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.

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 β-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.

EGF-R regulates cell proliferation, migration, and invasion in fibroblasts. However, the connection of EGF-R with downstream signaling pathways mediating these responses has remained elusive. Here we provide genetic and biochemical evidence that EGF-R- and AP-1-mediated signals are required for MMP expression and collagen contraction in fibroblasts. In EGF-R (-/-) mouse embryonal fibroblasts, basal and inducible expression of several MMPs, including MMP-2, -3, and -14 is impaired in comparison to wild-type counterparts. The loss of MMP expression is associated with a suppression of EGF-induced Erk and Jnk activities, and AP-1 DNA-binding and transactivation capacities. While inhibition of Jnk mainly prevents EGF-induced phosphorylation of c-Jun, inhibition of Erk pathway suppresses both the expression and phosphorylation of c-Jun and c-Fos proteins. Moreover, the expression of MMP-3 and -14, and collagen contraction is partially prevented by Mek/Erk and Jnk inhibitors. However, Jnk inhibitor also suppresses cell growth independently of EGF-R activity. The central role of AP-1 as a mediator of EGF-R signaling in fibroblasts is emphasized by the finding that expression of a dominant negative c-Jun downregulates the expression of MMP-3. Conversely, expression of a constitutively active Mek1 can induce MMP-3 expression independently of upstream signals. The results indicate that ERK pathway and AP-1 are downstream effectors of the EGF-R-mediated MMP-3 expression and collagen contraction in fibroblasts.

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.