👤 Anisley Martinez

Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) and its substrates (extracellular signal-regulated kinase [ERK] and p38) play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT) and MKP-3 knockout (KO) mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2) inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08 g day 1), which resolved normally by postoperative day 12 (0.46±0.08 g, N=6). In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6). KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm Show less

A high intake of n-3 PUFA provides health benefits via changes in the n-6/n-3 ratio in blood. In addition to such dietary PUFAs, variants in the fatty acid desaturase 1 (FADS1) gene are also associated with altered PUFA profiles. We used mathematical modeling to predict levels of PUFA in whole blood, based on multiple hypothesis testing and bootstrapped LASSO selected food items, anthropometric and lifestyle factors, and the rs174546 genotypes in FADS1 from 1607 participants (Food4Me Study). The models were developed using data from the first reported time point (training set) and their predictive power was evaluated using data from the last reported time point (test set). Among other food items, fish, pizza, chicken, and cereals were identified as being associated with the PUFA profiles. Using these food items and the rs174546 genotypes as predictors, models explained 26-43% of the variability in PUFA concentrations in the training set and 22-33% in the test set. Selecting food items using multiple hypothesis testing is a valuable contribution to determine predictors, as our models' predictive power is higher compared to analogue studies. As unique feature, we additionally confirmed our models' power based on a test set. Show less

The mechanisms that drive the normal resolution of acute postoperative pain are not completely understood. We hypothesize a pivotal role of a major spinal mitogen-activated protein kinase (MAPKs) regulator, MAPK phosphatase (MKP)-3, in the resolution of postoperative pain. We used wild-type and MKP-3 knock-out (KO) mice, a paw incision model of acute postoperative pain, and behavioral and molecular biology experiments. We observed persistent mechanical allodynia in mice lacking MKP-3 (postoperative day 21), concurrently with persistent phosphorylation of spinal p38 and extracellular signal-regulated kinases (ERK)-1/2 on postoperative day 12, while both MAPK phosphorylation and allodynia resolved on postoperative day 7 in wild-type mice. Spinal p-ERK was expressed mainly in neurons and microglia, while spinal p-p38 was expressed mostly in microglia in MKP-3 KO mice, and their selective pharmacological inhibition reduced the persistent allodynia observed in these mice. Our findings strongly suggest that dysregulation of MKP-3 prevents spontaneous resolution of acute postoperative pain and drives its transition to persistent pain via persistent neuronal and microglial MAPK phosphorylation in the spinal cord. Show less

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function. Show less

Severe hypertriglyceridemia (HTG) has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis) were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family. We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel). Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives. A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter) found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls. The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family. Show less

Peripheral nerve injury generally results in spinal neuronal and glial plastic changes associated with chronic behavioral hypersensitivity. Spinal mitogen-activated protein kinases (MAPKs), eg, p38 or extracellular signal-regulated kinases (ERKs), are instrumental in the development of chronic allodynia in rodents, and new p38 inhibitors have shown potential in acute and neuropathic pain patients. We have previously shown that the cannabinoid type 2 receptor agonist JWH015 inhibits ERK activity by inducing MAPK phosphatase (MKP)-1 and MKP-3 (the major regulators of MAPKs) in vitro in microglial cells. Therefore, we decided to investigate the role of these phosphatases in the mechanisms of action of JWH015 in vivo using the rat L5 nerve transection model of neuropathic pain. We observed that peripheral nerve injury reduced spinal MKP-1/3 expression and activity and that intrathecal JWH015 reduced established L5 nerve injury-induced allodynia, enhanced spinal MKP-1/3 expression and activity, and reduced the phosphorylated form of p38 and ERK-1/2. Triptolide, a pharmacological blocker of MKP-1 and MKP-3 expression, inhibited JWH015's effects, suggesting that JWH015 exerts its antinociceptive effects by modulating MKP-1 and MKP-3. JWH015-induced antinociception and MKP-1 and MKP-3 expression were inhibited by the cannabinoid type 2 receptor antagonist AM630. Our data suggest that MKP-1 and MKP-3 are potential targets for novel analgesic drugs. MAPKs are pivotal in the development of chronic allodynia in rodent models of neuropathic pain. A cannabinoid type 2 receptor agonist, JWH015, reduced neuropathic allodynia in rats by reducing MAPK phosphorylation and inducing spinal MAPK phosphatases 1 and 3, the major regulators of MAPKs. Show less

Differences in the interindividual response to dietary intervention could be modified by genetic variation in nutrient-sensitive genes. This study examined single nucleotide polymorphisms (SNPs) in presumed nutrient-sensitive candidate genes for obesity and obesity-related diseases for main and dietary interaction effects on weight, waist circumference, and fat mass regain over 6 mo. In total, 742 participants who had lost ≥ 8% of their initial body weight were randomly assigned to follow 1 of 5 different ad libitum diets with different glycemic indexes and contents of dietary protein. The SNP main and SNP-diet interaction effects were analyzed by using linear regression models, corrected for multiple testing by using Bonferroni correction and evaluated by using quantile-quantile (Q-Q) plots. After correction for multiple testing, none of the SNPs were significantly associated with weight, waist circumference, or fat mass regain. Q-Q plots showed that ALOX5AP rs4769873 showed a higher observed than predicted P value for the association with less waist circumference regain over 6 mo (-3.1 cm/allele; 95% CI: -4.6, -1.6; P/Bonferroni-corrected P = 0.000039/0.076), independently of diet. Additional associations were identified by using Q-Q plots for SNPs in ALOX5AP, TNF, and KCNJ11 for main effects; in LPL and TUB for glycemic index interaction effects on waist circumference regain; in GHRL, CCK, MLXIPL, and LEPR on weight; in PPARC1A, PCK2, ALOX5AP, PYY, and ADRB3 on waist circumference; and in PPARD, FABP1, PLAUR, and LPIN1 on fat mass regain for dietary protein interaction. The observed effects of SNP-diet interactions on weight, waist, and fat mass regain suggest that genetic variation in nutrient-sensitive genes can modify the response to diet. This trial was registered at clinicaltrials.gov as NCT00390637. Show less

Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [(14)C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [(14)C]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 μM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7-12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA. Show less

For patients with diabetes, insulin resistance and hyperglycemia both contribute to increased serum triglyceride in the form of very low-density lipoprotein (VLDL). Our objective was to define the insulin conditions in which hyperglycemia promotes increased serum VLDL in vivo. We performed hyperglycemic-hyperinsulinemic clamp studies and hyperglycemic-hypoinsulinemic clamp studies in rats, with metabolic tracers for glucose flux and de novo fatty acid synthesis. When blood glucose was clamped at hyperglycemia (17 mm) for 2 h under hyperinsulinemic conditions (4 mU/kg . min), serum VLDL levels were not increased compared with baseline. We speculated that hyperinsulinemia minimized glucose-mediated VLDL changes and performed hyperglycemic-hypoinsulinemic clamp studies in which insulin was clamped near fasting levels with somatostatin (17 mm blood glucose, 0.25 mU/kg . min insulin). Under low-insulin conditions, serum VLDL levels were increased 4.7-fold after hyperglycemia, and forkhead box O1 (FoxO1) was not excluded from the nucleus of liver cells. We tested the extent that impaired inactivation of FoxO1 by insulin was sufficient for glucose to promote increased serum VLDL. We found that, when the ability of insulin to inactivate FoxO1 is blocked after adenoviral delivery of constitutively active FoxO1, glucose increased serum VLDL triglyceride when given both by ip glucose tolerance testing (3.5-fold increase) and by a hyperglycemic clamp (4.6-fold). Under both experimental conditions in which insulin signaling to FoxO1 was impaired, we found increased activation of carbohydrate response element binding protein. These data suggest that glucose more potently promotes increased serum VLDL when insulin action is impaired, with either low insulin levels or disrupted downstream signaling to the transcription factor FoxO1. Show less

Habenular nuclei play a key role in the control of motor and cognitive behavior, processing emotion, motivation, and reward values in the brain. Thus, analysis of the molecular and cellular mechanisms underlying the development and evolution of this region will contribute to a better understanding of brain function. The Fgf8 gene is expressed in the dorsal midline of the diencephalon, close to the area in which the habenular region will develop. Given that Fgf8 is an important morphogenetic signal, we decided to investigate the role of Fgf8 signaling in diencephalic development. To this end, we analyzed the effects of altered Fgf8 expression in the mouse embryo, using molecular and cellular markers. Decreasing Fgf8 activity in the diencephalon was found to be associated with dosage-dependent alterations in the epithalamus: the habenular region and pineal gland are reduced or lacking in Fgf8 hypomorphic mice. Actually, our findings indicate that Fgf8 may be the master gene for these diencephalic domains, acting as an inductive and morphogenetic regulator. Therefore, the emergence of the habenular region in vertebrates could be understood in terms of a phylogenetic territorial addition caused by de novo expression of Fgf8 in the diencephalic alar plate. This region specializes to permit the development of adaptive control of the motor function in the vertebrate brain. Show less

The effect of a common apolipoprotein (apo) A-IV polymorphism (substitution of histidine for glutamine at position 360) on plasma lipid, lipoprotein cholesterol and lipoprotein(a) (Lp(a)) levels, and on low-density lipoprotein (LDL) particle size was examined by genotyping in 2322 Caucasian men and women (mean age: 48.9+/-10.1 years) participating in the Framingham Offspring Study (FOS). The relative frequencies of the apo A-IV-Gln (apo A-IV-1) and the apo A-IV-His (apo A-IV-2) alleles were 0.932 and 0.068, respectively, and were in Hardy-Weinberg equilibrium. No effect of the apo A-IV-2 genotype was observed on plasma triglyceride, total and lipoprotein cholesterol, and LDL particle size in either men or women after adjustment for age and body mass index. To avoid a possible interaction between the apo E genotype and the apo A-IV genotype, subgroup analyses were undertaken in 1,414 male and female subjects with the apo E3/3 genotype. Among women in this group there was a significant effect of the apo A-IV-2 allele on triglyceride levels (p=0.046). This effect was no longer significant after adjustment for age and BMI (p=0.074). No significant allele effect on other lipoprotein levels, including Lp(a), was noted in apo E3/3 men or women. We have also conducted a meta-analysis of our own data and of other studies found in the literature, indicating a significant lowering effect of apo A-IV-2 on plasma triglycerides, but no effects on other parameters. In conclusion, the apo A-IV-2 allele is associated with a modest reduction in plasma triglyceride levels in the general population. Show less

Single-nucleotide polymorphisms in genes involved in lipoprotein and adipocyte metabolism may explain why dyslipidemia and lipoatrophy occur in some but not all antiretroviral therapy (ART)-treated individuals. We evaluated the contribution of APOC3 -482C-->T, -455T-->C, and 3238C-->G; epsilon 2 and epsilon 4 alleles of APOE; and TNF -238G-->A to dyslipidemia and lipoatrophy by longitudinally modeling >2600 lipid determinations and 2328 lipoatrophy assessments in 329 ART-treated patients during a median follow-up period of 3.4 years. In human immunodeficiency virus (HIV)-infected individuals, the effects of variant alleles of APOE on plasma cholesterol and triglyceride levels and of APOC3 on plasma triglyceride levels were comparable to those reported in the general population. However, when treated with ritonavir, individuals with unfavorable genotypes of APOC3 and [corrected] APOE were at risk of extreme hypertriglyceridemia. They had median plasma triglyceride levels of 7.33 mmol/L, compared with 3.08 mmol/L in the absence of ART. The net effect of the APOE*APOC3*ritonavir interaction was an increase in plasma triglyceride levels of 2.23 mmol/L. No association between TNF -238G-->A and lipoatrophy was observed. Variant alleles of APOE and APOC3 contribute to an unfavorable lipid profile in patients with HIV. Interactions between genotypes and ART can lead to severe hyperlipidemia. Genetic analysis may identify patients at high risk for severe ritonavir-associated hypertriglyceridemia. Show less

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium. Show less

Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder most frequently caused by mutations in the EXT1 gene. MHE affects proper development of endochondral bones, such that all affected individuals present with exostoses adjacent to the growth plate of long bones, while some individuals exhibit additional bone deformities. EXT1 functions as a heparan sulfate (HS) co-polymerase, and when defective causes improper elongation of glycosaminoglycan side chains on core proteins of HS proteoglycans. Although analysis of heterozygous EXT1-deficient mice has failed to reveal any significant gross morphological variations in skeletal development, significant alterations in molecular signaling occur in the developing long bones. Our results indicate that defects in EXT1 and the resulting reduction in HS lead to enhanced Indian Hedgehog diffusion causing an increase in chondrocyte proliferation and delayed hypertrophic differentiation. Show less