Lipid receptors and islet function: therapeutic implications?

G-protein coupled receptors (GPCRs) are targets of approximately 30% of currently marketed drugs. Over the last few years, a number of GPCRs expressed in pancreatic beta-cells and activated by lipids have been discovered. GPR40 was shown to be activated by medium- to long-chain fatty acids (FAs). It has since been shown that GPR40 contributes to FA amplification of glucose-induced insulin secretion. Although some controversy still exists as to whether GPR40 agonists or antagonists should be designed as novel type 2 diabetes drugs, data obtained in our laboratory and others strongly suggest that GPR40 agonism might represent a valuable therapeutic approach. GPR119 is expressed in pancreatic beta-cells and enteroendocrine L-cells, and augments circulating insulin levels both through its direct insulinotropic action on beta-cells and through FA stimulation of glucagon-like peptide 1 (GLP-1) secretion. GPR120 is expressed in L-cells and was also shown to mediate FA-stimulated GLP-1 release. Finally, GPR41 and GPR43 are receptors for short-chain FAs and may indirectly regulate beta-cell function via adipokine secretion. Although the discovery of these various lipid receptors opens new and exciting avenues of research for drug development, a number of questions regarding their mechanisms of action and physiological roles remain to be answered.

Modulation of central leptin sensitivity and energy balance in a rat model of diet-induced obesity.

AIM: The aim of this study was to further explore the time-dependent changes in leptin sensitivity using a rat model of dietary fat-induced obesity and to investigate the potential mechanisms governing these changes. METHODS: We used male, adult Sprague-Dawley rats that were fed either a standard laboratory chow diet (3% fat) or a high-saturated fat (HF) diet (60% fat) for 2 or 5 weeks. Energy balance (body weight, energy intake and energy expenditure); sensitivity to central leptin and central alpha-melanin stimulating hormone (alpha-MSH) administration and expression levels of hypothalamic ObRb, signal transducers and activators of transcription factor (STAT)-3 phosphorylation, suppressor of cytokine signalling-3 (SOCS-3), proopiomelanocortin (POMC) processing hormones (prohormone convertase-1 and prohormone convertase-2) and neuropeptide Y (NPY) were measured. RESULTS: After 2 weeks of feeding HF diet, there was an increase in total energy intake (TEI) but a reduction in food intake as measured by the mass of food ingested. Body weight at this time was not significantly different between the two diet groups; however, white adipose tissue (WAT) weight was significantly greater in the HF-fed rats than in the chow-fed rats. In addition, spontaneous physical activity levels were increased, but no changes were observed in resting energy expenditure. Furthermore, chow-fed lean rats responded to central leptin administration by reducing the energy intake by approximately 67 kJ compared with saline treatment (p < 0.05), while the HF-fed diet-induced obese (DIO) rats responded by reducing their energy intake by approximately 197 kJ compared with saline treatment (p < 0.05). After 5 weeks of feeding HF diet, TEI remained significantly higher, body weight was significantly increased by 5% in the HF-fed rats and WAT weight was significantly heavier in HF-fed rats than in the chow-fed lean rats. After leptin treatment, the chow-fed lean rats reduced their energy intake by approximately 97 kJ (p < 0.05); yet, leptin had no significant effect in the HF-fed DIO rats. ObRb protein expression, STAT-3 phosphorylation levels, content and messenger RNA (mRNA) expression of NPY, SOCS-3 mRNA and protein expression and energy intake response to central alpha-MSH administration were not altered after HF diet feeding. CONCLUSION: These results suggest that early in the course of HF diet-induced weight gain, there was a period of central leptin hypersensitivity, and as the obesity progresses, central leptin insensitivity develops. This insensitivity does not appear to be explained by a downregulation of ObRb protein levels, reduced leptin signalling, an increase in either SOCS-3 or NPY expression or reduced function of the melanocortin system. The effect of an HF diet on other actions of leptin such as its effect on the endocannabinoid system should be investigated.

Increased nicotinamide nucleotide transhydrogenase levels predispose to insulin hypersecretion in a mouse strain susceptible to diabetes.

AIMS/HYPOTHESIS: Insulin hypersecretion may be an independent predictor of progression to type 2 diabetes. Identifying genes affecting insulin hypersecretion are important in understanding disease progression. We have previously shown that diabetes-susceptible DBA/2 mice congenitally display high insulin secretion. We studied this model to map and identify the gene(s) responsible for this trait. METHODS: Intravenous glucose tolerance tests followed by a genome-wide scan were performed on 171 (C57BL/6 x DBA/2) x C57BL/6 backcross mice. RESULTS: A quantitative trait locus, designated hyperinsulin production-1 (Hip1), was mapped with a logarithm of odds score of 7.7 to a region on chromosome 13. Production of congenic mice confirmed that Hip1 influenced the insulin hypersecretion trait. By studying appropriate recombinant inbred mouse strains, the Hip1 locus was further localised to a 2 Mb interval, which contained only nine genes. Expression analysis showed that the only gene differentially expressed in islets isolated from the parental strains was Nnt, which encodes the mitochondrial proton pump, nicotinamide nucleotide transhydrogenase (NNT). We also found in five mouse strains a positive correlation (r2 = 0.90, p < 0.01) between NNT activity and first-phase insulin secretion, emphasising the importance of this enzyme in beta cell function. Furthermore, of these five strains, only those with high NNT activity are known to exhibit severe diabetes after becoming obese. CONCLUSIONS/INTERPRETATION: Insulin hypersecretion is associated with increased Nnt expression. We suggest that NNT must play an important role in beta cell function and that its effect on the high insulin secretory capacity of the DBA/2 mouse may predispose beta cells of these mice to failure.

Effect of season and supplementation on percentage live sperm and sperm abnormalities in Horro (Zebu) bulls in sub-humid environment in Ethiopia.

A total of 32 Horro (Bos indicus) bulls, with an average initial body weight and age of approximately 211 kg and 6 years were evaluated. The bulls were divided into two treatment groups. One group was given a supplementary concentrate at a rate of 1.5 kg/day and the second group served as the control and received no supplementation. The observation period lasted for 50 weeks. Semen was collected every two weeks by mean of electrical stimulation with the aid of an electro-ejaculator. The percentage of abnormal sperm (total abnormalities, head, midpiece and tail) remained lower in the supplemented group than in the non-supplemented group. The percentage of live sperm tended to be higher in the supplemented group throughout the 50-week trial period. Until week 17 of the trial there were no significant differences; however, starting at week 18 there were significant differences between the two treatment groups except for week 26. Thereafter, significant differences were recorded until week 38; from week 40 no significant differences were observed between the two groups in respect of percentage of live sperm (except for week 48). Season (week) during which the semen samples were collected had no significant effect on the percentage of abnormal sperm in both treatment groups (supplemented and non-supplemented). At the start of the trial the nutritionally supplemented group had a higher occurrence of sperm abnormalities (11.4% +/- 1.1%) compared to 8.3% +/- 1.3% in the control group. Thereafter, sperm abnormalities declined rapidly in the supplemented group (except for week 50). The sperm abnormalities for the non-supplemented group showed a tendency to increase during the entire observation period.

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets.

AIMS/HYPOTHESIS: We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure. MATERIALS AND METHODS: Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes. RESULTS: Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-L: -cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels. CONCLUSIONS/INTERPRETATION: Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.

[Evaluation of lipoperoxidation in hemodialysis patients with a simple test using the "Free Oxygen Radical Monitor"]. / Evaluation de la peroxydation lipidique chez le patient hémodialysé par un test rapide utilisant le free oxygen radical monitor.

Incidence of cardiovascular events is higher in hemodialysis (HD) patients than in general population. Oxidative stress represents a major specific risk factor of accelerated atheroma particularly in association with inflammation and malnutrition. The aim of our study is to evaluate a simple test of lipid peroxidation measurement using the "Free Oxygen Radical Monitor" (FORM) (Callegari, Italy). The results obtained in HD patients were compared to standard oxidative stress markers, such as thiobarbituric acid reacting substances, carbonyls and vitamin E in plasma, and glutathione, oxidized to reduced form ratio, in erythrocytes. In conclusion, the FORM system presents no sufficient sensibility and specificity to determine oxidative stress in HD patients.

Oxidative stress and haemodialysis: role of inflammation and duration of dialysis treatment.

BACKGROUND: Oxidative stress has long been demonstrated in haemodialysis patients. However, the factors influencing their oxidative status have not been characterized extensively in these patients. Therefore, the present study was designed to investigate the influence of a large number of factors known to be associated with oxidative stress. METHODS: In the present cross-sectional study, we determined the plasma levels of lipid and protein oxidation markers in 31 non-smoking haemodialysis patients and 18 non-smoking healthy subjects, together with various components of the antioxidant system at the plasma and erythrocyte level. RESULTS: No influence of age, diabetes or iron overload on oxidative markers and plasma and erythrocyte antioxidant systems was detected in these haemodialysis patients. The lack of an association between iron overload and oxidative status may be related to the lower level of plasma ascorbate in haemodialysis patients, since ascorbate favours the generation of free iron from ferritin-bound iron. Interestingly, plasma C reactive protein (CRP) levels measured by highly sensitive CRP assay were correlated positively with plasma levels of thiobarbituric acid reactive substances (r=0.38, P<0.04) and negatively with plasma alpha-tocopherol levels (r=-0.46, P<0.01). Moreover, significant inverse correlations were observed between duration of dialysis treatment and plasma levels of alpha-tocopherol (r=-0.49, P<0.02) and ubiquinol (r=-0.40, P<0.05). CONCLUSIONS: Our results suggest that inflammatory status and duration of dialysis treatment are the most important factors relating to oxidative stress in haemodialysis patients.

Oxidized low-density lipoprotein induces macrophage respiratory burst via its protein moiety: A novel pathway in atherogenesis?

Oxidized low-density lipoproteins (oxLDL) play a crucial role in atherogenesis mainly via their capacity to bind and to activate macrophages. However, the role of the protein LDL moiety in this process is not yet established. In this study, human LDL were exposed to hypochlorous acid (HOCl), a selective protein oxidant, or copper sulfate (CuSO(4)), a major lipid oxidant, and tested for their capacity to activate the NADPH-oxidase of human THP-1- and U937-derived macrophages as measured by lucigenin chemiluminescence (CL). Compared to native LDL which had no effect, HOCl-oxLDL triggered potent CL responses in both U937 and THP-1 cells but only when these were fully differentiated into macrophages by phorbol myristate acetate. In contrast, Cu-oxLDL only triggered a moderate CL response of U937 cells and had little effect on THP-1 cells. While delipidation did not affect HOCl-oxLDL-induced CL response it abolished that induced by Cu-oxLDL. Interestingly, U937 cells showed higher CL responses to both types of oxLDL than THP-1 cells, a finding which could be related to their higher expression of the scavenger receptor CD36. Taken together these results strongly support the role of the protein moiety in oxLDL-induced macrophage activation.

Alpha-1-microglobulin: inhibitory effect on calcium oxalate crystallization in vitro and decreased urinary concentration in calcium oxalate stone formers.

In the past few years, alpha-1-microglobulin (alpha1m) has been copurified from human urine with bikunin, a potent inhibitor of calcium oxalate (CaOx) crystallization in vitro. In this study, we have purified alpha1m without bikunin contamination and investigated its possible role in CaOx crystallization by in vitro and in vivo studies. Alpha-1m was purified with an anti-alpha1m antibodies CNBr-activated sepharose column. Two molecular species of alpha1m of respectively 30 and 60 kDa were purified. For each protein, two blots of 30 and 60 kDa cross-reacted with anti-alpha1m antibodies, suggesting that these two forms were derived one from the other. Both protein species inhibited CaOx crystallization in a dose-dependent manner in two in vitro tests. In the first test, the presence of alpha1m of 30 kDa (8 microg/ml) in a medium containing 0. 76 mM CaCl(2) (with (45)Ca) and 0.76 mM Ox(NH(4))(2) inhibited CaOx crystallization by 38% as estimated by supernatant radioactivity after 1 h of agitation. In the second test, CaOx kinetics were examined for 3 to 10 min in a turbidimetric model at 620 nm. The presence of alpha1m of 30 kDa in a medium containing 4 mM CaCl(2) and 0.5 mM Na(2)Ox inhibited CaOx crystallization by 41.5%, as estimated by the slope modification of turbidimetric curve. Alpha-1m can be considered as another inhibitor of urinary CaOx crystal formation, as shown by the present in vitro studies. Using an ELISA assay, we found that urinary alpha1m concentration was significantly lower in 31 CaOx stone formers than in 18 healthy subjects (2.95 +/- 0.29 vs 5.34 +/- 1.08 mg/l respectively, P = 0.01). The decreased concentration of alpha1m in CaOx stone formers could be responsible in these patients, at least in part, for an increased risk of CaOx crystalluria.

Do low-risk prenatal patients really need a screening glucose challenge test?

BACKGROUND: It is common practice to routinely screen pregnant women for gestational diabetes. The screening technique typically used is the 1-hour 50-g oral glucose tolerance test (OGTT), with a subsequent 3-hour 100-g OGTT for women whose 1-hour test was positive. This process can be both time-consuming and inconvenient for patients. Additionally, its sensitivity and specificity are estimated to be 70% and 87% respectively, and data about the effect of screening and treatment on low-risk pregnancy outcomes are limited. The objective of this study was to reassess the value of routine screening of all pregnant patients with a 1-hour glucose challenge test. METHODS: At a university-based family practice center with a predominantly low-risk population, a retrospective analysis was performed of all patients (n = 595) who received prenatal care and gave birth between January 1988 and December 1993. Among women in whom gestational diabetes was diagnosed on the basis of glucose tolerance testing, we identified those with risk factors for the disease, and examined whether a selective screening program based on risk factors alone would have resulted in correct diagnoses of gestational diabetes. RESULTS: Of the 595 patients, 544 (91.4%) were screened with a 1-hour 50-g OGTT. This initial screening test was positive in 76 women (12.8%). Of these, 58 (76.3%) then had a 3-hour 100-g OGTT, and 13 received a diagnosis of gestational diabetes. Nine of these 13 women had risk factors for gestational diabetes. We determined that less than 1% of prenatal patients without risk factors for gestational diabetes were ultimately found to have gestational diabetes. CONCLUSIONS: Screening with a 1-hour 50-g OGTT only those women who have identifiable risk factors for gestational diabetes is a reasonable approach to identifying the disease in a low-risk population. All pregnant women should have a thorough history taken to determine whether they have risk factors for gestational diabetes.

In vitro susceptibilities of Bartonella henselae, B. quintana, B. elizabethae, Rickettsia rickettsii, R. conorii, R. akari, and R. prowazekii to macrolide antibiotics as determined by immunofluorescent-antibody analysis of infected Vero cell monolayers.

The in vitro susceptibilities of Bartonella (Rochalimaea) henselae, B. quintana, B. elizabethae, Rickettsia akari, R. conorii, R. prowazekii, and R. rickettsii to different concentrations of azithromycin, clarithromycin, dirithromycin, erythromycin, and roxithromycin in Vero cell cultures were evaluated. Bartonella and Rickettsia spp. were allowed to initiate infection of the antibiotic-free Vero cell monolayers, which were maintained in 16-chamber microscope slides in the absence of antibiotics at 32 degrees C in a CO2-enriched atmosphere. The monolayers were then incubated for 3 h to allow for initial host cell intracellular penetration by infecting species. After inoculation, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate (10 wells of each antibiotic dilution) for each species, and the monolayers were reincubated. Tetracycline served as the control. Growth status of Bartonella spp. and Rickettsia spp. was determined by evaluation of immunofluorescent staining bacilli. Five days later, when antibiotic-free, control-infected cell monolayers demonstrated significant fluorescence, media were removed for all cell monolayers, the monolayers were fixed, and all specimens were stained with standard indirect immunofluorescent antibody reagents. Fluorescent foci were enumerated by counting such foci on random fields visualized with an epifluorescence microscope. The extent of antibiotic-induced focus inhibition was recorded for each dilution of antibiotic and compared with that of an antibiotic-negative control. Effective antibiotic dilution endpoints for inhibition of Bartonella and Rickettsia proliferation, as judged by absence of increase of significant fluorescence (as compared with no-growth controls), were enumerated by determining the number of cell culture chambers at various antibiotic dilutions that were negative or positive for significant Bartonella- or Rickettsia-specific fluorescence. All of the macrolide agents tested were readily active against all three Bartonella organisms, and azithromycin, clarithromycin, and roxithromycin may have potential in the treatment of Rickettsia infections. Animal model-based clinical trials are warranted to define the specific treatment role of the newer macrolide antibiotics.

[Urinary calculi and crystalluria in HIV+ patients treated with indinavir sulfate]. / Calculs urinaires et cristalluries chez les patients VIH+ traités par le sulfate d'indinavir.

OBJECTIVES: Anti-proteases, a new class of anti-HIV drugs used in combination with reverse transcriptase inhibitors have led to spectacular improvement in the patients' clinical status. Since April 1996, indinavir is the most widely prescribed anti-protease in France. PATIENTS AND METHODS: From July 1996 to July 1997, we analyzed 46 spontaneously expulsed stones in 45 HIV+ patients (35 men and 10 women; age range 25 to 64 years) given indinavir in combination with other drugs since one week to ten months. Only six patients were known to have a past history of renal lithiasis. RESULTS: Forty-one calculi contained indinavir monohydrate (INDM) identified by mass spectrometry and infrared spectrophotometry. INDM was the only component excepting proteins in 39/45 calculi. In the 12 others, other compounds were also identified. Among the 114 urine samples collected 2 to 3 hours after an 800 mg dose of indinavir, 38 (33%) monohydrate indinavir crystals, identified by infrared microscopy. Mean urinary pH was significantly higher than in samples without INDM crystals (6.53 +/- 0.68 versus 5.96 +/- 0.71, p < 0.001). CONCLUSION: Two measures could possibly reduce the risk of crystalization: administration of urine acidifiers and increased fluid intake to raise diuresis. Alkalinisation is not indicated. Long-term increased fluid intake should be preferred over acidification which could be reserved solely for the treatment of drug-induced lithiasis.

Papanicolaou smear quality assurance: providing feedback to physicians.

BACKGROUND: The effective management of Papanicolaou (Pap) smears depends on the reliability and accuracy of obtaining and interpreting the specimen. Provider sampling error is one of the important factors contributing to inadequate specimens. Feedback on provider performance may be an effective way to improve the quality of Pap smears. METHODS: A pilot study in a university-based residency program involving resident and faculty physicians was initiated to assess the impact of feedback on performance of Pap smears. After establishing adequacy and inadequacy criteria and recording adequacy rates for 3 months, individual and group feedback was implemented. No formal educational intervention on Pap smear technique was undertaken. RESULTS: The quality of 836 Pap smears performed by 9 faculty and 13 resident physicians showed continued improvement in both sampling and slide preparation to 90% adequacy over a 9-month period. This improvement, though clinically useful, was not statistically significant owing to the relatively small numbers of smears performed by each physician. This form of feedback may be useful in both practice and educational settings. CONCLUSIONS: Feedback without any formal educational intervention led to a clinically useful trend of improvement in the quality of Pap smears, which has been sustained since the study began. This type of simple feedback may be useful in practice settings and particularly valuable in pinpointing areas for improvement for learners in residency programs.

Development of a Pap smear quality-assurance system in family practice.

BACKGROUND AND OBJECTIVES: To improve the effectiveness of cervical cancer screening, quality assurance programs can be designed to ensure that normal Pap smear results are dealt with appropriately and that Pap smears yielding inadequate specimen material are brought to a physician's attention. The objective of this study was to use a new Pap Smear Quality Assurance (PAPQA) system to determine and monitor the performance of physicians in a family practice over a two-year period. METHODS: We developed a PAPQA system designed to gather data and report on: 1) Pap smear adequacy, 2) reporting of abnormal results to physicians, and 3) follow-up of patients who had abnormal Pap smear results. We followed these parameters for two years. RESULTS: Over a two-year period, 2,771 cervical Pap smears were performed, of which 64% were normal. The percentage of Pap smears that yielded adequate specimen material improved from 82% to 91%, an improvement we attributed to feedback the system provided to physicians. Overall, Pap smear results and follow-up appeared in the medical record of 94% of patients who had abnormal findings. However, during the second year, the quality assurance system detected a deterioration in documentation of results and follow-up plans that coincided with moving the practice to a new facility. The operating cost for this program was approximately $950 per year. CONCLUSIONS: Quality assurance programs can effectively monitor physicians' performance in dealing with abnormal Pap smears, can detect deteriorations in performance, and can improve some aspects of performance through feedback reporting to physicians.

Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice.

A model of experimental Streptococcus suis infection was developed in young mice. Minimum lethal dose (MLD) values were calculated for four virulent serotypes (1/2, 1, 2, 3) of S. suis using this model. Temperature-sensitive (ts) mutants of S. suis serotypes 1/2 and 1-8 were isolated and characterized on the basis of their growth kinetics and reversion rates. Ts mutants of S. suis 1/2, 1, 2, and 3 were tested as vaccines against the virulent homologous and heterologous challenges in mice. The protection provided was evaluated by analyzing the clinical signs, death or survival. Homologous but not heterologous protection was noted in all mice vaccinated with the mutant strains. Ts mutants of S. suis 1/2 provided 100% protection against challenge by virulent strains of S. suis 1/2, 1, and 2.