Faculty Bibliography

Diabetes is the greatest risk factor for the development of cardiovascular disease, which, in turn, is the most prevalent cause of mortality and morbidity in diabetics. These patients have elevations in inflammatory monocytes, a factor consistently reported to drive the development of atherosclerosis. In preclinical models of both type 1 and type 2 diabetes, studies have demonstrated that the increased production and activation of monocytes is driven by enhanced myelopoiesis, promoted by factors, including hyperglycemia, impaired cholesterol efflux, and inflammasome activation, that affect the proliferation of bone marrow precursor cells. This suggests that continued mechanistic investigations of the enhanced myelopoiesis and the generation of inflammatory monocytes are timely, from the dual perspectives of understanding more deeply the underlying bases of diabetes pathophysiology and identifying therapeutic targets to reduce cardiovascular risk in these patients.

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.

Lipid accumulation is a pathological feature of every type of kidney injury. Despite this striking histological feature, physiological accumulation of lipids in the kidney is poorly understood. We studied whether the accumulation of lipids in the fasted kidney are derived from lipoproteins or non-esterified fatty acids (NEFAs). With overnight fasting, kidneys accumulated triglyceride but had reduced levels of ceramide and glycosphingolipid species. Fasting led to a nearly 5-fold increase in kidney uptake of plasma [14C]oleic acid. Increasing circulating NEFAs using a beta adrenergic receptor agonist caused a 15-fold greater accumulation of lipid in the kidney, while mice with reduced NEFAs due to adipose tissue deficiency of adipose triglyceride lipase had reduced triglycerides. Cd36 mRNA increased 2-fold, and Angptl4, an LpL inhibitor, increased 10-fold. Fasting-induced kidney lipid accumulation was not affected by inhibition of LpL with poloxamer 407 or by use of mice with induced genetic LpL deletion. Despite the increase in CD36 expression with fasting, genetic loss of CD36 did not alter fatty acid uptake or triglyceride accumulation. Our data demonstrate that fasting-induced triglyceride accumulation in the kidney correlates with the plasma concentrations of NEFAs, but is not due to uptake of lipoprotein lipids and does not involve the fatty acid transporter CD36.

Abnormal lipid metabolism may contribute to myocardial injury and remodeling. To determine whether accumulation of very long-chain ceramides occurs in human failing myocardium, we analyzed myocardial tissue and serum from patients with severe heart failure (HF) undergoing placement of left ventricular assist devices and controls. Lipidomic analysis revealed increased total and very long-chain ceramides in myocardium and serum of patients with advanced HF. After unloading, these changes showed partial reversibility. Following myocardial infarction (MI), serine palmitoyl transferase (SPT), the rate-limiting enzyme of the de novo pathway of ceramide synthesis, and ceramides were found increased. Blockade of SPT by the specific inhibitor myriocin reduced ceramide accumulation in ischemic cardiomyopathy and decreased C16, C24:1, and C24 ceramides. SPT inhibition also reduced ventricular remodeling, fibrosis, and macrophage content following MI. Further, genetic deletion of the SPTLC2 gene preserved cardiac function following MI. Finally, in vitro studies revealed that changes in ceramide synthesis are linked to hypoxia and inflammation. In conclusion, cardiac ceramides accumulate in the failing myocardium, and increased levels are detectable in circulation. Inhibition of de novo ceramide synthesis reduces cardiac remodeling. Thus, increased de novo ceramide synthesis contributes to progressive pathologic cardiac remodeling and dysfunction.

The pharmacological effects of type 2 diabetes (T2DM) medications on lipoprotein metabolism are difficult to assess in preclinical models because those created failure to replicate the human condition in which insulin deficiency is superimposed on obesity-related insulin resistance. To create a better model, we fed mice with high fat (HF) diet and treated the animals with low dose streptozotocin (STZ) to mimic T2DM. We used this model to evaluate the effects of canagliflozin (CANA), a drug that reduces plasma glucose by inhibiting the sodium-glucose transporter 2 (SGLT2), which mediates ~90% of renal glucose reabsorption] on lipid and lipoprotein metabolism. After 6 weeks of CANA (30 mg/kg/day) treatment, the increase in total plasma cholesterol in HF-STZ diabetic mice was reversed, but plasma triglycerides were not affected. Lipoprotein fractionation and cholesterol distribution analysis showed that CANA kept HDL-Cholesterol, LDL-Cholesterol, and IDL-Cholesterol levels steady while these lipoprotein species were increased in placebo- and insulin-treated control groups. CANA treatment of HF-STZ mice reduced post-heparin plasma lipoprotein lipase (LPL) activity at 2 (-40%) and 5 (-30%) weeks compared to placebo. Tissue-specific LPL activity following CANA treatment showed similar reduction. In summary, CANA prevented the total cholesterol increase in HF-STZ mice without effects on plasma lipids or lipoproteins, but did decrease LPL, implying a potential role of LPL-dependent lipoprotein metabolism in CANA action. These effects did not recapitulate the effect of SGLT2 inhibitors on lipids and lipoproteins in human, suggesting that a better murine T2DM model (such as the ApoB100 humanized CETP-overexpressing mouse) is needed next.

Background A protein that is expressed on capillary endothelial cells, called GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1), binds lipoprotein lipase and shuttles it to its site of action in the capillary lumen. A deficiency in GPIHBP1 prevents lipoprotein lipase from reaching the capillary lumen. Patients with GPIHBP1 deficiency have low plasma levels of lipoprotein lipase, impaired intravascular hydrolysis of triglycerides, and severe hypertriglyceridemia (chylomicronemia). During the characterization of a monoclonal antibody-based immunoassay for GPIHBP1, we encountered two plasma samples (both from patients with chylomicronemia) that contained an interfering substance that made it impossible to measure GPIHBP1. That finding raised the possibility that those samples might contain GPIHBP1 autoantibodies. Methods Using a combination of immunoassays, Western blot analyses, and immunocytochemical studies, we tested the two plasma samples (as well as samples from other patients with chylomicronemia) for the presence of GPIHBP1 autoantibodies. We also tested the ability of GPIHBP1 autoantibodies to block the binding of lipoprotein lipase to GPIHBP1. Results We identified GPIHBP1 autoantibodies in six patients with chylomicronemia and found that these autoantibodies blocked the binding of lipoprotein lipase to GPIHBP1. As in patients with GPIHBP1 deficiency, those with GPIHBP1 autoantibodies had low plasma levels of lipoprotein lipase. Three of the six patients had systemic lupus erythematosus. One of these patients who had GPIHBP1 autoantibodies delivered a baby with plasma containing maternal GPIHBP1 autoantibodies; the infant had severe but transient chylomicronemia. Two of the patients with chylomicronemia and GPIHBP1 autoantibodies had a response to treatment with immunosuppressive agents. Conclusions In six patients with chylomicronemia, GPIHBP1 autoantibodies blocked the ability of GPIHBP1 to bind and transport lipoprotein lipase, thereby interfering with lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and causing severe hypertriglyceridemia. (Funded by the National Heart, Lung, and Blood Institute and the Leducq Foundation.).

BACKGROUND & AIMS: CD36 has immuno-metabolic actions and is abundant in the small intestine on epithelial, endothelial and immune cells. We examined the role of CD36 in gut homeostasis 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 using immunohistochemistry and electron microscopy (EM). Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors and COX-2. Barrier integrity was assessed from circulating lipopolysaccharide (LPS) and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized using two photon microscopy. RESULTS: The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix (ECM) accumulation with increased expression of ECM proteins, evidence of neutrophil infiltration, inflammation and compromised barrier function. EM 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.

The number of individuals with prediabetes is expected to grow substantially and estimated to globally affect 482 million people by 2040. Therefore, effective methods for diagnosing prediabetes will be required to reduce the risk of progressing to diabetes and its complications. The current biomarkers, glycated hemoglobin (HbA1c), fructosamine, and glycated albumin have limitations including moderate sensitivity and specificity and are inaccurate in certain clinical conditions. Therefore, identification of additional biomarkers is being explored recognizing that any single biomarker will also likely have inherent limitations. Therefore, combining several biomarkers may more precisely identify those at high risk for developing prediabetes and subsequent progression to diabetes. This review describes recently identified biomarkers and their potential utility for addressing the burgeoning epidemic of dysglycemic disorders.