Adiposity, immunity, and inflammation: interrelationships in health and disease: a report from 24th Annual Harvard Nutrition Obesity Symposium, June 2023.

The rapidly evolving field of immunometabolism explores how changes in local immune environments may affect key metabolic and cellular processes, including that of adipose tissue. Importantly, these changes may contribute to low-grade systemic inflammation. In turn, chronic low-grade inflammation affecting adipose tissue may exacerbate the outcome of metabolic diseases. Novel advances in our understanding of immunometabolic processes may critically lead to interventions to reduce disease severity and progression. An important example in this regard relates to obesity, which has a multifaceted effect on immunity, activating the proinflammatory pathways such as the inflammasome and disrupting cellular homeostasis. This multifaceted effect of obesity can be investigated through study of downstream conditions using cellular and systemic investigative techniques. To further explore this field, the National Institutes of Health P30 Nutrition Obesity Research Center at Harvard, in partnership with Harvard Medical School, assembled experts to present at its 24th Annual Symposium entitled "Adiposity, Immunity, and Inflammation: Interrelationships in Health and Disease" on 7 June, 2023. This manuscript seeks to synthesize and present key findings from the symposium, highlighting new research and novel disease-specific advances in the field. Better understanding the interaction between metabolism and immunity offers promising preventative and treatment therapies for obesity-related immunometabolic diseases.

Spatial mapping of hepatic ER and mitochondria architecture reveals zonated remodeling in fasting and obesity.

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility.

Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.

Obesity alters levels of pituitary hormones that govern hepatic immune-metabolic homeostasis, dysregulation of which leads to nonalcoholic fatty liver disease (NAFLD). However, the impact of obesity on intra-pituitary homeostasis is largely unknown. Here, we uncovered a blunted unfolded protein response (UPR) but elevated inflammatory signatures in pituitary glands of obese mice and humans. Furthermore, we found that obesity inflames the pituitary gland, leading to impaired pituitary inositol-requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) UPR branch, which is essential for protecting against pituitary endocrine defects and NAFLD progression. Intriguingly, pituitary IRE1-deletion resulted in hypothyroidism and suppressed the thyroid hormone receptor B (THRB)-mediated activation of Xbp1 in the liver. Conversely, activation of the hepatic THRB-XBP1 axis improved NAFLD in mice with pituitary UPR defect. Our study provides the first evidence and mechanism of obesity-induced intra-pituitary cellular defects and the pathophysiological role of pituitary-liver UPR communication in NAFLD progression.

An Organism-Level Quantitative Flux Model of Mammalian Energy Metabolism.

Mammalian tissues feed on nutrients in the blood circulation. At the organism-level, mammalian energy metabolism comprises of oxidation, interconverting, storing and releasing of circulating nutrients. Though much is known about the individual processes and nutrients, a holistic and quantitative model describing these processes for all major circulating nutrients is lacking. Here, by integrating isotope tracer infusion, mass spectrometry, and isotope gas analyzer measurement, we developed a framework to systematically quantify fluxes through these processes for 10 major circulating energy nutrients in mice, resulting in an organism-level quantitative flux model of energy metabolism. This model revealed in wildtype mice that circulating nutrients' metabolic cycling fluxes are more dominant than their oxidation fluxes, with distinct partition between cycling and oxidation flux for individual circulating nutrients. Applications of this framework in obese mouse models showed on a per animal basis extensive elevation of metabolic cycling fluxes in ob/ob mice, but not in diet-induced obese mice. Thus, our framework describes quantitatively the functioning of energy metabolism at the organism-level, valuable for revealing new features of energy metabolism in physiological and disease conditions.

Endothelial-derived FABP4 constitutes the majority of basal circulating hormone and regulates lipolysis-driven insulin secretion.

Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with obesity and metabolic pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with Fabp4 deletion in cells known to express the gene - adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) - to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was not significantly reduced in Adipo-KO mice, whereas Endo-KO mice showed ~87% reduction versus WT controls. In contrast, Adipo-KO mice exhibited ~62% decreased induction of FABP4 responses to lipolysis, while Endo-KO mice showed only mildly decreased induction, indicating that adipocytes are the main source of increases in FABP4 during lipolysis. We did not detect any myeloid contribution to circulating FABP4. Surprisingly, despite the nearly intact induction of FABP4, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis.

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion.

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by ß-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGLAdpKO). Here, we find that upon activation of ß-adrenergic receptors in vivo, ATGLAdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGLfl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGLAdpKO mice was indeed from the adipocytes. ATGLAdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGLAdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.

Ubiquinone deficiency drives reverse electron transport to disrupt hepatic metabolic homeostasis in obesity.

Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generation in vivo remain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH 2 /Q ratio, driving excessive mROS production via reverse electron transport (RET) from site I Q in complex I. Using multiple complementary genetic and pharmacological models in vivo we demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH 2 /Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology.

Fatty acid-binding protein 4 (FABP4) is a critical immune-metabolic modulator, mainly expressed in adipocytes and macrophages, secreted from adipocytes in association with lipolysis, and plays essential pathogenic roles in cardiovascular and metabolic diseases. We previously reported Chlamydia pneumoniae infecting murine 3T3-L1 adipocytes and causing lipolysis and FABP4 secretion in vitro. However, it is still unknown whether C. pneumoniae intranasal lung infection targets white adipose tissues (WATs), induces lipolysis, and causes FABP4 secretion in vivo. In this study, we demonstrate that C. pneumoniae lung infection causes robust lipolysis in WAT. Infection-induced WAT lipolysis was diminished in FABP4-/- mice or FABP4 inhibitor-pretreated wild-type mice. Infection by C. pneumoniae in wild-type but not FABP4-/- mice induces the accumulation of TNF-α- and IL-6-producing M1-like adipose tissue macrophages in WAT. Infection-induced WAT pathology is augmented by endoplasmic reticulum (ER) stress/the unfolded protein response (UPR), which is abrogated by treatment with azoramide, a modulator of the UPR. C. pneumoniae lung infection is suggested to target WAT and induce lipolysis and FABP4 secretion in vivo via ER stress/UPR. FABP4 released from infected adipocytes may be taken up by other neighboring intact adipocytes or adipose tissue macrophages. This process can further induce ER stress activation and trigger lipolysis and inflammation, followed by FABP4 secretion, leading to WAT pathology. A better understanding of the role of FABP4 in C. pneumoniae infection-induced WAT pathology will provide the basis for rational intervention measures directed at C. pneumoniae infection and metabolic syndrome, such as atherosclerosis, for which robust epidemiologic evidence exists.

Differential IL18 signaling via IL18 receptor and Na-Cl co-transporter discriminating thermogenesis and glucose metabolism regulation.

White adipose tissue (WAT) plays a role in storing energy, while brown adipose tissue (BAT) is instrumental in the re-distribution of stored energy when dietary sources are unavailable. Interleukin-18 (IL18) is a cytokine playing a role in T-cell polarization, but also for regulating energy homeostasis via the dimeric IL18 receptor (IL18r) and Na-Cl co-transporter (NCC) on adipocytes. Here we show that IL18 signaling in metabolism is regulated at the level of receptor utilization, with preferential role for NCC in brown adipose tissue (BAT) and dominantly via IL18r in WAT. In Il18r-/-Ncc-/- mice, high-fat diet (HFD) causes more prominent body weight gain and insulin resistance than in wild-type mice. The WAT insulin resistance phenotype of the double-knockout mice is recapitulated in HFD-fed Il18r-/- mice, whereas decreased thermogenesis in BAT upon HFD is dependent on NCC deletion. BAT-selective depletion of either NCC or IL18 reduces thermogenesis and increases BAT and WAT inflammation. IL18r deletion in WAT reduces insulin signaling and increases WAT inflammation. In summary, our study contributes to the mechanistic understanding of IL18 regulation of energy metabolism and shows clearly discernible roles for its two receptors in brown and white adipose tissues.

Regulation of liver subcellular architecture controls metabolic homeostasis.

Cells display complex intracellular organization by compartmentalization of metabolic processes into organelles, yet the resolution of these structures in the native tissue context and their functional consequences are not well understood. Here we resolved the three-dimensional structural organization of organelles in large (more than 2.8 × 105 µm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) at high resolution (8 nm isotropic pixel size) using enhanced focused ion beam scanning electron microscopy1,2 imaging followed by deep-learning-based automated image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese mice and found substantial alterations, particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural reorganization characterized by marked disorganization of stacks of ER sheets3 and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes by monitoring the effects of experimental recovery of the subcellular organization on cellular and systemic metabolism. We conclude that the hepatic subcellular organization of the ER architecture are highly dynamic, integrated with the metabolic state and critical for adaptive homeostasis and tissue health.

Aberrant Ca2+ signaling by IP3Rs in adipocytes links inflammation to metabolic dysregulation in obesity.

Chronic metabolic inflammation is a key feature of obesity, insulin resistance, and diabetes. Here, we showed that altered regulation of the Ca2+ channel inositol trisphosphate receptor (IP3R) was an adipocyte-intrinsic event involved in the emergence and propagation of inflammatory signaling and the resulting insulin resistance. Inflammation induced by cytokine exposure in vitro or by obesity in vivo led to increases in the abundance and activity of IP3Rs and in the phosphorylation of the Ca2+-dependent kinase CaMKII in adipocytes in a manner dependent on the kinase JNK. In mice, adipocyte-specific loss of IP3R1/2 protected against adipose tissue inflammation and insulin resistance, despite the mice exhibiting substantial diet-induced weight gain. Thus, this work suggests that increased IP3R activity is a key link between obesity, inflammation, and insulin resistance. These data also suggest that approaches to target IP3R-mediated Ca2+ homeostasis in adipocytes may offer new therapeutic opportunities against metabolic diseases, especially because GWAS studies also implicate this locus in human obesity.

A hormone complex of FABP4 and nucleoside kinases regulates islet function.

The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis1,2. Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4)3. Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans4-7, no mechanism of action has yet been described8-10. Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease.

Metabolic Messengers: tumour necrosis factor.

Tumour necrosis factor (TNF) is a classical, pleiotropic pro-inflammatory cytokine. It is also the first 'adipokine' described to be produced from adipose tissue, regulated in obesity and proposed to contribute to obesity-associated metabolic disease. In this review, we provide an overview of TNF in the context of metabolic inflammation or metaflammation, its discovery as a metabolic messenger, its sites and mechanisms of action and some critical considerations for future research. Although we focus on TNF and the studies that elucidated its immunometabolic actions, we highlight a conceptual framework, generated by these studies, that is equally applicable to the complex network of pro-inflammatory signals, their biological activity and their integration with metabolic regulation, and to the field of immunometabolism more broadly.

The unfolded protein response regulates hepatic autophagy by sXBP1-mediated activation of TFEB.

Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyzes of both primary hepatocytes and whole livers further showed that sXBP1 occupies the -743 to -523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction.Abbreviations: AAV8: adeno-associated virus serotype 8; ACTB: actin, beta; ANOVA: analysis of variance; ATF6: activating transcription factor-6; ATG: autophagy related; BECN1: beclin 1; BMI: body mass index; ChIP: chromatin immunoprecipitation; CLEAR: coordinated lysosomal expression and regulation; Cre: cre recombinase; DIO: diet-induced obesity; EBSS: Earle's balanced salt solution; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HFD: high-fat diet; h: hours; HSCs: hepatic stellate cells; INS: insulin; L/A: ammonium chloride and leupeptin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; RD: regular diet; RFP: red fluorescent protein; SERPINA7/TBG: serpin family A member 7; SQSTM1/p62: sequestome 1; sXbp1 LOE: liver-specific overexpression of spliced Xbp1; TFEB: transcription factor EB; TG: thapsigargin; TN: tunicamycin; UPR: unfolded protein response; wks: weeks; WT: wild type; XBP1: X-box binding protein 1; xbp1 LKO: liver-specific Xbp1 knockout.

Cardiolipin deficiency in Barth syndrome is not associated with increased superoxide/H2 O2 production in heart and skeletal muscle mitochondria.

Barth syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in the gene encoding the transacylase tafazzin and characterized by loss of cardiolipin and severe cardiomyopathy. Mitochondrial oxidants have been implicated in the cardiomyopathy in BTHS. Eleven mitochondrial sites produce superoxide/hydrogen peroxide (H2 O2 ) at significant rates. Which of these sites generate oxidants at excessive rates in BTHS is unknown. Here, we measured the maximum capacity of superoxide/H2 O2 production from each site and the ex vivo rate of superoxide/H2 O2 production in the heart and skeletal muscle mitochondria of the tafazzin knockdown mice (tazkd) from 3 to 12 months of age. Despite reduced oxidative capacity, superoxide/H2 O2 production was indistinguishable between tazkd mice and wild-type littermates. These observations raise questions about the involvement of mitochondrial oxidants in BTHS pathology.

Intercellular Transmission of Hepatic ER Stress in Obesity Disrupts Systemic Metabolism.

Endoplasmic reticulum stress (ERS) has a pathophysiological role in obesity-associated insulin resistance. Yet, the coordinated tissue response to ERS remains unclear. Increased connexin 43 (Cx43)-mediated intercellular communication has been implicated in tissue-adaptive and -maladaptive response to various chronic stresses. Here, we demonstrate that in hepatocytes, ERS results in increased Cx43 expression and cell-cell coupling. Co-culture of ER-stressed "donor" cells resulted in intercellular transmission of ERS and dysfunction to ERS-naive "recipient" cells ("bystander response"), which could be prevented by genetic or pharmacologic suppression of Cx43. Hepatocytes from obese mice were able to transmit ERS to hepatocytes from lean mice, and mice lacking liver Cx43 were protected from diet-induced ERS, insulin resistance, and hepatosteatosis. Taken together, our results indicate that in obesity, the increased Cx43-mediated cell-cell coupling allows intercellular propagation of ERS. This novel maladaptive response to over-nutrition exacerbates the tissue ERS burden, promoting hepatosteatosis and impairing whole-body glucose metabolism.

Adipocytes promote interleukin-18 binding to its receptors during abdominal aortic aneurysm formation in mice.

AIMS: Obesity is a risk factor of abdominal aortic aneurysm (AAA). Inflammatory cytokine interleukin-18 (IL18) has two receptors: IL18 receptor (IL18r) and Na-Cl co-transporter (NCC). In human and mouse AAA lesions, IL18 colocalizes to its receptors at regions rich in adipocytes, suggesting a role of adipocytes in promoting IL18 actions in AAA development. METHODS AND RESULTS: We localized both IL18r and NCC in human and mouse AAA lesions. Murine AAA development required both receptors. In mouse AAA lesions, IL18 binding to these receptors increased at regions enriched in adipocytes or adjacent to perivascular adipose tissue. 3T3-L1 adipocytes enhanced IL18 binding to macrophages, aortic smooth muscle cells (SMCs), and endothelial cells by inducing the expression of both IL18 receptors on these cells. Adipocytes also enhanced IL18r and IL18 expression from T cells and macrophages, AAA-pertinent protease expression from macrophages, and SMC apoptosis. Perivascular implantation of adipose tissue from either diet-induced obese mice or lean mice but not that from leptin-deficient ob/ob mice exacerbated AAA development in recipient mice. Further experiments established an essential role of adipocyte leptin and fatty acid-binding protein 4 (FABP4) in promoting IL18 binding to macrophages and possibly other inflammatory and vascular cells by inducing their expression of IL18, IL18r, and NCC. CONCLUSION: Interleukin-18 uses both IL18r and NCC to promote AAA formation. Lesion adipocyte and perivascular adipose tissue contribute to AAA pathogenesis by releasing leptin and FABP4 that induce IL18, IL18r, and NCC expression and promote IL18 actions.