Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs.

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.

FOXK1 and FOXK2 regulate aerobic glycolysis.

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility1. A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients2. Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

Combination of phosphodiesterase-5-inhibitors and beta blockers improves experimental portal hypertension and erectile dysfunction.

BACKGROUND & AIMS: Phosphodiesterase-5 inhibitors (PDE-5-I) are used for treatment of erectile dysfunction (ED), which is common in patients with cirrhosis. They may improve portal hypertension (PH), but contradictory data on efficacy and side-effects have been reported. Non-selective beta blockers (NSBB) reduce portal pressure, but might aggravate ED. Thus, we evaluated the combination of PDE-5-I with NSBB and its impact on PH and ED in experimental cirrhosis. METHODS: ED was assessed in cirrhotic patients (n = 86) using standardized questionnaire. Experimental cirrhosis was induced by bile-duct-ligation or carbon-tetrachloride intoxication in rats. Corpus cavernosum pressure - a surrogate of ED -, as well as systemic and portal haemodynamics, were measured in vivo and in situ after acute administration of udenafil alone or in combination with propranolol. mRNA and protein levels of PDE-5 signalling were analysed using PCR and western Blot. RESULTS: ED in humans was related to severity of liver disease and to NSBB treatment. PDE-5 was mainly expressed in hepatic stellate cells and upregulated in human and experimental cirrhosis. Propranolol reduced corpus cavernosum pressure in cirrhotic rats and it was restored by udenafil. Even though udenafil treatment improved PH, it led to a reduction of mean arterial pressure. The combination of udenafil and propranolol reduced portal pressure and hepatic resistance without systemic side-effects. CONCLUSIONS: ED is common with advanced cirrhosis and concomitant NSBB treatment. The combination of PDE-5-I and NSBB improves ED and PH in experimental cirrhosis.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of ß-adrenergic receptors. Because BAT therapies based on cold exposure or ß-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

A2A R-induced transcriptional deregulation in astrocytes: An in vitro study.

Adenosine A2A receptors (A2A R) are modulators of various physiological processes essential for brain homeostasis and fine synaptic tuning. In certain neurodegenerative conditions, notably Alzheimer's disease (AD), A2A Rs are pathologically upregulated in neurons but also in astrocytes. In that context, the use of A2A Rs inhibitors, normalizing impaired receptor function, is seen as a potential therapeutic strategy. However, the impact of A2A R alterations, particularly in astrocytes, is not fully understood. Here, we investigated the effect of A2A R overexpression on transcriptional deregulation in primary astrocytic cultures. By performing whole transcriptome analysis, we found that A2A R overexpression promotes robust transcriptional changes, mostly affecting immune response, angiogenesis, and cell activation-related genes. Importantly, we observed that treatment with SCH58261, a selective A2A R antagonist, restored the expression levels of several inflammatory and astrocytic activation-related genes, such as Interleukin-1beta and vimentin. This supports the notion that A2A R blockade could restore some astrocytic dysfunctions associated with abnormal A2A R expression, further arguing for a potential beneficial impact of receptor antagonists in A2A R-induced transcriptional deregulation, inflammation, and astrogliosis. Overall, our findings provide novel insights into the putative impact of A2A R overexpression on transcriptional deregulation in astrocytes, thereby opening novel avenues for the use of A2A R antagonists as potential therapeutic strategy in neurodegenerative diseases.

Regulation of human brown adipose tissue by adenosine and A2A receptors - studies with [15O]H2O and [11C]TMSX PET/CT.

PURPOSE: Brown adipose tissue (BAT) has emerged as a potential target to combat obesity and diabetes, but novel strategies to activate BAT are needed. Adenosine and A2A receptor (A2AR) agonism activate BAT in rodents, and endogenous adenosine is released locally in BAT as a by-product of noradrenaline, but physiological data from humans is lacking. The purpose of this pilot study was to investigate the effects of exogenous adenosine on human BAT perfusion, and to determine the density of A2ARs in human BAT in vivo for the first time, using PET/CT imaging. METHODS: Healthy, lean men (n = 10) participated in PET/CT imaging with two radioligands. Perfusion of BAT, white adipose tissue (WAT) and muscle was quantified with [15O]H2O at baseline, during cold exposure and during intravenous administration of adenosine. A2AR density of the tissues was quantified with [11C]TMSX at baseline and during cold exposure. RESULTS: Adenosine increased the perfusion of BAT even more than cold exposure (baseline 8.3 ± 4.5, cold 19.6 ± 9.3, adenosine 28.6 ± 7.9 ml/100 g/min, p < 0.01). Distribution volume of [11C]TMSX in BAT was significantly lower during cold exposure compared to baseline. In cold, low [11C]TMSX binding coincided with high concentrations of noradrenaline. CONCLUSIONS: Adenosine administration caused a maximal perfusion effect in human supraclavicular BAT, indicating increased oxidative metabolism. Cold exposure increased noradrenaline concentrations and decreased the density of A2AR available for radioligand binding in BAT, suggesting augmented release of endogenous adenosine. Our results show that adenosine and A2AR are relevant for activation of human BAT, and A2AR provides a future target for enhancing BAT metabolism.

cGMP manipulation in cardiometabolic disease: chances and challenges.

PURPOSE OF REVIEW: Cardiovascular and metabolic diseases are closely linked and commonly occur in the same patients. This review focuses on the cyclic guanosine monophosphate (cGMP) system and its crosstalk between metabolism and the cardiovascular system. RECENT FINDINGS: Recent studies suggest that cGMP, which serves as second messenger for nitric oxide and for natriuretic peptides, improves oxidative metabolism and insulin signaling. The clinical evidence is particularly strong for the natriuretic peptide branch of the cGMP system. Clinical trials suggested improvements in insulin sensitivity and reductions in the risk of progressing to type 2 diabetes mellitus. However, further studies are needed. SUMMARY: Enhancing cGMP signaling through nonpharmacological or pharmacological means may improve glucose metabolism in addition to affecting the cardiovascular system. However, excessive cGMP production could have significant unwanted cardiovascular and metabolic effects.

BAT Exosomes: Metabolic Crosstalk with Other Organs and Biomarkers for BAT Activity.

In the last decade, exosomes have gained interest as a new type of intercellular communication between cells and tissues. Exosomes are circulating, cell-derived lipid vesicles smaller than 200 nm that contain proteins and nucleic acids, including microRNAs (miRNAs), and are able to modify cellular targets. Exosomal miRNAs function as signalling molecules that regulate the transcription of their target genes and can cause phenotypic transformation of recipient cells. Recent studies have shown that brown fat secretes exosomes as a form of communication with other metabolic organs such as the liver. Moreover, it has been shown that levels of miRNAs in BAT-derived exosomes change after BAT activation in vitro and in vivo. Thus, BAT-derived exosomes can be used as potential biomarkers of BAT activity. Here, we review the present knowledge about BAT-derived exosomes and their role in metabolism.

Role of cAMP and cGMP Signaling in Brown Fat.

Cold-induced activation of brown adipose tissue (BAT) is mediated by norepinephrine and adenosine that are released during sympathetic nerve activation. Both signaling molecules induce an increase in intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) in murine and human BAT. In brown adipocytes, cAMP plays a central role, because it activates lipolysis, glucose uptake, and thermogenesis. Another well-studied intracellular second messenger is 3',5'-cyclic guanosine monophosphate (cGMP), which closely resembles cAMP. Several studies have shown that intact cGMP signaling is essential for normal adipogenic differentiation and BAT-mediated thermogenesis in mice. This chapter highlights recent observations, demonstrating the physiological significance of cyclic nucleotide signaling in BAT as well as their potential to induce browning of white adipose tissue (WAT) in mice and humans.

The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity.

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

Brown, beige, and white: the new color code of fat and its pharmacological implications.

Brown adipose tissue (BAT) was previously regarded as a special type of fat relevant only for defending hibernating animals and newborns against a cold environment. Recently, BAT has received considerable attention following its (re)discovery in humans. Using glucose tracers, multiple laboratories independently found metabolically active BAT in adults. The enormous metabolic powers of BAT in animal models could make it an attractive target for antiobesity therapies in humans. Here, we review the present knowledge on the role of BAT in energy homeostasis and metabolism, focusing on signaling pathways and potential targets for novel therapeutics. We also shine light on ongoing debates, including those about the true color of brown fat in adults, as well as on the requirements for translation of basic research on BAT into clinical medicine.

Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871.

Correction to: Regulation of human brown adipose tissue by adenosine and A2A receptors - studies with [15O]H2O and [11C]TMSX PET/CT.

The original version of this article contained a mistake in the first sentence of the Results section of the Abstract.

NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal Spreading: A Novel Role for Cx37 (Connexin 37) and the Protein Tyrosine Phosphatase SHP-2.

OBJECTIVE: Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. APPROACH AND RESULTS: Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). CONCLUSIONS: Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.

HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

Fat tissues, the brite and the dark sides.

Fat tissue is well known for its capacity to store energy and its detrimental role in obesity and metaflammation. However, humans possess different types of fat that have different functions in physiology and metabolic diseases. Apart from white adipose tissue (WAT), the body's main energy storage, there is also brown adipose tissue (BAT) that dissipates energy as a defense against cold and maintains energy balance for the whole body. BAT is present not only in newborns but also in adult humans and its mass correlates with leanness. Moreover, "brown-like" adipocytes have been detected in human WAT. These "brown-in-white" (brite) or beige cells can be induced by cold and a broad spectrum of pharmacological substances and, therefore, they are also known as "inducible brown adipocytes." Activation of brown and/or brite adipocytes reduces metabolic diseases, at least in murine models of obesity. Thus, brown/brite adipocytes represent the "brite" side of fat and are potential targets for novel therapeutic approaches for treatment of obesity and obesity-associated diseases.