Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

Objective: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. Method: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). Result: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. Conclusion: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications

THP-1 Macrophages and SGBS Adipocytes – A New Human in vitro Model System of Inflamed Adipose Tissue

Obesity is associated with an accumulation of macrophages in adipose tissue. This inflammation of adipose tissue is a key event in the pathogenesis of several obesity-related disorders, particularly insulin resistance. Here, we summarized existing model systems that mimic the situation of inflamed adipose tissue in vitro, most of them being murine. Importantly, we introduce our newly established human model system which combines the THP-1 monocytic cell line and the preadipocyte cell strain Simpson–Golabi–Behmel syndrome (SGBS). THP-1 cells, which originate from an acute monocytic leukemia, differentiate easily into macrophages in vitro. The human preadipocyte cell strain SGBS was recently introduced as a unique tool to study human fat cell functions. SGBS cells are characterized by a high capacity for adipogenic differentiation. SGBS adipocytes are capable of fat cell-specific metabolic functions such as insulin-stimulated glucose uptake, insulin-stimulated de novo lipogenesis and β-adrenergic-stimulated lipolysis and they secrete typical adipokines including leptin, adiponectin, and RBP4. Applying either macrophage-conditioned medium or a direct co-culture of macrophages and fat cells, our model system can be used to distinguish between paracrine and cell-contact dependent effects. In conclusion, we propose this model as a useful tool to study adipose inflammation in vitro. It represents an inexpensive, highly reproducible human system. The methods described here can be easily extended for usage of primary human macrophages and fat cells

The role of fat cell apoptosis during obesity-associated adipose tissue inflammation

Obesity-associated macrophage infiltration into adipose tissue is responsible for both local and systemic inflammation. The initial event, however, that triggers macrophage infiltration, has yet to be identified. We hypothesize that apoptosis is involved in the inflammatory process of adipose tissue and persued an in vivo as well as an in vitro approach to elucidate a possible connection of fat cell apoptosis and macrophage accumulation in the human adipose tissue. For the in vivo study we subjected serial sections of human subcutaneous and visceral adipose tissue immunohistochemical analysis. We localized CD11c+-macrophages around apoptotic adipocytes in both fat depots. Additionally, the percentage of apoptotic adipocytes strongly correlated with the percentage of macrophages (sc: r = 0.888; p < 0.001; visc: r = 0.560, p = 0.003), supporting the idea of a connection between macrophage infiltration and fat cell apoptosis. To investigate the effects of an inflammatory micro-environment on fat cells, we established a human in vitro model system of inflamed adipose tissue by using THP-1 macrophages and SGBS adipocytes. Macrophage-secreted factors (MacCM) induced insulin resistance and inhibited insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, macrophage-secreted factors induced apoptosis of fat cells. This apoptosis-inducing effect was even more pronounced in direct co-cultures of adipocytes and macrophages. Our data suggest a link between insulin resistance and apoptosis sensitivity. Accordingly, pharmacological and genetic inhibition of insulin signaling at the level of Akt2 sensitized adipocytes to apoptosis induction by macrophage-secreted factors. In conclusion, we describe here a novel interaction of macrophages and fat cells, i.e. induction of apoptosis. Our data suggest a feed-forward cycle in which macrophages further drive the inflammatory process by inducing insulin resistance and concomitant apoptosis of adipocytes

Interleukin-1β downregulates RBP4 secretion in human adipocytes.

AIMS/HYPOTHESIS: The excessive accumulation of adipose tissue in the obese state is linked to an altered secretion profile of adipocytes, chronic low-grade inflammation and metabolic complications. RBP4 has been implicated in these alterations, especially insulin resistance. The aim of the present study was to determine if a local inflammatory micro-environment in adipose tissue regulates RBP4 expression and secretion. METHODS: Human SGBS and primary adipocytes cultured with conditioned media from human THP-1 macrophages were used as an in vitro model for adipose inflammation. Adipocytes were exposed to recombinant TNF-α, IL-1β, IL-6 or IL-8. In addition, coexpression of IL-1β and RBP4 was measured in adipose tissue samples from 18 healthy females. RBP4 expression was studied by quantitative PCR and ELISA. RESULTS: RBP4 mRNA expression and secretion was significantly reduced upon incubation with macrophage-conditioned media in SGBS adipocytes and human primary adipocytes. Out of several factors studied we identified IL-1β as a new factor regulating RBP4. IL-1β significantly downregulated RBP4 mRNA and secretion in a time- and dose-dependent manner. IL-1β mediated its inhibitory effects on RBP4 expression via IL-1 receptor and NF-κB, as incubation with the IL-1 receptor blocking antibody and the NF-κB inhibitors CAPE and SC-514 reversed its effect. Most interestingly, RBP4 mRNA was negatively correlated with IL-1β mRNA in subcutaneous adipose tissue. CONCLUSIONS: Adipose tissue inflammation as found in the obese state might lead to a downregulation in local RBP4 levels. IL-1β was identified as a major factor contributing to the decrease in RBP4. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to inflammatory processes in adipose tissue

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

Objective: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. Method: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). Result: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. Conclusion: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications

Microstructural Comparison of Porous Oxide Ceramics from the System Al2O3–ZrO2 Prepared with Starch as a Pore-Forming Agent

In this paper we show examples of microstructures of porous oxide ceramics prepared by traditional slip casting (TSC) and starch consolidation casting (SCC) and present results obtained using different microstructural characterization techniques; Archimedes method (open and total porosity), shrinkage measurement, mercury intrusion porosimetry (pore size distribution) and microscopic methods – optical microscopy with microscopic image analysis (pore size distribution) and scanning electron microscopy (detailed investigation of the local microstructure). In particular, microstructures are compared for porous ceramics from the system Al2O3–ZrO2 prepared with rice and corn starch. It is shown that maximum values of the total porosity of porous ceramics prepared with starch as a pore-forming agent were approx. 50%. A major finding by using SEM with respect to starch-produced porous ceramics is the existence of pore fillings in the form of small sintered ceramic shell inside the pores, as a result of starch granule shrinkage during the drying and burn-out steps

IL-1β decreases RBP4 production.

<p>SGBS adipocytes were treated with increasing doses of IL-1β (0.05, 0.5, 5, 50 ng/ml) or the corresponding vehicle control. (A) RBP4 mRNA expression was measured by quantitative PCR. RBP4 expression was normalized to succinate dehydrogenase complex subunit A (SDHA) and related to medium control using 2^−ΔΔCT method. Data are presented as mean+SEM of three independent experiments. *p<0.05 (treatment vs. vehicle). (B) Accumulation of RBP4 in the medium supernatant was measured by ELISA. Measurements were related to untreated medium control. Data are presented as mean+SEM of three independent experiments. *p<0.05 (treatment vs. vehicle). (C, D) Human primary adipocytes obtained from three different donors were treated with 5 ng/ml of IL-1β or vehicle. (C) mRNA expression and (D) secretion of RBP4 was performed as described above.*p<0.05 (treatment vs. vehicle). (E) SGBS adipocytes were treated with 5 ng/ml of IL-1β for 24, 48, and 72 hours. mRNA expression of RBP4 was measured by quantitative PCR. RBP4 expression was normalized to SDHA and related to medium control at 0 hours using 2^−ΔΔCT method. *p<0.05 (treatment vs. vehicle).</p

Macrophage-secreted factors decrease RBP4 production.

<p>SGBS adipocytes (A, B, C) were treated with increasing concentrations of MacCM (5, 10, 20, 50%) or the corresponding vehicle control. (A) Representative pictures of cells treated for 48 hours with 10% of MacCM. (B) RBP4 mRNA expression was measured by quantitative PCR. RBP4 expression was normalized to succinate dehydrogenase complex subunit A (SDHA) and related to medium control using 2^−ΔΔCT method. Data are presented as mean+SEM of three independent experiments. *p<0.05 (treatment vs. vehicle). (C) Accumulation of RBP4 in the medium supernatant was measured by ELISA. Measurements were related to untreated medium control. Data are presented as mean+SEM of three independent experiments. *p<0.05 (treatment vs. vehicle). (D, E) Human primary adipocytes obtained from three different donors were treated with 10% of MacCM or vehicle. (D) mRNA expression and (E) secretion of RBP4 was performed as described above. *p<0.05 (treatment vs. vehicle). (F) SGBS adipocytes were treated with 10% MacCM or vehicle for 24, 48, and 72 hours. mRNA expression of RBP4 was measured by quantitative PCR. RBP4 expression was normalized to SDHA and related to medium control at 0 hours using 2^−ΔΔCT method. *p<0.05 (treatment vs. vehicle).</p