeting diabetic symptoms and its chronic complications. Among different treatment options for diabetes, adipose-derived mesenchymal stem cells (ADMSCs) therapy attract the most attention. The therapeutic effects of ADMSCs are based primarily on their paracrine release of immunomodulatory, anti-inflammatory, and trophic factors. Animal models of diabetes as well as human clinical trials have shown that ADMSCs can effectively facilitate endogenous ß cell regeneration, preserve residual ß cell mass, reduce islet graft rejection, regulate the immune system, and ultimately improve insulin sensitivity or ameliorate insulin resistance in peripheral tissues. Nevertheless, transplantation of mesenchymal stem cells is associated with certain risks; therefore recently much attention has been devoted to ADMSCs derivatives, such as exosomes or conditioned media, as therapeutic agents for the treatment of diabetes. Compared to ADMSCs, cell-free therapy has even better therapeutic potential. This narrative review summarizes recent outcomes and molecular mechanisms of ADMSCs action in the treatment for both type 1 DM and type 2 DM, as well as shows their feasibility, benefits, and current limitations.

The Phenotype of the Adipocytes Derived from Subcutaneous and Visceral ADMSCs Is Altered When They Originate from Morbidly Obese Women: Is There a Memory Effect?

Adipose tissue is an abundant source of mesenchymal stem cells (ADMSCs). Evidence has suggested that depot-specific ADMSCs (obtained from subcutaneous or visceral adipose tissue-subADMSCs or visADMSCs, respectively) account for differential responses of each depot to metabolic challenges. However, little is known about the phenotype and changes in metabolism of the adipocytes derived from ADMSCs of obese individuals. Therefore, we investigated the phenotypic and metabolic characteristics, particularly the lipid profile, of fully differentiated adipocytes derived from ADMSCs of lean and obese (with/without metabolic syndrome) postmenopausal women. We observed a depot-specific pattern, with more pronounced changes present in the adipocytes obtained from subADMSCs. Namely, chronic oversupply of fatty acids (present in morbid obesity) triggered an increase in CD36/SR-B2 and FATP4 protein content (total and cell surface), which translated to an increased LCFA influx (3H-palmitate uptake). This was associated with the accumulation of TAG and DAG in these cells. Furthermore, we observed that the adipocytes of visADMSCs origin were larger and showed smaller granularity than their counterparts of subADMSCs descent. Although ADMSCs were cultured in vitro, in a fatty acids-deprived environment, obesity significantly influenced the functionality of the progenitor adipocytes, suggesting the existence of a memory effect.

The influence of the pH on the incorporation of caffeic acid into biomimetic membranes and cancer cells.

Caffeic acid (CA) is a phenolic compound synthesized by all plant species. It constitutes the main hydroxycinnamic acid found in human diet and presents a variety of beneficial effects including anticancer activity. Current data suggests essential role of the interplay between anticancer drugs and the cell membrane. Given this, biophysical interactions between CA and cancer cells or biomimetic membranes were investigated. Glioblastoma cell line U118MG and colorectal adenocarcinoma cell line DLD-1, as well as lipid bilayers and liposomes, were used as in vitro models. Electrophoretic light scattering was used to assess the effect of CA on the surface charge of cancer cells and liposomal membranes. Electrochemical impedance spectroscopy was chosen to evaluate CA-dependent modulatory effect on the electrical capacitance and electrical resistance of the bilayers. Our results suggest that CA fulfills physicochemical criteria determining drug-like properties of chemical compounds, and may serve as a potential cytostatic agent in cancer treatment.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

Using adipose-derived mesenchymal stem cells to fight the metabolic complications of obesity: Where do we stand?

Obesity is a critical risk factor for the development of metabolic diseases, and its prevalence is increasing worldwide. Stem cell-based therapies have become a promising tool for therapeutic intervention. Among them are adipose-derived mesenchymal stem cells (ADMSCs), secreting numerous bioactive molecules, like growth factors, cytokines, and chemokines. Their unique features, including immunosuppressive and immunomodulatory properties, make them an ideal candidates for clinical applications. Numerous experimental studies have shown that ADMSCs can improve pancreatic islet cell viability and function, ameliorate hyperglycemia, improve insulin sensitivity, restore liver function, counteract dyslipidemia, lower pro-inflammatory cytokines, and reduce oxidative stress in the animal models. These results prompted scientists to use ADMSCs clinically. However, up to date, there have been few clinical studies or ongoing trails using ADMSCs to treat metabolic disorders such as type 2 diabetes mellitus (T2DM) or liver cirrhosis. Most human studies have implemented autologous ADMSCs with minimal risk of cellular rejection. Because the functionality of ADMSCs is significantly reduced in subjects with obesity and/or metabolic syndrome, their efficacy is questioned. ADMSCs transplantation may offer a potential therapeutic approach for the treatment of metabolic complications of obesity, but randomized controlled trials are required to establish their safety and efficacy in humans prior to routine clinical use.

Does TBC1D4 (AS160) or TBC1D1 Deficiency Affect the Expression of Fatty Acid Handling Proteins in the Adipocytes Differentiated from Human Adipose-Derived Mesenchymal Stem Cells (ADMSCs) Obtained from Subcutaneous and Visceral Fat Depots?

TBC1D4 (AS160) and TBC1D1 are Rab GTPase-activating proteins that play a key role in the regulation of glucose and possibly the transport of long chain fatty acids (LCFAs) into muscle and fat cells. Knockdown (KD) of TBC1D4 increased CD36/SR-B2 and FABPpm protein expressions in L6 myotubes, whereas in murine cardiomyocytes, TBC1D4 deficiency led to a redistribution of CD36/SR-B2 to the sarcolemma. In our study, we investigated the previously unexplored role of both Rab-GAPs in LCFAs uptake in human adipocytes differentiated from the ADMSCs of subcutaneous and visceral adipose tissue origin. To this end we performed a single- and double-knockdown of the proteins (TBC1D1 and TBC1D4). Herein, we provide evidence that AS160 mediates fatty acid entry into the adipocytes derived from ADMSCs. TBC1D4 KD resulted in quite a few alterations to the cellular phenotype, the most obvious of which was the shift of the CD36/SR-B2 transport protein to the plasma membrane. The above translated into an increased uptake of saturated long-chain fatty acid. Interestingly, we observed a tissue-specific pattern, with more pronounced changes present in the adipocytes derived from subADMSCs. Altogether, our data show that in human adipocytes, TBC1D4, but not TBC1D1, deficiency increases LCFAs transport via CD36/SR-B2 translocation.

Does supplementation with leucine-enriched protein alone and in combination with fish-oil-derived n-3 PUFA affect muscle mass, strength, physical performance, and muscle protein synthesis in well-nourished older adults? A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Leucine-enriched protein (LEU-PRO) and long-chain (LC) n-3 (ω-3) PUFAs have each been proposed to improve muscle mass and function in older adults, whereas their combination may be more effective than either alone. OBJECTIVE: The impact of LEU-PRO supplementation alone and combined with LC n-3 PUFAs on appendicular lean mass, strength, physical performance and myofibrillar protein synthesis (MyoPS) was investigated in older adults at risk of sarcopenia. METHODS: This 24-wk, 3-arm parallel, randomized, double-blind, placebo-controlled trial was conducted in 107 men and women aged ≥65 y with low muscle mass and/or strength. Twice daily, participants consumed a supplement containing either LEU-PRO (3 g leucine, 10 g protein; n = 38), LEU-PRO plus LC n-3 PUFAs (0.8 g EPA, 1.1 g DHA; LEU-PRO+n-3; n = 38), or an isoenergetic control (CON; n = 31). Appendicular lean mass, handgrip strength, leg strength, physical performance, and circulating metabolic and renal function markers were measured pre-, mid-, and postintervention. Integrated rates of MyoPS were assessed in a subcohort (n = 28). RESULTS: Neither LEU-PRO nor LEU-PRO+n-3 supplementation affected appendicular lean mass, handgrip strength, knee extension strength, physical performance or MyoPS. However, isometric knee flexion peak torque (treatment effect: -7.1 Nm; 95% CI: -12.5, -1.8 Nm; P < 0.01) was lower postsupplementation in LEU-PRO+n-3 compared with CON. Serum triacylglycerol and total adiponectin concentrations were lower, and HOMA-IR was higher, in LEU-PRO+n-3 compared with CON postsupplementation (all P < 0.05). Estimated glomerular filtration rate was higher and cystatin c was lower in LEU-PRO and LEU-PRO+n-3 postsupplementation compared with CON (all P < 0.05). CONCLUSIONS: Contrary to our hypothesis, we did not observe a beneficial effect of LEU-PRO supplementation alone or combined with LC n-3 PUFA supplementation on appendicular lean mass, strength, physical performance or MyoPS in older adults at risk of sarcopenia. This trial was registered at clinicaltrials.gov as NCT03429491.

Challenging of AS160/TBC1D4 Alters Intracellular Lipid milieu in L6 Myotubes Incubated With Palmitate.

The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160- /PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160- ). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG, and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG, and PL contents, but increased FFA content (PA/AS160- vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160- /PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. J. Cell. Physiol. 232: 2373-2386, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.