Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

Myeloid AMPK signaling restricts fibrosis but is not required for metformin improvements during CDAHFD-induced NASH in mice.

Metabolic programming underpins inflammation and liver macrophage activation in the setting of chronic liver disease. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1fl/fl/Prkaa2fl/fl (Flox) control and Flox-LysM-Cre+ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high-fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, and decreased expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Thermoneutral housing does not accelerate metabolic dysfunction-associated fatty liver disease in male or female C57Bl/6J mice fed a Western diet.

Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. Although dozens of preclinical models have been developed to recapitulate stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (TN) housing and consumption of a classical Western diet (WD) would accelerate the onset and progression of MAFLD. Male and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (WD) for 16 wk. Mice were housed with littermates at either standard temperature (TS; 22°C) or thermoneutral-like conditions (TN; ∼29°C). Male, but not female, mice housed at TN and fed a WD were significantly heavier than TS-housed control animals. WD-fed mice housed under TN conditions had lower levels of circulating glucose compared with TS mice; however, there were select but minimal differences in other circulating markers. Although WD-fed TN males had higher liver enzyme and higher liver triglyceride levels, no differences in markers of liver injury or hepatic lipid accumulation were observed in females. Housing temperature had little effect on histopathological scoring of MAFLD progression in males; however, although female mice retained a level of protection, WD-TN conditions trended toward a worsened hepatic phenotype, which was associated with higher macrophage transcript expression and content. Our results indicate that interventions coupling TN housing and WD-induced MAFLD should be longer than 16 wk to accelerate hepatic steatosis and increase inflammation in both sexes of mice.NEW & NOTEWORTHY Mouse models leading to accelerated fatty liver onset are a useful translational tool. Here we show that coupling thermoneutral-like housing and Western diet feeding in mice for 16 wk does not lead to significant disease progression in either sex, though the molecular phenotype indicates priming of immune-related and fibrotic pathways.

Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments.

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

Myeloid deletion and therapeutic activation of AMPK do not alter atherosclerosis in male or female mice.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

The citrus flavonoid nobiletin confers protection from metabolic dysregulation in high-fat-fed mice independent of AMPK.

Obesity, dyslipidemia, and insulin resistance, the increasingly common metabolic syndrome, are risk factors for CVD and type 2 diabetes that warrant novel therapeutic interventions. The flavonoid nobiletin displays potent lipid-lowering and insulin-sensitizing properties in mice with metabolic dysfunction. However, the mechanisms by which nobiletin mediates metabolic protection are not clearly established. The central role of AMP-activated protein kinase (AMPK) as an energy sensor suggests that AMPK is a target of nobiletin. We tested the hypothesis that metabolic protection by nobiletin required phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in mouse hepatocytes, in mice deficient in hepatic AMPK (Ampkß1-/-), in mice incapable of inhibitory phosphorylation of ACC (AccDKI), and in mice with adipocyte-specific AMPK deficiency (iß1ß2AKO). We fed mice a high-fat/high-cholesterol diet with or without nobiletin. Nobiletin increased phosphorylation of AMPK and ACC in primary mouse hepatocytes, which was associated with increased FA oxidation and attenuated FA synthesis. Despite loss of ACC phosphorylation in Ampkß1-/- hepatocytes, nobiletin suppressed FA synthesis and enhanced FA oxidation. Acute injection of nobiletin into mice did not increase phosphorylation of either AMPK or ACC in liver. In mice fed a high-fat diet, nobiletin robustly prevented obesity, hepatic steatosis, dyslipidemia, and insulin resistance, and it improved energy expenditure in Ampkß1-/-, AccDKI, and iß1ß2AKO mice to the same extent as in WT controls. Thus, the beneficial metabolic effects of nobiletin in vivo are conferred independently of hepatic or adipocyte AMPK activation. These studies further underscore the therapeutic potential of nobiletin and begin to clarify possible mechanisms.

Interleukin-18 up-regulates amino acid transporters and facilitates amino acid-induced mTORC1 activation in natural killer cells.

Upon inflammation, natural killer (NK) cells undergo metabolic changes to support their high energy demand for effector function and proliferation. The metabolic changes are usually accompanied by an increase in the expression of nutrient transporters, leading to increased nutrient uptake. Among various cytokines inducing NK cell proliferation, the mechanisms underlying the effect of interleukin (IL)-18 in promoting NK cell proliferation are not completely understood. Here, we demonstrate that IL-18 is a potent cytokine that can enhance the expression of the nutrient transporter CD98/LAT1 for amino acids independently of the mTORC1 pathway and thereby induce a dramatic metabolic change associated with increased proliferation of NK cells. Notably, treatment of IL-18-stimulated NK cells with leucine activates the metabolic sensor mTORC1, indicating that the high expression of amino acid transporters induces amino acid-driven mTORC1 activation. Inhibition of the amino acid transporter CD98/LAT1 abrogated the leucine-driven mTORC1 activation and reduced NK cell effector function. Taken together, our study identified a novel role of IL-18 in up-regulating nutrient transporters on NK cells and thereby inducing metabolic changes, including the mTORC1 activation by amino acids.

Maternal diet-induced obesity alters muscle mitochondrial function in offspring without changing insulin sensitivity.

In utero overnutrition can predispose offspring to metabolic disease. Although the mechanisms are unclear, increased oxidative stress accelerating cellular aging has been shown to play a role. Mitochondria are the main site of reactive oxygen species (ROS) production in most cell types. Levels of ROS and the risk for oxidative damage are dictated by the balance between ROS production and antioxidant defense mechanisms. Originally considered as toxic species, physiologic levels of ROS are now known to be essential cell signaling molecules. Using a model of maternal overnutrition in C57BL6N mice, we investigate the mechanisms involved in the development of insulin resistance (IR) in muscle. In red and white gastrocnemius muscles of offspring, we are the first to report characteristics of oxidative phosphorylation, H2O2 production, activity of mitoflashes, and electron transport chain supercomplex formation. Results demonstrate altered mitochondrial function with reduced response to glucose in offspring of mice fed a high-fat and high-sucrose diet, increases in mitochondrial leak respiration, and a reduction in ROS production in red gastrocnemius in response to palmitoyl carnitine. We also demonstrate differences in supercomplex formation between red and white gastrocnemius, which may be integral to fiber-type specialization. We conclude that in this model of maternal overnutrition, mitochondrial alterations occur before the development of IR.-McMurray, F., MacFarlane, M., Kim, K., Patten, D. A., Wei-LaPierre, L., Fullerton, M. D., Harper, M. E. Maternal diet-induced obesity alters muscle mitochondrial function in offspring without changing insulin sensitivity.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Does prenylation predict progression in NAFLD?

Non-alcoholic fatty liver disease (NAFLD) often develops in concert with related metabolic diseases, such as obesity, dyslipidemia and insulin resistance. Prolonged lipid accumulation and inflammation can progress to non-alcoholic steatohepatitis (NASH). Although factors associated with the development of NAFLD are known, triggers for the progression of NAFLD to NASH are poorly understood. Recent findings published in The Journal of Pathology reveal the possible regulation of NASH progression by metabolites of the mevalonate pathway. Mevalonate can be converted into the isoprenoids farnesyldiphosphate (FPP) and geranylgeranyl diphosphate (GGPP). GGPP synthase (GGPPS), the enzyme that converts FPP to GGPP, is dysregulated in humans and mice during NASH. Both FPP and GGPP can be conjugated to proteins through prenylation, modifying protein function and localization. Deletion or knockdown of GGPPS favors FPP prenylation (farnesylation) and augments the function of liver kinase B1, an upstream kinase of AMP-activated protein kinase (AMPK). Despite increased AMPK activation, livers in Ggpps-deficient mice on a high-fat diet poorly oxidize lipids due to mitochondrial dysfunction. Although work from Liu et al provides evidence as to the potential importance of the prenylation portion of the mevalonate pathway during NAFLD, future studies are necessary to fully grasp any therapeutic or diagnostic potential. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lysine acetyltransferase NuA4 and acetyl-CoA regulate glucose-deprived stress granule formation in Saccharomyces cerevisiae.

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules.

Foam Cell Induction Activates AMPK But Uncouples Its Regulation of Autophagy and Lysosomal Homeostasis.

The dysregulation of macrophage lipid metabolism drives atherosclerosis. AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics and plays essential roles regulating macrophage lipid dynamics. Here, we investigated the consequences of atherogenic lipoprotein-induced foam cell formation on downstream immunometabolic signaling in primary mouse macrophages. A variety of atherogenic low-density lipoproteins (acetylated, oxidized, and aggregated forms) activated AMPK signaling in a manner that was in part due to CD36 and calcium-related signaling. In quiescent macrophages, basal AMPK signaling was crucial for maintaining markers of lysosomal homeostasis as well as levels of key components in the lysosomal expression and regulation network. Moreover, AMPK activation resulted in targeted upregulation of members of this network via transcription factor EB. However, in lipid-induced macrophage foam cells, neither basal AMPK signaling nor its activation affected lysosomal-associated programs. These results suggest that while the sum of AMPK signaling in cultured macrophages may be anti-atherogenic, atherosclerotic input dampens the regulatory capacity of AMPK signaling.

Choline transport links macrophage phospholipid metabolism and inflammation.

Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine. Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well-known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline are important for macrophage inflammation. Polarization of primary bone marrow macrophages with lipopolysaccharide (LPS) resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased levels of diacylglycerol and activation of protein kinase C. These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.

Characterization of Redox-Responsive LXR-Activating Nanoparticle Formulations in Primary Mouse Macrophages.

Activation of the transcription factor liver X receptor (LXR) has beneficial effects on macrophage lipid metabolism and inflammation, making it a potential candidate for therapeutic targeting in cardiometabolic disease. While small molecule delivery via nanomedicine has promising applications for a number of chronic diseases, questions remain as to how nanoparticle formulation might be tailored to suit different tissue microenvironments and aid in drug delivery. In the current study, we aimed to compare the in vitro drug delivering capability of three nanoparticle (NP) formulations encapsulating the LXR activator, GW-3965. We observed little difference in the base characteristics of standard PLGA-PEG NP when compared to two redox-active polymeric NP formulations, which we called redox-responsive (RR)1 and RR2. Moreover, we also observed similar uptake of these NP into primary mouse macrophages. We used the transcript and protein expression of the cholesterol efflux protein and LXR target ATP-binding cassette A1 (ABCA1) as a readout of GW-3956-induced LXR activation. Following an initial acute uptake period that was meant to mimic circulating exposure in vivo, we determined that although the induction of transcript expression was similar between NPs, treatment with the redox-sensitive RR1 NPs resulted in a higher level of ABCA1 protein. Our results suggest that NP formulations responsive to cellular cues may be an effective tool for targeted and disease-specific drug release.

The apolipoprotein C-III (Gln38Lys) variant associated with human hypertriglyceridemia is a gain-of-function mutation.

Recent cell culture and animal studies have suggested that expression of human apo C-III in the liver has a profound impact on the triacylglycerol (TAG)-rich VLDL1 production under lipid-rich conditions. The apoC-III Gln38Lys variant was identified in subjects of Mexican origin with moderate hypertriglyceridemia. We postulated that Gln38Lys (C3QK), being a gain-of-function mutation, promotes hepatic VLDL1 assembly/secretion. To test this hypothesis, we expressed C3QK in McA-RH7777 cells and apoc3-null mice to contrast its effect with WT apoC-III (C3WT). In both model systems, C3QK expression increased the secretion of VLDL1-TAG (by 230%) under lipid-rich conditions. Metabolic labeling experiments with C3QK cells showed an increase in de novo lipogenesis (DNL). Fasting plasma concentration of TAG, cholesterol, cholesteryl ester, and FA were increased in C3QK mice as compared with C3WT mice. Liver of C3QK mice also displayed an increase in DNL and expression of lipogenic genes as compared with that in C3WT mice. These results suggest that C3QK variant is a gain-of-function mutation that can stimulate VLDL1 production, through enhanced DNL.

AMP-activated protein kinase and its multifaceted regulation of hepatic metabolism.

PURPOSE OF REVIEW: The current review summarizes recent advancements in our mechanistic and physiological understanding of the energy sensing AMP-activated protein kinase (AMPK) and its regulation of select aspects of hepatic metabolism. RECENT FINDINGS: A highly conserved serine/threonine kinase, AMPK governs a multitude of cellular process to activate catabolic and inhibit anabolic pathways. Recent work has provided clarity as to the importance and contribution of the AMPK signaling cascade to various aspects of cellular metabolism, including lipid homeostasis, hepatic glucose production, mitochondrial metabolism, and autophagy. SUMMARY: With more than 60 confirmed substrates, the physiological significance of AMPK signaling has been difficult to ascertain. The generation of targeted knock-in mutations on key AMPK substrates has begun to shed light on this complex system. Future studies are needed to further decipher the complexity, significance, and potential therapeutic targeting of hepatic AMPK signaling.

Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity.

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK ß1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK ß1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

Salicylate improves macrophage cholesterol homeostasis via activation of Ampk.

Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk ß1-deficient (ß1(-/-)) mice. Macrophages from Ampk ß1(-/-) mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk ß1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk ß1(-/-) macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk ß1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk ß1(-/-) macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis.

PPARδ activation attenuates hepatic steatosis in Ldlr-/- mice by enhanced fat oxidation, reduced lipogenesis, and improved insulin sensitivity.

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr(-/-) mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA ß-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKß1(-/-) hepatocytes. However, FA oxidation was only partially reduced in AMPKß1(-/-) hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKß1(-/-) hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.

AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice.

AIMS/HYPOTHESIS: Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK-ACC2-malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice. METHODS: Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates. RESULTS: ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance. CONCLUSIONS/INTERPRETATION: These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.