Extracellular metallothionein as a therapeutic target in the early progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by lymphocyte infiltration into the pancreatic islets of Langerhans, leading to the destruction of insulin-producing beta cells and uncontrolled hyperglycemia. In the nonobese diabetic (NOD) murine model of T1D, the onset of this infiltration starts several weeks before glucose dysregulation and overt diabetes. Recruitment of immune cells to the islets is mediated by several chemotactic cytokines, including CXCL10, while other cytokines, including SDF-1α, can confer protective effects. Global gene expression studies of the pancreas from prediabetic NOD mice and single-cell sequence analysis of human islets from prediabetic, autoantibody-positive patients showed an increased expression of metallothionein (MT), a small molecular weight, cysteine-rich metal-binding stress response protein. We have shown that beta cells can release MT into the extracellular environment, which can subsequently enhance the chemotactic response of Th1 cells to CXCL10 and interfere with the chemotactic response of Th2 cells to SDF-1α. These effects can be blocked in vitro with a monoclonal anti-MT antibody, clone UC1MT. When administered to NOD mice before the onset of diabetes, UC1MT significantly reduces the development of T1D. Manipulation of extracellular MT may be an important approach to preserving beta cell function and preventing the development of T1D.

A CRISPR Screen Identifies the E3 Ubiquitin Ligase Rfwd2 as a Negative Regulator of Glucose Uptake in Brown Adipocytes.

Brown adipose tissue activation increases energy expenditure and has been shown to improve glucose tolerance, making it a promising target for the treatment of obesity and type 2 diabetes. Brown adipocytes differentiate into cells with multilocular lipid droplets, which can efficiently absorb and oxidize glucose; however, the mechanisms regulating these processes are not completely understood. We conducted a genome-wide loss-of-function screen using a CRISPR-based approach to identify genes that promote or inhibit adipogenesis and glucose uptake in brown adipocytes. We validated genes that negatively or positively regulated these pathways and verified that the E3-ubiquitin ligase Rfwd2 suppressed brown adipocyte glucose uptake. Brown adipocytes with CRISPR-targeted Rfwd2 deletion showed an altered proteomic landscape and increased basal, as well as insulin-stimulated, glucose uptake. These data reveal the complexity of genetic regulation of brown adipogenesis and glucose metabolism.

ScreenDMT reveals linoleic acid diols replicably associate with BMI and stimulate adipocyte calcium fluxes.

Activating brown adipose tissue (BAT) improves systemic metabolism, making it a promising target for metabolic syndrome. BAT is activated by 12, 13-dihydroxy-9Z-octadecenoic acid (12, 13-diHOME), which we previously identified to be inversely associated with BMI and which directly improves metabolism in multiple tissues. Here we profile plasma lipidomics from a cohort of 83 people and test which lipids' association with BMI replicates in a concordant direction using our novel tool ScreenDMT, whose power and validity we demonstrate via mathematical proofs and simulations. We find that the linoleic acid diols 12, 13-diHOME and 9, 10-diHOME both replicably inversely associate with BMI and mechanistically activate calcium fluxes in mouse brown and white adipocytes in vitro, which implicates this pathway and 9, 10-diHOME as candidate therapeutic targets. ScreenDMT can be applied to test directional mediation, directional replication, and qualitative interactions, such as identifying biomarkers whose association is shared (replication) or opposite (qualitative interaction) across diverse populations.

ADRA1A-Gαq signalling potentiates adipocyte thermogenesis through CKB and TNAP.

Noradrenaline (NA) regulates cold-stimulated adipocyte thermogenesis1. Aside from cAMP signalling downstream of ß-adrenergic receptor activation, how NA promotes thermogenic output is still not fully understood. Here, we show that coordinated α1-adrenergic receptor (AR) and ß3-AR signalling induces the expression of thermogenic genes of the futile creatine cycle2,3, and that early B cell factors, oestrogen-related receptors and PGC1α are required for this response in vivo. NA triggers physical and functional coupling between the α1-AR subtype (ADRA1A) and Gαq to promote adipocyte thermogenesis in a manner that is dependent on the effector proteins of the futile creatine cycle, creatine kinase B and tissue-non-specific alkaline phosphatase. Combined Gαq and Gαs signalling selectively in adipocytes promotes a continual rise in whole-body energy expenditure, and creatine kinase B is required for this effect. Thus, the ADRA1A-Gαq-futile creatine cycle axis is a key regulator of facultative and adaptive thermogenesis.

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure.

Objective: Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete "classical" depots, or interspersed in white adipose tissue (WAT), termed "inducible/recruitable" BAT, or 'beige/brite' adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. Methods: In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for in vivo delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior in vitro brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive in vivo whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Results: Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in "browning", including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). Conclusions: In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments.

Endothelial Cells Induced Progenitors Into Brown Fat to Reduce Atherosclerosis.

BACKGROUND: Insulin resistance (IR) can increase atherosclerotic and cardiovascular risk by inducing endothelial dysfunction, decreasing nitric oxide (NO) production, and accelerating arterial inflammation. The aim is to determine the mechanism by which insulin action and NO production in endothelial cells can improve systemic bioenergetics and decrease atherosclerosis via differentiation of perivascular progenitor cells (PPCs) into brown adipocytes (BAT). METHODS: Studies used various endothelial transgenic and deletion mutant ApoE-/- mice of insulin receptors, eNOS (endothelial NO synthase) and ETBR (endothelin receptor type B) receptors for assessments of atherosclerosis. Cells were isolated from perivascular fat and micro-vessels for studies on differentiation and signaling mechanisms in responses to NO, insulin, and lipokines from BAT. RESULTS: Enhancing insulin's actions on endothelial cells and NO production in ECIRS1 transgenic mice reduced body weight and increased systemic energy expenditure and BAT mass and activity by inducing differentiation of PPCs into beige/BAT even with high-fat diet. However, positive changes in bioenergetics, BAT differentiation from PPCs and weight loss were inhibited by N(gamma)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of eNOS, in ECIRS1 mice and eNOSKO mice. The mechanism mediating NO's action on PPC differentiation into BAT was identified as the activation of solubilized guanylate cyclase/PKGIα (cGMP protein-dependent kinase Iα)/GSK3ß (glycogen synthase kinase 3ß) pathways. Plasma lipidomics from ECIRS1 mice with NO-induced increased BAT mass revealed elevated 12,13-diHOME production. Infusion of 12,13-diHOME improved endothelial dysfunction and decreased atherosclerosis, whereas its reduction had opposite effects in ApoE-/-mice. CONCLUSIONS: Activation of eNOS and endothelial cells by insulin enhanced the differentiation of PPC to BAT and its lipokines and improved systemic bioenergetics and atherosclerosis, suggesting that endothelial dysfunction is a major contributor of energy disequilibrium in obesity.

Telomerase Reverse Transcriptase Expression Marks a Population of Rare Adipose Tissue Stem Cells.

In adult tissues such as adipose tissue, post-mitotic cells like adipocytes can be replaced by differentiation of a population of tissue-resident stem cells. Expression of mouse telomerase reverse transcriptase (mTert) is a hallmark of stem cell populations, and previous efforts to identify tissue-resident adult stem cells by measuring mTert expression have increased our understanding of stem cell biology significantly. Here, we used a doxycycline-inducible mouse model to perform longitudinal, live-animal lineage-tracing of mTert-expressing cells for more than 1 year. We identified a rare (<2%) population of stem cells in different fat depots that express putative preadipocyte markers. The adipose-derived mTert-positive cells are capable of self-renewal and possess adipogenic potential. Finally, we demonstrate that high-fat diet (HFD) can initiate differentiation of these cells in vivo. These data identify a population of adipose stem cells that contribute to the depot-specific response to HFD.

Vascular smooth muscle-derived Trpv1+ progenitors are a source of cold-induced thermogenic adipocytes.

Brown adipose tissue (BAT) and beige fat function in energy expenditure in part due to their role in thermoregulation, making these tissues attractive targets for treating obesity and metabolic disorders. While prolonged cold exposure promotes de novo recruitment of brown adipocytes, the exact sources of cold-induced thermogenic adipocytes are not completely understood. Here, we identify transient receptor potential cation channel subfamily V member 1 (Trpv1)+ vascular smooth muscle (VSM) cells as previously unidentified thermogenic adipocyte progenitors. Single-cell RNA sequencing analysis of interscapular brown adipose depots reveals, in addition to the previously known platelet-derived growth factor receptor (Pdgfr)α-expressing mesenchymal progenitors, a population of VSM-derived adipocyte progenitor cells (VSM-APC) expressing the temperature-sensitive cation channel Trpv1. We demonstrate that cold exposure induces the proliferation of Trpv1+ VSM-APCs and enahnces their differentiation to highly thermogenic adipocytes. Together, these findings illustrate the landscape of the thermogenic adipose niche at single-cell resolution and identify a new cellular origin for the development of brown and beige adipocytes.

Defining the lineage of thermogenic perivascular adipose tissue.

Brown adipose tissue can expend large amounts of energy, and therefore increasing its size or activity is a promising therapeutic approach to combat metabolic disease. In humans, major deposits of brown fat cells are found intimately associated with large blood vessels, corresponding to perivascular adipose tissue (PVAT). However, the cellular origins of PVAT are poorly understood. Here, we determine the identity of perivascular adipocyte progenitors in mice and humans. In mice, thoracic PVAT develops from a fibroblastic lineage, consisting of progenitor cells (Pdgfra+, Ly6a+ and Pparg-) and preadipocytes (Pdgfra+, Ly6a+ and Pparg+), which share transcriptional similarity with analogous cell types in white adipose tissue. Interestingly, the aortic adventitia of adult animals contains a population of adipogenic smooth muscle cells (Myh11+, Pdgfra- and Pparg+) that contribute to perivascular adipocyte formation. Similarly, human PVAT contains presumptive fibroblastic and smooth muscle-like adipocyte progenitor cells, as revealed by single-nucleus RNA sequencing. Together, these studies define distinct populations of progenitor cells for thermogenic PVAT, providing a foundation for developing strategies to augment brown fat activity.

Endogenous Fatty Acid Synthesis Drives Brown Adipose Tissue Involution.

Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.

Brown Fat-Activating Lipokine 12,13-diHOME in Human Milk Is Associated With Infant Adiposity.

CONTEXT: Little is known about the specific breastmilk components responsible for protective effects on infant obesity. Whether 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME), an oxidized linoleic acid metabolite and activator of brown fat metabolism, is present in human milk, or linked to infant adiposity, is unknown. OBJECTIVE: To examine associations between concentrations of 12,13-diHOME in human milk and infant adiposity. DESIGN: Prospective cohort study from 2015 to 2019, following participants from birth to 6 months of age. SETTING: Academic medical centers. PARTICIPANTS: Volunteer sample of 58 exclusively breastfeeding mother-infant pairs; exclusion criteria included smoking, gestational diabetes, and health conditions with the potential to influence maternal or infant weight gain. MAIN OUTCOME MEASURES: Infant anthropometric measures including weight, length, body mass index (BMI), and body composition at birth and at 1, 3, and 6 months postpartum. RESULTS: We report for the first time that 12,13-diHOME is present in human milk. Higher milk 12,13-diHOME level was associated with increased weight-for-length Z-score at birth (ß = 0.5742, P = 0.0008), lower infant fat mass at 1 month (P = 0.021), and reduced gain in BMI Z-score from 0 to 6 months (ß = -0.3997, P = 0.025). We observed similar associations between infant adiposity and milk abundance of related oxidized linoleic acid metabolites 12,13-Epoxy-9(Z)-octadecenoic acid (12,13-epOME) and 9,10-Dihydroxy-12-octadecenoic acid (9,10-diHOME), and metabolites linked to thermogenesis including succinate and lyso-phosphatidylglycerol 18:0. Milk abundance of 12,13-diHOME was not associated with maternal BMI, but was positively associated with maternal height, milk glucose concentration, and was significantly increased after a bout of moderate exercise. CONCLUSIONS: We report novel associations between milk abundance of 12,13-diHOME and adiposity during infancy.

CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice.

Brown and brown-like beige/brite adipocytes dissipate energy and have been proposed as therapeutic targets to combat metabolic disorders. However, the therapeutic effects of cell-based therapy in humans remain unclear. Here, we created human brown-like (HUMBLE) cells by engineering human white preadipocytes using CRISPR-Cas9-SAM-gRNA to activate endogenous uncoupling protein 1 expression. Obese mice that received HUMBLE cell transplants showed a sustained improvement in glucose tolerance and insulin sensitivity, as well as increased energy expenditure. Mechanistically, increased arginine/nitric oxide (NO) metabolism in HUMBLE adipocytes promoted the production of NO that was carried by S-nitrosothiols and nitrite in red blood cells to activate endogenous brown fat and improved glucose homeostasis in recipient animals. Together, these data demonstrate the utility of using CRISPR-Cas9 technology to engineer human white adipocytes to display brown fat-like phenotypes and may open up cell-based therapeutic opportunities to combat obesity and diabetes.

Integrated metabolomics reveals altered lipid metabolism in adipose tissue in a model of extreme longevity.

Adipose tissue plays an essential role in metabolic health. Ames dwarf mice are exceptionally long-lived and display metabolically beneficial phenotypes in their adipose tissue, providing an ideal model for studying the intersection between adipose tissue and longevity. To this end, we assessed the metabolome and lipidome of adipose tissue in Ames dwarf mice. We observed distinct lipid profiles in brown versus white adipose tissue of Ames dwarf mice that are consistent with increased thermogenesis and insulin sensitivity, such as increased cardiolipin and decreased ceramide concentrations. Moreover, we identified 5-hydroxyeicosapentaenoic acid (5-HEPE), an ω-3 fatty acid metabolite, to be increased in Ames dwarf brown adipose tissue (BAT), as well as in circulation. Importantly, 5-HEPE is increased in other models of BAT activation and is negatively correlated with body weight, insulin resistance, and circulating triglyceride concentrations in humans. Together, these data represent a novel lipid signature of adipose tissue in a mouse model of extreme longevity.

FGF6 and FGF9 regulate UCP1 expression independent of brown adipogenesis.

Uncoupling protein-1 (UCP1) plays a central role in energy dissipation in brown adipose tissue (BAT). Using high-throughput library screening of secreted peptides, we identify two fibroblast growth factors (FGF), FGF6 and FGF9, as potent inducers of UCP1 expression in adipocytes and preadipocytes. Surprisingly, this occurs through a mechanism independent of adipogenesis and involves FGF receptor-3 (FGFR3), prostaglandin-E2 and interaction between estrogen receptor-related alpha, flightless-1 (FLII) and leucine-rich-repeat-(in FLII)-interacting-protein-1 as a regulatory complex for UCP1 transcription. Physiologically, FGF6/9 expression in adipose is upregulated by exercise and cold in mice, and FGF9/FGFR3 expression in human neck fat is significantly associated with UCP1 expression. Loss of FGF9 impairs BAT thermogenesis. In vivo administration of FGF9 increases UCP1 expression and thermogenic capacity. Thus, FGF6 and FGF9 are adipokines that can regulate UCP1 through a transcriptional network that is dissociated from brown adipogenesis, and act to modulate systemic energy metabolism.

Cell-autonomous light sensitivity via Opsin3 regulates fuel utilization in brown adipocytes.

Opsin3 (Opn3) is a transmembrane heptahelical G protein-coupled receptor (GPCR) with the potential to produce a nonvisual photoreceptive effect. Interestingly, anatomical profiling of GPCRs reveals that Opn3 mRNA is highly expressed in adipose tissue. The photosensitive functions of Opn3 in mammals are poorly understood, and whether Opn3 has a role in fat is entirely unknown. In this study, we found that Opn3-knockout (Opn3-KO) mice were prone to diet-induced obesity and insulin resistance. At the cellular level, Opn3-KO brown adipocytes cultured in darkness had decreased glucose uptake and lower nutrient-induced mitochondrial respiration than wild-type (WT) cells. Light exposure promoted mitochondrial activity and glucose uptake in WT adipocytes but not in Opn3-KO cells. Brown adipocytes carrying a defective mutation in Opn3's putative G protein-binding domain also exhibited a reduction in glucose uptake and mitochondrial respiration in darkness. Using RNA-sequencing, we identified several novel light-sensitive and Opn3-dependent molecular signatures in brown adipocytes. Importantly, direct exposure of brown adipose tissue (BAT) to light in living mice significantly enhanced thermogenic capacity of BAT, and this effect was diminished in Opn3-KO animals. These results uncover a previously unrecognized cell-autonomous, light-sensing mechanism in brown adipocytes via Opn3-GPCR signaling that can regulate fuel metabolism and mitochondrial respiration. Our work also provides a molecular basis for developing light-based treatments for obesity and its related metabolic disorders.

Lipokines and Thermogenesis.

Adaptive thermogenesis is a catabolic process that consumes energy-storing molecules and expends that energy as heat in response to environmental changes. This process occurs primarily in brown and beige adipose tissue. Thermogenesis is regulated by many factors, including lipid derived paracrine and endocrine hormones called lipokines. Recently, technologic advances for identifying new lipid biomarkers of thermogenic activity have shed light on a diverse set of lipokines that act through different pathways to regulate energy expenditure. In this review, we highlight a few examples of lipokines that regulate thermogenesis. The biosynthesis, regulation, and effects of the thermogenic lipokines in several families are reviewed, including oloeylethanolamine, endocannabinoids, prostaglandin E2, and 12,13-diHOME. These thermogenic lipokines present potential therapeutic targets to combat states of excess energy storage, such as obesity and related metabolic disorders.

12-Lipoxygenase Regulates Cold Adaptation and Glucose Metabolism by Producing the Omega-3 Lipid 12-HEPE from Brown Fat.

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and ß3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway.

TGF-ß2 is an exercise-induced adipokine that regulates glucose and fatty acid metabolism.

Exercise improves health and well-being across diverse organ systems, and elucidating mechanisms underlying the beneficial effects of exercise can lead to new therapies. Here, we show that transforming growth factor-ß2 (TGF-ß2) is secreted from adipose tissue in response to exercise and improves glucose tolerance in mice. We identify TGF-ß2 as an exercise-induced adipokine in a gene expression analysis of human subcutaneous adipose tissue biopsies after exercise training. In mice, exercise training increases TGF-ß2 in scWAT, serum, and its secretion from fat explants. Transplanting scWAT from exercise-trained wild type mice, but not from adipose tissue-specific Tgfb2-/- mice, into sedentary mice improves glucose tolerance. TGF-ß2 treatment reverses the detrimental metabolic effects of high fat feeding in mice. Lactate, a metabolite released from muscle during exercise, stimulates TGF-ß2 expression in human adipocytes. Administration of the lactate-lowering agent dichloroacetate during exercise training in mice decreases circulating TGF-ß2 levels and reduces exercise-stimulated improvements in glucose tolerance. Thus, exercise training improves systemic metabolism through inter-organ communication with fat via a lactate-TGF-ß2-signaling cycle.

Cold-Activated Lipid Dynamics in Adipose Tissue Highlights a Role for Cardiolipin in Thermogenic Metabolism.

Thermogenic fat expends energy during cold for temperature homeostasis, and its activity regulates nutrient metabolism and insulin sensitivity. We measured cold-activated lipid landscapes in circulation and in adipose tissue by MS/MSALL shotgun lipidomics. We created an interactive online viewer to visualize the changes of specific lipid species in response to cold. In adipose tissue, among the approximately 1,600 lipid species profiled, we identified the biosynthetic pathway of the mitochondrial phospholipid cardiolipin as coordinately activated in brown and beige fat by cold in wild-type and transgenic mice with enhanced browning of white fat. Together, these data provide a comprehensive lipid bio-signature of thermogenic fat activation in circulation and tissue and suggest pathways regulated by cold exposure.