Search

Summary: Coronary artery disease (CAD) remains a leading cause of disease burden globally, and there is a persistent need for new therapeutic targets. Instrumental variable (IV) and genetic colocalization analyses can help identify novel therapeutic targets for human disease by nominating causal genes in genome-wide association study (GWAS) loci. We conducted cis-IV analyses for 20,125 genes and 1,746 plasma proteins with CAD using molecular trait quantitative trait loci variant (QTLs) data from three different studies. 19 proteins and 119 genes were significantly associated with CAD risk by IV analyses and demonstrated evidence of genetic colocalization. Notably, our analyses validated well-established targets such as PCSK9 and ANGPTL4 while also identifying HTRA1 and endotrophin (a cleavage product of COL6A3) as proteins whose levels are causally associated with CAD risk. Further experimental studies are needed to confirm the causal role of the genes and proteins identified through our multiomic cis-IV analyses on human disease.

The disease progression of the metabolic syndrome is associated with prolonged hyperlipidemia and insulin resistance, eventually giving rise to impaired insulin secretion, often concomitant with hypoadiponectinemia. As an adipose tissue derived hormone, adiponectin is beneficial for insulin secretion and β cell health and differentiation. However, the down-stream pathway of adiponectin in the pancreatic islets has not been studied extensively. Here, along with the overall reduction of endocrine pancreatic function in islets from adiponectin KO mice, we examine PPARα and HNF4α as additional down-regulated transcription factors during a prolonged metabolic challenge. To elucidate the function of β cell-specific PPARα and HNF4α expression, we developed doxycycline inducible pancreatic β cell-specific PPARα (β-PPARα) and HNF4α (β-HNF4α) overexpression mice. β-PPARα mice exhibited improved protection from lipotoxicity, but elevated β-oxidative damage in the islets, and also displayed lowered phospholipid levels and impaired glucose-stimulated insulin secretion. β-HNF4α mice showed a more severe phenotype when compared to β-PPARα mice, characterized by lower body weight, small islet mass and impaired insulin secretion. RNA-sequencing of the islets of these models highlights overlapping yet unique roles of β-PPARα and β-HNF4α. Given that β-HNF4α potently induces PPARα expression, we define a novel adiponectin-HNF4α-PPARα cascade. We further analyzed downstream genes consistently regulated by this axis. Among them, the islet amyloid polypeptide (IAPP) gene is an important target and accumulates in adiponectin KO mice. We propose a new mechanism of IAPP aggregation in type 2 diabetes through reduced adiponectin action.

Summary: Genetic loss of Agpat2 in humans and mice results in congenital generalized lipodystrophy with near-total loss of adipose tissue and predisposition to develop insulin resistance, diabetes mellitus, hepatic steatosis, and hypertriglyceridemia. The mechanism by which Agpat2 deficiency results in loss of adipose tissue remains unknown. We studied this by re-expressing human AGPAT2 (hAGPAT2) in Agpat2-null mice, regulated by doxycycline. In both sexes of Agpat2-null mice, adipose-tissue-specific re-expression of hAGPAT2 resulted in partial regeneration of both white and brown adipose tissue (but only 30%–50% compared with wild-type mice), which had molecular signatures of adipocytes, including leptin secretion. Furthermore, the stromal vascular fraction cells of regenerated adipose depots differentiated ex vivo only with doxycycline, suggesting the essential role of Agpat2 in adipocyte differentiation. Turning off expression of hAGPAT2 in vivo resulted in total loss of regenerated adipose tissue, clear evidence that Agpat2 is essential for adipocyte differentiation in vivo.

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β3-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

Objective: Renal fibrosis is a hallmark for chronic kidney disease (CKD), and often leads to end stage renal disease (ESRD). However, limited interventions are available clinically to ameliorate or reverse renal fibrosis. Methods: Herein, we evaluated whether blockade of endotrophin through neutralizing antibodies protects from renal fibrosis in the podocyte insult model (the “POD-ATTAC” mouse). We determined the therapeutic effects of endotrophin targeted antibody through assessing renal function, renal inflammation and fibrosis at histological and transcriptional levels, and podocyte regeneration. Results: We demonstrated that neutralizing endotrophin antibody treatment significantly ameliorates renal fibrosis at the transcriptional, morphological, and functional levels. In the antibody treatment group, expression of pro-inflammatory and pro-fibrotic genes was significantly reduced, normal renal structures were restored, collagen deposition was decreased, and proteinuria and renal function were improved. We further performed a lineage tracing study confirming that podocytes regenerate as de novo podocytes upon injury and loss, and blockade of endotrophin efficiently enhances podocyte-specific marker expressions. Conclusion: Combined, we provide pre-clinical evidence supporting neutralizing endotrophin as a promising therapy for intervening with renal fibrosis in CKD, and potentially in other chronic fibro-inflammatory diseases.

Background: Chronic disease appears connected to obesity. However, evidence suggests that chronic metabolic diseases are more specifically related to adipose dysfunction rather than to body weight itself. Scope of review: Further study of the first generation “insulin sensitizer” pioglitazone and molecules based on its structure suggests that is possible to decouple body weight from the metabolic dysfunction that drives adverse outcomes. The growing understanding of the mechanism of action of these agents together with advances in the pathophysiology of chronic metabolic disease offers a new approach to treat chronic conditions, such as type 2 diabetes, fatty liver disease, and their common organ and vascular sequelae. Major conclusions: We hypothesize that treating adipocyte dysfunction with new insulin sensitizers might significantly impact the interface of infectious disease and chronic metabolic disease.

The breakthrough discoveries of leptin and adiponectin more than two decades ago led to a widespread recognition of adipose tissue as an endocrine organ. Many more adipose tissue-secreted signaling mediators (adipokines) have been identified since then, and much has been learned about how adipose tissue communicates with other organs of the body to maintain systemic homeostasis. Beyond proteins, additional factors, such as lipids, metabolites, noncoding RNAs, and extracellular vesicles (EVs), released by adipose tissue participate in this process. Here, we review the diverse signaling mediators and mechanisms adipose tissue utilizes to relay information to other organs. We discuss recently identified adipokines (proteins, lipids, and metabolites) and briefly outline the contributions of noncoding RNAs and EVs to the ever-increasing complexities of adipose tissue inter-organ communication. We conclude by reflecting on central aspects of adipokine biology, namely, the contribution of distinct adipose tissue depots and cell types to adipokine secretion, the phenomenon of adipokine resistance, and the capacity of adipose tissue to act both as a source and sink of signaling mediators.

Objective: The sympathetic nervous system (SNS) is a key regulator of the metabolic and endocrine functions of adipose tissue. Increased SNS outflow promotes fat mobilization, stimulates non-shivering thermogenesis, promotes browning, and inhibits leptin production. Most of these effects are attributed to norepinephrine activation of the Gs-coupled beta adrenergic receptors located on the surface of the adipocytes. Evidence suggests that other adrenergic receptor subtypes, including the Gi-coupled alpha 2 adrenergic receptors might also mediate the SNS effects on adipose tissue. However, the impact of acute stimulation of adipocyte Gs and Gi has never been reported. Methods: We harness the power of chemogenetics to develop unique mouse models allowing the specific and spatiotemporal stimulation of adipose tissue Gi and Gs signaling. We evaluated the impact of chemogenetic stimulation of these pathways on glucose homeostasis, lipolysis, leptin production, and gene expression. Results: Stimulation of Gs signaling in adipocytes induced rapid and sustained hypoglycemia. These hypoglycemic effects were secondary to increased insulin release, likely consequent to increased lipolysis. Notably, we also observed differences in gene regulation and ex vivo lipolysis in different adipose depots. In contrast, acute stimulation of Gi signaling in adipose tissue did not affect glucose metabolism or lipolysis, but regulated leptin production. Conclusion: Our data highlight the significance of adipose Gs signaling in regulating systemic glucose homeostasis. We also found previously unappreciated heterogeneity across adipose depots following acute stimulation. Together, these results highlight the complex interactions of GPCR signaling in adipose tissue and demonstrate the usefulness of chemogenetic technology to better understand adipocyte function. Keywords: Adipose tissue, DREADD, Glucose, Insulin, Leptin, Lipolysis

The pleiotropic actions of adiponectin in improving cell survival and metabolism have motivated the development of small-molecule therapeutic agents for treating diabetes and lipotoxicity. AdipoRon is a synthetic agonist of the adiponectin receptors, yet is limited by its poor solubility and bioavailability. In this work, we expand on the protective effects of AdipoRon in pancreatic β-cells and examine how structural modifications could affect the activity, pharmacokinetics, and bioavailability of this small molecule. We describe a series of AdipoRon analogs containing amphiphilic ethylene glycol (PEG) chains. Among these, AdipoRonPEG5 induced pleiotropic effects in mice under insulinopenic and high-fat diet (HFD) conditions. While both AdipoRon and AdipoRonPEG5 substantially attenuate palmitate-induced lipotoxicity in INS-1 cells, only AdipoRonPEG5 treatment is accompanied by a significant reduction in cytotoxic ceramides. In vivo, AdipoRonPEG5 can substantially reduce pancreatic, hepatic, and serum ceramide species, with a concomitant increase in the corresponding sphingoid bases and improves insulin sensitivity of mice under HFD feeding conditions. Furthermore, hyperglycemia in streptozotocin (STZ)-induced insulinopenic adiponectin-null mice is also attenuated upon AdipoRonPEG5 treatment. Our results suggest that AdipoRonPEG5 is more effective in reducing ceramides and dihydroceramides in the liver of HFD-fed mice than AdipoRon, consistent with its potent activity in activating ceramidase in vitro in INS-1 cells. Additionally, these results indicate that the beneficial effects of AdipoRonPEG5 can be partially attributed to improved pharmacokinetics as compared with AdipoRon, thus suggesting that further derivatization may improve affinity and tissue-specific targeting.

Objective: Obesity-induced insulin resistance is closely associated with chronic subclinical inflammation in white adipose tissue. However, the mechanistic involvement of adipocyte-derived inflammation under these disease conditions remains unclear. Our aim was to investigate the relative inflammation-related contributions of adipocytes and macrophages to insulin sensitivity. Methods: RIDα/β is an adenoviral protein complex that inhibits several inflammatory pathways, including TLR4, TNFα, and IL1β signaling. We generated novel mouse models with adipocyte-specific and macrophage-specific doxycycline (dox)-inducible RIDα/β-transgenic mice (RIDad and RIDmac mice, respectively). Results: RIDα/β induction significantly reduced LPS-stimulated inflammatory markers, such as Tnf, Il1b, and Saa3 in adipose tissues. Surprisingly, RIDad mice had elevated levels of postprandial glucose and insulin and exhibited glucose intolerance and insulin resistance, even under chow-fed conditions. Moreover, the RIDad mice displayed further insulin resistance under obesogenic (high-fat diet, HFD) conditions despite reduced weight gain. In addition, under pre-existing obese and inflamed conditions on an HFD, subsequent induction of RIDα/β in RIDad mice reduced body weight gain, further exacerbating glucose tolerance, enhancing insulin resistance and fatty liver, and reducing adiponectin levels. This occurred despite effective suppression of the inflammatory pathways (including TNFα and IL1β). In contrast, RIDmac mice, upon HFD feeding, displayed similar weight gain, comparable adiponectin levels, and insulin sensitivity, suggesting that the inflammatory properties of macrophages did not exert a negative impact on metabolic readouts. RIDα/β expression and the ensuing suppression of inflammation in adipocytes enhanced adipose tissue fibrosis and reduced vascularization. Conclusion: Our novel findings further corroborate our previous observations suggesting that suppressing adipocyte inflammation impairs adipose tissue function and promotes insulin resistance, despite beneficial effects on weight gain.

Objective: Hyperleptinemia per se is sufficient to promote leptin resistance in the obese state. Leptin sensitivity can be restored by reducing circulating leptin levels within a physiologically healthy range and is a viable antiobesity and antidiabetic strategy. However, a previous study suggests that partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice, arguing that a lower leptin level may indeed promote diet-induced obesity and its associated metabolic disorders. Here, we aim to elucidate what the impact of partial leptin deficiency is on fat mass and insulin sensitivity. Methods: We used two different mouse models of partial leptin deficiency: an adipocyte-specific congenital heterozygous leptin knockout mouse line (LepHZ) and the well-established whole body heterozygous leptin knockout mouse (OBHZ). The metabolic studies of OBHZ and LepHZ mice were performed both on normal carbohydrate-rich chow diet and on a high-fat diet (HFD). Male and female mice were included in the study to account for sex-specific differences. Body weight, food intake, glucose tolerance, and insulin tolerance were tested. Histology of adipose tissue and liver tissue allowed insights into adipose tissue inflammation and hepatic triglyceride content. Immunohistochemistry was paired with RT-PCR analysis for expression levels of inflammatory markers. Results: Both OBHZ and LepHZ mice displayed reduced circulating leptin levels on the chow diet and HFD. On chow diet, male OBHZ and LepHZ mice showed elevated fat mass and body weight, while their glucose tolerance and insulin sensitivity remained unchanged. However, the inability in partially leptin-deficient mice to fully induce circulating leptin during the development of diet-induced obesity results in reduced food intake and leaner mice with lower body weight compared to their littermate controls. Importantly, a strong reduction of adipose tissue inflammation is observed along with improvements in insulin sensitivity and enhanced glucose tolerance. Additionally, partial leptin deficiency protects the mice from fatty liver and liver fibrosis. Chronically HFD-fed OBHZ and LepHZ mice remain more sensitive to exogenous leptin injection, as reflected by their reduced food intake upon an acute leptin treatment. Conclusion: In response to HFD feeding, the inability to upregulate leptin levels due to partial leptin deficiency protects mice from diet-induced obesity and metabolic dysregulation. Thus, in an obesogenic environment, maintaining lower leptin levels is highly beneficial for both obesity and diabetes management. Chronic leptin reduction represents a viable preventive strategy whose efficacy awaits clinical testing.

Objective: TANK Binding Kinase 1 (TBK1) has been implicated in the regulation of metabolism through studies with the drug amlexanox, an inhibitor of the IκB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since amlexanox is a dual IKKε/TBK1 inhibitor, the specific metabolic contribution of TBK1 is not clear. Methods: To distinguish metabolic functions unique to TBK1, we examined the metabolic profile of global Tbk1 mutant mice challenged with an obesogenic diet and investigated potential mechanisms for the improved metabolic phenotype. Results and conclusion: We report that systemic loss of TBK1 kinase function has an overall protective effect on metabolic readouts in mice on an obesogenic diet, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor. Keywords: TBK1, IKKε, Obesity, Insulin, Insulin resistance, Metabolism

Objective: Recent studies have suggested a critical role for toll-like receptor 4 (TLR4) in the development of alcoholic liver disease. As TLR4 is widely expressed throughout the body, it is unclear which TLR4-expressing cell types contribute to alcohol-induced liver damage. Methods: We selectively ablated TLR4 in hepatocytes and myeloid cells. Male mice were fed a liquid diet containing either 5% alcohol or pair-fed a control diet for 4 weeks to examine chronic alcohol intake-induced liver damage and inflammation. In addition, mice were administered a single oral gavage of alcohol to investigate acute alcohol drinking-associated liver injury. Results: We found that selective hepatocyte TLR4 deletion protected mice from chronic alcohol-induced liver injury and fatty liver. This result was in part due to decreased expression of endogenous lipogenic genes and enhanced expression of genes involved in fatty acid oxidation. In addition, mice lacking hepatocyte TLR4 exhibited reduced mRNA expression of inflammatory genes in white adipose tissue. Furthermore, in an acute alcohol binge model, hepatocyte TLR4 deficient mice had significantly decreased plasma alanine transaminase (ALT) levels and attenuated hepatic triglyceride content compared to their alcohol-gavaged control mice. In contrast, deleting TLR4 in myeloid cells did not affect the development of chronic-alcohol induced fatty liver, despite the finding that mice lacking myeloid cell TLR4 had significantly reduced circulating ALT concentrations. Conclusions: These findings suggest that hepatocyte TLR4 plays an important role in regulating alcohol-induced liver damage and fatty liver disease. Keywords: Alcoholic fatty liver disease, Toll-like receptor 4, Hepatocyte, Myeloid cell, Liver injury, Inflammation

Objective: The spliced transcription factor Xbp1 (Xbp1s), a transducer of the unfolded protein response (UPR), regulates lipolysis. Lipolysis is stimulated by fasting when uridine synthesis is also activated in adipocytes. Methods: Here we have examined the regulatory role Xbp1s in stimulation of uridine biosynthesis in adipocytes and triglyceride mobilization using inducible mouse models. Results: Xbp1s is a key molecule involved in adipocyte uridine biosynthesis and release by activation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD), the rate-limiting enzyme for UMP biosynthesis. Adipocyte Xbp1s overexpression drives energy mobilization and protects mice from obesity through activation of the pyrimidine biosynthesis pathway. Conclusion: These observations reveal that Xbp1s is a potent stimulator of uridine production in adipocytes, enhancing lipolysis and invoking a potential anti-obesity strategy through the induction of a futile biosynthetic cycle. Keywords: Obesity, Xbp1, ER stress, UPR, Pyrimidine, Uridine, CAD

Objective and methods: Metabolic viscera and their vasculature are richly innervated by peripheral sensory neurons. Here, we examined the metabolic and inflammatory profiles of mice with selective ablation of all Nav1.8-expressing primary afferent neurons. Results: While mice lacking sensory neurons displayed no differences in body weight, food intake, energy expenditure, or body composition compared to controls on chow diet, ablated mice developed an exaggerated inflammatory response to high-fat feeding characterized by bouts of weight loss, splenomegaly, elevated circulating interleukin-6 and hepatic serum amyloid A expression. This phenotype appeared to be directly mediated by the ingestion of saturated lipids. Conclusions: These data demonstrate that the Nav1.8-expressing afferent neurons are not essential for energy balance but are required for limiting the acute phase response caused by an obesogenic diet.

Objective: Adiponectin and the signaling induced by its cognate receptors, AdipoR1 and AdipoR2, have garnered attention for their ability to promote insulin sensitivity and oppose steatosis. Activation of these receptors promotes the deacylation of ceramide, a lipid metabolite that appears to play a causal role in impairing insulin signaling. Methods: Here, we have developed transgenic mice that overexpress AdipoR1 or AdipoR2 under the inducible control of a tetracycline response element. These represent the first inducible genetic models that acutely manipulate adiponectin receptor signaling in adult mouse tissues, which allows us to directly assess AdipoR signaling on glucose and lipid metabolism. Results: Overexpression of either adiponectin receptor isoform in the adipocyte or hepatocyte is sufficient to enhance ceramidase activity, whole body glucose metabolism, and hepatic insulin sensitivity, while opposing hepatic steatosis. Importantly, metabolic improvements fail to occur in an adiponectin knockout background. When challenged with a leptin-deficient genetic model of type 2 diabetes, AdipoR2 expression in adipose or liver is sufficient to reverse hyperglycemia and glucose intolerance. Conclusion: These observations reveal that adiponectin is critical for AdipoR-induced ceramidase activation which enhances hepatic glucose and lipid metabolism via rapidly acting “cross-talk” between liver and adipose tissue sphingolipids. Keywords: Sphingolipid, Insulin resistance, NAFLD

Objective: Adiponectin receptors (AdipoRs) are located on neurons of the hypothalamus involved in metabolic regulation – including arcuate proopiomelanocortin (Pomc) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. AdipoRs play a critical role in regulating glucose and fatty acid metabolism by initiating several signaling cascades overlapping with Leptin receptors (LepRs). However, the mechanism by which adiponectin regulates cellular activity in the brain remains undefined. Methods: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify Pomc and NPY/AgRP neurons which express LepRs for patch-clamp electrophysiology experiments. Results: We found that leptin and adiponectin synergistically activated melanocortin neurons in the arcuate nucleus. Conversely, NPY/AgRP neurons were inhibited in response to adiponectin. The adiponectin-induced depolarization of arcuate Pomc neurons occurred via activation of Phosphoinositide-3-kinase (PI3K) signaling, independent of 5′ AMP-activated protein kinase (AMPK) activity. Adiponectin also activated melanocortin neurons at various physiological glucose levels. Conclusions: Our results demonstrate a requirement for PI3K signaling in the acute adiponectin-induced effects on the cellular activity of arcuate melanocortin neurons. Moreover, these data provide evidence for PI3K as a substrate for both leptin and adiponectin to regulate energy balance and glucose metabolism via melanocortin activity. Author Video: Author Video Watch what authors say about their articles Keywords: Melanocortin, Obesity, Diabetes, Energy balance, Patch-clamp, Electrophysiology

Background: Evidence hints at the ability of β-cells to emerge from non-β-cells upon genetic or pharmacological interventions. However, their quantitative contributions to the process of autonomous β-cell regeneration without genetic or pharmacological manipulations remain to be determined. Methods & results: Using PANIC-ATTAC mice, a model of titratable, acute β-cell apoptosis capable of autonomous, and effective islet mass regeneration, we demonstrate that an extended washout of residual tamoxifen activity is crucial for β-cell lineage tracing studies using the tamoxifen-inducible Cre/loxP systems. We further establish a doxycycline-inducible system to label different cell types in the mouse pancreas and pursued a highly quantitative assessment to trace adult β-cells after various metabolic challenges. Beyond proliferation of pre-existing β-cells, non-β-cells contribute significantly to the post-challenge regenerated β-cell pool. α-cell trans-differentiation is the predominant mechanism upon post-apoptosis regeneration and multiparity. No contributions from exocrine acinar cells were observed. During diet-induced obesity, about 25% of α-cells arise de novo from β-cells. Ectopic expression of Nkx6.1 promotes α-to-β conversion and insulin production. Conclusions: We identify the origins and fates of adult β-cells upon post-challenge upon autonomous regeneration of islet mass and establish the quantitative contributions of the different cell types using a lineage tracing system with high temporal resolution. Author Video: Author Video Watch what authors say about their articles Keywords: Lineage tracing, Adult β-cell origins, Nkx6.1, Tamoxifen artifacts

Background: The selective estrogen receptor modulator tamoxifen, in combination with the Cre-ERT2 fusion protein, has been one of the mainstream methods to induce genetic recombination and has found widespread application in lineage tracing studies. Methods & results: Here, we report that tamoxifen exposure at widely used concentrations remains detectable by mass-spectrometric analysis in adipose tissue after a washout period of 10 days. Surprisingly, its ability to maintain nuclear translocation of the Cre-ERT2 protein is preserved beyond 2 months of washout. Tamoxifen treatment acutely leads to transient lipoatrophy, followed by de novo adipogenesis that reconstitutes the original fat mass. In addition, we find a “synthetically lethal” phenotype for adipocytes when tamoxifen treatment is combined with adipocyte-specific loss-of-function mutants, such as an adipocyte-specific PPARγ knockout. This is observed to a lesser extent when alternative inducible approaches are employed. Conclusions: These findings highlight the potential for tamoxifen-induced adipogenesis, and the associated drawbacks of the use of tamoxifen in lineage tracing studies, explaining the discrepancy in lineage tracing results from different systems with temporal control of gene targeting.

Background/Purpose: Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. Methods: We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. Results: Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. Conclusion: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

Adipocyte differentiation and function have become areas of intense focus in the field of energy metabolism; however, understanding the role of specific genes in the establishment and maintenance of fat cell function can be challenging and complex. In this review, we offer practical guidelines for the study of adipocyte development and function. We discuss improved cellular and genetic systems for the study of adipose biology and highlight recent insights gained from these new approaches.

We previously suggested that, in obese animals and humans, white adipose tissue inflammation results from the death of hypertrophic adipocytes; these are then cleared by macrophages, giving rise to distinctive structures we denominated crown-like structures. Here we present evidence that subcutaneous and visceral hypertrophic adipocytes of leptin-deficient (ob/ob and db/db) obese mice exhibit ultrastructural abnormalities (including calcium accumulation and cholesterol crystals), many of which are more common in hyperglycemic db/db versus normoglycemic ob/ob mice and in visceral versus subcutaneous depots. Degenerating adipocytes whose intracellular content disperses in the extracellular space were also noted in obese mice; in addition, increased anti-reactive oxygen species enzyme expression in obese fat pads, documented by RT-PCR and immunohistochemistry, suggests that ultrastructural changes are accompanied by oxidative stress. RT-PCR showed NLRP3 inflammasome activation in the fat pads of both leptin-deficient and high-fat diet obese mice, in which formation of active caspase-1 was documented by immunohistochemistry in the cytoplasm of several hypertrophic adipocytes. Notably, caspase-1 was not detected in FAT-ATTAC transgenic mice, where adipocytes die of apoptosis. Thus, white adipocyte overexpansion induces a stress state that ultimately leads to death. NLRP3-dependent caspase-1 activation in hypertrophic adipocytes likely induces obese adipocyte death by pyroptosis, a proinflammatory programmed cell death.

Subclinical inflammation is frequently associated with obesity. Here, we aim to better define the acute inflammatory response during fasting. To do so, we analyzed representatives of immune-related proteins in circulation and in tissues as potential markers for adipose tissue inflammation and modulation of the immune system. Lipopolysaccharide treatment or high-fat diet led to an increase in circulating serum amyloid (SAA) and α1-acid glycoprotein (AGP), whereas adipsin levels were reduced. Mouse models that are protected against diet-induced challenges, such as adiponectin-overexpressing animals or mice treated with PPARγ agonists, displayed lower SAA levels and higher adip-sin levels. An oral lipid gavage, as well as prolonged fasting, increased circulating SAA concurrent with the elevation of free FA levels. Moreover, prolonged fasting was associated with an increased number of Mac2-positive crown-like structures, an increased capillary permeability, and an increase in several M2-type macrophage markers in adipose tissue. This fasting-induced increase in SAA and M2-type macrophage markers was impaired in metabolically challenged animals. These data suggest that metabolic inflexibility is associated with a lack of “immunological fitness.”

Adipose tissue metabolism exerts a profound impact on whole-body metabolism. We review how fuel partitioning between adipocytes and other tissues affects insulin signaling pathways. We discuss the role of adipose tissue inflammation in adipocyte metabolism and whole-body insulin sensitivity. Finally, we mention the role of adipokines in autocrine and paracrine signaling.