👤 Paul Lewandowski

The incretin peptides glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptors coordinate β cell secretion that is proportional to nutrient intake. This effect permits consistent and restricted glucose excursions across a range of carbohydrate intake. The canonical signaling downstream of ligand-activated incretin receptors involves coupling to Gαs protein and generation of intracellular cAMP. However, recent reports have highlighted the importance of additional signaling nodes engaged by incretin receptors, including other G proteins and β-arrestin proteins. Here, the importance of Gαs signaling was tested in mice with conditional, postdevelopmental β cell deletion of Gnas (encoding Gαs) under physiological and pharmacological conditions. Deletion of Gαs/cAMP signaling induced immediate and profound hyperglycemia that responded minimally to incretin receptor agonists, a sulfonylurea, or bethanechol. While islet area and insulin content were not affected in Gnasβcell-/-, perifusion of isolated islets demonstrated impaired responses to glucose, incretins, acetylcholine, and IBMX In the absence of Gαs, incretin-stimulated insulin secretion was impaired but not absent, with some contribution from Gαq signaling. Collectively, these findings validate a central role for cAMP in mediating incretin signaling, but also demonstrate broad impairment of insulin secretion in the absence of Gαs that causes both fasting hyperglycemia and glucose intolerance. Show less

Movement of circulating lipids into tissues and arteries requires transfer across the endothelial cell (EC) barrier. This process allows the heart to obtain fatty acids, its chief source of energy, and apoB-containing lipoproteins to cross the arterial endothelial barrier, leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial TRLs (triglyceride-rich lipoproteins) as an independent risk factor for cardiovascular disease. We explored how chylomicrons affect ECs and transfer their fatty acids across the EC barrier. We had reported that media from chylomicron-treated ECs lead to lipid droplet formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid, and microRNA content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of SR-BI (scavenger receptor-BI) altered these responses. In addition, using chylomicrons labeled with [ Chylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage lipid droplet accumulation. The EVs contained little free fatty acids and triglycerides, but abundant phospholipids and diacylglycerols. In concert with this, [ EC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. Fatty acids within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition. Show less

Movement of circulating lipids into tissues and arteries requires transfer across the endothelial cell barrier. This process allows the heart to obtain fatty acids (FAs), its chief source of energy and apolipoprotein B (apoB)-containing lipoproteins to cross the arterial endothelial barrier leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial triglyceride-rich lipoproteins (TRLs) as an independent risk factor for cardiovascular disease (CVD). We explored how chylomicrons affect ECs and transfer their FAs across the EC barrier. We had reported that media from chylomicron-treated ECs leads to lipid droplet (LD) formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid and microRNA (miR) content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of scavenger receptor-BI (SR-BI) altered these responses. In addition, using chylomicrons labeled with [ Chylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage LD accumulation. The EVs contained little free fatty acids and triglyceride, but abundant phospholipids and diacylglycerols. In concert with this, [ EC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. FAs within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition. Show less

The effects of supplementing diets with n-3 alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) on plasma metabolites, carcass yield, muscle n-3 fatty acids and liver messenger RNA (mRNA) in lambs were investigated. Lambs (n = 120) were stratified to 12 groups based on body weight (35 ± 3.1 kg), and within groups randomly allocated to four dietary treatments: basal diet (BAS), BAS with 10.7 % flaxseed supplement (Flax), BAS with 1.8 % algae supplement (DHA), BAS with Flax and DHA (FlaxDHA). Lambs were fed for 56 days. Blood samples were collected on day 0 and day 56, and plasma analysed for insulin and lipids. Lambs were slaughtered, and carcass traits measured. At 30 min and 24 h, liver and muscle samples, respectively, were collected for determination of mRNA (FADS1, FADS2, CPT1A, ACOX1) and fatty acid composition. Lambs fed Flax had higher plasma triacylglycerol, body weight, body fat and carcass yield compared with the BAS group (P < 0.001). DHA supplementation increased carcass yield and muscle DHA while lowering plasma insulin compared with the BAS diet (P < 0.01). Flax treatment increased (P < 0.001) muscle ALA concentration, while DHA treatment increased (P < 0.001) muscle DHA concentration. Liver mRNA FADS2 was higher and CPT1A lower in the DHA group (P < 0.05). The FlaxDHA diet had additive effects, including higher FADS1 and ACOX1 mRNA than for the Flax or DHA diet. In summary, supplementation with ALA or DHA modulated plasma metabolites, muscle DHA, body fat and liver gene expression differently. Show less

The aim of this study was to determine the effects of high-glucose, high-fructose and high-sucrose diets on weight gain, liver lipid metabolism and gene expression of proteins involved with hepatic fat metabolism. Rats were fed a diet containing either 60% glucose, 60% fructose, 60% sucrose, or a standard chow for 28 days. Results indicated that high-fructose and high-sucrose diets were associated with higher mRNA levels of gene transcripts involved with fat synthesis; ACC, FAS and ChREBP, with no change in SREBP-1C mRNA. The protein level of ChREBP and SREBP1c was similar in liver homogenates from all groups, but were higher in nuclear fractions from the liver of high-fructose and high-sucrose fed rats. The mRNA level of gene transcripts involved with fat oxidation was the same in all three diets, whilst a high-fructose diet was associated with greater amount of mRNA of the fat transporter CD36. Despite the changes in mRNA of lipogenic proteins, the body weight of animals from each group was the same and the livers from rats fed high-fructose and high-sucrose diets did not contain more fat than control diet livers. In conclusion, changing the composition of the principal monosaccharide in the diet to a fructose containing sugar elicits changes in the level of hepatic mRNA of lipogenic and fat transport proteins and protein levels of their transcriptional regulators; however this is not associated with any changes in body weight or liver fat content. Show less

The oxysterol receptors [liver X receptors (LXRalpha and LXRbeta)] regulate cholesterol and lipid biosynthesis and several studies link dysregulation of these metabolic pathways to aberrant cell growth. Here, we show that activation of LXR significantly reduced proliferation in several human breast cancer cells lines. LXR suppressed messenger RNA and/or protein expression of Skp2, cyclin A2, cyclin D1 and estrogen receptor (ER) alpha, whereas it increased the expression of p53 at the protein level and maintained the retinoblastoma protein in a hypophosphorylated active form. These changes may constitute part of the molecular mechanisms behind the antiproliferative effect of LXR. Furthermore, activation of LXR induced expression of key lipogenic genes including sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase and stearoyl-coenzyme A desaturase 1, leading to increased triglyceride production in MCF7 cells. Small interfering RNA knockdown of SREBP1c, a master regulator of the lipid biosynthesis, did not abolish the antiproliferative effect of LXR in these cells. Combined these studies identify LXRs as both antiproliferative and lipogenic factors in breast cancer cells and indicate that the antiproliferative effect of LXRs is independent of lipid biosynthesis. Show less