Saturated fatty acid (SFA) high-fat diets (HFDs) enhance interleukin (IL)-1β–mediated adipose inflammation and insulin resistance. However, the mechanisms by which different fatty acids regulate IL-1β and the subsequent effects on adipose tissue biology and insulin sensitivity in vivo remain elusive. We hypothesized that the replacement of SFA for monounsaturated fatty acid (MUFA) in HFDs would reduce pro-IL-1β priming in adipose tissue and attenuate insulin resistance via MUFA-driven AMPK activation. MUFA-HFD–fed mice displayed improved insulin sensitivity coincident with reduced pro-IL-1β priming, attenuated adipose IL-1β secretion, and sustained adipose AMPK activation compared with SFA-HFD–fed mice. Furthermore, MUFA-HFD–fed mice displayed hyperplastic adipose tissue, with enhanced adipogenic potential of the stromal vascular fraction and improved insulin sensitivity. In vitro, we demonstrated that the MUFA oleic acid can impede ATP-induced IL-1β secretion from lipopolysaccharide- and SFA-primed cells in an AMPK-dependent manner. Conversely, in a regression study, switching from SFA- to MUFA-HFD failed to reverse insulin resistance but improved fasting plasma insulin levels. In humans, high-SFA consumers, but not high-MUFA consumers, displayed reduced insulin sensitivity with elevated pycard-1 and caspase-1 expression in adipose tissue. These novel findings suggest that dietary MUFA can attenuate IL-1β–mediated insulin resistance and adipose dysfunction despite obesity via the preservation of AMPK activity.

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The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Preliminary evidence has suggested that high-fat diets (HFD) enriched with SFA, but not MUFA, promote hyperinsulinaemia and pancreatic hypertrophy with insulin resistance. The objective of this study was to determine whether the substitution of dietary MUFA within a HFD could attenuate the progression of pancreatic islet dysfunction seen with prolonged SFA-HFD. For 32 weeks, C57BL/6J mice were fed either: (1) low-fat diet, (2) SFA-HFD or (3) SFA-HFD for 16 weeks, then switched to MUFA-HFD for 16 weeks (SFA-to-MUFA-HFD). Fasting insulin was assessed throughout the study; islets were isolated following the intervention. Substituting SFA with MUFA-HFD prevented the progression of hyperinsulinaemia observed in SFA-HFD mice (P < 0·001). Glucose-stimulated insulin secretion from isolated islets was reduced by SFA-HFD, yet not fully affected by SFA-to-MUFA-HFD. Markers of β-cell identity (Ins2, Nkx6.1, Ngn3, Rfx6, Pdx1 and Pax6) were reduced, and islet inflammation was increased (IL-1β, 3·0-fold, P = 0·007; CD68, 2·9-fold, P = 0·001; Il-6, 1·1-fold, P = 0·437) in SFA-HFD – effects not seen with SFA-to-MUFA-HFD. Switching to MUFA-HFD can partly attenuate the progression of SFA-HFD-induced hyperinsulinaemia, pancreatic inflammation and impairments in β-cell function. While further work is required from a mechanistic perspective, dietary fat may mediate its effect in an IL-1β–AMP-activated protein kinase α1-dependent fashion. Future work should assess the potential translation of the modulation of metabolic inflammation in man.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Abstract. A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied prospectively to examine the relationship between circulating autoantibodies, islet insulin secretion, pancreatic infiltration, and islet cell replication during the pre-diabetic period. Although a higher incidence of islet cell surface (ICSA) and insulin autoantibodies (IAA) was detected in the diabetes-prone than in the low diabetic-incidence BB/E rats there was no correlation between the two antibodies in individual animals. Moreover, ICSA, but not IAA, were associated with loss of first phase islet insulin release. Between 75 and 105 days of age the number of diabetes-prone rats with ICSA and impaired islet insulin secretory function increased. Over the same period, there was a concomitant increase in the proportion of diabetes-prone animals with pancreatic infiltration, and increased islet endocrine cell proliferation. All these interrelated phenomena were observed in diabetes-prone BB/E rats at a time when the animals were normoglycaemic.

AMPK, a critical metabolic sensor exhibits anti‐inflammatory properties. Recently we demonstrated that AMPK played an important role in modulating IL‐1β‐induced inflammation in response to different dietary fatty acids, wherein oleic acid (OA) impeded both ATP and palmitic acid (PA) induced IL‐1β secretion from macrophages coincident with increased levels of phosphorylated AMPK. The aim of this study is to further define the macrophage inflammatory and metabolic phenotype in the presence of monounsaturated fatty acids (MUFA) and to determine how MUFA‐activated AMPK impedes saturated fatty acid (SFA)‐induced inflammation.The inflammatory and metabolic phenotype of bone marrow derived macrophages treated with OA and PA or a combination of both, with or without LPS stimulation was measured by ELISA and seahorse XF24 analyser. Western blot analysis was performed on adipose tissue harvested from mice fed a 45% SFA (palm oil) or 45% MUFA (olive oil) –high‐fat diet for 16–32 weeks.The magnitude of inflammation in M1 polarised macrophages was reduced when cultured in the presence of OA compared to PA as demonstrated by reduced TNFα cytokine secretion from OA treated cells. On the other hand, lack of AMPK abolished the OA effect. OA impeded LPS‐induced NFκB activation and reduced pro‐IL‐1β levels similar to the AMPK agonist AICAR. Corresponding alterations were observed in the macrophage metabolic phenotype wherein cells treated with PA had reduced ATP production and increased glycolysis and glycolytic capacity compared to cells treated with OA. Additionally macrophages treated with PA had reduced coupling efficiency compared to those treated with OA. Importantly, pre‐treatment with OA reduced PA induced glycolysis and glycolytic capacity and increased cell coupling efficiency. In mice switched from a SFA to a MUFA high fat diet, the levels of phosphorylated AMPK were increased in the adipose tissue compared to animals maintained on a SFA high‐fat diet. Correspondingly both immature and secreted forms of adipose IL‐1β were reduced in these mice, and hyperinsulinemia was also reduced.In conclusion, macrophages exposed to MUFA exhibit alleviated inflammation in response to pro‐inflammatory stimuli. Preliminary data point towards a critical role for AMPK in this process whereby, AMPK activation may mitigate inflammation, by inhibiting NFκB activity and consequently impeding priming of pro‐IL‐1β. Critically, MUFA‐treated cells indicate a greater capacity to respond to stress, exemplified by reduced glycolysis and increased oxidative phosphorylation compared to PA treated cells. Overall, these results indicate that MUFA may have the ability to both impede and reverse SFA induced inflammation, via AMPK. This may shed light into new possibilities for both the prevention and treatment of chronic inflammatory related diseases, such as obesity‐induced insulin resistance and type 2 diabetes.This research is funded by the Science Foundation Principal Investigator Award (11/PI/1119). Orla F Finucane was awarded an Albert Reynold Travel Fellowship from the European Foundation for the study of Diabetes for research conducted in BIDMC, Harvard Medical School, Boston, MA, Boston, USA.

Previous work has established that different dietary fats can have varying impacts on metabolism and insulin sensitivity (1) . Specifically, saturated fatty acids (SFA) drive insulin resistance and inflammation, while dietary monounsaturated fatty acids (MUFA) do not disrupt metabolism to the same extent (2) . Moreover, feeding a high-fat diet (HFD) enriched with MUFA, rather than SFA-HFD, causes less IL-1β mediated inflammation, insulin resistance and hyperinsulinemia (3) . Our aim was to establish whether switching to a MUFA-based diet after previously feeding a SFA-based diet could ameliorate hyperinsulinemia, inflammatory markers and pancreatic islet function.A total of 30 male C57BL/J6 mice were assigned one of three diets: i) a low fat diet for 32 weeks (LFD; 10 % kcal; n = 10), ii) a SFA-based HFD for 32 weeks (SFA-HFD; 45 % kcal; n = 10) or iii) a SFA-based diet for 16 weeks followed by a MUFA-based diet for an additional 16 weeks (MUFA-HFD; 45 % kcal; n = 10). Fasting glucose and insulin levels were used to calculate HOMA-IR and HOMA-%β. Glucose-stimulated insulin secretion response (1·5 g/kg intraperitoneal glucose) was assessed at baseline and following the 32-week diets. Following the diets, mice were killed and metabolic tissues (e.g., liver, adipose tissue, and pancreas) were harvested. Pancreatic islets were isolated by collagenase digestion and Ficoll separation, and were subsequently used to assess glucose-stimulated insulin secretion. Islet gene expression was analyzed by real-time RT-PCR, and pancreatic immunostaining was used to assess expression of insulin, IL-1β as well as macrophage infiltration. Statistical analysis included 1-way or 2-way ANOVA, where appropriate, with Bonferroni post-hoc comparisons.HOMA-IR was elevated in both SFA-HFD and MUFA-HFD groups; however, the degree of insulin resistance was significantly lower in the MUFA-HFD group at 20, 24 and 32 weeks. Furthermore, the MUFA-HFD group exhibited reduced hyperinsulinemia compared to the SFA-HFD group (P < 0·001). Islets isolated from the SFA-HFD group had a significantly lower (∼50 %, P = 0·029) insulin stimulatory index compared to the LFD group. Interestingly, this decline was partially reduced in the MUFA-HFD group. Pancreatic macrophage infiltration and IL-1β content were higher in the SFA-HFD group compared to both the LFD and MUFA-HFD groups. Finally, gene expression analysis of markers of β-cell development and function (e.g., insulin, Nkx6·1, Pdk1) revealed significant declines in the SFA-HFD group compared to the LFD and MUFA-HFD groups (27 %, 36 %, and 15 %, respectively). Ampk expression was also reduced in islets isolated following the SFA-HFD but was preserved in MUFA-HFD islets. Moreover, there was a 57 % reduction in IL-1β in islets from the MUFA-HFD group compared to both LFD and SFA-HFD fed mice. In conclusion, this work suggests that changing from a SFA-HFD to a MUFA-HFD can partially prevent pancreatic dysfunction, and emphasizes the varying effects of different dietary fats on metabolic health.