Inhibition of VEGF-B signaling prevents non-alcoholic fatty liver disease development by targeting lipolysis in the white adipose tissue.

BACKGROUND & AIMS: Hepatic steatosis is a hallmark of non-alcoholic fatty liver disease (NAFLD), a common comorbidity in type 2 diabetes mellitus (T2DM). The pathogenesis of NAFLD is complex and involves the crosstalk between the liver and the white adipose tissue (WAT). Vascular endothelial growth factor B (VEGF-B) has been shown to control tissue lipid accumulation by regulating the transport properties of the vasculature. The role of VEGF-B signaling and the contribution to hepatic steatosis and NAFLD in T2DM is currently not understood. METHODS: C57BL/6 J mice treated with a neutralizing antibody against VEGF-B, or mice with adipocyte-specific overexpression or under-expression of VEGF-B (AdipoqCre+/VEGF-BTG/+ mice and AdipoqCre+/Vegfbfl/+mice) were subjected to a 6-month high-fat diet (HFD), or chow-diet, whereafter NAFLD development was assessed. VEGF-B expression was analysed in WAT biopsies from patients with obesity and NAFLD in a pre-existing clinical cohort (n = 24 patients with NAFLD and n = 24 without NAFLD) and correlated to clinicopathological features. RESULTS: Pharmacological inhibition of VEGF-B signaling in diabetic mice reduced hepatic steatosis and NAFLD by blocking WAT lipolysis. Mechanistically we show, by using HFD-fed AdipoqCre+/VEGF-BTG/+ mice and HFD-fed AdipoqCre+/Vegfbfl/+mice, that inhibition of VEGF-B signaling targets lipolysis in adipocytes. Reducing VEGF-B signaling ameliorated NAFLD by decreasing WAT inflammation, resolving WAT insulin resistance, and lowering the activity of the hormone sensitive lipase. Analyses of human WAT biopsies from individuals with NAFLD provided evidence supporting the contribution of VEGF-B signaling to NAFLD development. VEGF-B expression levels in adipocytes from two WAT depots correlated with development of dysfunctional WAT and NAFLD in humans. CONCLUSIONS: Taken together, our data from mouse models and humans suggest that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. IMPACT AND IMPLICATIONS: Non-alcoholic fatty liver disease (NAFLD) is a common comorbidity in type 2 diabetes mellitus (T2DM) and has a global prevalence of between 25-29%. There are currently no approved drugs for NAFLD, and given the scale of the ongoing diabetes epidemics, there is an urgent need to identify new treatment options. Our work suggests that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. The neutralizing anti-VEGF-B antibody, which was used in this study, has already entered clinical trials for patients with diabetes. Therefore, we believe that our results are of great general interest to a broad audience, including patients and patient organizations, the medical community, academia, the life science industry and the public.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Expression of vascular endothelial growth factor (VEGF)-B and its receptor (VEGFR1) in murine heart, lung and kidney.

Metabolic diseases, such as obesity and diabetes, are a serious burden for the health system. Vascular endothelial growth factor (VEGF)-B has been shown to regulate tissue uptake and accumulation of fatty acids and is thus involved in these metabolic diseases. However, the cell-type-specific expression pattern of Vegfb and its receptor (VEGFR1, gene Flt1) remains unclear. We explore the expression of Vegfb and Flt1 in the murine heart, lung and kidney by utilizing ß-galactosidase knock-in mouse models and combining the analysis of reporter gene expression and immunofluorescence microscopy. Furthermore, Flt1 heterozygous mice were analyzed with regard to muscular fatty acid accumulation and peripheral insulin sensitivity. Throughout the heart, Vegfb expression was found in cardiomyocytes with a postnatal ventricular shift corresponding to known changes in energy requirements. Vegfb expression was also found in the pulmonary myocardium of the lung and in renal epithelial cells of the thick ascending limb of Henle's loop, the connecting tubule and the collecting duct. In all analyzed organs, VEGFR1 expression was restricted to endothelial cells. We also show that reduced expression of VEGFR1 resulted in decreased cardiac fatty acid accumulation and increased peripheral insulin sensitivity, possibly as a result of attenuated VEGF-B/VEGFR1 signaling. Our data therefore support a tightly controlled, paracrine signaling mechanism of VEGF-B to VEGFR1. The identified cell-specific expression pattern of Vegfb and Flt1 might form the basis for the development of cell-type-targeted research models and contributes to the understanding of the physiological and pathological role of VEGF-B/VEGFR1 signaling.

Vascular endothelial growth factor B controls endothelial fatty acid uptake.

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb(-/-) mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Endothelial fatty acid transport: role of vascular endothelial growth factor B.

Dietary lipids present in the circulation have to be transported through the vascular endothelium to be utilized by tissue cells, a vital mechanism that is still poorly understood. Vascular endothelial growth factor B (VEGF-B) regulates this process by controlling the expression of endothelial fatty acid transporter proteins (FATPs). Here, we summarize research on the role of the vascular endothelium in nutrient transport, with emphasis on VEGF-B signaling.

Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms.

BACKGROUND: New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. METHODS AND RESULTS: We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-alpha interacting protein, terminus C (GIPC) and upregulation of G-protein-alpha interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium. CONCLUSIONS: VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.

Visualizing Fatty Acid Flux.

In a recent issue of Cell Metabolism, He et al. (2018) describes a novel technique to visualize cardiac intravascular lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and then follow the flux of released fatty acids across the endothelium to the underlying cardiomyocytes at high spatial resolution. This allows for detailed analyses of this clearly complex process.

Reducing VEGF-B Signaling Ameliorates Renal Lipotoxicity and Protects against Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.

PGC-1α Coordinates Mitochondrial Respiratory Capacity and Muscular Fatty Acid Uptake via Regulation of VEGF-B.

Vascular endothelial growth factor (VEGF) B belongs to the VEGF family, but in contrast to VEGF-A, VEGF-B does not regulate blood vessel growth. Instead, VEGF-B controls endothelial fatty acid (FA) uptake and was identified as a target for the treatment of type 2 diabetes. The regulatory mechanisms controlling Vegfb expression have remained unidentified. We show that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) together with estrogen-related receptor α (ERR-α) regulates expression of Vegfb Mice overexpressing PGC-1α under the muscle creatine kinase promoter (MPGC-1αTG mice) displayed increased Vegfb expression, and this was accompanied by increased muscular lipid accumulation. Ablation of Vegfb in MPGC-1αTG mice fed a high-fat diet (HFD) normalized glucose intolerance, insulin resistance, and dyslipidemia. We suggest that VEGF-B is the missing link between PGC-1α overexpression and the development of the diabetes-like phenotype in HFD-fed MPGC-1αTG mice. The findings identify Vegfb as a novel gene regulated by the PGC-1α/ERR-α signaling pathway. Furthermore, the study highlights the role of PGC-1α as a master metabolic sensor that by regulating the expression levels of Vegfa and Vegfb coordinates blood vessel growth and FA uptake with mitochondrial FA oxidation.

Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease.

Excess lipid accumulation in peripheral tissues is a key feature of many metabolic diseases. Therefore, techniques for imaging and quantifying lipids in various tissues are important for understanding and evaluating the overall metabolic status of a research subject. Here we present a protocol that detects neutral lipids and lipid droplet (LD) morphology by oil red O (ORO) staining of sections from frozen tissues. The method allows for easy estimation of tissue lipid content and distribution using only basic laboratory and computer equipment. Furthermore, the procedure described here is well suited for the comparison of different metabolically challenged animal models. As an example, we include data on muscular and hepatic lipid accumulation in diet-induced and genetically induced diabetic mice. The experimental description presents details for optimal staining of lipids using ORO, including tissue collection, sectioning, staining, imaging and measurements of tissue lipids, in a time frame of less than 2 d.

Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis.

BACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing ß-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.

Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.