Parabrachial Interleukin-6 reduces body weight and food intake and increases thermogenesis to regulate energy metabolism

Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6This research was funded by the Swedish Research Council ( 2014-2945 to K.P.S.; 2017-00792 to I.W.A.; and 2013-7107 to Patrik Rorsman), the Novo Nordisk Foundation Excellence project grant (to K.P.S. and I.W.A.), the Ragnar Söderberg Foundation (to K.P.S.), Harald Jeanssons Stiftelse and Greta Jeanssons Stiftelse (to K.P.S.), Magnus Bergvalls Stiftelse (to K.P.S.), the Wallenberg Foundation and the Center for Molecular and Translational Medicine (to K.P.S.), postdoctoral stipendium from The Swedish Brain Foundation (to D.M.), the ERC ( BFU2015-70664-R and StG-281408 ) (to R.N.), and the NIH ( DK-21397 ) (to H.J.G.)S

Hindbrain insulin controls feeding behavior

Objective: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice. Methods: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used.Results: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and meta-bolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis. Conclusions: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis. (c) 2022 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</p

Pathophysiological regulation of white adipocyte exocytosis of different adiponectin molecular forms

In this thesis we have identified mechanisms involved in the exocytosis of different adiponectin molecular forms in health and in metabolic disease. We have also studied similarities and differences in depot-specific adipocyte adiponectin release. In paper I we show that the physiological regulation of subcutaneous white adipocyte adiponectin exocytosis involves β3 adrenergic receptors (β3ARs) and Exchange Protein directly Activated by cAMP isoform 1 (Epac1) signalling. Furthermore, we show that adiponectin secretion is disturbed in obesity/type 2 diabetes induced catecholamine resistance due to reduced abundance of the key proteins β3ARs and Epac1. This condition of catecholamine resistance is further associated with a ~50% reduction of circulating high-molecular weight (HMW) adiponectin. In paper II we show that β3AR-activation rapidly triggers the release of HMW adiponectin-containing vesicle whereas insulin induces release of smaller molecular forms, with delayed time-kinetics. We moreover demonstrate that both catecholamine-triggered exocytosis of HMW adiponectin and the insulin-induced secretion of smaller adiponectin forms is entirely diminished in adipocytes from obese/type 2 diabetic mice. The equivalent regulation of secretion of different adiponectin molecular forms by catecholamines and insulin was confirmed in human adipocytes, thus defining a novel role of β3ARs in human adipocyte function. In paper III we propose that adiponectin exocytosis is regulated by sympathetic nerve endings, co-releasing noradrenaline and ATP within the adipose tissue. Secretion measurements confirmed that noradrenaline (elevates cAMP), like adrenaline in paper I, triggers adiponectin exocytosis. Extracellular ATP was shown to augment the exocytotic process, largely due to its elevation of intracellular Ca2+. We also show that defect purinergic signalling together with reduced white adipose tissue noradrenaline content likely aggravates the catecholamine resistance observed in paper I and II. Finally we describe regulation of mouse visceral adipocyte adiponectin secretion in paper IV. As demonstrated in subcutaneous adipocytes (paper I-III), visceral adipocyte adiponectin secretion is also stimulated by activation of β3AR and Epac1. In obese/diabetic conditions, visceral adipocytes are likewise unresponsive to stimulation with catecholamines, but the underlying molecular defect does not involve reduced levels of neither β3AR nor Epac1, thus differing from observations in subcutaneous adipocytes. In conclusion, our results suggest that secretory defects in obesity/type 2 diabetes, attributed to catecholamine resistance, underlie the reduced levels of HMW adiponectin in metabolic disease

Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis

Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca2+ and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca2+-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof

Adipocyte-specific ablation of the Ca2+pump SERCA2 impairs whole-body metabolic function and reveals the diverse metabolic flexibility of white and brown adipose tissue

Objective: Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the endoplasmic retitculum (ER) and is essential for appropriate regulation of intracellular Ca2+ homeostasis. The objective of this study was to test the hypothesis that SERCA pumps are involved in the regulation of white adipocyte hormone secretion and other aspects of adipose tissue function and that this control is disturbed in obesity-induced type-2 diabetes. Methods: SERCA expression was measured in isolated human and mouse adipocytes as well as in whole mouse adipose tissue by Western blot and RT-qPCR. To test the significance of SERCA2 in adipocyte functionality and whole-body metabolism, we generated adipocyte-specific SERCA2 knockout mice. The mice were metabolically phenotyped by glucose tolerance and tracer studies, histological analyses, measurements of glucose-stimulated insulin release in isolated islets, and gene/protein expression analyses. We also tested the effect of pharmacological SERCA inhibition and genetic SERCA2 ablation in cultured adipocytes. Intracellular and mitochondrial Ca2+ levels were recorded with dualwavelength ratio imaging and mitochondrial function was assessed by Seahorse technology. Results: We demonstrate that SERCA2 is downregulated in white adipocytes from patients with obesity and type-2 diabetes as well as in adipocytes from diet-induced obese mice. SERCA2-ablated adipocytes display disturbed Ca2+ homeostasis associated with upregulated ER stress markers and impaired hormone release. These adipocyte alterations are linked to mild lipodystrophy, reduced adiponectin levels, and impaired glucose tolerance. Interestingly, adipocyte-specific SERCA2 ablation leads to increased glucose uptake in white adipose tissue while the glucose uptake is reduced in brown adipose tissue. This dichotomous effect on glucose uptake is due to differently regulated mitochondrial function. In white adipocytes, SERCA2 deficiency triggers an adaptive increase in fibroblast growth factor 21 (FGF21), increased mitochondrial uncoupling protein 1 (UCP1) levels, and increased oxygen consumption rate (OCR). In contrast, brown SERCA2 null adipocytes display reduced OCR despite increased mitochondrial content and UCP1 levels compared to wild type controls. Conclusions: Our data suggest causal links between reduced white adipocyte SERCA2 levels, deranged adipocyte Ca2+ homeostasis, adipose tissue dysfunction and type-2 diabetes. (c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Funding Agencies|Swedish Research Council [2013-07107, 2017-00792, 2019-01239, 2020-01463]; Magnus Bergvall Foundation [2016-01711]; Novo Nordisk Foundation [NNF19OC0056601]; Swedish Diabetes Foundation [DIA2016-127, DIA2018-358, DIA2019-419, DIA2014-074, DIA2015-062, DIA2017- 273, DIA2018-354]</p