Arijeet K. Gattu

Anti-angiogenic pigment epithelium-derived factor regulates hepatocyte triglyceride content through adipose triglyceride lipase (ATGL) ☆

BACKGROUND/AIMS Anti-angiogenic pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that is highly expressed in hepatocytes. Adipose triglyceride lipase (ATGL), a novel lipase critical for triglyceride metabolism, is a receptor for PEDF. We postulated that hepatocyte triglyceride metabolism was dependent on interactions between PEDF and ATGL, and loss of PEDF would impair mobilization of triglycerides in the liver. METHODS Immunoprecipitation studies were performed in PEDF null and control hepatocytes with recombinant PEDF (rPEDF) as bait. Immunofluorescent microscopy was used to localize ATGL. Triglyceride content was analyzed in hepatocytes and in whole liver with and without rPEDF. ATGL was blocked using an inhibitor, (R)-bromoenol lactone. RESULTS PEDF co-immunoprecipitated with ATGL in hepatic and HCC lysates. All PEDF deficient livers demonstrated steatosis. Triglyceride content was significantly increased in PEDF null livers compared to wildtype (p<0.05) and in isolated hepatocytes (p<0.01). Treatment of PEDF null hepatocytes with rPEDF decreased TG content (p<0.05) and this activity was dependent on ATGL. CONCLUSIONS Our results identify a novel role for PEDF in hepatic triglyceride homeostasis through binding to ATGL and demonstrate that rPEDF and ATGL localize to adiposomes in hepatocytes. Dysregulation of this pathway may be one mechanism underlying fatty liver disease.

Insulin-independent regulation of hepatic triglyceride synthesis by fatty acids

Significance The paradox of selective hepatic insulin resistance, wherein the insulin-resistant liver fails to suppress glucose production but continues to produce triglycerides, is central to the pathophysiology of type 2 diabetes. We hypothesized that hepatic triglyceride synthesis is regulated mostly by fatty acid delivery to the liver and independent of changes in hepatic insulin signaling. To examine this hypothesis, we used a novel LC-MS/MS method to measure rates of hepatic fatty acid esterification in vivo. In contrast to hepatic de novo hepatic lipogenesis, rates of hepatic esterification of fatty acids into triglyceride was primarily dependent on fatty acid delivery and independent of hepatic insulin action, providing an explanation for increased hepatic triglyceride synthesis in the presence of hepatic insulin resistance. A central paradox in type 2 diabetes is the apparent selective nature of hepatic insulin resistance—wherein insulin fails to suppress hepatic glucose production yet continues to stimulate lipogenesis, resulting in hyperglycemia, hyperlipidemia, and hepatic steatosis. Although efforts to explain this have focused on finding a branch point in insulin signaling where hepatic glucose and lipid metabolism diverge, we hypothesized that hepatic triglyceride synthesis could be driven by substrate, independent of changes in hepatic insulin signaling. We tested this hypothesis in rats by infusing [U-13C] palmitate to measure rates of fatty acid esterification into hepatic triglyceride while varying plasma fatty acid and insulin concentrations independently. These experiments were performed in normal rats, high fat-fed insulin-resistant rats, and insulin receptor 2′-O-methoxyethyl chimeric antisense oligonucleotide-treated rats. Rates of fatty acid esterification into hepatic triglyceride were found to be dependent on plasma fatty acid infusion rates, independent of changes in plasma insulin concentrations and independent of hepatocellular insulin signaling. Taken together, these results obviate a paradox of selective insulin resistance, because the major source of hepatic lipid synthesis, esterification of preformed fatty acids, is primarily dependent on substrate delivery and largely independent of hepatic insulin action.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content

Pigment epithelium‐derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF‐directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator‐activated receptor‐γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control‐treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt‐β‐catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt‐β‐catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium‐derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J. 27, 4384–4394 (2013). www.fasebj.org

Ethanol Exposure Depletes Hepatic Pigment Epithelium-Derived Factor, a Novel Lipid Regulator

BACKGROUND & AIMS Ethanol abuse can lead to hepatic steatosis and evolve into cirrhosis and hepatocellular carcinoma. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that is expressed by hepatocytes. Proteomic, experimental, and clinical studies implicate PEDFs role in lipid regulation. Because matrix metalloproteinase (MMP)-2/9 activity regulates PEDF levels, we investigated whether PEDF degradation by MMPs has a permissive role in ethanol-induced hepatic steatosis. METHODS PEDF levels were examined in liver biopsy specimens from patients with ethanol-induced steatosis. Hepatic PEDF levels and MMP activity were assessed in 2 animal models of ethanol feeding (rats on an alcohol-containing liquid diet and mice given intragastric infusion of ethanol). The consequences of PEDF depletion in the liver were examined in PEDF-null mice. RESULTS Liver biopsy samples from patients with ethanol-induced steatosis had reduced PEDF levels, compared with normal liver samples. Ethanol-fed animals had histologic steatosis and increased liver triglyceride content (P< .05), as well as reduced levels of hepatic PEDF and increased MMP-2/9 activity. Ethanol-exposed hepatic lysates degraded PEDF in a MMP-2/9-dependent manner, and liver sections demonstrated abundant MMP-2/9 activity in situ. Addition of recombinant PEDF to PEDF-null hepatocytes, reduced their triglyceride content. CONCLUSIONS Ethanol exposure leads to marked loss of hepatic PEDF in human livers and in 2 animal models of ethanol feeding. Loss of PEDF contributes to the accumulation of lipids in ethanol-induced hepatic steatosis.

Pigment Epithelium-Derived Factor Regulates Early Pancreatic Fibrotic Responses and Suppresses the Profibrotic Cytokine Thrombospondin-1

Pigment epithelium-derived factor (PEDF) is important for maintaining the normal extracellular matrix. We hypothesized that the initiation of pancreatic fibrosis is dependent on the loss of PEDF. Pancreatic PEDF expression was assessed in wild-type mice fed either a control or ethanol diet using an intragastric feeding model. Pancreatitis responses were elicited with either a single episode or a repetitive cerulein-induced (50 μg/kg, 6 hourly i.p. injections) protocol in wild-type and PEDF-null mice. Quantitative real-time PCR and immunoblotting were performed to assess fibrogenic responses. In wild-type animals, PEDF expression increased with pancreatitis and was more pronounced in mice fed ethanol. Compared with wild-type mice, α-smooth muscle actin staining and expression levels of fibrogenic markers (eg, transforming growth factor-β1, platelet-derived growth factor, collagen I, and thrombospondin-1) were higher in PEDF-null mice at baseline. Sirius red staining revealed more fibrosis in PEDF-null versus wild-type pancreas 1 week after pancreatitis. Differences in tissue fibrosis resolved with longer recovery periods. PEDF overexpression suppressed thrombospondin-1 levels in vitro. Ethanol feeding and experimental pancreatitis increased PEDF expression in wild-type mice. PEDF-null mice, however, demonstrated enhanced early fibrotic responses compared with wild-type mice with pancreatitis. These findings indicate that PEDF acts as a compensatory antifibrotic cytokine in pancreatitis.

Pigment Epithelium-Derived Factor (PEDF) Suppresses IL-1β-Mediated c-Jun N-Terminal Kinase (JNK) Activation to Improve Hepatocyte Insulin Signaling

Pigment epithelium-derived factor (PEDF) is an antiinflammatory protein that circulates at high levels in the metabolic syndrome. Metabolic studies of PEDF knockout (KO) mice were conducted to investigate the relationship between PEDF, inflammatory markers, and metabolic homeostasis. Male PEDF KO mice demonstrated a phenotype consisting of increased adiposity, glucose intolerance, and elevated serum levels of metabolites associated with the metabolic syndrome. Genome expression analysis revealed an increase in IL-1β signaling in the livers of PEDF KO mice that was accompanied by impaired IRS and Akt signaling. In human hepatocytes, PEDF blocked the effects of an IL-1β challenge by suppressing activation of the inflammatory mediator c-Jun N-terminal kinase while restoring Akt signaling. RNA interference of PEDF in human hepatocytes was permissive for c-Jun N-terminal kinase activation and decreased Akt signaling. A metabolomics profile identified elevated circulating levels of tricarboxyclic acid cycle intermediates including succinate, an inducer of IL-1β, in PEDF KO mice. Succinate-dependent IL-1β expression was blocked by PEDF in PEDF KO, but not wild-type hepatocytes. In vivo, PEDF restoration reduced hyperglycemia and improved hepatic insulin signaling in PEDF KO mice. These findings identify elevated PEDF as a homeostatic mechanism in the human metabolic syndrome.

Muscle-specific activation of Ca 2+ /calmodulin-dependent protein kinase IV increases whole-body insulin action in mice

Aims/hypothesisAerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca2+/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo.MethodsThe effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4●) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic–euglycaemic clamp and isotopic measurements of glucose metabolism.ResultsThe rate of insulin-stimulated whole-body glucose uptake was increased by ∼25% in CAMK4● mice. This was largely attributed to an increase of ∼60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4● mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4● skeletal muscle.Conclusions/interpretationActivation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.

Targeting steroid receptor coactivator 1 with antisense oligonucleotides increases insulin-stimulated skeletal muscle glucose uptake in chow-fed and high-fat-fed male rats.

The steroid receptor coactivator 1 (SRC1) regulates key metabolic pathways, including glucose homeostasis. SRC1(-/-) mice have decreased hepatic expression of gluconeogenic enzymes and a reduction in the rate of endogenous glucose production (EGP). We sought to determine whether decreasing hepatic and adipose SRC1 expression in normal adult rats would alter glucose homeostasis and insulin action. Regular chow-fed and high-fat-fed male Sprage-Dawley rats were treated with an antisense oligonucleotide (ASO) against SRC1 or a control ASO for 4 wk, followed by metabolic assessments. SRC1 ASO did not alter basal EGP or expression of gluconeogenic enzymes. Instead, SRC1 ASO increased insulin-stimulated whole body glucose disposal by ~30%, which was attributable largely to an increase in insulin-stimulated muscle glucose uptake. This was associated with an approximately sevenfold increase in adipose expression of lipocalin-type prostaglandin D2 synthase, a previously reported regulator of insulin sensitivity, and an approximately 70% increase in plasma PGD2 concentration. Muscle insulin signaling, AMPK activation, and tissue perfusion were unchanged. Although GLUT4 content was unchanged, SRC1 ASO increased the cleavage of tether-containing UBX domain for GLUT4, a regulator of GLUT4 translocation. These studies point to a novel role of adipose SRC1 as a regulator of insulin-stimulated muscle glucose uptake.