Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis.

As chronic inflammation is a hallmark of obesity, pathways that integrate nutrient- and pathogen sensing pathways are of great interest in understanding the mechanisms of insulin resistance, type 2 diabetes, and other chronic metabolic pathologies. Here, we provide evidence that double-stranded RNA-dependent protein kinase (PKR) can respond to nutrient signals as well as endoplasmic reticulum (ER) stress and coordinate the activity of other critical inflammatory kinases such as the c-Jun N-terminal kinase (JNK) to regulate insulin action and metabolism. PKR also directly targets and modifies insulin receptor substrate and hence integrates nutrients and insulin action with a defined pathogen response system. Dietary and genetic obesity features marked activation of PKR in adipose and liver tissues and absence of PKR alleviates metabolic deterioration due to nutrient or energy excess in mice. These findings demonstrate PKR as a critical component of an inflammatory complex that responds to nutrients and organelle dysfunction.

Notch ligand delta-like 4 blockade attenuates atherosclerosis and metabolic disorders.

Atherosclerosis and insulin resistance are major components of the cardiometabolic syndrome, a global health threat associated with a systemic inflammatory state. Notch signaling regulates tissue development and participates in innate and adaptive immunity in adults. The role of Notch signaling in cardiometabolic inflammation, however, remains obscure. We noted that a high-fat, high-cholesterol diet increased expression of the Notch ligand Delta-like 4 (Dll4) in atheromata and fat tissue in LDL-receptor-deficient mice. Blockade of Dll4-Notch signaling using neutralizing anti-Dll4 antibody attenuated the development of atherosclerosis, diminished plaque calcification, improved insulin resistance, and decreased fat accumulation. These changes were accompanied by decreased macrophage accumulation, diminished expression of monocyte chemoattractant protein-1 (MCP-1), and lower levels of nuclear factor-κB (NF-κB) activation. In vitro cell culture experiments revealed that Dll4-mediated Notch signaling increases MCP-1 expression via NF-κB, providing a possible mechanism for in vivo effects. Furthermore, Dll4 skewed macrophages toward a proinflammatory phenotype ("M1"). These results suggest that Dll4-Notch signaling plays a central role in the shared mechanism for the pathogenesis of cardiometabolic disorders.

Neuronal PTP1B regulates body weight, adiposity and leptin action.

Obesity is a major health problem and a risk factor for type 2 diabetes. Leptin, an adipocyte-secreted hormone, acts on the hypothalamus to inhibit food intake and increase energy expenditure. Most obese individuals develop hyperleptinemia and leptin resistance, limiting the therapeutic efficacy of exogenously administered leptin. Mice lacking the tyrosine phosphatase PTP1B are protected from diet-induced obesity and are hypersensitive to leptin, but the site and mechanism for these effects remain controversial. We generated tissue-specific PTP1B knockout (Ptpn1(-/-)) mice. Neuronal Ptpn1(-/-) mice have reduced weight and adiposity, and increased activity and energy expenditure. In contrast, adipose PTP1B deficiency increases body weight, whereas PTP1B deletion in muscle or liver does not affect weight. Neuronal Ptpn1(-/-) mice are hypersensitive to leptin, despite paradoxically elevated leptin levels, and show improved glucose homeostasis. Thus, PTP1B regulates body mass and adiposity primarily through actions in the brain. Furthermore, neuronal PTP1B regulates adipocyte leptin production and probably is essential for the development of leptin resistance.

Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2.

Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.

Macrophage Mal1 deficiency suppresses atherosclerosis in low-density lipoprotein receptor-null mice by activating peroxisome proliferator-activated receptor-γ-regulated genes.

OBJECTIVE: The adipocyte/macrophage fatty acid-binding proteins aP2 (FABP4) and Mal1 (FABP5) are intracellular lipid chaperones that modulate systemic glucose metabolism, insulin sensitivity, and atherosclerosis. Combined deficiency of aP2 and Mal1 has been shown to reduce the development of atherosclerosis, but the independent role of macrophage Mal1 expression in atherogenesis remains unclear. METHODS AND RESULTS: We transplanted wild-type (WT), Mal1(-/-), or aP2(-/-) bone marrow into low-density lipoprotein receptor-null (LDLR(-/-)) mice and fed them a Western diet for 8 weeks. Mal1(-/-)→LDLR(-/-) mice had significantly reduced (36%) atherosclerosis in the proximal aorta compared with control WT→LDLR(-/-) mice. Interestingly, peritoneal macrophages isolated from Mal1-deficient mice displayed increased peroxisome proliferator-activated receptor-γ (PPARγ) activity and upregulation of a PPARγ-related cholesterol trafficking gene, CD36. Mal1(-/-) macrophages showed suppression of inflammatory genes, such as COX2 and interleukin 6. Mal1(-/-)→LDLR(-/-) mice had significantly decreased macrophage numbers in the aortic atherosclerotic lesions compared with WT→LDLR(-/-) mice, suggesting that monocyte recruitment may be impaired. Indeed, blood monocytes isolated from Mal1(-/-)→LDLR(-/-) mice on a high-fat diet had decreased CC chemokine receptor 2 gene and protein expression levels compared with WT monocytes. CONCLUSION: Taken together, our results demonstrate that Mal1 plays a proatherogenic role by suppressing PPARγ activity, which increases expression of CC chemokine receptor 2 by monocytes, promoting their recruitment to atherosclerotic lesions.

Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

Adipocyte/macrophage fatty acid-binding proteins contribute to metabolic deterioration through actions in both macrophages and adipocytes in mice.

Adipose tissue inflammation is a characteristic of obesity. However, the mechanisms that regulate this inflammatory response and link adipose inflammation to systemic metabolic consequences are not fully understood. In this study, we have taken advantage of the highly restricted coexpression of adipocyte/macrophage fatty acid-binding proteins (FABPs) aP2 (FABP4) and mal1 (FABP5) to examine the contribution of these lipid chaperones in macrophages and adipocytes to local and systemic inflammation and metabolic homeostasis in mice. Deletion of FABPs in adipocytes resulted in reduced expression of inflammatory cytokines in macrophages, whereas the same deletion in macrophages led to enhanced insulin signaling and glucose uptake in adipocytes. Using radiation chimerism through bone marrow transplantation, we generated mice with FABP deficiency in bone marrow and stroma-derived elements in vivo and studied the impact of each cellular target on local and systemic insulin action and glucose metabolism in dietary obesity. The results of these experiments indicated that neither macrophages nor adipocytes individually could account for the total impact of FABPs on systemic metabolism and suggest that interactions between these 2 cell types, particularly in adipose tissue, are critical for the inflammatory basis of metabolic deterioration.

Adipocyte/macrophage fatty acid binding proteins control integrated metabolic responses in obesity and diabetes.

Fatty acid binding proteins (FABPs) are cytosolic fatty acid chaperones whose biological role and mechanisms of action are not well understood. Here, we developed mice with targeted mutations in two related adipocyte FABPs, aP2 and mal1, to resolve their role in systemic lipid, glucose, and energy metabolism. Mice lacking aP2 and mal1 exhibited a striking phenotype with strong protection from diet-induced obesity, insulin resistance, type 2 diabetes, and fatty liver disease. These mice have altered cellular and systemic lipid transport and composition, leading to enhanced insulin receptor signaling, enhanced muscle AMP-activated kinase (AMP-K) activity, and dramatically reduced liver stearoyl-CoA desaturase-1 (SCD-1) activity underlying their phenotype. Taken together with the previously reported strong protection against atherosclerosis, these results demonstrate that adipocyte/macrophage FABPs have a robust impact on multiple components of metabolic syndrome, integrating metabolic and inflammatory responses in mice and constituting a powerful target for the treatment of these diseases.

The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation.

The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma.

Regulation of metabolic responses by adipocyte/macrophage Fatty Acid-binding proteins in leptin-deficient mice.

Fatty acid-binding proteins (FABPs) are cytosolic fatty acid chaperones that play a critical role in systemic regulation of lipid and glucose metabolism. In animals lacking the adipocyte/macrophage FABP isoforms aP2 and mal1, there is strong protection against diet-induced obesity, insulin resistance, type 2 diabetes, fatty liver disease, and hypercholesterolemic atherosclerosis. On high-fat diet, FABP-deficient mice also exhibit enhanced muscle AMP-activated kinase (AMPK) and reduced liver stearoyl-CoA desaturase-1 (SCD-1) activities. Here, we performed a cross between aP2(-/-), mal1(-/-), and leptin-deficient (ob/ob) mice to elucidate the role of leptin action on the metabolic phenotype of aP2-mal1 deficiency. The extent of obesity in the ob/ob-aP2-mal1(-/-) mice was comparable with ob/ob mice. However, despite severe obesity, ob/ob-aP2-mal1(-/-) mice remained euglycemic and demonstrated improved peripheral insulin sensitivity. There was also a striking protection from liver fatty infiltration in the ob/ob-aP2-mal1(-/-) mice with strong suppression of SCD-1 activity. On the other hand, the enhanced muscle AMPK activity in aP2-mal1(-/-) mice was lost in the ob/ob background. These results indicated that both decreased body weight and enhanced muscle AMPK activity in aP2-mal1(-/-) mice are potentially leptin dependent but improved systemic insulin sensitivity and protection from liver fatty infiltration are largely unrelated to leptin action and that insulin-sensitizing effects of FABP deficiency are, at least in part, independent of its effects on total-body adiposity.

Role of the fatty acid binding protein mal1 in obesity and insulin resistance.

The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.

Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice.

Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

Deficiency of fatty acid-binding proteins in mice confers protection from development of experimental autoimmune encephalomyelitis.

Fatty acid-binding proteins (FABPs) act as intracellular receptors for a variety of hydrophobic compounds, enabling their diffusion within the cytoplasmic compartment. Recent studies have demonstrated the ability of FABPs to simultaneously regulate metabolic and inflammatory pathways. We investigated the role of adipocyte FABP and epithelial FABP in the development of experimental autoimmune encephalomyelitis to test the hypothesis that these FABPs impact adaptive immune responses and contribute to the pathogenesis of autoimmune disease. FABP-deficient mice exhibited a lower incidence of disease, reduced clinical symptoms of experimental autoimmune encephalomyelitis and dramatically lower levels of proinflammatory cytokine mRNA expression in CNS tissue as compared with wild-type mice. In vitro Ag recall responses of myelin oligodendrocyte glycoprotein 35-55-immunized FABP(-/-) mice showed reduced proliferation and impaired IFN-gamma production. Dendritic cells deficient for FABPs were found to be poor producers of proinflammatory cytokines and Ag presentation by FABP(-/-) dendritic cells did not promote proinflammatory T cell responses. This study reveals that metabolic-inflammatory pathway cross-regulation by FABPs contributes to adaptive immune responses and subsequent autoimmune inflammation.

Coordinated regulation of nutrient and inflammatory responses by STAMP2 is essential for metabolic homeostasis.

Metabolic and inflammatory pathways crosstalk at many levels, and, while required for homeostasis, interaction between these pathways can also lead to metabolic dysregulation under conditions of chronic stress. Thus, we hypothesized that mechanisms might exist to prevent overt inflammatory responses during physiological fluctuations in nutrients or under nutrient-rich conditions, and we identified the six-transmembrane protein STAMP2 as a critical modulator of this integrated response system of inflammation and metabolism in adipocytes. Lack of STAMP2 in adipocytes results in aberrant inflammatory responses to both nutrients and acute inflammatory stimuli. Similarly, in whole animals, visceral adipose tissue of STAMP2(-/-) mice exhibits overt inflammation, and these mice develop spontaneous metabolic disease on a regular diet, manifesting insulin resistance, glucose intolerance, mild hyperglycemia, dyslipidemia, and fatty liver disease. We conclude that STAMP2 participates in integrating inflammatory and metabolic responses and thus plays a key role in systemic metabolic homeostasis.

Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes.

Endoplasmic reticulum (ER) stress is a key link between obesity, insulin resistance, and type 2 diabetes. Here, we provide evidence that this mechanistic link can be exploited for therapeutic purposes with orally active chemical chaperones. 4-Phenyl butyric acid and taurine-conjugated ursodeoxycholic acid alleviated ER stress in cells and whole animals. Treatment of obese and diabetic mice with these compounds resulted in normalization of hyperglycemia, restoration of systemic insulin sensitivity, resolution of fatty liver disease, and enhancement of insulin action in liver, muscle, and adipose tissues. Our results demonstrate that chemical chaperones enhance the adaptive capacity of the ER and act as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes.

Regulation of dendritic cell function and T cell priming by the fatty acid-binding protein AP2.

The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IkappaBalpha was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IkappaB kinase-NF-kappaB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-gamma by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-gamma by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.

A central role for JNK in obesity and insulin resistance.

Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus, yet the molecular mechanisms of this association are poorly understood. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin action in cultured cells and are activated by inflammatory cytokines and free fatty acids, molecules that have been implicated in the development of type 2 diabetes. Here we show that JNK activity is abnormally elevated in obesity. Furthermore, an absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signalling capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and a potential target for therapeutics.

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.