Fatty acid binding protein 5 suppression attenuates obesity-induced hepatocellular carcinoma by promoting ferroptosis and intratumoral immune rewiring.

Due to the rise in overnutrition, the incidence of obesity-induced hepatocellular carcinoma (HCC) will continue to escalate; however, our understanding of the obesity to HCC developmental axis is limited. We constructed a single-cell atlas to interrogate the dynamic transcriptomic changes during hepatocarcinogenesis in mice. Here we identify fatty acid binding protein 5 (FABP5) as a driver of obesity-induced HCC. Analysis of transformed cells reveals that FABP5 inhibition and silencing predispose cancer cells to lipid peroxidation and ferroptosis-induced cell death. Pharmacological inhibition and genetic ablation of FABP5 ameliorates the HCC burden in male mice, corresponding to enhanced ferroptosis in the tumour. Moreover, FABP5 inhibition induces a pro-inflammatory tumour microenvironment characterized by tumour-associated macrophages with increased expression of the co-stimulatory molecules CD80 and CD86 and increased CD8+ T cell activation. Our work unravels the dual functional role of FABP5 in diet-induced HCC, inducing the transformation of hepatocytes and an immunosuppressive phenotype of tumour-associated macrophages and illustrates FABP5 inhibition as a potential therapeutic approach.

Loss of cis-PTase function in the liver promotes a highly penetrant form of fatty liver disease that rapidly transitions to hepatocellular carcinoma.

Obesity-linked fatty liver is a significant risk factor for hepatocellular carcinoma (HCC)1,2; however, the molecular mechanisms underlying the transition from non-alcoholic fatty liver disease (NAFLD) to HCC remains unclear. The present study explores the role of the endoplasmic reticulum (ER)-associated protein NgBR, an essential component of the cis-prenyltransferases (cis-PTase) enzyme3, in chronic liver disease. Here we show that genetic depletion of NgBR in hepatocytes of mice (N-LKO) intensifies triacylglycerol (TAG) accumulation, inflammatory responses, ER/oxidative stress, and liver fibrosis, ultimately resulting in HCC development with 100% penetrance after four months on a high-fat diet. Comprehensive genomic and single cell transcriptomic atlas from affected livers provides a detailed molecular analysis of the transition from liver pathophysiology to HCC development. Importantly, pharmacological inhibition of diacylglycerol acyltransferase-2 (DGAT2), a key enzyme in hepatic TAG synthesis, abrogates diet-induced liver damage and HCC burden in N-LKO mice. Overall, our findings establish NgBR/cis-PTase as a critical suppressor of NAFLD-HCC conversion and suggests that DGAT2 inhibition may serve as a promising therapeutic approach to delay HCC formation in patients with advanced non-alcoholic steatohepatitis (NASH).

MKP1 promotes nonalcoholic steatohepatitis by suppressing AMPK activity through LKB1 nuclear retention.

Nonalcoholic steatohepatitis (NASH) is triggered by hepatocyte death through activation of caspase 6, as a result of decreased adenosine monophosphate (AMP)-activated protein kinase-alpha (AMPKα) activity. Increased hepatocellular death promotes inflammation which drives hepatic fibrosis. We show that the nuclear-localized mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP1) is upregulated in NASH patients and in NASH diet fed male mice. The focus of this work is to investigate whether and how MKP1 is involved in the development of NASH. Under NASH conditions increased oxidative stress, induces MKP1 expression leading to nuclear p38 MAPK dephosphorylation and decreases liver kinase B1 (LKB1) phosphorylation at a site required to promote LKB1 nuclear exit. Hepatic deletion of MKP1 in NASH diet fed male mice releases nuclear LKB1 into the cytoplasm to activate AMPKα and prevents hepatocellular death, inflammation and NASH. Hence, nuclear-localized MKP1-p38 MAPK-LKB1 signaling is required to suppress AMPKα which triggers hepatocyte death and the development of NASH.

MKP1 promotes nonalcoholic steatohepatitis by suppressing AMPK activity through LKB1 nuclear retention.

Nonalcoholic steatohepatitis (NASH) is triggered by hepatocyte death through activation of caspase 6, as a result of decreased adenosine monophosphate (AMP)-activated protein kinase-alpha (AMPKα) activity. Increased hepatocellular death promotes inflammation which drives hepatic fibrosis. We show that the nuclear-localized mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP1) is upregulated in NASH patients and in NASH diet fed mice. The focus of this work was to investigate whether and how MKP1 is involved in the development of NASH. Under NASH conditions increased oxidative stress, induces MKP1 expression leading to nuclear p38 MAPK dephosphorylation and decreased liver kinase B1 (LKB1) phosphorylation at a site required to promote LKB1 nuclear exit. Hepatic deletion of MKP1 in NASH diet fed mice released nuclear LKB1 into the cytoplasm to activate AMPKα and prevent hepatocellular death, inflammation and NASH. Hence, nuclear-localized MKP1-p38 MAPK-LKB1 signaling is required to suppress AMPKα which triggers hepatocyte death and the development of NASH.

The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques.

Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin ß3 resulting in increased tumor necrosis factor [TNF]-α signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin ß3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Hepatocyte-specific miR-33 deletion attenuates NAFLD-NASH-HCC progression.

The complexity of the multiple mechanisms underlying non-alcoholic fatty liver disease (NAFLD) progression remains a significant challenge for the development of effective therapeutics. miRNAs have shown great promise as regulators of biological processes and as therapeutic targets for complex diseases. Here, we study the role of hepatic miR-33, an important regulator of lipid metabolism, during the progression of NAFLD. We report that miR-33 is overexpressed in hepatocytes isolated from mice with NAFLD and demonstrate that its specific suppression in hepatocytes (miR-33 HKO ) improves multiple aspects of the disease, including insulin resistance, steatosis, and inflammation and limits the progression to non-alcoholic steatohepatitis (NASH), fibrosis and hepatocellular carcinoma (HCC). Mechanistically, we find that hepatic miR-33 deficiency reduces lipid biosynthesis and promotes mitochondrial fatty acid oxidation to reduce lipid burden in hepatocytes. Additionally, miR-33 deficiency improves mitochondrial function, reducing oxidative stress. In miR-33 deficient hepatocytes, we found an increase in AMPKα activation, which regulates several pathways resulting in the attenuation of liver disease. The reduction in lipid accumulation and liver injury resulted in decreased transcriptional activity of the YAP/TAZ pathway, which may be involved in the reduced progression to HCC in the HKO livers. Together, these results suggest suppressing hepatic miR-33 may be an effective therapeutic approach at different stages of NAFLD/NASH/HCC disease progression.

microRNA-33 deficiency in macrophages enhances autophagy, improves mitochondrial homeostasis, and protects against lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein (SREBP) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.

Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.

BACKGROUND: Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. METHODS: The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h-/- mice to lethally irradiated Ldlr-/- mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr-/- or Ch25h-/-;Ldlr-/- mice. The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage-tracing mouse model, Myh11ERT2CREmT/mG;Ldlr-/-, adoptively transferred with wild-type or Ch25h-/- mice bone marrow followed by 12 weeks of Western diet feeding. RESULTS: We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB-mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC-mediated modulation of liver X receptor or SREBP (sterol regulatory element-binding protein) transcriptional activity. CONCLUSIONS: Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression.

BACKGROUND: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. METHODS: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. RESULTS: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Col1a2, Fn1, etc) and tissue inhibitor of metalloproteinase 3 (Timp3) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. CONCLUSIONS: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.

MMAB promotes negative feedback control of cholesterol homeostasis.

Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product.

Desmosterol suppresses macrophage inflammasome activation and protects against vascular inflammation and atherosclerosis.

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3ß-hydroxysterol Δ24-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45+CD11b+ cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

PCSK9 Activity Is Potentiated Through HDL Binding.

Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

Liver injury in COVID-19 and IL-6 trans-signaling-induced endotheliopathy.

BACKGROUND AND AIMS: COVID-19 is associated with liver injury and elevated interleukin-6 (IL-6). We hypothesized that IL-6 trans-signaling in liver sinusoidal endothelial cells (LSECs) leads to endotheliopathy (a proinflammatory and procoagulant state) and liver injury in COVID-19. METHODS: Coagulopathy, endotheliopathy, and alanine aminotransferase (ALT) were retrospectively analyzed in a subset (n = 68), followed by a larger cohort (n = 3,780) of patients with COVID-19. Liver histology from 43 patients with COVID-19 was analyzed for endotheliopathy and its relationship to liver injury. Primary human LSECs were used to establish the IL-6 trans-signaling mechanism. RESULTS: Factor VIII, fibrinogen, D-dimer, von Willebrand factor (vWF) activity/antigen (biomarkers of coagulopathy/endotheliopathy) were significantly elevated in patients with COVID-19 and liver injury (elevated ALT). IL-6 positively correlated with vWF antigen (p = 0.02), factor VIII activity (p = 0.02), and D-dimer (p <0.0001). On liver histology, patients with COVID-19 and elevated ALT had significantly increased vWF and platelet staining, supporting a link between liver injury, coagulopathy, and endotheliopathy. Intralobular neutrophils positively correlated with platelet (p <0.0001) and vWF (p <0.01) staining, and IL-6 levels positively correlated with vWF staining (p <0.01). IL-6 trans-signaling leads to increased expression of procoagulant (factor VIII, vWF) and proinflammatory factors, increased cell surface vWF (p <0.01), and increased platelet attachment in LSECs. These effects were blocked by soluble glycoprotein 130 (IL-6 trans-signaling inhibitor), the JAK inhibitor ruxolitinib, and STAT1/3 small-interfering RNA knockdown. Hepatocyte fibrinogen expression was increased by the supernatant of LSECs subjected to IL-6 trans-signaling. CONCLUSION: IL-6 trans-signaling drives the coagulopathy and hepatic endotheliopathy associated with COVID-19 and could be a possible mechanism behind liver injury in these patients. LAY SUMMARY: Patients with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection often have liver injury, but why this occurs remains unknown. High levels of interleukin-6 (IL-6) and its circulating receptor, which form a complex to induce inflammatory signals, have been observed in patients with COVID-19. This paper demonstrates that the IL-6 signaling complex causes harmful changes to liver sinusoidal endothelial cells and may promote blood clotting and contribute to liver injury.

Deficiency of histone lysine methyltransferase SETDB2 in hematopoietic cells promotes vascular inflammation and accelerates atherosclerosis.

Epigenetic modifications of the genome, including DNA methylation, histone methylation/acetylation, and noncoding RNAs, have been reported to play a fundamental role in regulating immune response during the progression of atherosclerosis. SETDB2 is a member of the KMT1 family of lysine methyltransferases, and members of this family typically methylate histone H3 Lys9 (H3K9), an epigenetic mark associated with gene silencing. Previous studies have shown that SETDB2 is involved in innate and adaptive immunity, the proinflammatory response, and hepatic lipid metabolism. Here, we report that expression of SETDB2 is markedly upregulated in human and murine atherosclerotic lesions. Upregulation of SETDB2 was observed in proinflammatory M1 but not antiinflammatory M2 macrophages. Notably, we found that genetic deletion of SETDB2 in hematopoietic cells promoted vascular inflammation and enhanced the progression of atherosclerosis in BM transfer studies in Ldlr-knockout mice. Single-cell RNA-Seq analysis in isolated CD45+ cells from atherosclerotic plaques from mice transplanted with SETDB2-deficient BM revealed a significant increase in monocyte population and enhanced expression of genes involved in inflammation and myeloid cell recruitment. Additionally, we found that loss of SETDB2 in hematopoietic cells was associated with macrophage accumulation in atherosclerotic lesions and attenuated efferocytosis. Overall, these studies identify SETDB2 as an important inflammatory cell regulator that controls macrophage activation in atherosclerotic plaques.

Loss of endothelial glucocorticoid receptor accelerates diabetic nephropathy.

Endothelial cells play a key role in the regulation of disease. Defective regulation of endothelial cell homeostasis may cause mesenchymal activation of other endothelial cells or neighboring cell types, and in both cases contributes to organ fibrosis. Regulatory control of endothelial cell homeostasis is not well studied. Diabetes accelerates renal fibrosis in mice lacking the endothelial glucocorticoid receptor (GR), compared to control mice. Hypercholesterolemia further enhances severe renal fibrosis. The fibrogenic phenotype in the kidneys of diabetic mice lacking endothelial GR is associated with aberrant cytokine and chemokine reprogramming, augmented Wnt signaling and suppression of fatty acid oxidation. Both neutralization of IL-6 and Wnt inhibition improve kidney fibrosis by mitigating mesenchymal transition. Conditioned media from endothelial cells from diabetic mice lacking endothelial GR stimulate Wnt signaling-dependent epithelial-to-mesenchymal transition in tubular epithelial cells from diabetic controls. These data demonstrate that endothelial GR is an essential antifibrotic molecule in diabetes.

miR-27b Modulates Insulin Signaling in Hepatocytes by Regulating Insulin Receptor Expression.

Insulin resistance (IR) is one of the key contributing factors in the development of type 2 diabetes mellitus (T2DM). However, the molecular mechanisms leading to IR are still unclear. The implication of microRNAs (miRNAs) in the pathophysiology of multiple cardiometabolic pathologies, including obesity, atherosclerotic heart failure and IR, has emerged as a major focus of interest in recent years. Indeed, upregulation of several miRNAs has been associated with obesity and IR. Among them, miR-27b is overexpressed in the liver in patients with obesity, but its role in IR has not yet been thoroughly explored. In this study, we investigated the role of miR-27b in regulating insulin signaling in hepatocytes, both in vitro and in vivo. Therefore, assessment of the impact of miR-27b on insulin resistance through the hepatic tissue is of special importance due to the high expression of miR-27b in the liver together with its known role in regulating lipid metabolism. Notably, we found that miR-27b controls post-transcriptional expression of numerous components of the insulin signaling pathway including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1) in human hepatoma cells. These results were further confirmed in vivo showing that overexpression and inhibition of hepatic miR-27 enhances and suppresses hepatic INSR expression and insulin sensitivity, respectively. This study identified a novel role for miR-27 in regulating insulin signaling, and this finding suggests that elevated miR-27 levels may contribute to early development of hepatic insulin resistance.

Disulfiram Treatment Normalizes Body Weight in Obese Mice.

Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy. Additionally, disulfiram treatment reversed established diet-induced obesity and metabolic dysfunctions in middle-aged mice. Reductions in feeding efficiency and increases in energy expenditure were associated with body weight regulation in response to long-term disulfiram treatment. Loss of fat tissue and an increase in liver fenestrations were also observed in rats on disulfiram. Given the potent anti-obesogenic effects in rodents, repurposing disulfiram in the clinic could represent a new strategy to treat obesity and its metabolic comorbidities.

ANGPTL4: a multifunctional protein involved in metabolism and vascular homeostasis.

PURPOSE OF REVIEW: Since the first discovery of Angiopoetin-like 4 (ANGPTL4) in 2000, the involvement of ANGPTL4 in different aspects of lipid metabolism and vascular biology has emerged as an important research field. In this review, we summarize the fundamental roles of ANGPTL4 in regulating metabolic and nonmetabolic functions and their implication in lipid metabolism and with several aspects of vascular function and dysfunction. RECENT FINDINGS: ANGPTL4 is a secreted glycoprotein with a physiological role in lipid metabolism and a predominant expression in adipose tissue and liver. ANGPTL4 inhibits the activity of lipoprotein lipase and thereby promotes an increase in circulating triglyceride levels. Therefore, ANGPTL4 has been highly scrutinized as a potential therapeutic target. Further involvement of ANGPTL4 has been shown to occur in tumorigenesis, angiogenesis, vascular permeability and stem cell regulation, which opens new opportunities of using ANGPTL4 as potential therapeutic targets for other pathophysiological conditions. SUMMARY: Further determination of ANGPTL4 regulatory circuits and defining specific molecular events that mediate its biological effects remain key to future ANGPTL4-based therapeutic applications in different disease settings. Many new and unanticipated roles of ANGPTL4 in the control of cell-specific functions will assist clinicians and researchers in developing potential therapeutic applications.

Suppressing miR-21 activity in tumor-associated macrophages promotes an antitumor immune response.

microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor- associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophage-mediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications.

MicroRNA 7 Impairs Insulin Signaling and Regulates Aß Levels through Posttranscriptional Regulation of the Insulin Receptor Substrate 2, Insulin Receptor, Insulin-Degrading Enzyme, and Liver X Receptor Pathway.

Brain insulin resistance is a key pathological feature contributing to obesity, diabetes, and neurodegenerative disorders, including Alzheimer's disease (AD). Besides the classic transcriptional mechanism mediated by hormones, posttranscriptional regulation has recently been shown to regulate a number of signaling pathways that could lead to metabolic diseases. Here, we show that microRNA 7 (miR-7), an abundant microRNA in the brain, targets insulin receptor (INSR), insulin receptor substrate 2 (IRS-2), and insulin-degrading enzyme (IDE), key regulators of insulin homeostatic functions in the central nervous system (CNS) and the pathology of AD. In this study, we found that insulin and liver X receptor (LXR) activators promote the expression of the intronic miR-7-1 in vitro and in vivo, along with its host heterogeneous nuclear ribonucleoprotein K (HNRNPK) gene, encoding an RNA binding protein (RBP) that is involved in insulin action at the posttranscriptional level. Our data show that miR-7 expression is altered in the brains of diet-induced obese mice. Moreover, we found that the levels of miR-7 are also elevated in brains of AD patients; this inversely correlates with the expression of its target genes IRS-2 and IDE. Furthermore, overexpression of miR-7 increased the levels of extracellular Aß in neuronal cells and impaired the clearance of extracellular Aß by microglial cells. Taken together, these results represent a novel branch of insulin action through the HNRNPK-miR-7 axis and highlight the possible implication of these posttranscriptional regulators in a range of diseases underlying metabolic dysregulation in the brain, from diabetes to Alzheimer's disease.