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Adipose tissue plays a crucial role in metabolic syndrome, autoimmune diseases, and many cancers. Because of adipose’s role in so many aspects of human health, there is a critical need for in vitro models that replicate adipose architecture and function. Traditional monolayer models, despite their convenience, are limited, showing heterogeneity and functional differences compared to 3D models. While monolayer cultures struggle with detachment and inefficient differentiation, healthy adipocytes in 3D culture accumulate large lipid droplets, secrete adiponectin, and produce low levels of inflammatory cytokines. The shift from monolayer models to more complex 3D models aims to better replicate the physiology of healthy adipose tissue in culture. This study introduces a simple and accessible protocol for generating adipose organoids using a scaffold-free spheroid model. The method, utilizing either 96-well spheroid plates or agarose micromolds, demonstrates increased throughput, uniformity, and ease of handling compared to previous techniques. This protocol allows for diverse applications, including drug testing, toxin screening, tissue engineering, and co-culturing. The choice between the two methods depends on the experimental goals, with the 96-well plate providing individualized control and the micromold offering scale advantages. The outlined protocol covers isolation, expansion, and characterization of stromal vascular fraction cells, followed by detailed steps for spheroid formation and optional downstream analyses.

Alongside the liver, white adipose tissue (WAT) is critical in regulating systemic energy homeostasis. Although each organ has its specialised functions, they must work coordinately to regulate whole-body metabolism. Adipose tissues and the liver are relatively resilient and can adapt to an energy surplus by facilitating triglyceride (TG) storage up to a certain threshold level without significant metabolic disturbances. However, lipid storage in WAT beyond a " personalised " adiposity threshold becomes dysfunctional, leading to metabolic inflexibility, progressive inflammation, and aberrant adipokine secretion. Moreover, the failure of adipose tissue to store and mobilise lipids results in systemic knock-on lipid overload, particularly in the liver. Factors contributing to hepatic lipid overload include lipids released from WAT, dietary fat intake, and enhanced de novo lipogenesis. In contrast, extrahepatic mechanisms counteracting toxic hepatic lipid overload entail coordinated compensation through oxidation of surplus fatty acids in brown adipose tissue and storage of fatty acids as TGs in WAT. Failure of these integrated homeostatic mechanisms leads to quantitative increases and qualitative alterations to the lipidome of the liver. Initially, hepatocytes preferentially accumulate TG species leading to a relatively " benign " non-alcoholic fatty liver. However, with time, inflammatory responses ensue, progressing into more severe conditions such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, in some individuals (often without an early prognostic clue). Herein, we highlight the pathogenic importance of obesity-induced " adipose tissue failure ", resulting in decreased adipose tissue functionality (i.e. fat storage capacity and metabolic flexibility), in the development and progression of NAFL/NASH. [ABSTRACT FROM AUTHOR]

Adipose tissue plays a crucial role in metabolic syndrome, autoimmune diseases, and many cancers. Because of adipose's role in so many aspects of human health, there is a critical need for in vitro models that replicate adipose architecture and function. Traditional monolayer models, despite their convenience, are limited, showing heterogeneity and functional differences compared to 3D models. While monolayer cultures struggle with detachment and inefficient differentiation, healthy adipocytes in 3D culture accumulate large lipid droplets, secrete adiponectin, and produce low levels of inflammatory cytokines. The shift from monolayer models to more complex 3D models aims to better replicate the physiology of healthy adipose tissue in culture. This study introduces a simple and accessible protocol for generating adipose organoids using a scaffold-free spheroid model. The method, utilizing either 96-well spheroid plates or agarose micromolds, demonstrates increased throughput, uniformity, and ease of handling compared to previous techniques. This protocol allows for diverse applications, including drug testing, toxin screening, tissue engineering, and co-culturing. The choice between the two methods depends on the experimental goals, with the 96-well plate providing individualized control and the micromold offering scale advantages. The outlined protocol covers isolation, expansion, and characterization of stromal vascular fraction cells, followed by detailed steps for spheroid formation and optional downstream analyses.

Dietary patterns high in fat influence local and systemic oxidative stress through adipose tissue (AT) accrual and increased reactive oxygen species generation. Lycopene, a carotenoid with antioxidant functionality, may mitigate excess oxidative stress, yet the lipophilic nature of this compound may limit its functionality if sequestered by AT. Thus, it is critical to elucidate whether lycopene's efficacy is limited based on adiposity. The purpose of this study was to investigate the influence of lycopene-supplemented normal- and high-fat diets on systemic and AT redox status. Male Sprague-Dawley rats (n = 18) were fed a 30% normal-fat (NFD) or 60% high-fat (HFD) purified diet supplemented with 100 mg of lycopene/day. Body weight and visceral AT mass, as well as serum and AT lycopene, lipid peroxides, and antioxidant capacity (AC), were assessed after 3, 7, and 10 weeks of supplementation. At week 10, AT mass was significantly higher (P = .028) in the HFD group, yet there were no significant differences in serum or AT lycopene concentrations or lipid peroxides between groups. Additionally, AT in the HFD group exhibited significantly greater lipophilic AC (27.6% higher, P = .031). Results suggest that excess adiposity did not negatively influence circulating lycopene, nor did it limit its antioxidant functionality. [ABSTRACT FROM AUTHOR]

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.

ObjectiveIn a phase II clinical trial in nine obese, insulin-resistant humans, we observed that treatment with KDT501, a novel isohumulone drug, increased total and high-molecular weight (HMW) adiponectin in plasma. The objective was to determine whether KDT501 increased adiponectin secretion from subcutaneous white adipose tissue (SC WAT) and the underlying mechanism(s).MethodsNine obese participants with either prediabetes or with normal glucose tolerance plus three features of metabolic syndrome were part of the study. SC WAT biopsies were performed before and after 28 days of KDT501 treatment in a clinical research setting. In addition, a cold stimulus was used to induce thermogenic gene expression. Adiponectin secretion was measured, and gene expression of 130 genes involved in adipose tissue function was determined. The effect of KDT501 on adipocyte mitochondrial function was analyzed in vitro.ResultsSC WAT explants secreted more total and HMW adiponectin after KDT501 treatment (P

Objective: In a phase II clinical trial in nine obese, insulin-resistant humans, we observed that treatment with KDT501, a novel isohumulone drug, increased total and high-molecular weight (HMW) adiponectin in plasma. The objective was to determine whether KDT501 increased adiponectin secretion from subcutaneous white adipose tissue (SC WAT) and the underlying mechanism(s). Methods: Nine obese participants with either prediabetes or with normal glucose tolerance plus three features of metabolic syndrome were part of the study. SC WAT biopsies were performed before and after 28 days of KDT501 treatment in a clinical research setting. In addition, a cold stimulus was used to induce thermogenic gene expression. Adiponectin secretion was measured, and gene expression of 130 genes involved in adipose tissue function was determined. The effect of KDT501 on adipocyte mitochondrial function was analyzed in vitro. Results: SC WAT explants secreted more total and HMW adiponectin after KDT501 treatment (P < 0.05). After KDT501 treatment, a number of genes involved in thermogenesis and lipolysis were induced by cold (P < 0.05). KDT501 also potentiated ß-adrenergic signaling (P < 0.001) and enhanced mitochondrial function in adipocytes (P < 0.001). Conclusion: KDT501 induced adiponectin secretion posttranscriptionally and increased gene expression of thermogenic and lipolytic genes in response to cold stimulation. These beneficial effects on SC WAT may be explained by the ability of KDT501 to potentiate ß-adrenergic signaling and enhance mitochondrial function in adipocytes. [ABSTRACT FROM AUTHOR]

Over the past 2 decades, the incidence of childhood obesity has risen dramatically. This recent rise in childhood obesity is particularly concerning as adults who were obese during childhood develop type II diabetes that is intractable to current forms of treatment compared with individuals who develop obesity in adulthood. While the mechanisms responsible for the exacerbated diabetic phenotype associated with childhood obesity is not clear, it is well known that childhood is an important time period for the establishment of normal white adipose tissue in humans. This association suggests that exposure to obesogenic stimuli during adipose development may have detrimental effects on adipose function and metabolic homeostasis. In this study, we identify the period of development associated with puberty, postnatal days 18–34, as critical for the establishment of normal adipose mass in mice. Exposure of mice to high fat diet only during this time period results in metabolic dysfunction, increased leptin expression, and increased adipocyte size in adulthood in the absence of sustained increased fat mass or body weight. These findings indicate that exposure to obesogenic stimuli during critical developmental periods have prolonged effects on adipose tissue function that may contribute to the exacerbated metabolic dysfunctions associated with childhood obesity. [ABSTRACT FROM PUBLISHER]

Compared to global estimates, Sub-Saharan Africa (SSA) has the highest projected rates of increase in type 2 diabetes (T2D) over the next 25years. This is attributed to the ageing population, increasing urbanisation and the associated lifestyle changes. Although the prevalence does not differ by gender, deaths attributable to T2D in SSA are greater in women, likely due to differences in beliefs and access to care. Women in SSA also have greater risk factor burden for T2D than men, in particular obesity, which is explained in part by sociocultural factors. The pathogenesis of diabetes differs between African and Caucasian women, with implications for risk assessment. African women are more insulin resistant than their Caucasian counterparts, despite a more 'favourable' body fat distribution. Notably, women in SSA face the dual burden of T2D and HIV/AIDS. HIV positive women in SSA are typically young and obese, with the latter being exacerbated by anti-retroviral therapy (ART). Cultural perceptions regarding weight loss and limited financial resources are the major limitations to the management of T2D. Hence prevention is vital. However, there is a paucity of studies examining the effectiveness and sustainability of interventions to reduce T2D in SSA. [ABSTRACT FROM AUTHOR]

Adiponectin and leptin are proteins secreted by the adipose tissue and have an influence on insulin sensitivity and on inflammatory markers. Altered levels could play a part in the pathogenesis of type 1 diabetes mellitus. We determined adiponectin and leptin levels over a nine-year period in children with type 1 diabetes mellitus (T1D) in relation to the increasing incidence of T1D, and studied the impact of patient status, age, gender and body mass index (BMI). Data were derived from a population-based registry of diabetic children (DanDiabKids) from 1997 to 2005. Children with newly diagnosed T1D ( n = 482) were included, and healthy siblings ( n = 479) were chosen as a control group. Leptin levels were significantly higher in recent years (in both patients and siblings), whereas for adiponectin, the levels were lower in recent years in the patient group. Leptin levels were lower in children with T1D (RR 0.74, p = 0.003) and in males (RR 0.52, p < 0.001) and increasing with age in both groups. For adiponectin, there was a negative association between level and age in patients. Both adipokines showed a significant correlation with BMI and lower levels in children with blood samples taken within the first 2 days after initiation of insulin treatment. There has been a change in leptin and adiponectin levels in children with or without T1D from 1997 to 2005. This is not explained by changes in BMI and may reflect changes in other factors like diet or physical activity. [ABSTRACT FROM AUTHOR]

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT. [ABSTRACT FROM AUTHOR]

Objective: Spinal epidural lipomatosis (EL) represents an excessive deposition of unencapsulated adipose tissue in the spinal canal that can result in chronic back pain in patients who are obese with and without diabetes. We aim to calculate the total volumetric epidural fat on lumbar spine MRI in a predominately obese population and correlate total epidural fat to lower back pain (LBP) and body mass index (BMI)., Research Design and Methods: We developed a program (Fat Finder) to quantify volumetric distribution of epidural fat throughout the lumbar spine. Eleven patients with LBP were imaged using two MRI protocols: parallel axial slices and conventional clinical protocol. The distribution of epidural fat per level was analyzed and normalized to the spinal canal size., Results: Our sample had an average age of 59.9 years and BMI of 31.57 kg/m 2 . EL subgroup consisted of seven patients. The L2-L5 total fat volume was 3477.6 mm 3 (1431.1-5595.9) in the EL group versus 1783.8 mm 3 (815.0-2717.5) in the age-similar non-EL group. A higher percentage of fat volume in the canal was associated with higher LBP scores. The fat percentage was 32.2% among patients with EL versus 15.4% for age-similar non-EL with LBP score of 6.1 and 4.0, respectively., Conclusions: The Fat Finder is a novel volumetric method to quantify epidural lumbar spinal fat. The epidural fat favors the lower spinal segment with direct proportionality between the fat volume and LBP score, independent of BMI., Competing Interests: Competing interests: None declared.

Flow cytometry and fluorescence-activated cell sorting (FACS) techniques have significantly advanced the characterization of adipocyte precursor cell (APC) populations. They allow immunophenotyping, quantification, and isolation of distinct populations, which is critical for understanding adipose tissue development and homeostasis. Here, we describe the identification and purification of adipocyte precursor cells using flow cytometry and FACS, defined by previously established surface marker profiles. In addition, we describe the mouse models and whole adipose tissue visualization techniques that will enable us to characterize the plasticity and the cellular origin of APCs.

Peri-adipocyte extracellular matrix (ECM) remodeling is a key biological process observed during adipose tissue development and expansion. The genetic loss of a pericellular collagenase, MMP14 (also known as MT1-MMP), renders mice lipodystrophic with the accumulation of undigested collagen fibers in adipose tissues. MMP14 is not necessary for adipocyte differentiation (adipogenesis) per se under a conventional two-dimensional (2-D) culture condition; however, MMP14 plays a critical role in adipogenesis in vivo. The role of MMP14 in adipogenesis and adipocyte gene expression was uncovered in vitro only when tested within a three-dimensional (3-D) collagen gel, which recapitulated the in vivo ECM-rich environment. Studying adipogenesis in 3-D may serve as an effective experimental approach to bridge gaps in our understanding of in vivo adipocyte biology. Moreover, by assessing the content of collagen family members and their rate of degradation in adipose tissues, we should be able to better define the role of dynamic ECM remodeling in the pathogenesis of obesity and diabetes., (© 2014 Elsevier Inc. All rights reserved.)