Obesity is characterized by a state of chronic inflammation in adipose tissue mediated by the secretion of a range of inflammatory cytokines. In comparison to WAT, relatively little is known about the inflammatory status of brown adipose tissue (BAT) in physiology and pathophysiology. Because BAT and brown/beige adipocytes are specialized in energy expenditure they have protective roles against obesity and associated metabolic diseases. BAT appears to be is less susceptible to developing inflammation than WAT. However, there is increasing evidence that inflammation directly alters the thermogenic activity of brown fat by impairing its capacity for energy expenditure and glucose uptake. The inflammatory microenvironment can be affected by cytokines secreted by immune cells as well as by the brown adipocytes themselves. Therefore, pro-inflammatory signals represent an important component of the thermogenic potential of brown and beige adipocytes and may contribute their dysfunction in obesity.

Cardiovascular diseases (CVDs) are a major healthcare burden on the population worldwide. Early detection of this disease is important in prevention and treatment to minimise morbidity and mortality. Biomarkers are a critical tool to either diagnose, screen, or provide prognostic information for pathological conditions. This review discusses the historical cardiac biomarkers used to detect these conditions, discussing their application and their limitations. Identification of new biomarkers have since replaced these and are now in use in routine clinical practice, but still do not detect all disease. Future cardiac biomarkers are showing promise in early studies, but further studies are required to show their value in improving detection of CVD above the current biomarkers. Additionally, the analytical platforms that would allow them to be adopted in healthcare are yet to be established. There is also the need to identify whether these biomarkers can be used for diagnostic, prognostic, or screening purposes, which will impact their implementation in routine clinical practice.

Background: The acquisition of brown-adipocyte-properties by white-adipocytes (BRITE-adipocytes) is an appealing-prospect to combat obesity and type-2-diabetes-Mellitus (T2DM). This may support counteracting the impact of inflammation and mitochondrial-dysfunction, which contribute to the obesity-pathogenesis. However, our previous-finding shave shown that the gut-derived-inflammatory-agent endotoxin can increase the inflammatory-response in white-adipose-tissue impacted by obesity and T2DM, although its effect on the browning-process is unknown. Therefore, the objective of this study was to (1) investigate the in-vitro-effect of endotoxin on the browning-process in human-adipocytes; (2) define the expression of brown-fat-genes and inflammatory-genes in human-abdominal-subcutaneous (AbdSc) and omental (AbdOm) adipose-tissue-cohort (AT; n=128 female; age: 31.6±0.63) to determine the influence of adiposity, AT-depot, inflammation and mitochondrial-function. Methods: Human-primary-adipocytes were differentiated with or without Rosiglitazone, which promotes adipocytes-browning, with or without endotoxin (100 ng/ml). Differentiated-cells were treated with isoproterenol to induce UCP1. AbdSc and AbdOm AT-biopsies were collected with ethical-approval during elective-surgeries. RNA from both cultured-adipocytes and AT was extracted and gene-expression was quantified by qRT-PCR. Results: Endotoxin significantly reduced the BRITE-phenotype in the Rosiglitazone-treated-cells by reducing the key-brown-fat-genes-expression UCP1 (P<0.05), CIDEA (P<0.05), ELOVL3 (P<0.05), PLIN5 (P<0.05), SLC27A2 (P<0.05). Furthermore, endotoxin reduced key-mitochondrial-gene-expression (FIS1, DRP1, OPA1, MFN2, P<0.05). In addition, adiposity was associated with significantly reduced-expression of the key-brown-fat-genes (≈40%, P<0.05) and there was strong negative-correlation between BMI and brown-fat-genes in AbdSc-AT and AbdOm-AT (P<0.05). As anticipated adiposity significantly-increased inflammatory-genes-expression: IL6, MCP1, TNFα, IL1ß (≈30%, P<0.05) in both AbdOm-AT and AbdSc-AT. Interestingly, there were negative-correlations between inflammatory-genes and brown-fat-genes in AbdOm-AT (P<0.05), and direct positive-correlation between mitochondrial and brown-fat-genes in AT (P<0.05). Conclusions: Endotoxin appears to reduce the in-vitro BRITE-phenotype-capacity in adipocytes due to enhanced-mitochondrial-dysfunction and inflammation, which is exacerbated with increasing adiposity. Taken together, subjects with obesity and T2DM would appear to have a reduced-capability to promote a BRITE-phenotype in WAT, as a means to reduce further weight-gain than lean non-diabetic-individuals.

Introduction: Central obesity is a significant risk factor for type 2 diabetes mellitus (T2DM), with omental adipose tissue (AT) particularly involved in such risk. Additionally, obesity can cause low-level gut-derived endotoxemia, which may drive metabolic dysfunction in AT through mitochondrial damage and reduced BRITE (brown-in-white) adipocytes. Bariatric surgery reduces obesity and may prevent such dysfunction. This study investigated whether endotoxin: 1) impairs mitochondrial function and reduces browning in human AT, 2) has a depot specific effect on pathogenic risk, 3) has reduced impact following bariatric surgery. Methods: Ex-vivo AT was collected from human abdominal subcutaneous (AbdSc) and omental (AbdOm) depots (n=134) from Caucasian women (lean:BMI=22.3±1.9 kg/m2, age=32.0±5.2, n=38; overweight (OW):BMI=27.3±1.4 kg/m2, age=31.5±7.5, n=40; obese (OB):BMI=38.1±6.1 kg/m2, age=40.8±12.9, n=63). Sub-cohort analysis of participants with severe obesity (BMI>35 kg/m2) examined AbdScAT pre- and post-bariatric surgery (BMI=42.2±5.6 kg/m2, age=54.5±5.9, n=26). Mitochondrial function and adipocyte browning genes, and serum endotoxin were assessed. Results: With increased weight, rising endotoxin levels correlated with reduced mitochondrial fission and fusion in AbdOmAT, noted by FIS1 (P<0.05, r= -0.37, OW), DRP1 (P<0.05, r=-0.43, OB), and OPA1 (P<0.05, r=-0.39, OB); no such associations were observed in AbdScAT depot. Rising endotoxin levels also correlated with reduced BRITE gene expression in AbdOm from participants classed as overweight, indicated by Cidea (P<0.05, r=-0.3962), ELOVL3 (P<0.05, r=-0.42), PLIN5 (P<0.01, r=-0.46) and SLC27A2 (P<0.01, r= -0.48); this was not observed in AbdScAT. Endotoxin change with bariatric surgery negatively correlated with brown gene change (P<0.05). Conclusion: These data suggest that gut-derived endotoxemia may impair mitochondrial function and reduce adipocyte browning in abdominal AT. This appears to preferentially impact AbdOmAT over AbdScAT, and may therefore have more profound metabolic consequences. Overall, these data highlight that reduced gut-derived endotoxin may improve AbdAT mitochondrial health and BRITE adipocyte development, and therefore lower metabolic risk.

Background: Dysfunctional adipose tissue in obesity is known to contribute to metabolic diseases, including type 2 diabetes mellitus (T2DM). This may be due to increased gut-derived endotoxemia (LPS) reducing brown adipose tissue (BAT) activity and altering mitochondrial function. However, the effect of LPS on BAT activity in 3D culture models has not been studied, despite giving a better representation of in-vivo tissue. Therefore, this study investigated the effects of LPS on 2D and 3D brown adipocyte biology and mitochondrial function. Methods: Immortalized murine brown adipocyte 2D and 3D (spheroid) cultures were differentiated with/without LPS (100 ng/ml). β3-adrenergic stimulator CL316,243 (CL) (10 μM), was used to induce BAT activity via UCP1. Gene and protein expression were analysed using qRT-PCR and immunohistochemistry. Results: LPS reduced expression of browning genes in 2D and 3D models: CIDEA (2D: FC=-14.11, P<0.0001; 3D: FC=-26.21, P<0.0001), ELOVL3 (2D: FC=-24.84, P<0.0001; 3D: FC=-59.82, P<0.001), PLIN5 (2D: FC=-24.18, P<0.01; 3D: FC=-20.32, P<0.0001). Moreover, LPS decreased UCP1 gene expression in basal conditions (2D: FC=-56.38, P<0.05; 3D: FC=-25, P<0.05) and following CL treatment (2D: FC=-2.82, P<0.01; 3D: FC= -7.41, P<0.0001). Immunohistochemistry identified reduced UCP1 expression in LPS-differentiated 3D spheroids (P<0.0001). Mitochondrial genes were also reduced with LPS including (a) dynamic genes: DRP1 and MFN2, with a maximum reduction of FC=-3.2 (P<0.001) in 2D and FC=-6.03 (P<0.0001) in 3D models, (b) biogenesis genes: PGC1α and POLG with a maximum reduction of FC=-3.31 (P<0.0001) in 2D and FC=-8.67 (P<0.0001) in 3D model, and other key functional genes CPT1β, CS, COX4I1. Conclusion: This study identified LPS as an inhibitor of brown adipocytes phenotype and mitochondrial function, with more potency in 3D spheroids. This highlights that gut-derived endotoxemia may contribute to individual brown adipocyte dysfunction and reduced browning capacity of the adipocyte cell population. Therefore, targeting this inflammatory pathway may therefore reduce obesity-associated metabolic dysfunction.

Background: The presence of brown adipose tissue (BAT) in adults offers an opportunity to examine inflammatory factors that may affect metabolic function in states of obesity. Gut-derived lipopolysaccharide (LPS), which is elevated in obesity, and initiates the innate immune response in white adipose tissue, has not been fully studied in BAT. The interactions between LPS, TLR4 and ß3-adrenergic receptors in BAT is unknown. ß3-adrenergic receptor ligands as CL 316,243 (CL) induce BAT activity through UCP1-stimulation. Therefore, the objective of this study was to investigate the effect of LPS on the CL response and examine how LPS may alter mitochondrial function in BAT. Methods: Immortalized brown adipocytes were differentiated with or without LPS (100 ng/ml, 1000 ng/ml). After treating cells with CL, RNA and protein were extracted for qRT-PCR and Western blot analysis. Mitochondrial respiration was assessed using Seahorse Bioscience XF24 extracellular flux. Mitochondrial membrane potential (ΔΨm) was assessed by confocal microscopy images. Reactive oxygen species (ROS) assay was performed to estimate the capacity to prevent cellular damage. Results: LPS significantly reduced BAT phenotype and mitochondrial function. LPS decreased key brown fat genes CIDEA (P<0.001), UCP1 (P<0.01), PGC-1a (P<0.01). Furthermore, LPS-treated cells showed significantly decreased UCP1-expression in response to CL at both protein (≈60%↓) and mRNA levels (≈65%↓). In addition, LPS significantly reduced key mitochondrial genes: ATPase8, CPT1B (P<0.05), CytC (P<0.05), and ND1 (P<0.05). Functional analysis highlighted that LPS impaired mitochondrial function through reduced O₂ consumption rate as well as loss of active membrane potential ΔΨm (≈65%). With ROS production also increased (P<0.001). Conclusions: These findings suggest that LPS poses a risk to damaging mitochondrial function in BAT. Overall, this current data indicates that blocking LPS-TLR4 signalling has potential to enhance BAT activity and mitigate inflammation to counteract obesity and metabolic diseases.