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1.
Diabetologia ; 61(5): 1155-1166, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29427237

RESUMEN

AIMS/HYPOTHESIS: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulatory macrophage-derived factor that increases with obesity and leads to a higher risk of cardiovascular disease (CVD). Despite this, its role in adipose tissue and the adipocyte is unknown. Therefore, the aims of this study were to clarify the expression of Lp-PLA2 in relation to different adipose tissue depots and type 2 diabetes, and ascertain whether markers of obesity and type 2 diabetes correlate with circulating Lp-PLA2. A final aim was to evaluate the effect of cholesterol on cellular Lp-PLA2 in an in vitro adipocyte model. METHODS: Analysis of anthropometric and biochemical variables from a cohort of lean (age 44.4 ± 6.2 years; BMI 22.15 ± 1.8 kg/m2, n = 23), overweight (age 45.4 ± 12.3 years; BMI 26.99 ± 1.5 kg/m2, n = 24), obese (age 49.0 ± 9.1 years; BMI 33.74 ± 3.3 kg/m2, n = 32) and type 2 diabetic women (age 53.0 ± 6.13 years; BMI 35.08 ± 8.6 kg/m2, n = 35), as part of an ethically approved study. Gene and protein expression of PLA2 and its isoforms were assessed in adipose tissue samples, with serum analysis undertaken to assess circulating Lp-PLA2 and its association with cardiometabolic risk markers. A human adipocyte cell model, Chub-S7, was used to address the intracellular change in Lp-PLA2 in adipocytes. RESULTS: Lp-PLA2 and calcium-independent PLA2 (iPLA2) isoforms were altered by adiposity, as shown by microarray analysis (p < 0.05). Type 2 diabetes status was also observed to significantly alter gene and protein levels of Lp-PLA2 in abdominal subcutaneous (AbdSc) (p < 0.01), but not omental, adipose tissue. Furthermore, multivariate stepwise regression analysis of circulating Lp-PLA2 and metabolic markers revealed that the greatest predictor of Lp-PLA2 in non-diabetic individuals was LDL-cholesterol (p = 0.004). Additionally, in people with type 2 diabetes, oxidised LDL (oxLDL), triacylglycerols and HDL-cholesterol appeared important predictors, accounting for 59.7% of the variance (p < 0.001). Subsequent in vitro studies determined human adipocytes to be a source of Lp-PLA2, as confirmed by mRNA expression, protein levels and immunochemistry. Further in vitro experiments revealed that treatment with LDL-cholesterol or oxLDL resulted in significant upregulation of Lp-PLA2, while inhibition of Lp-PLA2 reduced oxLDL production by 19.8% (p < 0.05). CONCLUSIONS/INTERPRETATION: Our study suggests adipose tissue and adipocytes are active sources of Lp-PLA2, with differential regulation by fat depot and metabolic state. Moreover, levels of circulating Lp-PLA2 appear to be influenced by unfavourable lipid profiles in type 2 diabetes, which may occur in part through regulation of LDL-cholesterol and oxLDL metabolism in adipocytes.


Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Perfilación de la Expresión Génica , Lípidos/sangre , Obesidad/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Adulto , Antropometría , Índice de Masa Corporal , Colesterol/sangre , Diabetes Mellitus Tipo 2/sangre , Endotoxinas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación , Lipoproteínas LDL/sangre , Persona de Mediana Edad , Obesidad/sangre , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Arriba
2.
iScience ; 26(1): 105822, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36636339

RESUMEN

Cultivated meat is a promising technology with the potential to mitigate the ethical and environmental issues associated with traditional meat. Fat plays a key role in the meat flavor; therefore, development of suitable adipogenic protocols for livestock is essential. The traditional adipogenic cocktail containing IBMX, dexamethasone, insulin and rosiglitazone is not food-compatible. Here, we demonstrate that of the four inducers only insulin and rosiglitazone are necessary in both serum-free (DMAD) and serum-containing media, with DMAD outperforming FBS. Two glucocorticoid receptor activators, progesterone and hydrocortisone, found in DMAD and FBS, affect differentiation homogeneity, without playing an essential role in activating adipogenic genes. Importantly, this protocol leads to mature adipocytes in 3D culture. This was demonstrated in both media types and in four species: ruminant and monogastric. We therefore propose a simplified one-step adipogenic protocol which, given the replacement of rosiglitazone by a food-compatible PPARγ agonist, is suitable for making cultivated fat.

3.
STAR Protoc ; 4(2): 102143, 2023 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-36897775

RESUMEN

Here, we present a cost-effective protocol to differentiate bovine fibro-adipogenic progenitors in a thin hydrogel sheet adherent to 96-well plates. We describe steps for the embedding and culturing of cells in alginate sheets, culture maintenance, and analysis. Compared to alternative three-dimensional (3D) models such as hydrogel-based microfibers, this approach simplifies automation while retaining efficient maturation of adipocytes. Embedded cells are still subjected to a 3D environment, but the sheets can be handled and analyzed like two-dimensional cultures.

4.
NPJ Sci Food ; 6(1): 6, 2022 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-35075125

RESUMEN

Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat.

5.
Free Radic Biol Med ; 175: 65-79, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34455039

RESUMEN

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.


Asunto(s)
Carnosina , Complicaciones de la Diabetes , Diabetes Mellitus Tipo 2 , Animales , Carnosina/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Productos Finales de Glicación Avanzada/metabolismo , Ratones , Estrés Oxidativo , Carbonilación Proteica
6.
J Clin Endocrinol Metab ; 105(9)2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32413131

RESUMEN

CONTEXT: Dysfunctional endoplasmic reticulum (ER) and mitochondria are known to contribute to the pathology of metabolic disease. This damage may occur, in part, as a consequence of ER-mitochondria cross-talk in conditions of nutrient excess such as obesity. To date, insight into this dynamic relationship has not been characterized in adipose tissue. Therefore, this study investigated whether ER stress contributes to the development of mitochondrial inefficiency in human adipocytes from lean and obese participants. METHODS: Human differentiated adipocytes from Chub-S7 cell line and primary abdominal subcutaneous adipocytes from lean and obese participants were treated with tunicamycin to induce ER stress. Key parameters of mitochondrial function were assessed, including mitochondrial respiration, membrane potential (MMP), and dynamics. RESULTS: ER stress led to increased respiratory capacity in a model adipocyte system (Chub-S7 adipocytes) in a concentration and time dependent manner (24 h: 23%↑; 48 h: 68%↑, P < 0.001; 72 h: 136%↑, P < 0.001). This corresponded with mitochondrial inefficiency and diminished MMP, highlighting the formation of dysfunctional mitochondria. Morphological analysis revealed reorganization of mitochondrial network, specifically mitochondrial fragmentation. Furthermore, p-DRP1, a key protein in fission, significantly increased (P < 0.001). Additionally, adipocytes from obese subjects displayed lower basal respiration (49%↓, P < 0.01) and were unresponsive to tunicamycin in contrast to their lean counterparts, demonstrating inefficient mitochondrial oxidative capacity. CONCLUSION: These human data suggest that adipocyte mitochondrial inefficiency is driven by ER stress and exacerbated in obesity. Nutrient excess-induced ER stress leads to mitochondrial dysfunction that may therefore shift lipid deposition ectopically and thus have further implications on the development of related metabolic disorders.


Asunto(s)
Adipocitos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Tunicamicina/farmacología , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Estudios de Cohortes , Estrés del Retículo Endoplásmico/fisiología , Femenino , Humanos , Mitocondrias/fisiología , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Adulto Joven
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