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.

Premature aging, as denoted by a reduced telomere length (TL), has been observed in several chronic inflammatory diseases, such as obesity and type 2 diabetes mellitus (T2DM). However, no study to date has addressed the potential inflammatory influence of the gut-derived Gram-negative bacterial fragments lipopolysaccharide, also referred to as endotoxin, and its influence on TL in low-grade inflammatory states such as type 2 diabetes mellitus (T2DM). The current study therefore investigated the influence of endotoxin and inflammatory factors on telomere length (TL) in adults with (T2DM: n = 387) and without (non-diabetic (ND) controls: n = 417) obesity and T2DM. Anthropometric characteristics were taken, and fasted blood samples were used to measure biomarkers, TL, and endotoxin. The findings from this study highlighted across all participants that circulating endotoxin (r = −0.17, p = 0.01) was inversely associated with TL, noting that endotoxin and triglycerides predicted 18% of the variance perceived in TL (p < 0.001). Further stratification of the participants according to T2DM status and sex highlighted that endotoxin significantly predicted 19% of the variance denoted in TL among male T2DM participants (p = 0.007), where TL was notably influenced. The influence on TL was not observed to be impacted by anti-T2DM medications, statins, or anti-hypertensive therapies. Taken together, these results show that TL attrition was inversely associated with circulating endotoxin levels independent of the presence of T2DM and other cardiometabolic factors, suggesting that low-grade chronic inflammation may trigger premature biological aging. The findings further highlight the clinical relevance of mitigating the levels of circulating endotoxin (e.g., manipulation of gut microbiome) not only for the prevention of chronic diseases but also to promote healthy aging.

Altered expression of receptor tyrosine kinases (RTKs) is a major driver of growth and metastasis of cancers. Recepteur d’origine nantais (RON) receptor is a single-pass transmembrane RTK aberrantly expressed in a number of cancers. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies are complicated by the presence of unknown number/types of isoforms of RON, which, despite having similar sequences, are localized differently and mediate varied functions. The objective of this study was to identify splice variants of RON transcripts between exons 1 and 10 that code for the extracellular region. Direct cDNA sequencing was performed for the transcript between exons 1–10 of RON by Sanger sequencing in various lung cancer cell lines. PCR amplification and bi-directional sequencing of cDNA for section between exons 1 and 10 from lung cancer cell lines revealed the presence of several splice variants of RON transcripts; the variants were formed by skipping of exons 2, 2–3, 5–6, 6 and 8–9. Each of these transcript variants were found in one or more cell lines. While the variants formed by skipping of exons 2, 2–3 and 5–6 resulted in loss of 63, 106 and 109 amino acids, respectively, and didn’t cause reading-frameshift, the transcripts formed by skipping of exons 6 and 8–9 caused reading-frameshift. Splice variant lacking exons 8–9 was found in 13 out of 23 cell lines tested. Lung cancer cell lines contain several splice variants of RON which involve skipping of exons coding for extracellular region. Some of the splicing changes result in reading-frameshift and the N-terminally truncated isoforms are expected to be secreted out. The ubiquitous nature of alternative splicing events in RON suggests the need for isoform specific approaches to functional analysis and therapeutic targeting of RON.

This protocol describes a robust method for the extraction of intra and extracellular metabolites of gut bacterial mono and co-cultures. In recent years, the co-culture techniques employed in the field of microbiology have demonstrated significant importance in regard to understanding cell–cell interactions, cross-feeding, and the metabolic interactions between different bacteria, fungi, and microbial consortia which enable the mimicking of complex co-habitant conditions. This protocol highlights a robust reproducible physiologically relevant culture and extraction protocol for the co-culture of gut bacterium. The novel extraction steps are conducted without using quenching and cell disruption through bead-cell methods, freeze–thaw cycles, and sonication, which tend to affect the physical and biochemical properties of intracellular metabolites and secretome. The extraction procedure of inoculated bacterial co-cultures and monocultures use fast vacuum filtration and centrifugation. The extraction methodology is fast, effective, and robust, requiring 4 h to complete.

Background: Dysfunctional endoplasmic reticula (ER) and mitochondria contribute to the pathogenesis of obesity and type 2 diabetes mellitus (T2DM). This may, in part, be facilitated by cross-talk between the two organelles during conditions of nutrient excess such as obesity, however the potential impact of ER stress on mitochondrial function has not been well studied. This study investigated whether induction of ER stress in human adipocytes may contribute to mitochondrial dysfunction. Methods: Differentiated human adipocytes from a cell line (Chub-S7) and primary abdominal subcutaneous (AbdSc) adipocytes from lean and obese donors were treated with Tunicamycin (Tn) to induce ER stress. Mitochondrial function was assessed via oxygen consumption rate (OCR), mitochondrial membrane potential (MMP), ATP concentration, mitochondrial dynamics and number. Results: Tunicamycin-induced ER stress increased respiratory capacity in a dose- and time-dependent manner (24 h:23%, (P<0.05); 48 h:68%, (P<0.01); 72 h:136%, (P<0.01)), with a preference for glycolysis after 72 h. Both ATP production and MMP were impaired in a corresponding manner (26%, P<0.001; 32%, P<0.0001 respectively), demonstrating that ER stress contributes to impaired mitochondrial function. Confocal microscopy highlighted that ER stress also resulted in reduced mitochondrial elongation (16%, P<0.05) and cellular area occupied by mitochondria (28%, P<0.05). Additionally, pDrp1, a key fission protein was significantly increased with Tn treatment. AbdSc adipocytes from lean subjects had a similar response to ER stress, with a 21% rise in OCR with Tn treatment (P<0.01), whilst adipocytes from obese subjects had a 33% reduced basal respiration than their lean counterparts (P<0.001), demonstrating an existing significantly impaired respiratory function. Summary: These data suggest that ER stress induces mitochondrial dysfunction and that this is exacerbated during obesity, contributing to metabolic pathologies such as T2DM. This highlights that therapies targeting ER stress in adipocytes may be beneficial in the treatment of chronic ER stress and mitochondrial dysfunction in metabolic disease.

Lipoprotein-associated phospholipase A2 (LpPLA2) is a member of the phospholipaseA2 super family of enzymes, and is upregulated in arterial inflammation, obesity and cardiovascular disease. The other isoforms, iPLA2 and cPLA2, appear to contribute to inflammation through production of lipid mediators. The role of PLA2 in human adipose tissue (AT) is unclear, therefore we sought to i) characterise PLA2 isoforms in lean, obese, T2DM abdominal subcutaneous (AbdSc) and omental (Om) AT ii) evaluate the role of lipids and inflammatory markers on circulating LpPLA2 levels and iii) determine the in vitro regulation of LpPLA2 in human adipocytes. AT and sera from lean, overweight, obese and T2DM subjects were taken. PLA2 gene expression was determined by microarray, RT-qPCR and Western Blot. Association between circulating LpPLA2 and metabolic parameters were investigated. The human adipocyte cell line, Chub-S7, was used to assess the effects of oxidized LDL on PLA2 expression. LpPLA2 mRNA levels were higher in AbdSc AT than Om AT in obesity by twofold (P<0.05). The cPLA2 protein expression was increased with obesity in AbdSc AT (P<0.01). T2DM showed increased LpPLA2 mRNA levels in AbdSc (P<0.001) and OmAT (P<0.01). Serum LpPLA2 showed positive correlations with cholesterol, TG, LDL, endotoxin and oxidized LDL (Ox-LDL) (P<0.001) in non-diabetic subjects and with Ox-LDL (P<0.001), LDL (P<0.01) and cholesterol (P<0.05) in T2DM. In differentiated pre-adipocytes, activation of LpPLA2 protein expression was noted in response to LDL and Ox-LDL (P<0.001). The adipocyte appears to be an active source of LpPLA2, altered by fat depot and metabolic state, with LpPLA2 protein expression being induced by LDL and Ox-LDL in vitro. As such, increased LpPLA2 protein from the adipocyte in obesity and/or T2DM may contribute to raise circulating Ox-LDL, which promotes further inflammation and atherosclerotic risk. Taken together, LpPLA2 in the adipocyte and AT represents an important therapeutic target to reduce inflammation, atherosclerotic risk and development of metabolic complications. Declaration of funding: Birminggham Science City and Thai Government.