Clinical significance of measuring soluble LR11, a circulating marker of atherosclerosis and HbA1c in familial hypercholesterolemia.

OBJECTIVES: The LDL receptor relative with 11 ligand-binding repeats (LR11) is closely related to atherosclerotic disease or diabetes. The aim of the study was to clarify how soluble LR11 was related to Achilles' tendon thickness (ATT) and HbA1c in familial hypercholesterolemia. DESIGN AND METHODS: The present study is a cross-sectional case-control study. We enrolled twenty-four patients with heterozygous FH (age 51.0±20.0 year; male, 50%; 20 cases with LDL receptor mutation, 1 case with proprotein convertase subtilisin/kexin type 9 (PCSK9) E32K and 3 cases without confirmed mutations). Soluble LR11 (sLR11) was measured using a sandwich enzyme-linked immunosorbent assay method. RESULTS: Univariate regression analysis showed that sLR11 had positive correlations with age and HbA1c, and inverse correlations with apoA1 in FH. There were also positive correlations of sLR11 with apoE, IDL-C and average ATT. Multivariate regression analysis showed that there were positive correlations of sLR11 to IDL-C and HbA1c independent of age and BMI. In another multivariate regression analysis on the relationships of average ATT as a dependent variable with age, BMI and sLR11 (IDL-C and HbA1c) as independent variables, sLR11 had a positive correlation with average ATT, independent of age and BMI. However, this independency did not persist after adding IDL-C and HbA1c as confounding factors. Of special note is that HbA1c showed a significant correlation with average ATT, independent of other parameters including sLR11. CONCLUSION: It is crucial to intervene in the existence of remnant lipoprotein as well as hypercholesterolemia from an early stage and conduct glycemic control to prevent the progression of atherosclerotic disease in FH.

Matrix metalloproteinase 2 improves the transplanted adipocyte survival in mice.

BACKGROUND: Fat tissue is a common material for autologous transplantation in plastic and reconstructive surgery. Basic fibroblast growth factor (bFGF) ameliorates the fat graft survival. A transplantation model has shown the gene expression of matrix metalloproteinases (MMPs) to increase in adipocytes. The aim of this study is to investigate the role of MMPs in the amelioration of survival by bFGF. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated with or without 10 microg mL(-1) bFGF for 8 h in the presence or absence of the MMP inhibitor GM6001, vascular endothelial growth factor (VEGF), MMP-2 or anti-bFGF antibody to study the effect of bFGF on MMP-2 mRNA expression, MMP-2 activity, fat accumulation or 2-deoxyglucose uptake. Collagen sheets containing l x l0(7) adipocytes with or without bFGF in the presence or absence of GM6001 were subcutaneously transplanted into mice, and the appearance, histology, mRNA expression and fat accumulation of the grafts were analysed 4 weeks after transplantation. RESULTS: The MMP-2 expression was drastically induced by bFGF among MMPs in 3T3-L1 adipocytes. MMP-2 accelerated fat accumulation, peroxisome proliferator-activated receptor gamma (PPAR gamma) mRNA expression, and glucose uptake to an extent similar to those induced by bFGF, respectively. The bFGF-induced increases were inhibited by the blocking of MMP-2. The transplantation of adipocytes into mice showed that bFGF ameliorates the appearance and fat accumulation, as well as mRNA expression in grafts. These effects were almost or partly inhibited by a MMP blockade. CONCLUSIONS: MMP-2 may be involved in the mechanism by which bFGF ameliorates the survival of fat grafts.

Macrophages regulate tumor necrosis factor-alpha expression in adipocytes through the secretion of matrix metalloproteinase-3.

OBJECTIVE: Adipocytes accumulated in the visceral area change their function to induce tumor necrosis factor-alpha (TNF-alpha) secretion with concomitant matrix metalloproteinase (MMP)-3 induction in mice. This study was performed to clarify the role of macrophages (Mphi)-secreted MMP on the functional changes in adipocytes using a culture system. DESIGN: Cultures of 3T3-L1 adipocytes with THP-1 Mphi or the Mphi-conditioned medium were used to investigate the role of Mphi-MMP on the TNF-alpha gene in 3T3-L1 adipocytes by the addition of MMP inhibitors. For animal experiments, male C57BL/6J mice were rendered insulin resistant by feeding a high-fat diet, and the expression of an Mphi marker F4/80, and MMP-3 genes in mesenteric and subcutaneous fat tissue specimens were examined. RESULTS: Mphi-conditioned media (Mphi-CM) increased the levels of TNF-alpha mRNA expression in 3T3-L1 adipocytes, and these adipocyte responses were abolished by treatment with GM6001, a broad-spectrum MMP inhibitor, or NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), an MMP-3 inhibitor. The activated form of MMP-3 enhanced glycerol release as well as TNF-alpha protein secretion from 3T3-L1 adipocytes. The incubation of adipocytes with MMP-3 inhibited insulin-induced glucose uptake in adipocytes. Furthermore, a high-fat intake increased the expression of MMP-3, decreased the insulin-induced glucose uptake of adipocytes and induced expression of F4/80 in mesenteric fat tissue of C57BL/6 mice. CONCLUSION: Mphi may cause a pathological link with surrounding adipocytes through the secretion of MMP-3 followed by TNF-alpha expression in adipocytes in visceral fat tissue.

Subcutaneous fat modulates insulin sensitivity in mice by regulating TNF-alpha expression in visceral fat.

The distribution of fat in obese persons is related to the risk of developing various metabolic disorders, such as glucose intolerance, dyslipidemia and hypertension, and the combination of these conditions is known as the metabolic syndrome. The aim of this study was to investigate the role of subcutaneous fat in regulating insulin resistance and its influence on TNF-alpha expression in visceral fat, by using mice that were subjected to subcutaneous lipectomy with or without subsequent fat transplantation. After partial subcutaneous lipectomy, mice showed significantly greater accumulation of visceral fat compared with sham-operated control mice. Lipectomy led to higher plasma insulin and lower plasma glucose levels after loading with glucose and insulin, respectively, compared with the levels in control mice. Insulin-induced phosphorylation of IRS-1 was decreased in the skeletal muscles of lipectomized mice. Subcutaneous transplantation of fat pads into lipectomized mice reversed the above-mentioned changes indicating insulin resistance in these animals. The fat storage area of adipocytes and TNF- alpha expression by adipocytes in visceral fat were significantly higher in the lipectomized mice than in controls, while subcutaneous transplantation of fat reduced both the fat storage area and TNF-alpha expression. The insulin resistance of lipectomized mice was also ameliorated by systemic neutralization of TNF-alpha activity using a specific antibody. These findings obtained in mice subjected to subcutaneous lipectomy with/without subsequent fat transplantation indicate that subcutaneous fat regulates systemic insulin sensitivity, possibly through altering fat storage and the expression of TNF-alpha by adipocytes in visceral fat. The balance between accumulation of subcutaneous fat and visceral fat may be important with respect to the occurrence of systemic insulin resistance in the metabolic syndrome.

Catalytically inactive lipoprotein lipase overexpression increases insulin sensitivity in mice.

Abnormalities in lipoprotein lipase (LPL) function contribute to the development of hypertriglyceridemia, one of the characteristic disorders observed in the metabolic syndrome. In addition to the hydrolyzing activity of triglycerides, LPL modulates various cellular functions via its binding ability to the cell surface. Here we show the effects of catalytically inactive LPL overexpression on high-fat diet (HFD)-induced decreased systemic insulin sensitivity in mice. The binding capacity of catalytically inactive G188E-LPL to C2C12 skeletal muscle cells was not significantly different from that of wild type LPL. Insulin-stimulated IRS-1 phosphorylation and glucose uptake were increased by addition of wild type or mutant LPL in C2C12 cells. After 10 weeks' of HFD feeding, mice had significantly higher blood glucose levels than chow-fed mice in insulin tolerance tests. The blood glucose levels after insulin injection was significantly decreased in mutated LPL-overexpressing mice (G188E mice), as well as in wild type LPL-overexpressing mice (WT mice). Overexpression of catalytically inactive LPL, as well as wild type LPL, improved impaired insulin sensitivity in mice. These results show that decreased expression of LPL possibly causes the insulin resistance, in addition to hypertriglyceridemia, in metabolic syndrome.

Implantation of primary cultured adipocytes that secrete insulin modifies blood glucose levels in diabetic mice.

AIM/HYPOTHESIS: In type 1 diabetic patients, basal insulin supplementation plays a central role in tight glycaemic control. Therefore, safe and steady supplementation of basal insulin is strongly desirable, despite the need for multiple injections. The aim of this study was to investigate a procedure for supplementation using genetically engineered, primary-cultured adipocytes in diabetic mice. METHODS: Furin-cleavable human proinsulin cDNA was transferred into murine primary-cultured adipocytes using a retroviral vector. The cells were implanted subcutaneously into streptozotocin-induced diabetic mice. RESULTS: The transfected cells secreted substantial amounts of mature insulin, as well as C-peptide, into conditioned medium. Syngeneic implantation of the cells significantly improved hyperglycaemia and blood HbA(1)c concentrations in a manner that was dependent on cell number, without causing hypoglycaemia. The plasma insulin concentration was dependent on the implanted cell number, and the systemic effect of the circulating insulin was confirmed by marked improvement of body weight reduction and liver glycogen content. Additionally, surgical resection of the implants, in which the insulin secretion was immunologically confirmed after transplantation, diminished the glucose-lowering effect, suggesting that in vivo expression could be eliminated if necessary. CONCLUSIONS/INTERPRETATION: These results indicate that the autotransplantation of functionalised adipocytes may lead to a clinical application in the treatment of diabetes.

Impaired glucose tolerance is accompanied by decreased insulin sensitivity in tissues of mice implanted with cells that overexpress resistin.

AIM/HYPOTHESIS: Resistin, the expression of which is suppressed by thiazolidinedione treatment in adipocytes, is one of the key molecules for the tight link between adiposity and insulin resistance. Here, we show the in vivo effects of resistin on insulin sensitivity in mature mice using a cell implantation method. METHODS: Resistin cDNA was transfected into 3T3-L1 pre-adipocytes, which were then implanted into subcutaneous areas of nude mice. Metabolic analyses were performed 4 or 6 weeks after implantation. RESULTS: The mice implanted with 3T3-L1 cells overexpressing resistin (R-mice) showed significantly (p<0.05) increased plasma resistin levels. After a glucose load plasma insulin levels were significantly greater in R-mice than in mice implanted with mock-transfected cells (M-mice). The AUC of insulin after glucose loading was positively correlated with circulating resistin levels. Significantly decreased glucose responses after insulin injection were observed in R-mice, compared to M-mice. The insulin-induced phosphorylation level of IRS-1 was significantly lower in muscles of R-mice than M-mice. The expression of TNF-alpha mRNA in intra-peritoneal fat tissues was significantly greater in R-mice than in M-mice, but there was no difference between the two groups with regard to subcutaneous fat tissues. The concentration of TNF-alpha in plasma was positively correlated with resistin levels in R-mice. CONCLUSIONS/INTERPRETATION: Resistin, when actually secreted from cells in mature mice, causes disturbed glucose metabolism, possibly based on decreased insulin sensitivity in muscle. The in vivo effects of resistin on insulin sensitivity might be in part mediated by increased TNF-alpha expression in visceral fat tissues.

Elevated serum vascular endothelial growth factor is associated with visceral fat accumulation in human obese subjects.

AIMS/HYPOTHESIS: Adipose tissue expresses some bioactive molecules, which may be involved in the development of obesity-associated metabolic disorders and cardiovascular diseases. Vascular endothelial growth factor (VEGF) an important angiogenic factor is implicated in normal and pathological vessel formation. The aim of this study is to investigate clinically the association between blood serum VEGF concentrations and body fat accumulation as well as distribution. The study also aims to show the effect of serum VEGF protein on gene expression of transcriptional factor E26 transformation-specific-1 (Ets-1) and matrix metalloproteinase (MMP)-3. METHODS: Serum VEGF concentrations were measured in 38 overweight or obese subjects. Fat distribution in the abdominal subcutaneous as well as visceral fat areas was assessed by computed tomography scans at umbilical level. Furthermore, the changes of serum VEGF concentrations following body weight reduction therapy were analyzed in eight subjects recruited from the original pool of subjects. Semi-purified circulating VEGF proteins were obtained by heparin-sepharose and its biological activities were shown to alter gene expressions in human aortic endothelial cells. RESULTS: Serum VEGF concentrations were positively correlated with BMI (r=0.433, p=0.007) and visceral fat area (r=0.488, p=0.002). Stepwise regression analysis showed the visceral fat area as the most important determinant factor for VEGF circulating levels. Following body weight reduction therapy, VEGF concentrations as well as visceral fat area were decreased. The serum semi-purified VEGF protein enhanced expressions of Ets-1 and MMP-3 in human aortic endothelial cells. CONCLUSION/INTERPRETATION: Increased serum VEGF concentrations associated with visceral fat accumulation could influence vascular endothelial function.

A PPAR agonist improves TNF-alpha-induced insulin resistance of adipose tissue in mice.

Thiazolidinediones (TZDs), agonists for PPARs, have been shown to block the inhibitory effects of TNF-alpha on insulin action using cultured cells. In order to clarify the in vivo effects of TZDs on the inhibition of insulin sensitivity by TNF-alpha, insulin action in muscles and adipose tissues was assessed in the TNF-alpha-overexpression mice model using transplantation of cells secreting the TNF-alpha protein. After the pioglitazone treatment for 4 weeks, glucose uptake, insulin-induced IRS-1 phosphorylation, and lipoprotein lipase mRNA levels were analyzed. Pioglitazone did not ameliorate TNF-alpha-induced hyperinsulinemia in this model, as assessed by the OGTT. Glucose uptake and lipoprotein lipase mRNA levels were decreased by TNF-alpha in adipose tissues from the TNF-alpha-overexpressing mice, and pioglitazone blocked these inhibitions by TNF-alpha. On the other hand, in muscles, pioglitazone did not reverse the effects of TNF-alpha on insulin-induced phosphorylation of IRS-1, glucose uptake, and lipoprotein lipase mRNA levels. Present study revealed the different sensitivities of pioglitazone for the recovery of decreased insulin action in a TNF-alpha-overexpressing model using cell transplantation. These results suggest that the effect of TZDs is dependent on the fat distribution and accumulation in humans.

Effects of nutrition on the cell survival and gene expression of transplanted fat tissues in mice.

Fat tissue transplantation is a useful and common clinical technique in the plastic and reconstructive surgeries. To know the nutritional effects on the survival and maintenance of fat grafts, the weights of tissues and cell sizes, and the gene expressions in the fat tissues were analyzed 14 days after transplantation. The body weight and the plasma insulin level in high nutritional group (HNG) were significantly higher (p<0.05) than those in low nutritional group (LNG), respectively. The measurements of cell size showed that there were 32.5% distributed in the diameter less than 2 microm in LNG, significantly higher than 28.5% in HNG. There were 7.5% distributed in the diameter more than 6 microm in LNG, significantly lower than 10.0% in HNG. The mRNA levels of leptin, lipoprotein lipase, and beta(3)-adrenergic receptor were 2.0-, 1.5-, and 1.7-fold higher in HNG than those in LNG, respectively. The levels of hormone sensitive lipase and hexokinase 2 transcripts were not significantly different in both groups. These results show that the systemic nutritional status in host causes the changes of cell size and tissue weight as well as gene expression in the transplanted fat using mice model. The nutritional condition is probably important for the fat graft clinically both as lipid-storage and functional cells.

Alterations of insulin sensitivity by the implantation of 3T3-L1 cells in nude mice. A role for TNF-alpha?

AIMS/HYPOTHESIS: Visceral adipocytes have different functions than those from the subcutaneous fat area. These differences could contribute to the pathological significance of excessive visceral fat accumulation for accompanying insulin resistance and hyperinsulinaemia. This study addresses this hypothesis and describes a unique method to clarify whether the functional differences between visceral and subcutaneous adipocytes depend on their anatomical location. METHODS: 3T3-L1 cells or TNF-alpha overexpressing CHO cells were implanted into subcutaneous fat area or mesenteric area as visceral fat area in athymic mice of BALB/C strain. Then, serum insulin, glucose, TNF-alpha, and several markers of lipid metabolism were measured in the fasting condition. OGTT was also analysed. RESULTS: During the course of glucose loading, the mice which had 3T3-L1 cells implanted into mesenteric area but not into subcutaneous fat area showed remarkably increased serum insulin and TMF-alpha concentrations, compared to the control mice. Moreover, serum insulin concentrations of the mice, implanted with TNF-alpha overexpressing cells into subcutaneous fat area, were apparently higher than that of control mice. CONCLUSION/INTERPRETATION: This method of implanting adipose cells into subcutaneous or visceral fat area showed high TNF-alpha concentration and insulin resistance by the adipose cells in visceral area of nude mice. Furthermore, we found that the functional significance of visceral fat accumulation for TNF-alpha-induced insulin resistance is partly caused by the interaction of adipocytes with surrounding conditions in mesenteric area.

Clinical characterization of a case with familial hypobetalipoproteinemia caused by apo B-76, a new truncation of apolipoprotein B, combined with apo E2/E2 phenotype.

We report a 43-year-old Japanese man with hypobetalipoproteinemia likely due to apolipoprotein (apo) B-76, a new truncation of apo B, and with homozygosity for the apo E2 isoform. He had no history suggestive of fat malabsorption and no sign of neurological disorder. His fasting baseline serum low-density lipoprotein (LDL) cholesterol and apo B levels were approximately half of normal. His plasma apo E level was elevated and its phenotype showed the E2/E2 homozygote. SDS-polyacrylamide gel electrophoresis of delipidated LDL fraction revealed a new truncated apo B, designated as apo B-76 according to the centile system of nomenclature. The postprandial lipid metabolism of the patient showed an almost normal response after fat loading.

Histological and functional analysis of vascular smooth muscle cells in a novel culture system with honeycomb-like structure.

Vascular smooth muscle cells (SMCs) undergo phenotype change with the development of atherosclerosis. The phenotype changes of SMCs have been observed in various culture conditions, such as collagen-coated dishes. Here, we report the morphological and functional features of SMCs in a novel culture system using type I-collagen in a characteristic three-dimensional structure designated as honeycombs. The number of ribosome and mitochondria in SMCs cultured in honeycombs was one half or third of those cultured on collagen-coated plastic plates. DNA and protein synthesis of SMCs cultured in honeycombs were less than 1 and 30-40%, respectively, of those cultured on plastic plates. In addition, PDGF-BB did not increase the amount of DNA synthesis in SMCs in honeycombs. SMCs in honeycombs were shown to express several proteins, which are known to express in SMCs in medial layers of arteries. Particularly, caldesmon heavy chain was expressed in SMCs cultured in honeycombs, whereas not in those on plastic plates. Although focal adhesion kinase (FAK) was clearly detected in SMCs in honeycomb, the phosphotyrosine content of focal adhesion kin ase decreased in the process of culture. Immunoblot analysis showed dear different expression of ERK1 and ERK2 of mitogen-activated protein kinase in SMCs. SMCs in honeycombs expressed ERK2, more abundantly compared to ERK1, whereas SMCs in plates show the same levels of expressions for both proteins. Thus, the histological and functional feature of SMCs in the novel culture system is different from SMCs in plastic plates. The three-dimensional culture system described here may be indicating that cultured SMCs are able to express different proteins responding to the surrounding structures.

LR11, a mosaic LDL receptor family member, mediates the uptake of ApoE-rich lipoproteins in vitro.

Since the molecular identification of the low density lipoprotein receptor (LDLR), an ever increasing number of related proteins have been discovered. These receptors belonging to the LDLR family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. We have discovered that the expression of a 250-kDa mosaic LDLR-related protein, which we termed LR11 for the presence of 11 LDLR ligand-binding repeats, is markedly induced in smooth muscle cells in the hyperplastic intima of animal models used for the study of atherosclerosis. Here, we demonstrate that the human LR11, when overexpressed in hamster cells, binds and internalizes 39-kDa receptor-associated protein (RAP), an in vitro ligand for all receptors belonging to the LDLR family. Furthermore, LR11 binds the apolipoprotein E (apoE)-rich lipoproteins, beta-very low density lipoproteins (VLDLs), with a high affinity similar to that of other members, such as the LDLR and VLDL receptor. RAP and beta-VLDL compete with each other; however, other serum lipoproteins are not able to inhibit their binding. LR11 shows specific binding of apoE-enriched HDL prepared from human cerebrospinal fluid as well as of beta-VLDL, suggesting that the apoE content of lipoproteins is most likely important for mediating the high-affinity binding to the receptor. LR11-overexpressing cells are able to internalize and degrade the bound beta-VLDL; these cells also show increased accumulation of cholesteryl esters when incubated with beta-VLDL. Incubation for 48 hours with beta-VLDL of LR11-overexpressing cells, but not of control cells, promotes the appearance of numerous intracellular lipid droplets. Taken together, LR11, a mosaic LDLR family member whose expression in smooth muscle cells is markedly induced in atheroma, has all the properties of a receptor for the endocytosis of lipoproteins, particularly for the incorporation of apoE-rich lipoproteins.

Pre-heparin plasma lipoprotein lipase mass: correlation with intra-abdominal visceral fat accumulation.

OBJECTIVE: To determine how lipoprotein lipase mass in the pre-heparin plasma is affected by body fat distribution, which is known to be closely related to lipid disorder, either directly or through insulin resistance. SUBJECTS: A total of 57 subjects consisting of 50 hyperlipidemic and 7 normolipidemic subjects (age 54 +/- IIy; 31 men, 26 women; body mass index 24+/- 2.5 kg/m2; serum total cholesterol 6.4+/-1.5 mmol/l; triglycerides, 2.4 +/- 1.7 mmol/l; HDL-cholesterol 1.3 +/- 0.5 mmol/l) were enrolled. MEASUREMENTS: We investigated the correlation between pre-heparin plasma LPL mass and intra-abdominal visceral fat area (or subcutaneous fat area) evaluated by computed tomography, and serum lipids and lipoproteins. RESULTS: Pre-heparin plasma LPL mass correlated inversely against intra-abdominal visceral fat area (r = - 0.51, p < 0.0001) and body mass index (r = - 0.46, p = 0.0003), but did not show any significant correlation with subcutaneous fat area. Pre-heparin plasma LPL mass had a positive correlation with serum high density lipoprotein cholesterol (r = 0.45, p = 0.0004) and a negative correlation against serum triglycerides (r = - 0.48, p = 0.0002). CONCLUSIONS: Pre-heparin plasma LPL mass is closely associated with intra-abdominal fat distribution, and the measurement of its value gives useful information concerning metabolic disorder.

Severe hypertriglyceridemia with plasma inhibitory factor(s) on lipoprotein lipase activity in a patient with a common Ser(447)-Ter LPL mutation.

Severe hypertriglyceridemia is a major risk for acute pancreatitis. So far, several mutations on the lipoprotein lipase (LPL) gene causing type I hyperlipidemia have been identified. However, the common mutation Ser(447)-Ter has been recently proposed to have a lowering effect on serum triglyceride concentrations in the general population. In this study, we analyzed blood from a patient suffering from severe hypertriglyceridemia and pancreatitis with the mutation on the lipoprotein lipase gene, Ser(447)-Ter. The patient's plasma showed inhibitory effects on the LPL activities from normal subjects. The bottom fraction separated by ultracentrifugation revealed enhanced effects as an inhibitory factor. The inhibitory effect observed in the bottom fraction was dose-dependent, stable at treatment of 65 degrees C for 30 min, and decreased significantly after being dialyzed using membranes with a cut-off molecular weight of 3500 or 6000 Da. The inhibitory effect was significantly higher when the post-heparin plasma was used from the patient or a subject with the same LPL mutation as an LPL source, compared to that from normal subjects. These results suggest that the patient has inhibitory factors in his plasma. Such inhibitory factors might cause severe hypertriglyceridemia in a case with the common mutation, which has been proposed to show the lowing effect on serum triglyceride concentrations in the general population.