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

Metabolic programming underpins inflammatory processes of immune cells. In the context of chronic liver disease, liver macrophage activation and response to hepatocellular damage is dependent on profound metabolic changes. 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 first line 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, decrease 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.

The aryl hydrocarbon receptor in ß-cells mediates the effects of TCDD on glucose homeostasis in mice.

OBJECTIVE: Chronic exposure to persistent organic pollutants (POPs) is associated with increased incidence of type 2 diabetes, hyperglycemia, and poor insulin secretion in humans. Dioxins and dioxin-like compounds are a broad class of POPs that exert cellular toxicity through activation of the aryl hydrocarbon receptor (AhR). We previously showed that a single high-dose injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, aka dioxin; 20 µg/kg) in vivo reduced fasted and glucose-stimulated plasma insulin levels for up to 6 weeks in male and female mice. TCDD-exposed male mice were also modestly hypoglycemic and had increased insulin sensitivity, whereas TCDD-exposed females were transiently glucose intolerant. Whether these effects are driven by AhR activation in ß-cells requires investigation. METHODS: We exposed female and male ß-cell specific Ahr knockout (ßAhrKO) mice and littermate Ins1-Cre genotype controls (ßAhrWT) to a single high dose of 20 µg/kg TCDD and tracked the mice for 6 weeks. RESULTS: Under baseline conditions, deleting AhR from ß-cells caused hypoglycemia in female mice, increased insulin secretion ex vivo in female mouse islets, and promoted modest weight gain in male mice. Importantly, high-dose TCDD exposure impaired glucose homeostasis and ß-cell function in ßAhrWT mice, but these phenotypes were largely abolished in TCDD-exposed ßAhrKO mice. CONCLUSION: Our study demonstrates that AhR signaling in ß-cells is important for regulating baseline ß-cell function in female mice and energy homeostasis in male mice. We also show that ß-cell AhR signaling largely mediates the effects of TCDD on glucose homeostasis in both sexes, suggesting that the effects of TCDD on ß-cell function and health are driving metabolic phenotypes in peripheral tissues.

Macrophage AMPK ß1 activation by PF-06409577 reduces the inflammatory response, cholesterol synthesis, and atherosclerosis in mice.

Atherosclerotic cardiovascular disease is characterized by both chronic low-grade inflammation and dyslipidemia. The AMP-activated protein kinase (AMPK) inhibits cholesterol synthesis and dampens inflammation but whether pharmacological activation reduces atherosclerosis is equivocal. In the current study, we found that the orally bioavailable and highly selective activator of AMPKß1 complexes, PF-06409577, reduced atherosclerosis in two mouse models in a myeloid-derived AMPKß1 dependent manner, suggesting a critical role for macrophages. In bone marrow-derived macrophages (BMDMs), PF-06409577 dose dependently activated AMPK as indicated by increased phosphorylation of downstream substrates ULK1 and acetyl-CoA carboxylase (ACC), which are important for autophagy and fatty acid oxidation/de novo lipogenesis, respectively. Treatment of BMDMs with PF-06409577 suppressed fatty acid and cholesterol synthesis and transcripts related to the inflammatory response while increasing transcripts important for autophagy through AMPKß1. These data indicate that pharmacologically targeting macrophage AMPKß1 may be a promising strategy for reducing atherosclerosis.

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.

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.

Pancreas-derived DPP4 is not essential for glucose homeostasis under metabolic stress.

Mice systemically lacking dipeptidyl peptidase-4 (DPP4) have improved islet health, glucoregulation, and reduced obesity with high-fat diet (HFD) feeding compared to wild-type mice. Some, but not all, of this improvement can be linked to the loss of DPP4 in endothelial cells (ECs), pointing to the contribution of non-EC types. The importance of intra-islet signaling mediated by α to ß cell communication is becoming increasingly clear; thus, our objective was to determine if ß cell DPP4 regulates insulin secretion and glucose tolerance in HFD-fed mice by regulating the local concentrations of insulinotropic peptides. Using ß cell double incretin receptor knockout mice, ß cell- and pancreas-specific Dpp4-/- mice, we reveal that ß cell incretin receptors are necessary for DPP4 inhibitor effects. However, although ß cell DPP4 modestly contributes to high glucose (16.7 mM)-stimulated insulin secretion in isolated islets, it does not regulate whole-body glucose homeostasis.

Hepatocyte-derived DPP4 regulates portal GLP-1 bioactivity, modulates glucose production, and when absent influences NAFLD progression.

Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase-positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4-/- mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.

Exercise training enhances muscle mitochondrial metabolism in diet-resistant obesity.

BACKGROUND: Current paradigms for predicting weight loss in response to energy restriction have general validity but a subset of individuals fail to respond adequately despite documented diet adherence. Patients in the bottom 20% for rate of weight loss following a hypocaloric diet (diet-resistant) have been found to have less type I muscle fibres and lower skeletal muscle mitochondrial function, leading to the hypothesis that physical exercise may be an effective treatment when diet alone is inadequate. In this study, we aimed to assess the efficacy of exercise training on mitochondrial function in women with obesity with a documented history of minimal diet-induced weight loss. METHODS: From over 5000 patient records, 228 files were reviewed to identify baseline characteristics of weight loss response from women with obesity who were previously classified in the top or bottom 20% quintiles based on rate of weight loss in the first 6 weeks during which a 900 kcal/day meal replacement was consumed. A subset of 20 women with obesity were identified based on diet-resistance (n=10) and diet sensitivity (n=10) to undergo a 6-week supervised, progressive, combined aerobic and resistance exercise intervention. FINDINGS: Diet-sensitive women had lower baseline adiposity, higher fasting insulin and triglycerides, and a greater number of ATP-III criteria for metabolic syndrome. Conversely in diet-resistant women, the exercise intervention improved body composition, skeletal muscle mitochondrial content and metabolism, with minimal effects in diet-sensitive women. In-depth analyses of muscle metabolomes revealed distinct group- and intervention- differences, including lower serine-associated sphingolipid synthesis in diet-resistant women following exercise training. INTERPRETATION: Exercise preferentially enhances skeletal muscle metabolism and improves body composition in women with a history of minimal diet-induced weight loss. These clinical and metabolic mechanism insights move the field towards better personalised approaches for the treatment of distinct obesity phenotypes. FUNDING: Canadian Institutes of Health Research (CIHR-INMD and FDN-143278; CAN-163902; CIHR PJT-148634).

Defective AMPK regulation of cholesterol metabolism accelerates atherosclerosis by promoting HSPC mobilization and myelopoiesis.

OBJECTIVES: Dysregulation of cholesterol metabolism in the liver and hematopoietic stem and progenitor cells (HSPCs) promotes atherosclerosis development. Previously, it has been shown that HMG-CoA-Reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, can be phosphorylated and inactivated by the metabolic stress sensor AMP-activated protein kinase (AMPK). However, the physiological significance of AMPK regulation of HMGCR to atherogenesis has yet to be elucidated. The aim of this study was to determine the role of AMPK/HMGCR axis in the development of atherosclerosis. METHODS: We have generated a novel atherosclerotic-prone mouse model with defects in the AMPK regulation of HMGCR (Apoe-/-/Hmgcr KI mice). Atherosclerotic lesion size, plaque composition, immune cell and lipid profiles were assessed in Apoe-/- and Apoe-/-/Hmgcr KI mice. RESULTS: In this study, we showed that both male and female atherosclerotic-prone mice with a disruption of HMGCR regulation by AMPK (Apoe-/-/Hmgcr KI mice) display increased aortic lesion size concomitant with an increase in plaque-associated macrophages and lipid accumulation. Consistent with this, Apoe-/-/Hmgcr KI mice exhibited an increase in total circulating cholesterol and atherogenic monocytes, Ly6-Chi subset. Mechanistically, increased circulating atherogenic monocytes in Apoe-/-/Hmgcr KI mice was associated with enhanced egress of bone marrow HSPCs and extramedullary myelopoiesis, driven by a combination of elevated circulating 27-hydroxycholesterol and intracellular cholesterol in HSPCs. CONCLUSIONS: Our results uncovered a novel signalling pathway involving AMPK-HMGCR axis in the regulation of cholesterol homeostasis in HSPCs, and that inhibition of this regulatory mechanism accelerates the development and progression of atherosclerosis. These findings provide a molecular basis to support the use of AMPK activators that currently undergoing Phase II clinical trial such as O-3O4 and PXL 770 for reducing atherosclerotic cardiovascular disease risks.

Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization.

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.

Salsalate reduces atherosclerosis through AMPKß1 in mice.

OBJECTIVE: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αßγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKß1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. METHODS: ApoE-/- and LDLr-/- mice with or without (-/-) germline or bone marrow AMPKß1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow-derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKß1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). RESULTS: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE-/- mice, but not ApoE-/- AMPKß1-/- mice. Similarly, salsalate reduced atherosclerosis in LDLr-/- mice receiving wild-type but not AMPKß1-/- bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. CONCLUSIONS: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKß1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.

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.

Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK.

BACKGROUND: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation. METHODS: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKß1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot. RESULTS: Genetic deletion of AMPKß1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the ß1 subunit. CONCLUSIONS: Together, these data suggest that the development of therapeutic agents targeting AMPKß1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease.

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.

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.

Hepatic Choline Transport Is Inhibited During Fatty Acid-Induced Lipotoxicity and Obesity.

Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate-choline or the phosphatidylethanolamine N-methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet-induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity-induced hepatic steatosis, male mice fed a 60% high-fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter-like 1 (CTL1) in the liver of mice fed a high-fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity-induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis.

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.

In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism.

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.

Adipose Tissue Inflammation Is Directly Linked to Obesity-Induced Insulin Resistance, while Gut Dysbiosis and Mitochondrial Dysfunction Are Not Required.

Obesity is associated with adipose tissue hypertrophy, systemic inflammation, mitochondrial dysfunction, and intestinal dysbiosis. Rodent models of high-fat diet (HFD)-feeding or genetic deletion of multifunctional proteins involved in immunity and metabolism are often used to probe the etiology of obesity; however, these models make it difficult to divorce the effects of obesity, diet composition, or immunity on endocrine regulation of blood glucose. We, therefore, investigated the importance of adipose inflammation, mitochondrial dysfunction, and gut dysbiosis for obesity-induced insulin resistance using a spontaneously obese mouse model. We examined metabolic changes in skeletal muscle, adipose tissue, liver, the intestinal microbiome, and whole-body glucose control in spontaneously hyperphagic C57Bl/6J mice compared to lean littermates. A separate subset of lean and obese mice was subject to 8 weeks of obesogenic HFD feeding, or to pair feeding of a standard rodent diet. Hyperphagia, obesity, adipose inflammation, and insulin resistance were present in obese mice despite consuming a standard rodent diet, and these effects were blunted with caloric restriction. However, hyperphagic obese mice had normal mitochondrial respiratory function in all tissues tested and no discernable intestinal dysbiosis relative to lean littermates. In contrast, feeding mice an obesogenic HFD altered the composition of the gut microbiome, impaired skeletal muscle mitochondrial bioenergetics, and promoted poor glucose control. These data show that adipose inflammation and redox stress occurred in all models of obesity, but gut dysbiosis and mitochondrial respiratory dysfunction are not always required for obesity-induced insulin resistance. Rather, changes in the intestinal microbiome and mitochondrial bioenergetics may reflect physiological consequences of HFD feeding.