Metabolic and behavioral alterations associated with viral vector-mediated toxicity in the paraventricular hypothalamic nucleus.

OBJECTIVE: Combining adeno-associated virus (AAV)-mediated expression of Cre recombinase with genetically modified floxed animals is a powerful approach for assaying the functional role of genes in regulating behavior and metabolism. Extensive research in diverse cell types and tissues using AAV-Cre has shown it can save time and avoid developmental compensation as compared to using Cre driver mouse line crossings. We initially sought to study the impact of ablation of corticotropin-releasing hormone (CRH) in the paraventricular hypothalamic nucleus (PVN) using intracranial AAV-Cre injection in adult animals. METHODS: In this study, we stereotactically injected AAV8-hSyn-Cre or a control AAV8-hSyn-GFP both Crh-floxed and wild-type mouse PVN to assess behavioral and metabolic impacts. We then used immunohistochemical markers to systematically evaluate the density of hypothalamic peptidergic neurons and glial cells. RESULTS: We found that delivery of one specific preparation of AAV8-hSyn-Cre in the PVN led to the development of obesity, hyperphagia, and anxiety-like behaviors. This effect occurred independent of sex and in both floxed and wild-type mice. We subsequently found that AAV8-hSyn-Cre led to neuronal cell death and gliosis at the site of viral vector injections. These behavioral and metabolic deficits were dependent on injection into the PVN. An alternatively sourced AAV-Cre did not reproduce the same results. CONCLUSIONS: Our findings reveal that delivery of a specific batch of AAV-Cre could lead to cellular toxicity and lesions in the PVN that cause robust metabolic and behavioral impacts. These alterations can complicate the interpretation of Cre-mediated gene knockout and highlight the need for rigorous controls.

Metabolic and behavioral alterations associated with viral vector-mediated toxicity in the paraventricular hypothalamic nucleus.

Objective: Combining adeno-associated virus (AAV)-mediated expression of Cre recombinase with genetically modified floxed animals is a powerful approach for assaying the functional role of genes in regulating behavior and metabolism. Extensive research in diverse cell types and tissues using AAV-Cre has shown it can save time and avoid developmental compensation as compared to using Cre driver mouse line crossings. We initially sought to study the impact of ablation of corticotropin-releasing hormone (CRH) in the paraventricular hypothalamic nucleus (PVN) using intracranial AAV-Cre injection in adult animals. Methods: In this study, we stereotactically injected AAV8-hSyn-Cre or a control AAV8-hSyn-GFP both Crh-floxed and wild-type mouse PVN to assess behavioral and metabolic impacts. We then used immunohistochemical markers to systematically evaluate the density of hypothalamic peptidergic neurons and glial cells. Results: We found that delivery of one specific preparation of AAV8-hSyn-Cre in the PVN led to the development of obesity, hyperphagia, and anxiety-like behaviors. This effect occurred independent of sex and in both floxed and wild-type mice. We subsequently found that AAV8-hSyn-Cre led to neuronal cell death and gliosis at the site of viral vector injections. These behavioral and metabolic deficits were dependent on injection into the PVN. An alternatively sourced AAV-Cre did not reproduce the same results. Conclusions: Our findings reveal that delivery of a specific batch of AAV-Cre could lead to cellular toxicity and lesions in the PVN that cause robust metabolic and behavioral impacts. These alterations can complicate the interpretation of Cre-mediated gene knockout and highlight the need for rigorous controls.

Glucagon changes substrate preference in gluconeogenesis.

Fasting hyperglycemia in diabetes mellitus is caused by unregulated glucagon secretion that activates gluconeogenesis (GNG) and increases the use of pyruvate, lactate, amino acids, and glycerol. Studies of GNG in hepatocytes, however, tend to test a limited number of substrates at nonphysiologic concentrations. Therefore, we treated cultured primary hepatocytes with three identical substrate mixtures of pyruvate/lactate, glutamine, and glycerol at serum fasting concentrations, where a different U-13C- or 2-13C-labeled substrate was substituted in each mix. In the absence of glucagon stimulation, 80% of the glucose produced in primary hepatocytes incorporated either one or two 13C-labeled glycerol molecules in a 1:1 ratio, reflecting the high overall activity of this pathway. In contrast, glucose produced from 13C-labeled pyruvate/lactate or glutamine rarely incorporated two labeled molecules. While glucagon increased the glycerol and pyruvate/lactate contributions to glucose carbon by 1.6- and 1.8-fold, respectively, the glutamine contribution to glucose carbon was increased 6.4-fold in primary hepatocytes. To account for substrate 13C carbon loss during metabolism, we also performed a metabolic flux analysis, which confirmed that the majority of glucose carbon produced by primary hepatocytes was from glycerol. In vivo studies using a PKA-activation mouse model that represents elevated glucagon activity confirmed that most circulating lactate carbons originated from glycerol, but very little glycerol was derived from lactate carbons, reflecting glycerol's importance as a carbon donor to GNG. Given the diverse entry points for GNG substrates, hepatic glucagon action is unlikely to be due to a single mechanism.

TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling.

Cholinergic and sympathetic counter-regulatory networks control numerous physiological functions, including learning/memory/cognition, stress responsiveness, blood pressure, heart rate, and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels, and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure.

Targeting hepatic kisspeptin receptor ameliorates nonalcoholic fatty liver disease in a mouse model.

Nonalcoholic fatty liver disease (NAFLD), the most common liver disease, has become a silent worldwide pandemic. The incidence of NAFLD correlates with the rise in obesity, type 2 diabetes, and metabolic syndrome. A hallmark featureof NAFLD is excessive hepatic fat accumulation or steatosis, due to dysregulated hepatic fat metabolism, which can progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Currently, there are no approved pharmacotherapies to treat this disease. Here, we have found that activation of the kisspeptin 1 receptor (KISS1R) signaling pathway has therapeutic effects in NAFLD. Using high-fat diet-fed mice, we demonstrated that a deletion of hepatic Kiss1r exacerbated hepatic steatosis. In contrast, enhanced stimulation of KISS1R protected against steatosis in wild-type C57BL/6J mice and decreased fibrosis using a diet-induced mouse model of NASH. Mechanistically, we found that hepatic KISS1R signaling activates the master energy regulator, AMPK, to thereby decrease lipogenesis and progression to NASH. In patients with NAFLD and in high-fat diet-fed mice, hepatic KISS1/KISS1R expression and plasma kisspeptin levels were elevated, suggesting a compensatory mechanism to reduce triglyceride synthesis. These findings establish KISS1R as a therapeutic target to treat NASH.

Deconstructing the origins of sexual dimorphism in sensory modulation of pancreatic ß cells.

The regulation of glucose-stimulated insulin secretion and glucose excursion has a sensory component that operates in a sex-dependent manner. OBJECTIVE: Here, we aim to dissect the basis of the sexually dimorphic interaction between sensory neurons and pancreatic ß cells and its overall impact on insulin release and glucose homeostasis. METHODS: We used viral retrograde tracing techniques, surgical and chemodenervation models, and primary cell-based co-culture systems to uncover the biology underlying sex differences in sensory modulation of pancreatic ß-cell activity. RESULTS: Retrograde transsynaptic labeling revealed a sex difference in the density of sensory innervation in the pancreas. The number of sensory neurons emanating from the dorsal root and nodose ganglia that project in the pancreas is higher in male than in female mice. Immunostaining and confocal laser scanning microscopy confirmed the higher abundance of peri-islet sensory axonal tracts in the male pancreas. Capsaicin-induced sensory chemodenervation concomitantly enhanced glucose-stimulated insulin secretion and glucose clearance in male mice. These metabolic benefits were blunted when mice were orchidectomized prior to the ablation of sensory nerves. Interestingly, orchidectomy also lowered the density of peri-islet sensory neurons. In female mice, capsaicin treatment did not affect glucose-induced insulin secretion nor glucose excursion and ovariectomy did not modify these outcomes. Interestingly, same- and opposite-sex sensory-islet co-culture paradigms unmasked the existence of potential gonadal hormone-independent mechanisms mediating the male-female difference in sensory modulation of islet ß-cell activity. CONCLUSION: Taken together, these data suggest that the sex-biased nature of the sensory control of islet ß-cell activity is a result of a combination of neurodevelopmental inputs, sex hormone-dependent mechanisms and the potential action of somatic molecules encoded by the sex chromosome complement.

Glycerol not lactate is the major net carbon source for gluconeogenesis in mice during both short and prolonged fasting.

OBJECTIVE: Fasting results in major metabolic changes including a switch from glycogenolysis to gluconeogenesis to maintain glucose homeostasis. However, the relationship between the length of fasting and the relative contribution of gluconeogenic substrates remains unclear. We investigated the relative contribution of glycogen, lactate, and glycerol in glucose production of male C57BL/6 J-albino mice after 6, 12, and 18 h of fasting. METHODS: We used non-perturbative infusions of 13C3 lactate, 13C3 glycerol, and 13C6 glucose combined with liquid chromatography mass spectrometry and metabolic flux analysis to study the contribution of substrates in gluconeogenesis (GNG). RESULTS: During infusion studies, both lactate and glycerol significantly label about 60% and 30-50% glucose carbon, respectively, but glucose labels much more lactate (∼90%) than glycerol carbon (∼10%). Our analyses indicate that lactate, but not glycerol is largely recycled during all fasting periods such that lactate is the largest direct contributor to GNG via the Cori cycle but a minor source of new glucose carbon (overall contribution). In contrast, glycerol is not only a significant direct contributor to GNG but also the largest overall contributor to GNG regardless of fasting length. Prolonged fasting decreases both the whole body turnover rate of glucose and lactate but increases that of glycerol, indicating that the usage of glycerol in GNG become more significant with longer fasting. CONCLUSION: Collectively, these findings suggest that glycerol is the dominant overall contributor of net glucose carbon in GNG during both short and prolonged fasting.

Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitro and in vivo.

Gluconeogenesis (GNG) is de novo production of glucose from endogenous carbon sources. Although it is a commonly studied pathway, particularly in disease, there is a lack of consensus about substrate preference. Moreover, primary hepatocytes are the current gold standard for in vitro liver studies, but no direct comparison of substrate preference at physiological fasting concentrations has been performed. We show that mouse primary hepatocytes prefer glycerol to pyruvate/lactate in glucose production assays and 13C isotope tracing studies at the high concentrations commonly used in the literature, as well as at more relevant fasting, physiological concentrations. In addition, when glycerol, pyruvate/lactate, and glutamine are all present, glycerol is responsible for over 75% of all glucose carbons labeled. We also found that glycerol can induce a rate-limiting enzyme of GNG, glucose-6-phosphatase. Lastly, we suggest that glycerol is a better substrate than pyruvate to test in vivo production of glucose in fasting mice. In conclusion, glycerol is the major carbon source for GNG in vitro and in vivo and should be compared with other substrates when studying GNG in the context of metabolic disease states.

Fast LC-MS quantitation of glucose and glycerol via enzymatic derivatization.

Glucose and glycerol are important circulating metabolites. Due to poor ionization and/or ion suppression, the liquid chromatography-mass spectrometry (LC-MS) detection of glucose and glycerol presents challenges. Here, we propose an efficient LC-MS method of quantitative glucose and glycerol detection via enzymatic derivatization to glucose-6-phosphate and sn-glycerol-3-phosphate, respectively. This derivatization protocol can be used to measure the concentrations of glucose production in a plethora of sample types for metabolic analysis and is compatible with the general metabolomics workflow. This novel approach allows us to quantitatively study glucose and glycerol metabolism using stable isotope tracers in vivo.

The subunit assembly state of the Mediator complex is nutrient-regulated and is dysregulated in a genetic model of insulin resistance and obesity.

The Mediator complex plays a critical role in the regulation of transcription by linking transcription factors to RNA polymerase II. By examining mouse livers, we have found that in the fasted state, the Mediator complex exists primarily as an approximately 1.2-MDa complex, consistent with the size of the large Mediator complex, whereas following feeding, it converts to an approximately 600-kDa complex, consistent with the size of the core Mediator complex. This dynamic change is due to the dissociation and degradation of the kinase module that includes the MED13, MED12, cyclin-dependent kinase 8 (CDK8), and cyclin C (CCNC) subunits. The dissociation and degradation of the kinase module are dependent upon nutrient activation of mTORC1 that is necessary for the induction of lipogenic gene expression because pharmacological or genetic inhibition of mTORC1 in the fed state restores the kinase module. The degradation but not dissociation of the kinase module depends upon the E3 ligase, SCFFBW7 In addition, genetically insulin-resistant and obese db/db mice in the fasted state displayed elevated lipogenic gene expression and loss of the kinase module that was reversed following mTORC1 inhibition. These data demonstrate that the assembly state of the Mediator complex undergoes physiologic regulation during normal cycles of fasting and feeding in the mouse liver. Furthermore, the assembly state of the Mediator complex is dysregulated in states of obesity and insulin resistance.

Skeletal muscle fibrosis is associated with decreased muscle inflammation and weakness in patients with chronic kidney disease.

Muscle dysfunction is an important cause of morbidity among patients with chronic kidney disease (CKD). Although muscle fibrosis is present in a CKD rodent model, its existence in humans and its impact on physical function are currently unknown. We examined isometric leg extension strength and measures of skeletal muscle fibrosis and inflammation in vastus lateralis muscle from CKD patients ( n = 10) and healthy, sedentary controls ( n = 10). Histochemistry and immunohistochemistry were used to assess muscle collagen and macrophage and fibro/adipogenic progenitor (FAP) cell populations, and RT-qPCR was used to assess muscle-specific inflammatory marker expression. Muscle collagen content was significantly greater in CKD compared with control (18.8 ± 2.1 vs. 11.7 ± 0.7% collagen area, P = 0.008), as was staining for collagen I, pro-collagen I, and a novel collagen-hybridizing peptide that binds remodeling collagen. Muscle collagen was inversely associated with leg extension strength in CKD ( r = -0.74, P = 0.01). FAP abundance was increased in CKD, was highly correlated with muscle collagen ( r = 0.84, P < 0.001), and was inversely associated with TNF-α expression ( r = -0.65, P = 0.003). TNF-α, CD68, CCL2, and CCL5 mRNA were significantly lower in CKD than control, despite higher serum TNF-α and IL-6. Immunohistochemistry confirmed fewer CD68+ and CD11b+ macrophages in CKD muscle. In conclusion, skeletal muscle collagen content is increased in humans with CKD and is associated with functional parameters. Muscle fibrosis correlated with increased FAP abundance, which may be due to insufficient macrophage-mediated TNF-α secretion. These data provide a foundation for future research elucidating the mechanisms responsible for this newly identified human muscle pathology.

The fructose-2,6-bisphosphatase TIGAR suppresses NF-κB signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC.

The systems integration of whole-body metabolism and immune signaling are central homeostatic mechanisms necessary for maintenance of normal physiology, and dysregulation of these processes leads to a variety of chronic disorders. However, the intracellular mechanisms responsible for cell-autonomous cross-talk between the inflammatory signaling pathways and metabolic flux have remained enigmatic. In this study, we discovered that the fructose-2,6-bisphosphatase TIGAR (Tp53-induced glycolysis and apoptosis regulator) critically regulates NF-κB activation. We found that TIGAR potently inhibits NF-κB-dependent gene expression by suppressing the upstream activation of IKKß phosphorylation and kinase activation. This inhibition occurred through a direct binding competition between NEMO and TIGAR for association with the linear ubiquitination assembly complex (LUBAC). This competition prevented linear ubiquitination of NEMO, which is required for activation of IKKß and other downstream targets. Furthermore, a TIGAR phosphatase activity-deficient mutant was equally effective as WT TIGAR in inhibiting NEMO linear ubiquitination, IKKß phosphorylation/activation, and NF-κB signaling, indicating that TIGAR's effect on NF-κB signaling is due to its interaction with LUBAC. Physiologically, TIGAR knockout mice displayed enhanced adipose tissue NF-κB signaling, whereas adipocyte-specific overexpression of TIGAR suppressed adipose tissue NF-κB signaling. Together, these results demonstrate that TIGAR has a nonenzymatic molecular function that modulates the NF-κB signaling pathway by directly inhibiting the E3 ligase activity of LUBAC.

Adipocyte-specific IKKß signaling suppresses adipose tissue inflammation through an IL-13-dependent paracrine feedback pathway.

Adipose tissue inflammation is one pathway shown to mediate insulin resistance in obese humans and rodents. Obesity induces dynamic cellular changes in adipose tissue to increase proinflammatory cytokines and diminish anti-inflammatory cytokines. However, we have found that anti-inflammatory interleukin-13 (IL-13) is unexpectedly induced in adipose tissue of obese humans and high-fat diet (HFD)-fed mice, and the source of IL-13 is primarily the adipocyte. Moreover, HFD-induced proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and IL-1ß mediate IL-13 production in adipocytes in an IKKß-dependent manner. In contrast, adipocyte-specific IKKß-deficient mice show diminished IL-13 expression and enhanced inflammation after HFD feeding, resulting in a worsening of the insulin-resistant state. Together these data demonstrate that although IKKß activates the expression of proinflammatory mediators, in adipocytes, IKKß signaling also induces the expression of the anti-inflammatory cytokine IL-13, which plays a unique protective role by limiting adipose tissue inflammation and insulin resistance.

EGR2 is critical for peripheral naïve T-cell differentiation and the T-cell response to influenza.

Early growth response 2 (EGR2) transcription factor negatively regulates T-cell activation, in contrast to the positive regulation of this process by EGR1. Here, we unexpectedly found that EGR2 promotes peripheral naïve T-cell differentiation, with delayed T-cell receptor-induced proliferation in naïve T cells from Egr2 conditional knockout (CKO) mice and decreased production of IFN-γ, IL-4, IL-9, and IL-17A in cells subjected to T-helper differentiation. Moreover, genes that promote T-cell activation, including Tbx21 and Notch1, had decreased expression in Egr2 CKO T cells and are direct EGR2 target genes. Following influenza infection, Egr2 CKO mice had delayed viral clearance, more weight loss, and more severe pathological changes in the lung than did WT and Egr1 KO mice, with decreased production of effector cytokines, increased infiltration of antigen-specific memory-precursor CD8(+) T cells, and lower numbers of lung-resident memory CD8(+) T cells. Thus, unexpectedly, EGR2 can function as a positive regulator that is essential for naïve T-cell differentiation and in vivo T-cell responses to a viral infection.

Adipokines mediate inflammation and insulin resistance.

For many years, adipose tissue was considered as an inert energy storage organ that accumulates and stores triacylglycerols during energy excess and releases fatty acids in times of systemic energy need. However, over the last two decades adipose tissue depots have been established as highly active endocrine and metabolically important organs that modulate energy expenditure and glucose homeostasis. In rodents, brown adipose tissue plays an essential role in non-shivering thermogenesis and in energy dissipation that can serve to protect against diet-induced obesity. White adipose tissue collectively referred too as either subcutaneous or visceral adipose tissue is responsible for the secretion of an array of signaling molecules, termed adipokines. These adipokines function as classic circulating hormones to communicate with other organs including brain, liver, muscle, the immune system, and adipose tissue itself. The dysregulation of adipokines has been implicated in obesity, type 2 diabetes, and cardiovascular disease. Recently, inflammatory responses in adipose tissue have been shown as a major mechanism to induce peripheral tissue insulin resistance. Although leptin and adiponectin regulate feeding behavior and energy expenditure, these adipokines are also involved in the regulation of inflammatory responses. Adipose tissue secretes various pro- and anti-inflammatory adipokines to modulate inflammation and insulin resistance. In obese humans and rodent models, the expression of pro-inflammatory adipokines is enhanced to induce insulin resistance. Collectively, these findings have suggested that obesity-induced insulin resistance may result, at least in part, from an imbalance in the expression of pro- and anti-inflammatory adipokines. Thus we will review the recent progress regarding the physiological and molecular functions of adipokines in the obesity-induced inflammation and insulin resistance with perspectives on future directions.

Fyn deficiency promotes a preferential increase in subcutaneous adipose tissue mass and decreased visceral adipose tissue inflammation.

Previous studies have demonstrated that Fyn knockout (FynKO) mice on a standard chow diet display increased glucose clearance and whole-body insulin sensitivity associated with decreased adiposity resulting from increased fatty acid use and energy expenditure. Surprisingly, however, despite a similar extent of adipose tissue (AT) mass accumulation on a high-fat diet, the FynKO mice remained fully glucose tolerant and insulin sensitive. Physiologic analyses demonstrated that the FynKO mice had a combination of skewed AT expansion into the subcutaneous compartment rather than to the visceral depot, reduced AT inflammation associated with reduced T-cell and macrophage infiltration, and increased proportion of anti-inflammatory M2 macrophages. These data demonstrate that Fyn is an important regulator of whole-body integrative metabolism that coordinates AT expansion, inflammation, and insulin sensitivity in states of nutrient excess. These data further suggest that inhibition of Fyn function may provide a novel target to prevent AT inflammation, insulin resistance, and the dyslipidemia components of the metabolic syndrome.

How does high-fat diet induce adipose tissue fibrosis?

Obesity is one of the most serious pandemic health problems in modern society and the predisposing factor for the type 2 diabetes mellitus. Chronic low-grade inflammation mediates the pathogenesis of insulin resistance in obese humans and rodents, and white adipose tissue is one of major tissues to modulate inflammation. Obese humans and rodents show dynamic changes of immunocellular compositions in white adipose tissue to induce inflammatory responses. Innate and adaptive immune responses mainly mediated by macrophages and T cells contribute insulin resistance. Recently, it has been shown that adipose tissue fibrosis is also enhanced in obese humans and rodents along with inflammatory responses, and suppression of adipose tissue fibrosis shows improved insulin sensitivity in rodent models, suggesting that adipose tissue fibrosis is involved in insulin resistance.

Loss of autophagy in hypothalamic POMC neurons impairs lipolysis.

Autophagy degrades cytoplasmic contents to achieve cellular homeostasis. We show that selective loss of autophagy in hypothalamic proopiomelanocortin (POMC) neurons decreases α-melanocyte-stimulating hormone (MSH) levels, promoting adiposity, impairing lipolysis and altering glucose homeostasis. Ageing reduces hypothalamic autophagy and α-MSH levels, and aged-mice phenocopy, the adiposity and lipolytic defect observed in POMC neuron autophagy-null mice. Intraperitoneal isoproterenol restores lipolysis in both models, demonstrating normal adipocyte catecholamine responsiveness. We propose that an unconventional, autophagosome-mediated form of secretion in POMC neurons controls energy balance by regulating α-MSH production. Modulating hypothalamic autophagy might have implications for preventing obesity and metabolic syndrome of ageing.

High-fat diet-induced adipocyte cell death occurs through a cyclophilin D intrinsic signaling pathway independent of adipose tissue inflammation.

OBJECTIVE: Previous studies have demonstrated that mice fed a high-fat diet (HFD) develop insulin resistance with proinflammatory macrophage infiltration into white adipose tissue. Concomitantly, adipocytes undergo programmed cell death with the loss of the adipocyte-specific lipid droplet protein perilipin, and the dead/dying adipocytes are surrounded by macrophages that are organized into crown-like structures. This study investigated whether adipocyte cell death provides the driving signal for macrophage inflammation or if inflammation induces adipocyte cell death. RESEARCH DESIGN AND METHODS: Two knockout mouse models were used: granulocyte/monocyte-colony stimulating factor (GM-CSF)-null mice that are protected against HFD-induced adipose tissue inflammation and cyclophilin D (CyP-D)-null mice that are protected against adipocyte cell death. Mice were fed for 4-14 weeks with a 60% HFD, and different markers of cell death and inflammation were analyzed. RESULTS: HFD induced a normal extent of adipocyte cell death in GM-CSF-null mice, despite a marked reduction in adipose tissue inflammation. Similarly, depletion of macrophages by clodronate treatment prevented HFD-induced adipose tissue inflammation without any affect on adipocyte cell death. However, CyP-D deficiency strongly protected adipocytes from HFD-induced cell death, without affecting adipose tissue inflammation. CONCLUSIONS: These data demonstrate that HFD-induced adipocyte cell death is an intrinsic cellular response that is CyP-D dependent but is independent of macrophage infiltration/activation.

Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors.

Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4(-/-) T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4(-/-) mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.