VEGFR-3 Signaling Regulates Triglyceride Retention and Absorption in the Intestine

The lymphatic system transports dietary lipids absorbed and packaged as chylomicrons by enterocytes, for delivery to the bloodstream. Once considered a passive drainage, chylomicron entry into intestinal lymphatic vessels, or lacteals, is now emerging to be an active process controlled by a dynamic and complex regulation. Vascular endothelial growth factor (VEGF)-C, a major lymphangiogenic factor, regulates lacteal maintenance and function. Little is known about the role of its cognate tyrosine kinase VEGF receptor 3 (VEGFR-3) during lipid absorption. Here we investigated role of VEGFR-3 signaling in triglyceride (TG) absorption and distribution into tissues using the Chy mouse model, which bears an inactivating mutation in the tyrosine kinase domain of VEGFR-3 (heterozygous A3157T mutation resulting in I1053F substitution). Our data show that inactivation of VEGFR-3 tyrosine kinase motif leads to retention of TGs in the enterocytes of the small intestine, decreased postprandial levels of TGs in the plasma and increased excretion of free fatty acids (FFAs) and TGs into their stools. We further show that levels of nitric oxide (NO), required for chylomicron mobilization into the bloodstream, are significantly reduced in the Chy intestine after a fat bolus suggesting a critical role for VEGFR-3 signaling in the generation of NO during lipid absorption. Our data support the hypothesis that VEGFR-3 signaling plays an important role in chylomicron-TG entry into lacteals, possibly affecting TG trafficking to peripheral tissues

Visceral obesity and insulin resistance associate with CD36 deletion in lymphatic endothelial cells

Disruption of lymphatic lipid transport is linked to obesity and type 2 diabetes (T2D), but regulation of lymphatic vessel function and its link to disease remain unclear. Here we show that intestinal lymphatic endothelial cells (LECs) have an increasing CD36 expression from lymphatic capillaries (lacteals) to collecting vessels, and that LEC CD36 regulates lymphatic integrity and optimizes lipid transport. Inducible deletion of CD36 in LECs in adult mice (Cd36(ΔLEC)) increases discontinuity of LEC VE-cadherin junctions in lacteals and collecting vessels. Cd36(ΔLEC) mice display slower transport of absorbed lipid, more permeable mesenteric lymphatics, accumulation of inflamed visceral fat and impaired glucose disposal. CD36 silencing in cultured LECs suppresses cell respiration, reduces VEGF-C-mediated VEGFR2/AKT phosphorylation and destabilizes VE-cadherin junctions. Thus, LEC CD36 optimizes lymphatic junctions and integrity of lymphatic lipid transport, and its loss in mice causes lymph leakage, visceral adiposity and glucose intolerance, phenotypes that increase risk of T2D

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. METHODS: Circulating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. RESULTS: In patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2-/-) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. CONCLUSIONS: These data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations

Multiple antisense oligonucleotides targeted against monoacylglycerol acyltransferase 1 (Mogat1) improve glucose metabolism independently of Mogat1

OBJECTIVE: Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high-fat diet (HFD)-fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway. METHODS: Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on an HFD, and glucose metabolism and insulin sensitivity were assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody. RESULTS: Genetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on an HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat-fed mice but did not cause insulin resistance. Multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off-target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs, but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis. CONCLUSION: These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling

CD36 Deficiency Impairs the Small Intestinal Barrier and Induces Subclinical Inflammation in MiceSummary

Background & Aims: CD36 has immunometabolic actions and is abundant in the small intestine on epithelial, endothelial, and immune cells. We examined the role of CD36 in gut homeostasis by using mice null for CD36 (CD36KO) and with CD36 deletion specific to enterocytes (Ent-CD36KO) or endothelial cells (EC-CD36KO). Methods: Intestinal morphology was evaluated by using immunohistochemistry and electron microscopy. Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors, and cyclooxygenase-2. Barrier integrity was assessed from circulating lipopolysaccharide and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized by using two-photon microscopy. Results: The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix accumulation with increased expression of extracellular matrix proteins, evidence of neutrophil infiltration, inflammation, and compromised barrier function. Electron microscopy showed shortened desmosomes with decreased desmocollin 2 expression. Systemically, leukocytosis and neutrophilia were present together with 80% reduction of anti-inflammatory Ly6Clow monocytes. Bone marrow transplants supported the primary contribution of non-hematopoietic cells to the inflammatory phenotype. Specific deletion of endothelial but not of enterocyte CD36 reproduced many of the gut phenotypes of germline CD36KO mice including fibronectin deposition, increased interleukin 6, neutrophil infiltration, desmosome shortening, and impaired epithelial barrier function. Conclusions: CD36 loss results in chronic neutrophil infiltration of the gut, impairs barrier integrity, and systemically causes subclinical inflammation. Endothelial cell CD36 deletion reproduces the major intestinal phenotypes. The findings suggest an important role of the endothelium in etiology of gut inflammation and loss of epithelial barrier integrity. Keywords: Neutrophils, Endothelium, Fibronectin, Collage

Diacylglycerol Enrichment of Endoplasmic Reticulum or Lipid Droplets Recruits Perilipin 3/TIP47 during Lipid Storage and Mobilization*

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF4− during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF4− activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF4− perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF4− redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell