Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.

BACKGROUND AND AIMS: A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial. METHODS: 19 healthy adults (age:28.4 ±â€¯1.7 years; BMI:22.7 ±â€¯0.3 kg/m2) received an overnight intravenous infusion of lipid (20% Intralipid) or saline followed by a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity using a randomized crossover design. Skeletal muscle biopsies were obtained after the overnight lipid infusion to evaluate activation of mitochondrial dynamics proteins, ex-vivo mitochondrial membrane potential, ex-vivo oxidative phosphorylation and electron transfer capacity, and mitochondrial ultrastructure. RESULTS: Overnight lipid infusion increased dynamin related protein 1 (DRP1) phosphorylation at serine 616 and PTEN-induced kinase 1 (PINK1) expression (P = 0.003 and P = 0.008, respectively) in skeletal muscle while reducing mitochondrial membrane potential (P = 0.042). The lipid infusion also increased mitochondrial-associated lipid droplet formation (P = 0.011), the number of dilated cristae, and the presence of autophagic vesicles without altering mitochondrial number or respiratory capacity. Additionally, lipid infusion suppressed peripheral glucose disposal (P = 0.004) and hepatic insulin sensitivity (P = 0.014). CONCLUSIONS: These findings indicate that activation of mitochondrial fission and quality control occur early in the onset of insulin resistance in human skeletal muscle. Targeting mitochondrial dynamics and quality control represents a promising new pharmacological approach for treating insulin resistance and type 2 diabetes. CLINICAL TRIAL REGISTRATION: NCT02697201, ClinicalTrials.gov.

Roux-en-Y gastric bypass restores islet function and morphology independent of body weight in ZDF rats.

Reductions in ß-cell number and function contribute to the onset type 2 diabetes (T2D). Roux-en-Y gastric bypass (RYGB) surgery can resolve T2D within days of operation, indicating a weight-independent mechanism of glycemic control. We hypothesized that RYGB normalizes glucose homeostasis by restoring ß-cell structure and function. Male Zucker Diabetic Fatty (fa/fa; ZDF) rats were randomized to sham surgery (n = 16), RYGB surgery (n = 16), or pair feeding (n = 16). Age-matched lean (fa/+) rats (n = 8) were included as a secondary control. Postprandial metabolism was assessed by oral glucose tolerance testing before and 27 days after surgery. Fasting and postprandial plasma GLP-1 was determined by mixed meal tolerance testing. Fasting plasma glucagon was also measured. ß-cell function was determined in isolated islets by a glucose-stimulated insulin secretion assay. Insulin and glucagon positive areas were evaluated in pancreatic sections by immunohistochemistry. RYGB reduced body weight (P < 0.05) and improved glucose tolerance (P < 0.05) compared with sham surgery. RYGB reduced fasting glucose compared with both sham (P < 0.01) and pair-fed controls (P < 0.01). Postprandial GLP-1 (P < 0.05) was elevated after RYGB compared with sham surgery. RYGB islets stimulated with 20 mM glucose had higher insulin secretion than both sham and pair-fed controls (P < 0.01) and did not differ from lean controls. Insulin content was greater after RYGB compared with the sham (P < 0.05) and pair-fed (P < 0.05) controls. RYGB improves insulin secretion and pancreatic islet function, which may contribute to the remission of type 2 diabetes following bariatric surgery.NEW & NOTEWORTHY The onset and progression of type 2 diabetes (T2D) results from failure to secrete sufficient amounts of insulin to overcome peripheral insulin resistance. Here, we demonstrate that Roux-en-Y gastric bypass (RYGB) restores islet function and morphology compared to sham and pair-fed controls in ZDF rats. The improvements in islet function were largely attributable to enhanced insulin content and secretory function in response to glucose stimulation.

Metabolic effects of duodenojejunal bypass surgery in a rat model of type 1 diabetes.

BACKGROUND: Metabolic surgery has beneficial metabolic effects, including remission of type 2 diabetes. We hypothesized that duodenojejunal bypass (DJB) surgery can protect against development of type 1 diabetes (T1D) by enhancing regulation of cellular and molecular pathways that control glucose homeostasis. METHODS: BBDP/Wor rats, which are prone to develop spontaneous autoimmune T1D, underwent loop DJB (n = 15) or sham (n = 15) surgery at a median age of 41 days, before development of diabetes. At T1D diagnosis, a subcutaneous insulin pellet was implanted, oral glucose tolerance test was performed 21 days later, and tissues were collected 25 days after onset of T1D. Pancreas and liver tissues were assessed by histology and RT-qPCR. Fecal microbiota composition was analyzed by 16S V4 sequencing. RESULTS: Postoperatively, DJB rats weighed less than sham rats (287.8 vs 329.9 g, P = 0.04). In both groups, 14 of 15 rats developed T1D, at similar age of onset (87 days in DJB vs 81 days in sham, P = 0.17). There was no difference in oral glucose tolerance, fasting and stimulated plasma insulin and c-peptide levels, and immunohistochemical analysis of insulin-positive cells in the pancreas. DJB rats needed 1.3 ± 0.4 insulin implants vs 1.9 ± 0.5 in sham rats (P = 0.002). Fasting and glucose stimulated glucagon-like peptide 1 (GLP-1) secretion was elevated after DJB surgery. DJB rats had reduced markers of metabolic stress in liver. After DJB, the fecal microbiome changed significantly, including increases in Akkermansia and Ruminococcus, while the changes were minimal in sham rats. CONCLUSION: DJB does not protect against autoimmune T1D in BBDP/Wor rats, but reduces the need for exogenous insulin and facilitates other metabolic benefits including weight loss, increased GLP-1 secretion, reduced hepatic stress, and altered gut microbiome.

Effects of gastric bypass surgery on expression of glucose transporters and fibrotic biomarkers in kidney of diabetic fatty rats.

BACKGROUND: Diabetic nephropathy is the leading cause of chronic kidney disease. Observational studies suggest Roux-en-Y gastric bypass (RYGB) reduces progression of diabetic nephropathy. OBJECTIVES: To unravel the mechanisms by which RYGB is beneficial and protective for diabetic nephropathy. SETTING: Academic laboratories. METHODS: Forty-eight Zucker diabetic fatty rats were randomized to RYGB, sham surgery (SHAM), or pair-fed (PF) groups. An oral glucose tolerance test was performed at 25 days post intervention and kidneys were harvested at 30 days. Primary outcome measures included expression of key genes and proteins in the glucose transport, oxidative stress, inflammation, and fibrosis pathways. RESULTS: Thirty days post intervention, RYGB rats weighed 349 ± 8 g, which was lower than SHAM (436 ± 14 g, P < .001), but not PF (374 ± 18 g) rats. RYGB rats had lower fasting glucose than PF animals and improved homeostatic model assessment of insulin resistance compared with PF and SHAM groups. These enhanced metabolic outcomes were accompanied by reduced sodium-glucose co-transporter 1 (Sglt1) gene expression (-23% versus PF, P = .01) in the kidney of RYGB rats. Expression of Sglt2, Glut1, or Glut2 mRNA, or oxidative stress and inflammation markers did not differ significantly. However, RYGB surgery induced a 19% lower expression of transforming growth factor (Tgfß) mRNA (P = .004) compared with SHAM treated animals. Notably, adenosine monophosphate-activated protein kinase phosphorylation was increased (P = .04) in kidneys of the RYGB surgery animals. CONCLUSIONS: Improvement of hyperglycemia after RYGB may reduce the glucose load on the kidney leading to a downregulation of specific glucose transporters. RYGB surgery may also attenuate kidney fibrosis through the adenosine monophosphate-activated protein kinase/TGFß pathway.

2-Octylcyanoacrylate for the prevention of anastomotic leak.

BACKGROUND: Anastomotic leak after colorectal surgery is a significant cause of morbidity and mortality. The aim of this study was to evaluate the impact of a reinforced colo-colonic anastomosis with tissue adhesive, 2-octylcyanoacrylate (2-OCA), on the integrity of anastomotic healing as measured by anastomotic bursting pressure. METHODS: Sixty-eight female Sprague-Dawley rats underwent a rectosigmoid colon transection and a sutured end-to-end anastomosis followed by randomization to receive no further intervention or reinforcement with the tissue adhesive, 2-OCA. After seven postoperative days, a macroscopic assessment of the anastomosis, mechanical assessment to determine anastomotic bursting pressure, and a detailed semi-quantitative histopathologic healing assessment were performed. RESULTS: Thirty-four animals were randomized to each group. Study characteristics did not differ between the groups. There was also no difference in the degree of adhesions present postoperatively. Although there was no difference between the net proximal and distal luminal areas in the two groups (0.37 cm2versus 0.55 cm2, P = 0.26), the 2-OCA group exhibited evidence of stricture in 15% of anastomoses as compared with 3% in the suture-only group (P < 0.0001). Histologically, the presence of only fibroblasts density was statistically more evident in the 2-OCA group compared with the sutured-only anastomosis (P = 0.0183). There was not a significant increase in mechanical strength in the 2-OCA group (238.9 mm Hg) versus in the suture-only group (231.8 mm Hg). There was no difference in the rate of anastomotic leak in the 2-OCA as compared with the suture-only group (9.1 versus 8.8%). CONCLUSIONS: Application of 2-OCA to reinforce a colo-colonic anastomosis clinically provides no benefit to its mechanical strength and detrimentally increases the rate of obstruction and/or stricture in this in vivo model.

Effect of Roux-en-Y gastric bypass on liver mitochondrial dynamics in a rat model of obesity.

Bariatric surgery provides significant and durable improvements in glycemic control and hepatic steatosis, but the underlying mechanisms that drive improvements in these metabolic parameters remain to be fully elucidated. Recently, alterations in mitochondrial morphology have shown a direct link to nutrient adaptations in obesity. Here, we evaluate the effects of Roux-en-Y gastric bypass (RYGB) surgery on markers of liver mitochondrial dynamics in a diet-induced obesity Sprague-Dawley (SD) rat model. Livers were harvested from adult male SD rats 90-days after either Sham or RYGB surgery and continuous high-fat feeding. We assessed expression of mitochondrial proteins involved in fusion, fission, mitochondrial autophagy (mitophagy) and biogenesis, as well as differences in citrate synthase activity and markers of oxidative stress. Gene expression for mitochondrial fusion genes, mitofusin 1 (Mfn1; P < 0.05), mitofusin 2 (Mfn2; P < 0.01), and optic atrophy 1 (OPA1; P < 0.05) increased following RYGB surgery. Biogenesis regulators, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; P < 0.01) and nuclear respiratory factor 1 (Nrf1; P < 0.05), also increased in the RYGB group, as well as mitophagy marker, BCL-2 interacting protein 3 (Bnip3; P < 0.01). Protein expression for Mfn1 (P < 0.001), PGC1α (P < 0.05), BNIP3 (P < 0.0001), and mitochondrial complexes I-V (P < 0.01) was also increased by RYGB, and Mfn1 expression negatively correlated with body weight, insulin resistance, and fasting plasma insulin. In the RYGB group, citrate synthase activity was increased (P < 0.02) and reactive oxygen species (ROS) was decreased compared to the Sham control group (P < 0.05), although total antioxidant capacity was unchanged between groups. These data are the first to show an association between RYGB surgery and improved markers of liver mitochondrial dynamics. These observed improvements may be related to weight loss and reduced energetic demand on the liver, which could facilitate normalization of glucose homeostasis and protect against hepatic steatosis.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Pancreatic Islets from Zucker Diabetic Fatty Rats.

PURPOSE: Diabetes and obesity are associated with inflammasome-mediated low-grade, chronic inflammation that may induce pancreatic beta-cell dysfunction and apoptosis. We examined the effects of Roux-en-Y gastric bypass (RYGB) surgery on NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome-related genes from pancreatic islets of Zucker diabetic fatty rats. MATERIALS AND METHODS: Islets were collected from Zucker diabetic fatty sham control and RYGB, 30 days after surgery. We assessed expression of genes that regulate glucose metabolism and the NLRP3 inflammasome (NLRP3, caspase-1, IL-1ß, IL-18, apoptosis-associated speck-like protein), IL-6, and monocyte chemoattractant protein-1. RESULTS: Gene expression for NLRP3 (p < 0.02), IL-1ß (p < 0.04), and IL-6 (p < 0.01) was reduced by RYGB and positively correlated with change in body weight. IL-1ß positively correlated with glucose AUC response. CONCLUSION: Suppression of the NLRP3 inflammasome in pancreatic islets may contribute to improved glycemic control after RYGB.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats.

OBJECTIVE: Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control. METHODS: Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase-1, apoptosis-associated speck-like protein (ASC), IL-1ß, IL-18, IL-6 and MCP-1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT). RESULTS: Compared to Sham surgery controls, RYGB surgery decreased IL-6, MCP-1, NLRP3, IL-18, caspase-1 and ASC in omental fat, and decreased IL-6, MCP1, IL-1ß, IL-18, caspase-1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL-6 and IL-1ß, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL-18, caspase-1 and IL-1ß protein expression in omental tissue. We found a positive correlation between caspase-1, ASC, MCP-1, IL-18 and IL-6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase-1 gene expression in subcutaneous fat. CONCLUSION: This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.

Differential regulation of inflammation and apoptosis in Fas-resistant hepatocyte-specific Bid-deficient mice.

BACKGROUND & AIMS: Activation of Fas death receptor results in apoptosis in multiple organs, particularly liver, in a process dependent on Bid cleavage. Mice injected with an anti-Fas antibody die within hours of acute liver failure associated with massive apoptosis and hemorrhage. Our aim was to investigate the crosstalk of apoptotic and inflammatory pathways and the contribution of selective hepatocellular apoptosis during in vivo Fas activation. METHODS: We generated hepatocyte-specific Bid deficient mice (hBid(-/-)). Acute liver injury was induced by Fas-activating antibody (Jo2) in a time-course study. RESULTS: In contrast to controls, nearly all Jo2 injected hBid(-/-) survived. Their livers showed complete protection against hepatocellular apoptosis with minimal focal hemorrhagic changes and mainly non-parenchymal cell apoptosis. In agreement, the hepatocytes had no mitochondrial cytochrome c release in cytosol, or caspase 3 activation. hBid(-/-) livers showed marked increase in acute inflammatory foci composed of neutrophils and monocytes associated with the increased expression of proinflammatory chemokines and cytokines, in the manner dependent on non-canonical interleukin-1ß activation and amplified in the absence of caspase-3 activation. In addition, hBid(-/-) mice were completely protected from hepatotoxicity and the infiltrated cells were cleared 2 weeks post single Jo2 injection. CONCLUSIONS: Hepatocyte Bid suppression is critical for the resistance to the lethal effects of Fas activation in vivo. Fas signaling induces differential activation of non-canonical interleukin-1ß maturation, amplified in the absence of apoptotic Bid-mitochondrial loop, in hepatocytes. These findings may have important pathophysiological and therapeutic implications in a variety of liver disorders associated with Fas activation.

Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells.

Angiogenesis is a key pathological feature of experimental and human steatohepatitis, a common chronic liver disease that is associated with obesity. We demonstrated that hepatocytes generated a type of membrane-bound vesicle, microparticles, in response to conditions that mimicked the lipid accumulation that occurs in the liver in some forms of steatohepatitis and that these microparticles promoted angiogenesis. When applied to an endothelial cell line, medium conditioned by murine hepatocytes or a human hepatocyte cell line exposed to saturated free fatty acids induced migration and tube formation, two processes required for angiogenesis. Medium from hepatocytes in which caspase 3 was inhibited or medium in which the microparticles were removed by ultracentrifugation lacked proangiogenic activity. Isolated hepatocyte-derived microparticles induced migration and tube formation of an endothelial cell line in vitro and angiogenesis in mice, processes that depended on internalization of microparticles. Microparticle internalization required the interaction of the ectoenzyme Vanin-1 (VNN1), an abundant surface protein on the microparticles, with lipid raft domains of endothelial cells. Large quantities of hepatocyte-derived microparticles were detected in the blood of mice with diet-induced steatohepatitis, and microparticle quantity correlated with disease severity. Genetic ablation of caspase 3 or RNA interference directed against VNN1 protected mice from steatohepatitis-induced pathological angiogenesis in the liver and resulted in a loss of the proangiogenic effects of microparticles. Our data identify hepatocyte-derived microparticles as critical signals that contribute to angiogenesis and liver damage in steatohepatitis and suggest a therapeutic target for this condition.

Lower dipeptidyl peptidase-4 following exercise training plus weight loss is related to increased insulin sensitivity in adults with metabolic syndrome.

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9±1.2 years, BMI: 34.2±1.1kg/m(2)) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60min/day for 5 days/week at ∼85% HRmax). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40mU/m(2)/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P<0.05). Exercise improved clamp-derived insulin sensitivity by 75% (P<0.001) and decreased HOMA-IR by 15% (P<0.05). Training decreased plasma DPP-4 by 10% (421.8±30.1 vs. 378.3±32.5ng/ml; P<0.04), and the decrease in DPP-4 was associated with clamp-derived insulin sensitivity (r=-0.59; P<0.04), HOMA-IR (r=0.59; P<0.04) and fat oxidation (r=-0.54; P<0.05). Increased fat oxidation also correlated with lower 2-h glucose levels (r=-0.64; P<0.02). Exercise training reduces plasma DPP-4, which may be linked to elevated insulin sensitivity and fat oxidation. Maintaining low plasma DPP-4 concentrations is a potential mechanism whereby exercise plus weight loss prevents/delays the onset of type 2 diabetes in adults with metabolic syndrome.

IL-4 and IL-13 employ discrete signaling pathways for target gene expression in alternatively activated monocytes/macrophages.

Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In the presence of the Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophages. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak/Stat-mediated signaling pathways that ultimately control expression of several inflammatory genes. Our data demonstrate that although the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that although IL-13 utilizes both IL-4Rα/Jak2/Stat3 and IL-13Rα1/Tyk2/Stat1/Stat6 signaling pathways, IL-4 can use only the IL-4Rα/Jak1/Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes, including 15-lipoxygenase, monoamine oxidase A (MAO-A), and the scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes such as dual-specificity phosphatase 1 and tissue inhibitor of metalloprotease-3 and do so through different Jaks. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species generation is influenced by different Jaks. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of various inflammatory diseases.

Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.

Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.

Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL.

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.

Reduction in ABCG1 in Type 2 diabetic mice increases macrophage foam cell formation.

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mM glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KK(ay) mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.