First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l. POPULATIONS: Interviews were conducted with dog owners to gather information on the history of dog treatment with conventional acaricides and IVM. The larval immersion test was used on the progeny of adult female ticks infesting dogs to test for susceptibility to IVM. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals, and slope were estimated by probit analysis. Ten tick populations (66.7%) were classified as resistant compared with the most susceptible population. A high inter-population variation in the phenotypic level of IVM resistance was evident (resistance ratio at LC50% and LC99% ranged from 1.0 to 30.5, and from 1.0 to 458.8, respectively). Tick populations classified as resistant were collected from dogs known to have been treated with IVM. To our knowledge, this is the first report of IVM resistance in R. sanguineus s.l. worldwide. Veterinary and pet owner education on integrated tick management practices is required to avoid widespread resistance and increased treatment failure with products containing IVM and other macrocyclic lactones used to control endo- and ectoparasites affecting dogs. Integrated tick management will also help mitigate the burden of brown dog tick-borne diseases on human and animal populations.

First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico.

Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose-mortality regressions, 50% lethal concentrations (LC50 ), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter-population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC50 : 1.0-13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter-population variation in the phenotypic level of resistance was evident (RRs at LC50 : 1.0-104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.

Ticks collected from humans, domestic animals, and wildlife in Yucatan, Mexico.

Domestic animals and wildlife play important roles as reservoirs of zoonotic pathogens that are transmitted to humans by ticks. Besides their role as vectors of several classes of microorganisms of veterinary and public health relevance, ticks also burden human and animal populations through their obligate blood-feeding habit. It is estimated that in Mexico there are around 100 tick species belonging to the Ixodidae and Argasidae families. Information is lacking on tick species that affect humans, domestic animals, and wildlife through their life cycle. This study was conducted to bridge that knowledge gap by inventorying tick species that infest humans, domestic animals and wildlife in the State of Yucatan, Mexico. Amblyomma ticks were observed as euryxenous vertebrate parasites because they were found parasitizing 17 animal species and human. Amblyomma mixtum was the most eryxenous species found in 11 different animal species and humans. Both A. mixtum and A. parvum were found parasitizing humans. Ixodes near affinis was the second most abundant species parasitizing six animal species (dogs, cats, horses, white-nosed coati, white-tail deer and black vulture) and was found widely across the State of Yucatan. Ixodid tick populations may increase in the State of Yucatan with time due to animal production intensification, an increasing wildlife population near rural communities because of natural habitat reduction and fragmentation. The diversity of ticks across host taxa documented here highlights the relevance of ecological information to understand tick-host dynamics. This knowledge is critical to inform public health and veterinary programs for the sustainable control of ticks and tick-borne diseases.

Lethal and Sublethal Effects of Ivermectin on Onthophagus landolti (Coleoptera: Scarabaeidae).

Previous work has documented toxic effects of ivermectin (IVM) on dung beetles from the Old World, but very little is known about this drug's effect on Neotropical dung beetles. Accordingly, we conducted a bioassay with dung spiked with IVM to assess its lethal and sublethal effects on the Neotropical dung beetle Onthophagus landolti Harold. The experimental design consisted of five treated groups G1, G2, G3, G4, and G5 receiving 0.001, 0.01, 0.1, 1.0, and 10 mg IVM/kg dung fresh weight, respectively, and two control groups (solvent control [CGA] and untreated control [CGU]). Adult survival and fecundity were measured throughout a 10-d period, and subsequent egg development and survival were monitored. Adult survival was only affected for treatment groups G4 and G5 groups (70 and 30%, respectively); groups G1, G2, G3 and both controls exhibited 100% survival. Fecundity was completely suppressed under treatment groups G4 and G5. Group G3 only had 1.7 and 2.1% brood mass production relative to CGA and CGU, respectively. Additionally, for groups G1 and G2 the proportion of adults emerging from brood masses was lower relative to CGA. Furthermore, development time for the second generation in groups G1 and G2 was 12.5% slower relative to control groups. Finally, dung removal by beetles from groups G3, G4, and G5 was significantly lower relative to control groups. In conclusion, toxic effects of IVM on O. landolti are associated mainly with reduced fecundity and lower dung-removal by adult beetles as well as reduced survival and slower development of offspring.

Use of Ivermectin as Endoparasiticide in Tropical Cattle Herds Generates Resistance in Gastrointestinal Nematodes and the Tick Rhipicephalus microplus (Acari: Ixodidae).

The objective of the present study was to determine simultaneously the status of resistance against ivermectin (IVM) in gastrointestinal nematodes (GIN) and Rhipicephalus microplus (Canestrini, 1888) ticks in 12 cattle farms where IVM was used for the control of GIN in the Mexican tropics. Six farms had frequent use of IVM (≥ 4 times per year) and six farms had low frequency of IVM use (1-2 times per year). The fecal egg count reduction test and the larval immersion test were used to determine the resistant status of GIN and R. microplus against IVM, respectively. The results indicated that 100% of the surveyed farms had IVM-resistant GIN (reduction % from 0 to 67%). The genera involved were Haemonchus, Cooperia, Ostertagia, Trichostrongylus, and Oesophagostomum. Although the IVM was never used for the control of ticks, 50% of the surveyed farms presented GIN and R. microplus simultaneously resistant to IVM. Furthermore, two R. microplus populations showed high resistance ratio (RR) to IVM (farm TAT: RR50% = 7 and RR99% = 40.1; and farm SLS: RR50% = 2.4; RR99% = 11.0). A high frequency of IVM use (≥ 4 times per year) seemed to promote IVM resistance amongst R. microplus ticks compared with the farms with low frequency of IVM use (1-2 times per year; 66.6 vs. 25.0%, respectively). However, the number of surveyed farms was insufficient to show clear statistical inferences (odds ratio = 6.00; 95% CI = 0.341-105.5). The use of IVM for the control of GIN promoted simultaneously the development of IVM resistance in the GIN and R. microplus populations of the cattle herds surveyed.

Efficacy of entomopathogenic fungi (Ascomycetes: Hypocreales) against adult Haematobia irritans (Diptera: Muscidae) under stable conditions in the Mexican dry tropics.

The purpose of this paper is to evaluate the effect of five strains of Metarhizium anisopliae (Ma) and three strains of Isaria fumosorosea (Ifr) at a concentration of 1×10(8)colony-forming units/ml applied by spraying onto bovines with controlled infestation of Haematobia irritans under stable conditions in the Mexican dry tropics. Four experiments were performed, in each of which three treatments (two fungal strains and one control) were evaluated with eight repetitions for each one, by carrying out a single application of the aqueous suspension of each strain. The animals were isolated in individual cages and direct counts of the infestation were carried out for 13 days. It was observed that strains Ma2, Ma6, Ma10, Ma14, and Ma34 caused 94-100% reduction in infestation between days 12 and 13 post-treatment, while strains Ifr19, Ifr11, and Ifr12 reduced infestation from 90% to 98% up to day 13 post-application. There was an effect in the generation of horn flies from the excrement of bovines that were treated with different strains, reducing the reproduction of subsequent generations. It was concluded that the strains of M. anisopliae and I. fumosorosea evaluated in this study can be used as biocontrol agents in infestations of H. irritans in stabled bovines.

Acaricide and ivermectin resistance in a field population of Rhipicephalus microplus (Acari: Ixodidae) collected from red deer (Cervus elaphus) in the Mexican tropics.

In the Neotropics the control of tick infestations in red deer (Cervus elaphus) is achieved primarily through the use of acaricides and macrocyclic lactones. In Mexico, resistance to one or multiple classes of acaricides has been reported in Rhipicephalus microplus infesting cattle, but information on acaricide susceptibility in R. microplus infesting red deer is lacking. In this study we report the level of resistance to different classes of acaricides and ivermectin in R. microplus collected from red deer in the Mexican tropics. Engorged R. microplus females were collected from a red deer farm in Yucatan, Mexico. The larval packet test was used to detect resistance to the organophosphates (OPs) chlorpyrifos and coumaphos, synthetic pyrethroids (SPs) cypermethrin and permethrin, and the phenylpyrazol, fipronil. Resistance to the formamidine amitraz (Am), and ivermectin was ascertained using the larval immersion test. Data were subjected to probit analysis to determine lethal concentrations and resistance ratios to kill 50% (RR50) and 99% (RR99) of the tick population under evaluation in relation to susceptible reference strains. Additionally, allele specific polymerase chain reaction was used to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus. The R. microplus population from red deer in Yucatan showed very high resistance to the two SPs evaluated (RRs>72.2 for cypermethrin; RR for permethrin resistance was so high a dose-response curve was not possible). All individual larvae tested to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus were homozygous. The same tick population showed different levels of resistance to OPs (chlorpyrifos: RR50=1.55, RR99=0.63; coumaphos: RR50=6.8, RR99=5.9), fipronil (RR50=1.8, RR99=0.9), and amitraz (RR50=2.3, RR99=4.4). Resistance to ivermectin was regarded as moderate (RR50=7.1, RR99=5.0). This is the first report of R. microplus ticks collected from red deer in Mexico with different levels of resistance to four acaricide groups and ivermectin.

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics.

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.

Red deer (Cervus elaphus) as a host for the cattle tick Rhipicephalus microplus (Acari: Ixodidae) in Yucatan, Mexico.

Rhipicephalus microplus is the most economically important cattle tick in the Mexican tropics. Wild ungulate species, including red deer (Cervus elaphus), are gaining popularity in diversified livestock ranching operations in Mexico. However, there is no information available on the susceptibility of red deer to infestation with the cattle tick, R. microplus, under hot, subhumid tropical conditions in Mexico. Biological data on R. microplus as an ectoparasite of cattle and red deer in a farm in the Mexican tropics are presented here. Ticks collected from red deer were identified as R. microplus (97 %) and Amblyomma cajennense (3 %), and tick species infesting cattle included R. microplus (95 %) and A. cajennense (5 %). Standard counts of R. microplus engorged females on red deer were 11 times higher than on cattle (428 ± 43 vs. 40 ± 18; p < 0.001). The reproductive efficiency index and larval hatching of R. microplus collected from cattle and red deer were similar (p > 0.05). Hemolymph samples of R. microplus collected from cattle were positive for Babesia spp. (10 %, 2/50) and all the samples from ticks infesting red deer were negative. Seventeen and ten percent of the blood samples from cattle and red deer were positive for Anaplasma marginale, respectively. The role of red deer as a host of R. microplus in Yucatan, Mexico and the importance of this host-parasite relationship relative to the epidemiology of R. microplus-borne diseases are discussed.

Prevalence of cattle herds with ivermectin resistant nematodes in the hot sub-humid tropics of Mexico.

The objective was to determine the prevalence of semi-intensive Bos indicus and Bos indicus × Bos taurus cattle herds with ivermectin (IVM) resistant nematodes in a sub-humid tropical zone of Mexico using the faecal egg count reduction test (FECRT). Thirty-three herds (28 beef and 5 dual purpose herds) were monitored in a period of 6 months (September 2008 to February, 2009). Only 14 of the 33 herds were included in the trial. The other herds had not enough animals with sufficient nematode eggs per gram of faeces (EPG) to be included in a FECRT. Some farms were visited twice trying to find more animals with egg counts higher than 150 EPG. In the 14 surveyed herds the calves were randomly distributed into two groups: (a) treatment group received 0.2mg of IVM/kg BW sc on day 0, and (b) control group without treatment. Faecal samples were obtained from each animal on days 0 and 14 post-treatment. Reduction percentages (% R) and 95% CI were calculated. The prevalence of cattle herds with IVM resistant nematodes was 78.6%. Those suspected of IVM resistance were 21.4%. All surveyed herds used IVM from two to three times a year (mainly beginning and end of the wet season) during 1-11 consecutive years. The farm with stronger resistance used IVM for 11 consecutive years (% R=0%; 95% CI=0-47%). Genera of nematodes resistant to IVM were: Ostertagia, Haemonchus, Cooperia and Trichostrongylus. A considerable effort is needed to perform FECRT in cattle herds under hot sub-humid tropical conditions.

Laboratory and field evaluation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) for the control of Rhipicephalus microplus (Acari: Ixodidae) in the Mexican tropics.

The aim of the present study was to evaluate the effect of Metarhizium anisopliae to control Rhipicephalus microplus under laboratory and field conditions (larvae on vegetation) in the Mexican tropics. In the laboratory study, Ma34, Ma14 and a mixture of Ma34+Ma14 strains of M. anisopliae were evaluated for their control of the adult and larval stages of R. microplus, using the adult and larval immersion test respectively. The reproductive efficiency index of engorged females was determined in the treated and control groups. In the adults, the Ma34 and MA14 strains both produced an efficacy of 100% on engorged females at 1 x 10(8), 1 x 10(7) and 1 x 10(6)conidia/ml; however, only Ma34 killed 100% at 1 x 10(6)conidia/ml dose. The mixture of both strains (Ma34+Ma14) produced an efficacy of 100% on engorged females at 1 x 10(8)conidia/ml. R. microplus engorged females treated with Ma34 and a mixture of strains Ma34 and Ma14 reduced egg oviposition by 55.5% and 39.1% respectively compared to treated controls (P<0.001). In the larval evaluation, Ma14 produced an efficacy of 45-62%; however, Ma34+Ma14 increased the efficacy reaching 90% (1 x 10(8)conidia/ml concentrations, P<0.05). In the field study, twelve 9 m(2) plots with vegetation were artificially infested with R. microplus larvae. Six plots were used as control and six as test areas. The treated plots received the Ma34+Ma14 (1 x 10(8)conidia/ml) by manual scattering at 0, 14 and 28 days post-treatment (PT). The number of larvae in the treated and control group was determined at 0, 7, 14, 21, 28 and 35 days PT by the tick drag method. This field study was carried out in two different seasons (January-March, wet, April-May, dry). In the wet season trial the efficacy of M. anisopliae to control R. microplus was 67.7% and 100% in the dry season trial .In conclusion, the mixture of Ma34 and Ma14 strains of M. anisopliae showed a high efficacy to control both larval and adult stages of R. microplus under laboratory conditions, and in field conditions these strains were efficient to control larval stages on vegetation.

Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53.

Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. To test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apoptosis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo studies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control glucose conditions. Blastocysts from p53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53+/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.

High insulin-like growth factor 1 (IGF-1) and insulin concentrations trigger apoptosis in the mouse blastocyst via down-regulation of the IGF-1 receptor.

Women with polycystic ovary syndrome have significantly higher rates of pregnancy loss, as well as elevated insulin and IGF-1 levels. In this study, preimplantation embryos exposed to high concentrations of IGF-1 or insulin undergo extensive apoptosis of the ICM nuclei. Lack of BAX expression, the caspase inhibitor, zVAD, or the ceramide synthase inhibitor, fumonisin B1, prevents this event, suggesting involvement of programmed cell death effector pathways. In other systems, the IGF-1 concentration regulates IGF-1R expression and thus high concentrations lead to down-regulation of the receptor. Here, data show a decrease in IGF-1 receptor protein expression, both by confocal immunofluorescent microscopy and by Western analysis upon exposure to 130 nM IGF-1. Insulin-stimulated glucose uptake, an event regulated via the IGF-1 receptor, is decreased upon exposure to excess IGF-1, suggesting decreased function of the receptor. The data also show that, by blocking receptor signal transduction or by decreasing receptor expression, the apoptotic event can be recreated, thus strongly suggesting that the mechanism of high IGF-1 induced apoptosis is decreased downstream IGF-1 receptor signaling. This embryotoxic insult by high IGF-1 levels may be responsible for the high incidence of pregnancy loss seen in women with polycystic ovary syndrome.

Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst.

We report that a decrease in facilitative glucose transporter (GLUT1) expression and reduced glucose transport trigger apoptosis in the murine blastocyst. Inhibition of GLUT1 expression either by high glucose conditions or with antisense oligodeoxynucleotides significantly lowers protein expression and function of GLUT1 and as a result induces a high rate of apoptosis at the blastocyst stage. Similar to wild-type mice, embryos from streptozotocin-induced diabetic Bax -/- mice experienced a significant decrease in glucose transport compared with embryos from non-diabetic Bax -/- mice. However, despite this decrease, these blastocysts demonstrate significantly fewer apoptotic nuclei as compared with blastocysts from hyperglycemic wild-type mice. This decrease in preimplantation apoptosis correlates with a decrease in resorptions and malformations among the infants of the hyperglycemic Bax -/- mice versus the Bax +/+ and +/- mice. These findings suggest that hyperglycemia by decreasing glucose transport acts as a cell death signal to trigger a BAX-dependent apoptotic cascade in the murine blastocyst. This work also supports the hypothesis that increased apoptosis at a blastocyst stage because of maternal hyperglycemia may result in loss of key progenitor cells and manifest as a resorption or malformation, two adverse pregnancy outcomes more common in diabetic women.

GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst.

Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits approximately 20-25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process that is inhibited by antisense oligoprobes. Presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. Moreover, the existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of GLUT4.

Myogenin induces a shift of enzyme activity from glycolytic to oxidative metabolism in muscles of transgenic mice.

Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3-0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle.

Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways.

Although perinatal mortality rates have improved for pregnant diabetic women because of insulin therapy and tight metabolic control, infants of diabetics still experience significantly higher rates of congenital malformations and spontaneous miscarriages compared with those of non-diabetic women. Our results here indicate that hyperglycemic conditions, either in vivo or in vitro, modulate the expression of an apoptosis regulatory gene as early as the pre-implantation blastocyst stage in the mouse. Apoptosis in the mammalian pre-implantation blastocyst is a normal process, thought to protect the early embryo by eliminating abnormal cells. Here we demonstrate that expression of Bax, a Bcl-2-like protein, is increased at the blastocyst stage in the presence of high concentrations of glucose, and that these changes correlate morphologically with increased DNA fragmentation. Expression of Bax and caspase are necessary for this in vitro glucose-induced apoptotic event, and ceramide is involved in mediating this embryotoxic effect of glucose. We also show that these apoptotic cellular changes can be prevented in vivo by treating hyperglycemic mice with insulin before and immediately after conception. These findings emphasize the importance of tight glycemic control in diabetic women at the earliest stages after conception.

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP.

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.

Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos.

Glucose utilization was studied in preimplantation embryos from normal and diabetic mice. With use of ultramicrofluorometric enzyme assays, intraembryonic free glucose in single embryos recovered from control and streptozotocin-induced hyperglycemic mice was measured at 24, 48, 72, and 96 h after mating. Free glucose concentrations dropped significantly in diabetics at 48 and 96 h, corresponding to the two-cell and blastocyst stages (48 h: diabetic 0.23 +/- 0.09 vs. control 2.30 +/- 0.43 mmol/kg wet wt; P < 0.001; 96 h: diabetic 0.31 +/- 0.29 vs. control 5.12 +/- 0.17 mmol/kg wet wt; P < 0.001). Hexokinase activity was not significantly different in the same groups. Transport was then compared using nonradioactive 2-deoxyglucose uptake and microfluorometric enzyme assays. The 2-deoxyglucose uptake was significantly lower at both 48 and 96 h in embryos from diabetic vs. control mice (48 h diabetic, 0.037 +/- 0. 003; control, 0.091 +/- 0.021 mmol . kg wet wt-1 . 10 min-1, P < 0. 05; 96 h diabetic, 0.249 +/- 0.008; control, 0.389 +/- 0.007 mmol . kg wet wt-1 . 10 min-1, P < 0.02). When competitive quantitative reverse transcription-polymerase chain reaction was used, there was 44 and 68% reduction in the GLUT-1 mRNA at 48 h (P < 0.001) and 96 h (P < 0.05), respectively, in diabetic vs. control mice. GLUT-2 and GLUT-3 mRNA values were decreased 63 and 77%, respectively (P < 0.01, P < 0.01) at 96 h. Quantitative immunofluorescence microscopy demonstrated 49 +/- 6 and 66 +/- 4% less GLUT-1 protein at 48 and 96 h and 90 +/- 5 and 84 +/- 6% less GLUT-2 and -3 protein, respectively, at 96 h in diabetic embryos. These findings suggest that, in response to a maternal diabetic state, preimplantation mouse embryos experience a decrease in glucose utilization directly related to a decrease in glucose transport at both the mRNA and protein levels.

Effect of centrifugation at 2G for 14 days on metabolic enzymes of the tibialis anterior and soleus muscles.

BACKGROUND: The enzyme composition of different muscle types vary greatly, leading to different changes of enzyme level caused by exposure to various stimuli. METHODS: Male Wistar rats were centrifuged at 2G in a 12-ft radius centrifuge for 14 d. Tibialis anterior (TA) and soleus muscles from four centrifuge and four control rats were analyzed for three enzymes characteristic of fast twitch muscles (phosphofructokinase, glycerol-3-phosphate dehydrogenase, and pyruvate kinase), and four enzymes characteristic of slow twitch muscles (hexokinase, mitochondrial thiolase, B-hydroxyacyl CoA dehydrogenase, and citrate synthase). RESULTS: The centrifuged TA muscles lost 15% of their weight; the corresponding soleus muscles gained 4%. Calculated on the basis of dry weight, the fast twitch enzyme activities were reduced 3-15% in the TA muscles but increased 10-23% in the soleus muscles. The slow twitch enzymes were reduced 18-30% in TA muscles but were almost unchanged in the soleus muscles. When calculated on the basis of total muscle weight, all of the enzymes in TA muscles were significantly reduced by centrifugation. In contrast, in soleus muscles, on the basis of total muscle weight, centrifugation caused an average increase of 22% in the fast twitch enzymes but only marginal changes in the slow twitch enzymes.