A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation.

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research.

Hepatocyte-secreted DPP4 in obesity promotes adipose inflammation and insulin resistance.

Obesity-induced metabolic disease involves functional integration among several organs via circulating factors, but little is known about crosstalk between liver and visceral adipose tissue (VAT). In obesity, VAT becomes populated with inflammatory adipose tissue macrophages (ATMs). In obese humans, there is a close correlation between adipose tissue inflammation and insulin resistance, and in obese mice, blocking systemic or ATM inflammation improves insulin sensitivity. However, processes that promote pathological adipose tissue inflammation in obesity are incompletely understood. Here we show that obesity in mice stimulates hepatocytes to synthesize and secrete dipeptidyl peptidase 4 (DPP4), which acts with plasma factor Xa to inflame ATMs. Silencing expression of DPP4 in hepatocytes suppresses inflammation of VAT and insulin resistance; however, a similar effect is not seen with the orally administered DPP4 inhibitor sitagliptin. Inflammation and insulin resistance are also suppressed by silencing expression of caveolin-1 or PAR2 in ATMs; these proteins mediate the actions of DPP4 and factor Xa, respectively. Thus, hepatocyte DPP4 promotes VAT inflammation and insulin resistance in obesity, and targeting this pathway may have metabolic benefits that are distinct from those observed with oral DPP4 inhibitors.

Paradoxical activation of transcription factor SREBP1c and de novo lipogenesis by hepatocyte-selective ATP-citrate lyase depletion in obese mice.

Hepatic steatosis associated with high-fat diet, obesity, and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl CoA (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high-fat feeding are unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to the generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, whereas in contrast, ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY-depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels because of its increased conversion to malonyl CoA and palmitate. Together, these data indicate that in fat diet-fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or malonyl CoA but rather by activities of DNL enzymes.

An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplant, with no approved therapeutics available. Although the exact molecular mechanism of NASH progression is not well understood, a widely held hypothesis is that fat accumulation is the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long-term gene silencing with single subcutaneous administration. Here, we identified a hyper-functional, fully chemically stabilized GalNAc-conjugated small interfering RNA (siRNA) targeting DGAT2 (Dgat2-1473) that, upon injection, elicits up to 3 months of DGAT2 silencing (>80%-90%, p < 0.0001) in wild-type and NSG-PiZ "humanized" mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (>85%, p < 0.0001) without increased accumulation of diglycerides, resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat did not translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Loss of Antigen Presentation in Adipose Tissue Macrophages or in Adipocytes, but Not Both, Improves Glucose Metabolism.

Macrophages, B cells, and adipocytes are among the adipose tissue (AT) APCs that differentiate and activate naive CD4+ T cells. Mice with adipocyte loss of MHC class II (MHC II) are more insulin sensitive. Because macrophages are professional APCs, mice with genetic myeloid MHC II depletion (myeloid MHC II knockout [mMHCII-/-]) were created and metabolically characterized. FITC+ glucan-coated particles (glucan-encapsulated small interfering RNA [siRNA] particles [GeRPs]) were also used to target MHC II knockout specifically in AT macrophages (ATMs). Mice with total body mMHCII-/- were generated by crossing LyzMCre with H2Ab1 floxed mice. For specific ATM depletion of H2Ab1, GeRPs containing H2Ab1 siRNA were administered to high-fat diet-fed C57BL/6 mice. Unexpectedly, mMHCII-/- mice had loss of both macrophage and adipocyte H2Ab1, one of only two Ag-presenting arms; thus, neither cell could present Ag and activate CD4+ T cells. This inability led to a reduction in AT immunosuppressive regulatory T cells, increased AT CD8+ T cells, and no improvement in systemic metabolism. Thus, with combined systemic myeloid and adipocyte MHC II loss, the impact of ATM-specific alterations in APC activity could not be delineated. Therefore, GeRPs containing H2Ab1 siRNA were administered to specifically reduce ATM H2Ab1 which, in contrast, revealed improved glucose tolerance. In conclusion, loss of either ATM or adipocyte APC function, but not both, improves systemic glucose metabolism because of maintenance of AT regulatory T cells.

Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes.

Acquired resistance to the action of insulin to stimulate glucose transport in skeletal muscle is associated with obesity and promotes the development of type 2 diabetes. In skeletal muscle, insulin resistance can result from high levels of circulating fatty acids that disrupt insulin signalling pathways. However, the severity of insulin resistance varies greatly among obese people. Here we postulate that this variability might reflect differences in levels of lipid-droplet proteins that promote the sequestration of fatty acids within adipocytes in the form of triglycerides, thereby lowering exposure of skeletal muscle to the inhibitory effects of fatty acids.

CRISPR-delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure.

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.

The Misshapen subfamily of Ste20 kinases regulate proliferation in the aging mammalian intestinal epithelium.

The intestinal epithelium has a high rate of cell turn over and is an excellent system to study stem cell-mediated tissue homeostasis. The Misshapen subfamily of the Ste20 kinases in mammals consists of misshapen like kinase 1 (MINK1), mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), and TRAF2 and NCK interacting kinase (TNIK). Recent reports suggest that this subfamily has a novel function equal to the Hippo/MST subfamily as upstream kinases for Warts/Large tumor suppressor kinase (LATS) to suppress tissue growth. To study the in vivo functions of Mink1, Map4k4, and Tnik, we generated a compound knockout of these three genes in the mouse intestinal epithelium. The intestinal epithelia of the mutant animals were phenotypically normal up to approximately 12 months. The older animals then exhibited mildly increased proliferation throughout the lower GI tract. We also observed that the normally spatially organized Paneth cells in the crypt base became dispersed. The expression of one of the YAP pathway target genes Sox9 was increased while other target genes including CTGF did not show a significant change. Therefore, the Misshapen and Hippo subfamilies may have highly redundant functions to regulate growth in the intestinal epithelium, as illustrated in recent tissue culture models.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced ß cell mass, fewer proliferating ß cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from ß cells in mice.

Map4k4 impairs energy metabolism in endothelial cells and promotes insulin resistance in obesity.

The blood vasculature responds to insulin, influencing hemodynamic changes in the periphery, which promotes tissue nutrient and oxygen delivery and thus metabolic function. The lymphatic vasculature regulates fluid and lipid homeostasis, and impaired lymphatic function can contribute to atherosclerosis and obesity. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and lymphangiogenesis as well as atherosclerosis. Here, we show that inducible EC Map4k4 deletion in adult mice ameliorates metabolic dysfunction in obesity despite the development of chylous ascites and a concomitant striking increase in adipose tissue lymphocyte content. Despite these defects, animals lacking endothelial Map4k4 were protected from skeletal muscle microvascular rarefaction in obesity, and primary ECs lacking Map4k4 displayed reduced senescence and increased metabolic capacity. Thus endothelial Map4k4 has complex and opposing functions in the blood and lymphatic endothelium postdevelopment. Whereas blood endothelial Map4k4 promotes vascular dysfunction and impairs glucose homeostasis in adult animals, lymphatic endothelial Map4k4 is required to maintain lymphatic vascular integrity and regulate immune cell trafficking in obesity.

Joint analysis of left ventricular expression and circulating plasma levels of Omentin after myocardial ischemia.

BACKGROUND: Omentin-1, also known as Intelectin-1 (ITLN1), is an adipokine with plasma levels associated with diabetes, obesity, and coronary artery disease. Recent studies suggest that ITLN1 can mitigate myocardial ischemic injury but the expression of ITLN1 in the heart itself has not been well characterized. The purpose of this study is to discern the relationship between the expression pattern of ITLN1 RNA in the human heart and the level of circulating ITLN1 protein in plasma from the same patients following myocardial ischemia. METHODS: A large cohort of patients (n = 140) undergoing elective cardiac surgery for aortic valve replacement were enrolled in this study. Plasma and left ventricular biopsy samples were taken at the beginning of cardiopulmonary bypass and after an average of 82 min of ischemic cross clamp time. The localization of ITLN1 in epicardial adipose tissue (EAT) was also further characterized with immunoassays and cell fate transition studies. RESULTS: mRNA expression of ITLN1 decreases in left ventricular tissue after acute ischemia in human patients (mean difference 280.48, p = 0.001) whereas plasma protein levels of ITLN1 increase (mean difference 5.24, p < 0.001). Immunohistochemistry localized ITLN1 to the mesothelium or visceral pericardium of EAT. Epithelial to mesenchymal transition in mesothelial cells leads to a downregulation of ITLN1 expression. CONCLUSIONS: Myocardial injury leads to a decrease in ITLN1 expression in the heart and a corresponding increase in plasma levels. These changes may in part be due to an epithelial to mesenchymal transition of the cells that express ITLN1 following ischemia. Trial Registration Clinicaltrials.gov ID: NCT00985049.

Coronin 2A mediates actin-dependent de-repression of inflammatory response genes.

Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A-actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis.

The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis.

The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.

Activated Kupffer cells inhibit insulin sensitivity in obese mice.

Obesity promotes insulin resistance associated with liver inflammation, elevated glucose production, and type 2 diabetes. Although insulin resistance is attenuated in genetic mouse models that suppress systemic inflammation, it is not clear whether local resident macrophages in liver, denoted Kupffer cells (KCs), directly contribute to this syndrome. We addressed this question by selectively silencing the expression of the master regulator of inflammation, NF-κB, in KCs in obese mice. We used glucan-encapsulated small interfering RNA particles (GeRPs) that selectively silence gene expression in macrophages in vivo. Following intravenous injections, GeRPs containing siRNA against p65 of the NF-κB complex caused loss of NF-κB p65 expression in KCs without disrupting NF-κB in hepatocytes or macrophages in other tissues. Silencing of NF-κB expression in KCs in obese mice decreased cytokine secretion and improved insulin sensitivity and glucose tolerance without affecting hepatic lipid accumulation. Importantly, GeRPs had no detectable toxic effect. Thus, KCs are key contributors to hepatic insulin resistance in obesity and a potential therapeutic target for metabolic disease.

Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA.

Translation of siRNA technology into the clinic is limited by the need for improved delivery systems that target specific cell types. Macrophages are particularly attractive targets for RNAi therapy because they promote pathogenic inflammatory responses in a number of important human diseases. We previously demonstrated that a multicomponent formulation of ß-1,3-d-glucan-encapsulated siRNA particles (GeRPs) can specifically and potently silence genes in mouse macrophages. A major advance would be to simplify the GeRP system by reducing the number of delivery components, thus enabling more facile manufacturing and future commercialization. Here we report the synthesis and evaluation of a simplified glucan-based particle (GP) capable of delivering siRNA in vivo to selectively silence macrophage genes. Covalent attachment of small-molecule amines and short peptides containing weak bases to GPs facilitated electrostatic interaction of the particles with siRNA and aided in the endosomal release of siRNA by the proton-sponge effect. Modified GPs were nontoxic and were efficiently internalized by macrophages in vitro. When injected intraperitoneally (i.p.), several of the new peptide-modified GPs were found to efficiently deliver siRNA to peritoneal macrophages in lean, healthy mice. In an animal model of obesity-induced inflammation, i.p. administration of one of the peptide-modified GPs (GP-EP14) bound to siRNA selectively reduced the expression of target inflammatory cytokines in the visceral adipose tissue macrophages. Decreasing adipose tissue inflammation resulted in an improvement of glucose metabolism in these metabolically challenged animals. Thus, modified GPs represent a promising new simplified system for the efficient delivery of therapeutic siRNAs specifically to phagocytic cells in vivo for modulation of inflammation responses.

Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice.

Adipose tissue (AT) inflammation and infiltration by macrophages is associated with insulin resistance and type 2 diabetes in obese humans, offering a potential target for therapeutics. However, whether AT macrophages (ATMs) directly contribute to systemic glucose intolerance has not been determined. The reason is the lack of methods to ablate inflammatory genes expressed in macrophages specifically localized within AT depots, leaving macrophages in other tissues unaffected. Here we report that i.p. administration of siRNA encapsulated by glucan shells in obese mice selectively silences genes in epididymal ATMs, whereas macrophages within lung, spleen, kidney, heart, skeletal muscle, subcutaneous (SubQ) adipose, and liver are not targeted. Such administration of GeRPs to silence the inflammatory cytokines TNF-α or osteopontin in epididymal ATMs of obese mice caused significant improvement in glucose tolerance. These data are consistent with the hypothesis that cytokines produced by ATMs can exacerbate whole-body glucose intolerance.

Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle-safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of beta1,3-D-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 microg kg(-1) siRNA directed against tumour necrosis factor alpha (Tnf-alpha) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-alpha levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-alpha and interleukin-1beta production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

ß3-Adrenergic receptor stimulation induces E-selectin-mediated adipose tissue inflammation.

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that ß(3)-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1ß, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that ß(3)-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.

Lipid storage by adipose tissue macrophages regulates systemic glucose tolerance.

Proinflammatory pathways in adipose tissue macrophages (ATMs) can impair glucose tolerance in obesity, but ATMs may also be beneficial as repositories for excess lipid that adipocytes are unable to store. To test this hypothesis, we selectively targeted visceral ATMs in obese mice with siRNA against lipoprotein lipase (LPL), leaving macrophages within other organs unaffected. Selective silencing of ATM LPL decreased foam cell formation in visceral adipose tissue of obese mice, consistent with a reduced supply of fatty acids from VLDL hydrolysis. Unexpectedly, silencing LPL also decreased the expression of genes involved in fatty acid uptake (CD36) and esterification in ATMs. This deficit in fatty acid uptake capacity was associated with increased circulating serum free fatty acids. Importantly, ATM LPL silencing also caused a marked increase in circulating fatty acid-binding protein-4, an adipocyte-derived lipid chaperone previously reported to induce liver insulin resistance and glucose intolerance. Consistent with this concept, obese mice with LPL-depleted ATMs exhibited higher hepatic glucose production from pyruvate and glucose intolerance. Silencing CD36 in ATMs also promoted glucose intolerance. Taken together, the data indicate that LPL secreted by ATMs enhances their ability to sequester excess lipid in obese mice, promoting systemic glucose tolerance.

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.