Highlights d A therapeutic dose (50 mg) of mirabegron does not stimulate human BAT thermogenesis d Human brown adipocytes lack b 3 -AR and do not respond to mirabegron in vitro d Norepinephrine-induced respiration is driven by b 2 -AR, which co-localizes with UCP1 d b 2 -AR is the main target for pharmacological activation of human brown adipocytes

High glucose and fatty acid levels impair pancreatic beta cell function. We have recently shown that palmitate-induced loss of INS-1E insulinoma cells is related to increased reactive oxygen species (ROS) production as both toxic effects are prevented by palmitoleate. Here we show that palmitate-induced ROS are mostly mitochondrial: oxidation of MitoSOX, a mitochondria-targeted superoxide probe, is increased by palmitate, whilst oxidation of the equivalent non-targeted probe is unaffected. Moreover, mitochondrial respiratory inhibition with antimycin A stimulates palmitate-induced MitoSOX oxidation. We also show that palmitate does not change the level of mitochondrial uncoupling protein-2 (UCP2) and that UCP2 knockdown does not affect palmitate-induced MitoSOX oxidation. Palmitoleate does not influence MitoSOX oxidation in INS-1E cells ±UCP2 and largely prevents the palmitate-induced effects. Importantly, UCP2 knockdown amplifies the preventive effect of palmitoleate on palmitate-induced ROS. Consistently, viability effects of palmitate and palmitoleate are similar between cells ±UCP2, but UCP2 knockdown significantly augments the palmitoleate protection against palmitate-induced cell loss at high glucose. We conclude that UCP2 neither mediates palmitate-induced mitochondrial ROS generation and the associated cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity.

It has been well established that excessive levels of glucose and palmitate lower glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells. This β-cell 'glucolipotoxicity' is possibly mediated by mitochondrial dysfunction, but involvement of bioenergetic failure in the pathological mechanism is the subject of ongoing debate. We show in the present study that increased palmitate levels impair GSIS before altering mitochondrial function. We demonstrate that GSIS defects arise from increased insulin release under basal conditions in addition to decreased insulin secretion under glucose-stimulatory conditions. Real-time respiratory analysis of intact mouse pancreatic islets reveals that mitochondrial ATP synthesis is not involved in the mechanism by which basal insulin is elevated. Equally, mitochondrial lipid oxidation and production of reactive oxygen species (ROS) do not contribute to increased basal insulin secretion. Palmitate does not affect KCl-induced insulin release at a basal or stimulatory glucose level, but elevated basal insulin release is attenuated by palmitoleate and associates with increased intracellular calcium. These findings deepen our understanding of β-cell glucolipotoxicity and reveal that palmitate-induced GSIS impairment is disconnected from mitochondrial dysfunction, a notion that is important when targeting β-cells for the treatment of diabetes and when assessing islet function in human transplants.

We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

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Adipose tissue type and distribution are major determinants of metabolic disease, warranting investigations of depot-defining mechanisms. Here we show that progenitor cells from four human brown and white adipose depots separate into two main cell fates, a metabolic and a structural branch, during early differentiation at single cell resolution. The metabolic cell type expresses Adiponectin and is driven by an adipogenic transcriptional network including PPARG. Halfway through maturation, these cells have a brown adipocyte signature regardless of adipose depot origin. The structural cell type arises from the same progenitors as the metabolic cell type but expresses extracellular matrix factors and is driven by a competing osteoblast transcription factor network. The metabolic adipocyte gene signature associates with traits for fat distribution. In conclusion, we provide a seamless differentiation map of human adipocytes, providing an insight in the WAT browning process and a long-term perspective of cell-type specific targeting of metabolic disease.

Pro-inflammatory cytokines contribute to beta cell dysfunction in type 1 diabetes (T1D), but the exact underlying mechanisms remain unclear. It was previously shown that cytokines inhibit mitochondrial glucose oxidation leading to impaired glucose-stimulated insulin secretion in rat INS-1E cells, however, the effect of cytokines on mitochondrial respiration in human islets has not been explored. In this study, we investigated mitochondrial dysfunction in human islets and rat beta cells following cytokine treatment. We exposed isolated human pancreatic islets and rat INS-1E cells for up to 24 h to IL-1β and IFN-γ to mimic early beta cell destruction in T1D. Mitochondrial bioenergetics were analyzed by Seahorse XFe24 technology. Human islets from three healthy organ donors exposed to cytokines showed a reduction in glucose-induced oxygen consumption and an increase in ATP-uncoupled respiration. Higher basal mitochondrial oxygen consumption was also observed, which was not associated with an increase in ATP production. In INS-1E cells, cytokines exerted a strong inhibitory effect on mitochondrial glucose oxidation in a dose-independent manner. In acute exposure experiments, no effect on oxygen consumption was observed up to eight hours after cytokine exposure. This finding proposes that cytokine-induced inhibition of mitochondrial respiration is dependent on secondary signaling mechanism and/or changes in gene transcription. To conclude, glucose-induced oxygen consumption is reduced by cytokines in both human islets and INS-1E cells indicative of impaired mitochondrial respiration. Moreover, cytokines augment mitochondrial uncoupled respiration in human islets, indicating mitochondrial dysfunction that may contribute to islet failure in T1D. Ongoing work is investigating gene expression of genes involved in mitochondrial respiration processes. Further studies are needed to clarify the possible pathogenic role of mitochondrial dysfunction in T1D and the underlying mechanisms. Disclosure J. Melo: None. V. Hirschberg Jensen: None. J. Størling: None.