Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

Aims/hypothesis

Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR^−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca^2 + handling in these islets.

Methods

Isolated islets from both LDLR^−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca^2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).

Results

The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR^−/− than in WT islets, paralleled by an impairment of Ca^2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR^−/− compared with WT islets. Removal of excess cholesterol from LDLR^−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca^2 + handling was also normalized in cholesterol-depleted LDLR^−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca^2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.

Conclusion

Abnormally high (LDLR^−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca^2 + handling. Normalization of cholesterol improves Ca^2 + handling and insulin secretion in LDLR^−/− islets.     

Uncleaved ApoM Signal Peptide Is Required for Formation of Large ApoM/Sphingosine 1-Phosphate (S1P)-enriched HDL Particles

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM^Q22A) introduces a functional signal peptidase cleavage site. Expression of apoM^Q22A in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM^WT). When apoM^Q22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM^WT. Hepatocytes isolated from both apoM^WT- and apoM^Q22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM^WT, apoM^Q22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM^Q22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM^WT and apoM^Q22A hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.     

Comprehensive lipidomics in apoM-/- mice reveals an overall state of metabolic distress and attenuated hepatic lipid secretion into the circulation

Apolipoprotein M (apoM) participates in both high-density lipoprotein and cholesterol metabolism. Little is known about how apoM affects lipid composition of the liver and serum. In this study, we systemically investigated the effects of apoM on liver and plasma lipidomes and how apoM participates in lipid cycling, via apoM knockout in mice and the human SMMC-7721 cell line. We used integrated mass spectrometry-based lipidomics approaches to semiquantify more than 600 lipid species from various lipid classes, which include free fatty acids, glycerolipids, phospholipids, sphingolipids, glycosphingolipids, cholesterol, and cholesteryl esters (CEs), in apoM^-/- mouse. Hepatic accumulation of neutral lipids, including CEs, triacylglycerols, and diacylglycerols, was observed in apoM^-/- mice; while serum lipidomic analyses showed that, in contrast to the liver, the overall levels of CEs and saturated/monounsaturated fatty acids were markedly diminished. Furthermore, the level of ApoB-100 was dramatically increased in the liver, whereas significant reductions in both ApoB-100 and low-density lipoprotein (LDL) cholesterol were observed in the serum of apoM^-/- mice, which indicated attenuated hepatic LDL secretion into the circulation. Lipid profiles and proinflammatory cytokine levels indicated that apoM^-/- leads to hepatic steatosis and an overall state of metabolic distress. Taken together, these results revealed that apoM knockout leads to hepatic steatosis, impaired lipid secretion, and an overall state of metabolic distress.     

Resistance exercise acutely enhances mesenteric artery insulin-induced relaxation in healthy rats

Aims

We evaluated the mechanisms involved in insulin-induced vasodilatation after acute resistance exercise in healthy rats.

Main methods

Wistar rats were divided into 3 groups: control (CT), electrically stimulated (ES) and resistance exercise (RE). Immediately after acute RE (15 sets with 10 repetitions at 70% of maximal intensity), the animals were sacrificed and rings of mesenteric artery were mounted in an isometric system. After this, concentration–response curves to insulin were performed in control condition and in the presence of LY294002 (PI3K inhibitor), L-NAME (NOS inhibitor), L-NAME + TEA (K⁺ channels inhibitor), LY294002 + BQ123 (ET-A antagonist) or ouabain (Na⁺/K⁺ ATPase inhibitor).

Key findings

Acute RE increased insulin-induced vasorelaxation as compared to control (CT: R_max = 7.3 ± 0.4% and RE: R_max = 15.8 ± 0.8%; p < 0.001). NOS inhibition reduced (p < 0.001) this vasorelaxation from both groups (CT: R_max = 2.0 ± 0.3%, and RE: R_max = − 1.2 ± 0.1%), while PI3K inhibition abolished the vasorelaxation in CT (R_max = − 0.1 ± 0.3%, p < 0.001), and caused vasoconstriction in RE (R_max = − 6.5 ± 0.6%). That insulin-induced vasoconstriction on PI3K inhibition was abolished (p < 0.001) by the ET-A antagonist (R_max = 2.9 ± 0.4%). Additionally, acute RE enhanced (p < 0.001) the functional activity of the ouabain-sensitive Na⁺/K⁺ ATPase activity (R_max = 10.7 ± 0.4%) and of the K⁺ channels (R_max = − 6.1 ± 0.5%; p < 0.001) in the insulin-induced vasorelaxation as compared to CT.

Significance

Such results suggest that acute RE promotes enhanced insulin-induced vasodilatation, which could act as a fine tuning to vascular tone.     

Leptin and leptin receptor genetic variants associate with habitual physical activity and the arm body composition response to resistance training

Purpose

We investigated the influence of Leptin (LEP) and leptin receptor (LEPR) SNPs on habitual physical activity (PA) and body composition response to a unilateral, upper body resistance training (RT) program.

Methods

European-derived American volunteers (men = 111, women = 131, 23.4 ± 5.4 yr, 24.4 ± 4.6 kg·m^− 2) were genotyped for LEP 19 G>A (rs2167270), and LEPR 326 A>G (rs1137100), 668 A>G (rs1137101), 3057 G>A (rs1805096), and 1968 G>C (rs8179183). They completed the Paffenbarger PA Questionnaire. Arm muscle and subcutaneous fat volumes were measured before and after 12 wk of supervised RT with MRI. Multivariate and repeated measures ANCOVA tested differences among phenotypes by genotype and gender with age and body mass index as covariates.

Results

Adults with the LEP 19 GG genotype reported more kcal/wk in vigorous intensity PA (1273.3 ± 176.8, p = 0.017) and sports/recreation (1922.8 ± 226.0, p < 0.04) than A allele carriers (718.0 ± 147.2, 1328.6 ± 188.2, respectively). Those with the LEP 19 GG genotype spent more h/wk in light intensity PA (39.7 ± 1.6) than A allele carriers (35.0 ± 1.4, p = 0.03). In response to RT, adults with the LEPR 668 G allele gained greater arm muscle volume (67,687.05 ± 3186.7 vs. 52,321.87 ± 5125.05 mm³, p = 0.01) and subcutaneous fat volume (10,599.89 ± 3683.57 vs. − 5224.73 ± 5923.98 mm³, p = 0.02) than adults with the LEPR 668 AA genotype, respectively.

Conclusion

LEP19 G>A and LEPR 668 A>G associated with habitual PA and the body composition response to RT. These LEP and LEPR SNPs are located in coding exons likely influencing LEP and LEPR function. Further investigation is needed to confirm our findings and establish mechanisms for LEP and LEPR genotype and PA and body composition associations we observed.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism

Objective

Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency.

Methods and results

HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL^−/− mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL^−/− background.

Conclusions

HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

Uncoupling protein 2 − 866G/A and uncoupling protein 3 − 55C/T polymorphisms in young South African Indian coronary artery disease patients

Background

Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Promoter polymorphisms of these genes have been linked to chronic diseases including heart disease and type II diabetes mellitus in several populations. This is the first investigation of the UCP2 − 866G/A rs659366 and UCP3 − 55C/T rs1800849 polymorphisms in young South African (SA) Indians with coronary artery disease (CAD).

Methods

A total of 300 subjects were recruited into this study of which 100 were SA Indian males with CAD, 100 age- (range 24–45 years), gender- and race-matched controls and 100 age-matched black SA males. The frequency of the UCP2 − 866G/A and UPC3 − 55C/T genotypes was assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP).

Results

The heterozygous UCP2 − 866G/A and homozygous UCP3 − 55C/C genotypes occurred at highest frequency in CAD patients (60% and 64%, respectively) compared to SA Indian controls (52% and 63%) and SA Black controls (50% and 58%). The UCP2 − 886G/A (OR = 1.110; 95% CI = 0.7438–1.655; p = 0.6835) and UCP3 − 55C/T (OR = 0.788; 95% CI = 0.482–1.289; p = 0.382) polymorphisms did not influence the risk of CAD.The rare homozygous UCP3 − 55T/T genotype was associated with highest fasting glucose (11.87 ± 3.7 mmol/L vs. C/C:6.11 ± 0.27 mmol/L and C/T:6.48 ± 0.57 mmol/L, p = 0.0025), HbA1c (10.05 ± 2.57% vs. C/C:6.44 ± 0.21% and C/T:6.76 ± 0.35%, p = 0.0006) and triglycerides (6.47 ± 1.7 mmol/L vs. C/C:2.33 ± 0.17 mmol/L and C/T:2.06 ± 0.25 mmol/L, p < 0.0001) in CAD patients.

Conclusion

The frequency of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms was similar in our SA Indian and SA Black groups. The presence of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms does not influence the risk of CAD in young South African Indian CAD patients.     

Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice

Objectives

To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development.

Methods and results

Chimeras with dysfunctional macrophage ABCA5 (ABCA5^−M/−M) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5^−/−) mice into irradiated LDLr^−/− mice. In vitro, bone marrow-derived macrophages from ABCA5^−M/−M chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr^−/− mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5^−M/−M chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5^−M/−M chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding.

Conclusions

ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr^−/− mice.     

Clinical value of plasma pentraxin 3 levels for predicting cardiac troponin elevation after percutaneous coronary intervention

Aims

Post-procedural myocardial necrosis manifested by elevated cardiac troponin T (cTnT) often complicates percutaneous coronary intervention (PCI). Plasma pentraxin 3 (PTX3) levels are increased in patients with arterial inflammation and especially unstable angina pectoris (UAP). This study tested whether plasma PTX3 levels can predict post-PCI cTnT elevation.

Main methods

We evaluated 94 consecutive patients with AP and normal pre-PCI cTnT levels who underwent PCI. Pre-PCI virtual histology-intravascular ultrasound was performed to assess culprit plaque composition. Plasma PTX3 and serum hs-CRP levels were measured pre-PCI. Patients were divided into 2 groups according to presence (Group I, n = 34) or absence (Group II, n = 60) of post-PCI cTnT elevation > 3 × the upper limit of normal at 24 h after PCI.

Key findings

Plasma PTX3 (4.06 ± 2.05 ng/ml vs 2.17 ± 1.02 ng/ml, p < 0.001), serum hs-CRP levels (0.25 ± 0.03 vs 0.16 ± 0.03 mg/dl, p = 0.048), plaque burden (80.9 ± 5.3 vs 75.4 ± 10.6%, p = 0.047), presence of positive remodeling (59 vs 25%, p = 0.034), and percent necrotic core area (19.0 ± 7.4 vs 14.0 ± 5.9%, p = 0.046) were significantly higher in Group I than in Group II. Receiver-operating characteristic curve analysis showed that with a best cut-off value of 2.83 ng/ml, plasma PTX3 level (AUC 0.823) predicted post-PCI cardiac TnT elevation better than did serum hs-CRP level (AUC 0.618). Multiple logistic regression analysis showed that plasma PTX3 level was the most independent predictor of post-PCI cardiac cTnT elevation (OR: 2.65; 95% CI: 1.56–10.1; p = 0.003).

Significance

Plasma PTX3 level may be a useful marker for predicting post-PCI cardiac cTnT elevation, which is associated with inflammatory status of culprit lesions.     

Hepatic lipase- and endothelial lipase-deficiency in mice promotes macrophage-to-feces RCT and HDL antioxidant properties

Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [³H]cholesterol-labeled mouse macrophages, after which the appearance of [³H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [³H]cholesterol 48 h after the label injection. The magnitude of macrophage-derived [³H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties.     

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice

The mutation L159R apoA-I or apoA-I_L159R (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL^−/−, apoA-I^−/−) (double knockout or DKO) were crossed > 9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr^−/−, ApoA-I^−/− (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12 weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α₃-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-I_L159R contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

The influence of 5-lipoxygenase on cigarette smoke-induced emphysema in mice

Background

Pulmonary emphysema is characterized by the loss of lung architecture. Our hypothesis is that the inhibition of 5-lipoxygenase (5-LO) production may be an important strategy to reduce inflammation, oxidative stress, and metalloproteinases in lung tissue resulting from cigarette smoke (CS)-induced emphysema.

Methods

5-LO knockout (129S2-Alox5^tm1Fun/J) and wild-type (WT) mice (129S2/SvPas) were exposed to CS for 60 days. Mice exposed to ambient air were used as Controls. Oxidative, inflammatory, and proteolytic markers were analyzed.

Results

The alveolar diameter was decreased in CS 5-LO^−/− mice when compared with the WT CS group. The CS exposure resulted in less pronounced pulmonary inflammation in the CS 5-LO^−/− group. The CS 5-LO^−/− group showed leukotriene B4 values comparable to those of the Control group. The expression of MMP-9 was decreased in the CS 5-LO^−/− group when compared with the CS WT group. The expression of superoxide dismutase, catalase, and glutathione peroxidase were decreased in the CS 5-LO^−/− group when compared with the Control group. The protein expression of nuclear factor (erythroid-derived 2)-like 2 was reduced in the CS 5-LO^−/− group when compared to the CS WT group.

Conclusion

In conclusion, we show for the first time that 5-LO deficiency protects 129S2 mice against emphysema caused by CS. We suggest that the main mechanism of pathogenesis in this model involves the imbalance between proteases and antiproteases, particularly the association between MMP-9 and TIMP-1.General significanceThis study demonstrates the influence of 5-LO mediated oxidative stress, inflammation, and proteolytic markers in CS exposed mice.     

A novel acylated form of (d-Ala)GIP with improved antidiabetic potential,lacking effect on body fat stores

Background

Rapid enzymatic degradation of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), limits therapeutic use of the native peptide for diabetes. However, enzymatically stable analogues of GIP, such as (d-Ala²)GIP, have been generated, but are still susceptible to renal filtration.

Methods

The present study examines the in vitro and in vivo biological actions of a novel, acylated GIP analogue, (d-Ala²)GIP[Lys³⁷PAL].

Results

In BRIN-BD11 cells, (d-Ala²)GIP[Lys³⁷PAL] concentration-dependently stimulated (p < 0.05 to p < 0.001) insulin secretion at 5.6 and 16.7 mM glucose. Intraperitoneal administration of (d-Ala²)GIP[Lys³⁷PAL] to normal mice 8 h prior to a glucose load significantly reduced (p < 0.05) the overall glycaemic excursion compared to controls, and increased (p < 0.001) the insulinotropic response compared to (d-Ala²)GIP and saline treated high fat control mice. Once daily administration of (d-Ala²)GIP[Lys³⁷PAL] for 21 days in high fat fed mice did not affect energy intake, body weight or fat deposition. However, circulating blood glucose was significantly lower (p < 0.05) accompanied by increased (p < 0.05) insulin concentrations by day 21. In addition, (d-Ala²)GIP[Lys³⁷PAL] treatment significantly (p < 0.01) reduced the overall glycaemic excursion and increased pancreatic insulin content (p < 0.05) and the insulinotropic response (p < 0.01) to an exogenous glucose challenge on day 21. Chronic treatment with (d-Ala²)GIP[Lys³⁷PAL] did not result in resistance to the metabolic effects of a bolus injection of native GIP. Finally, insulin sensitivity was significantly improved (p < 0.001) in (d-Ala²)GIP[Lys³⁷PAL] treated mice compared to high fat controls.

Conclusions

These data confirm that (d-Ala²)GIP[Lys³⁷PAL] is a stable, long-acting potent GIP agonist.

General significance

(d-Ala²)GIP[Lys³⁷PAL] may be suitable for further evaluation and future clinical development.     

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter,Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1^−/−), and subsequently in Cav-1^−/− mice that also lacked the lysosomal cholesterol transporter Niemann–Pick C1 (Npc1) (Cav-1^−/−:Npc1^−/−). In 50-day-old Cav-1^−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1^+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1^−/−:Npc1^−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1^+/+:Npc1^+/+ controls (0.137 ± 0.009), as well as in their Cav-1^−/−:Npc1^+/+ (0.191 ± 0.013) and Cav-1^+/+:Npc1^−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1^−/−:Npc1^−/− and Cav-1^+/+:Npc1^−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1^−/−:Npc1^−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.     

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.     

Synergistic effect of anti and pro-inflammatory cytokine genes and their promoter polymorphism with ST-elevation of myocardial infarction

Background

The single-gene approach in association studies of polygenic diseases such as acute myocardial infarction (AMI) is likely to provide limited value. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) plasma levels may be genetically influenced.

Aim

We evaluate the impact of single nucleotide polymorphism of the promoter region of these genes, as well as reciprocal interaction of these genes with ST-elevation of myocardial infarction (STEMI).

Methods

In a case–control study 500 STEMI patients and 500 age- and sex-matched controls were studied. Three single-nucleotide polymorphism genotypes were evaluated by polymerase chain reaction and restriction enzyme analysis and assessed their association with STEMI. The synergistic effects of IL-6, TNF-α and IL-10 gene polymorphisms were evaluated by using logistic regression analysis.

Results

We found that IL-6 and TNF-α concentrations of studied population were significantly different (p < 0.0001) in each genotype of IL-6 − 174G>C and TNF-α − 308G>A gene polymorphisms respectively. A significant association was found in multivariate analysis for the IL-6 − 174G>C [odds ratio (OR): 0.390; 95% confidence interval (CI): 0.176–0.865, p = 0.020] and TNF-α − 308G>A [OR: 0.372; 95% CI: 0.171–808, p = 0.012] gene polymorphisms with STEMI. In contrast, IL-10 − 592C>A gene polymorphism was no longer significant in the multivariate model (OR: 0.678; 95% CI: 0.288 to 1.594, p = 0.373) whereas significant in univariate analysis (OR: 0.697; 95% CI: 0.523–0.929, p = 0.014).

Conclusions

Our findings suggest that IL-6, TNF-α and IL-10 gene polymorphisms all contribute in the association with STEMI whereas the association persisted only for IL-6 and TNF-α but not for IL-10 gene polymorphism with this disease in the multivariate analysis.     

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout (Apom^−/−) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom^−/− mice excreted significant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 (Clcn5^−/−), or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5^−/− mice. In contrast to Apom^−/− mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however, with no impact on S1P plasma concentrations.