An orally available compound suppresses glucagon hypersecretion and normalizes hyperglycemia in type 1 diabetes.

Suppression of glucagon hypersecretion can normalize hyperglycemia during type 1 diabetes (T1D). Activating erythropoietin-producing human hepatocellular receptor type-A4 (EphA4) on α cells reduced glucagon hypersecretion from dispersed α cells and T1D islets from both human donor and mouse models. We synthesized a high-affinity small molecule agonist for the EphA4 receptor, WCDD301, which showed robust plasma and liver microsome metabolic stability in both mouse and human preparations. In islets and dispersed islet cells from nondiabetic and T1D human donors, WCDD301 reduced glucagon secretion comparable to the natural EphA4 ligand, Ephrin-A5. In diabetic NOD and streptozotocin-treated mice, once-daily oral administration of WCDD301 formulated with a time-release excipient reduced plasma glucagon and normalized blood glucose for more than 3 months. These results suggest that targeting the α cell EphA4 receptor by sustained release of WCDD301 is a promising pharmacologic pathway for normalizing hyperglycemia in patients with T1D.

Sex-dependent Effect of In-utero Exposure to Δ9-Tetrahydrocannabinol on Glucagon and Stathmin-2 in Adult Rat Offspring.

OBJECTIVES: Administration of Δ9-tetrahydrocannabinol (Δ9-THC) to pregnant rats results in glucose intolerance, insulin resistance and reduced islet mass in female, but not male, offspring. The effects of Δ9-THC on other islet hormones is not known. One downstream target of the cannabinoid receptor, stathmin-2 (Stmn2), has recently been shown to suppress glucagon secretion, thereby suggesting Δ9-THC may also affect alpha-cell function. The aim of the present study was to determine the effects of in-utero Δ9-THC exposure on the profile of glucagon, insulin and Stmn2 in the rat offspring islet and serum. METHODS: Pregnant Wistar rat dams were injected with Δ9-THC (3 mg/kg per day, intraperitoneally) or vehicle from gestational day 6 to birth. Offspring were euthanized at postnatal day 21 (PND21) or at 5 months (adult) to collect blood and pancreata. RESULTS: At PND21, control and Δ9-THC-exposed offspring showed that Stmn2 had a strong colocalization with glucagon (Pearson's correlation coefficient ≥0.6), and a weak colocalization with insulin (Pearson's correlation coefficient <0.4) in both males and females, with no changes by either treatment or sex. In adult female offspring in the Δ9-THC group, intensity analysis indicated an increased insulin-to-glucagon (I/G; p<0.05) ratio and a decreased glucagon-to-Stmn2 (G/S; p<0.01) ratio, and no changes in these ratios in adult males. Furthermore, Δ9-THC did not alter fasting blood glucose and serum insulin levels in either male or female adult offspring. However, female Δ9-THC-exposed offspring exhibited an increased I/G ratio (p<0.05) and decreased G/S ratio in serum by adulthood (p<0.05). CONCLUSION: Collectively, the reduced G/S ratio in both islet and serum in association with an increased serum I/G ratio has direct correlations with early glucose intolerance and insulin resistance observed exclusively in females' offspring in this prenatal cannabinoid model.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Misrouting of glucagon and stathmin-2 towards lysosomal system of α-cells in glucagon hypersecretion of diabetes.

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as an α-cell protein that regulates glucagon secretion by directing glucagon toward the endolysosomal system in αTC1-6 cells. We hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes in diabetes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ), glucagon secretion and cellular content were augmented, but cellular Stmn2 levels were reduced (p < .01), as measured by both ELISA and immunofluorescence intensity. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p < .001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p < .01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for 2 weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome toward the secretory Lamp1+ lysosome.

Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia.

Patients with diabetes mellitus exhibit hyperglucagonemia, or excess glucagon secretion, which may be the underlying cause of the hyperglycemia of diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may drive the development of hyperglucagonemia. Therefore, uncovering the mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus.

Addition of olive oil to diet of rats with mild pre-gestational diabetes impacts offspring ß-cell development.

Maternal diabetes impairs fetal development and increases the risk of metabolic diseases in the offspring. Previously, we demonstrated that maternal dietary supplementation with 6% of olive oil prevents diabetes-induced embryo and fetal defects, in part, through the activation of peroxisome proliferator-activated receptors (PPARs). In this study, we examined the effects of this diet on neonatal and adult pancreatic development in male and female offspring of mothers affected with pre-gestational diabetes. A mild diabetic model was developed by injecting neonatal rats with streptozotocin (90 mg/kg). During pregnancy, these dams were fed a chow diet supplemented or not with 6% olive oil. Offspring pancreata was examined at day 2 and 5 months of age by immunohistochemistry followed by morphometric analysis to determine number of islets, α and ß cell clusters and ß-cell mass. At 5 months, male offspring of diabetic mothers had reduced ß-cell mass that was prevented by maternal supplementation with olive oil. PPARα and PPARγ were localized mainly in α cells and PPARß/δ in both α and ß cells. Although Pparß/δ and Pparγ RNA expression showed reduction in 5-month-old male offspring of diabetic rats, Pparß/δ expression returned to control levels after olive-oil supplementation. Interestingly, in vitro exposure to oleic acid (major component of olive oil) and natural PPAR agonists such as LTB4, CPC and 15dPGJ2 also significantly increased expression of all Ppars in αTC1-6 cells. However, only oleic acid and 15dPGJ2 increased insulin and Pdx-1 expression in INS-1E cells suggesting a protective role in ß-cells. Olive oil may be considered a dietary supplement to improve islet function in offspring of affected mothers with pre-gestational diabetes.

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA-mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells.

Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-specific proteins. Isolated enriched secretory granules were then used for affinity purification with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was confirmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunofluorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon "interactome."

Latrunculin A-Induced Perturbation of the Actin Cytoskeleton Mediates Pap1p-Dependent Induction of the Caf5p Efflux Pump in Schizosaccharomyces pombe.

As part of an earlier study aimed at uncovering gene products with roles in defending against latrunculin A (LatA)-induced cytoskeletal perturbations, we identified three members of the oxidative stress response pathway: the Pap1p AP-1-like transcription factor, the Imp1p α-importin, and the Caf5p efflux pump. In this report, we characterize the pathway further and show that Pap1p translocates from the cytoplasm to the nucleus in an Imp1p-dependent manner upon LatA treatment. Moreover, preventing this translocation, through the addition of a nuclear export signal (NES), confers the same characteristic LatA-sensitive phenotype exhibited by pap1Δ cells. Lastly, we show that the caf5 gene is induced upon exposure to LatA and that Pap1p is required for this transcriptional upregulation. Importantly, the expression of trr1, a Pap1p target specifically induced in response to oxidative stress, is not significantly altered by LatA treatment. Taken together, these results suggest a model in which LatA-mediated cytoskeletal perturbations are sensed, triggering the Imp1p-dependent translocation of Pap1p to the nucleus and the induction of the caf5 gene (independently of oxidative stress).

A Genetic Screen for Fission Yeast Gene Deletion Mutants Exhibiting Hypersensitivity to Latrunculin A.

Fission yeast cells treated with low doses of the actin depolymerizing drug, latrunculin A (LatA), delay entry into mitosis via a mechanism that is dependent on both the Clp1p and Rad24p proteins. During this delay, cells remain in a cytokinesis-competent state that is characterized by continuous repair and/or reestablishment of the actomyosin ring. In this manner, cells ensure the faithful completion of the preceding cytokinesis in response to perturbation of the cell division machinery. To uncover other genes with a role in this response, or simply genes with roles in adapting to LatA-induced stress, we carried out a genome-wide screen and identified a group of 38 gene deletion mutants that are hyper-sensitive to the drug. As expected, we found genes affecting cytokinesis and/or the actin cytoskeleton within this set (ain1, acp2, imp2). We also identified genes with roles in histone modification (tra1, ngg1), intracellular transport (apl5, aps3), and glucose-mediated signaling (git3, git5, git11, pka1, cgs2). Importantly, while the identified gene deletion mutants are prone to cytokinesis failure in the presence of LatA, they are nevertheless fully capable of cell division in the absence of the drug. These results indicate that fission yeast cells make use of a diverse set of regulatory modules to counter abnormal cytoskeletal perturbations, and furthermore, that these modules act redundantly to ensure cell survival and proliferation.

Treatment of clinical endometritis in dairy cows by previously used controlled internal drug release devices.

Postpartum endometritis is considered as one of the diseases that lead to a potential profit reduction in dairy cows. The aims of the present study were to promote follicle growth by a previously used controlled internal drug release (CIDR) device and to evaluate its effect on the likelihood of recovery and the reproductive performance of clinical endometritis (CE) cows. Endometritis was diagnosed using ultrasonographic examination at 31 ± 3 (Day 0 of the experiment) days in milk, and CE cows were included in one of the three experimental groups according to the presence of a CL on their ovaries. Cows without CL on their ovaries received a reused CIDR device, which was previously used for 14 days (CIDR-14, n = 108), or PGF2α (PG-1, n = 112) on Day 0. In the third group, those with CL on their ovaries received PGF2α (PG-2, n = 107) at the same time. Ovarian structures, serum estradiol and progesterone concentrations were measured on Days 0, 7, and 14. Controlled internal drug release devices were removed, and response to treatment was evaluated in all treated cows on Day 14. Diameters of ovarian follicles were 11.61 ± 0.50, 12.46 ± 0.25, and 18.36 ± 0.60 mm on Day 7 and 11.63 ± 0.58, 14.35 ± 0.40, and 21.96 ± 0.77 mm on Day 14 in PG-1, PG-2, and CIDR-14 cows, respectively (P < 0.05). Serum estradiol concentrations were higher in CIDR-14 cows (141.17 ± 1.04 pg/mL) than in PG-1 (116.85 ± 1.05 pg/mL) and PG-2 (119.10 ± 1.05 pg/mL) cows on Day 7 (P < 0.05). Higher progesterone concentrations were observed in PG-2 cows than in PG-1 and CIDR-14 cows on Days 0, 7, and 14 (P < 0.001). The likelihood of clinical cure was 54.46%, 62.61%, and 64.81% in PG-1, PG-2, and CIDR-14 cows, respectively (P = 0.11). First-service conception risk, days to the first service, calving to conception interval, proportion of cows bred and pregnant by 120 days in milk did not differ among the treated groups (P > 0.05). The cumulative pregnancy risk was lower in PG-1 (77.67%) cows than in CIDR-14 (87.07%) and PG-2 (87.85%) cows (P = 0.02). In conclusion, reused CIDR would be contributed to the treatment of CE by promotion of follicle growth and induction of sustainable sources of endogenic estrogen secreted by the dominant follicle.

Selection of potential therapeutic human single-chain Fv antibodies against cholecystokinin-B/gastrin receptor by phage display technology.

BACKGROUND AND OBJECTIVE: Gastric/gastrointestinal cancers are associated with high mortality worldwide. G-protein coupled receptor (GPCR) superfamily members such as gastrin/cholecystokinin-B receptor (CCK-BR) are involved in progression of gastric tumors, thus CCK-BR is considered as a potential target for immunotherapy. However, production of functional monoclonal antibodies (mAbs) against GPCR seems to be very challenging, in part due to its integration in cell membranes and inaccessibility for selection. To tackle this problem, we implemented phage display technology and a solution-phase biopanning (SPB) scheme for production of mAbs specific to the native conformation of CCK-BR. METHODS: To perform the SPB process, we utilized a synthetic biotinylated peptide corresponding to the second extracellular loop (ECL2) of CCK-BR and a semi-synthetic phage antibody library. After enzyme-linked immunosorbent assay (ELISA) screening, the CCK-BR specificity of the selected single-chain variable fragments (scFvs) were further examined using immunoblotting, whole-cell ELISA, and flow cytometry assays. RESULTS: After performing four rounds of selection, we identified nine antibody clones which showed positive reactivity with the CCK-BR peptide in an ELISA assay. Of these, eight clones were unique scFv antibodies and one was a V(L) single domain antibody. Specificity analysis of the selected scFvs revealed that five of the selected scFvs recognized a denatured form of CCK-BR, while the majority of the selected scFvs were able to recognize the native conformation of CCK-BR on the surface of human gastric adenocarcinoma cells and cervical carcinoma HeLa cells. CONCLUSION: For the first time, we report on the establishment of a diverse panel of scFv antibody fragments that are specific to the native conformation of CCK-BR. Based on these results, we suggest the selected scFv antibody fragments as potential agents for diagnosis, imaging, targeting, and/or immunotherapy of cancers that overexpress CCK-BR.

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Comparative evaluation of two reconstructive methods following laparoscopic assisted subtotal gastrectomy in dogs.

BACKGROUND: Laparoscopic gastrectomy is a new and technically challenging surgical procedure with potential benefit. The objective of this study was to investigate clinical and para-clinical consequences following Roux-en-Y and Jejunal Loop interposition reconstructive techniques for subtotal gastrectomy using laparoscopic assisted surgery. RESULTS: Following resection of the stomach attachments through a laparoscopic approach, stomach was removed and reconstruction was performed with either standard Roux-en-Y (n = 5) or Jejunal Loop interposition (n = 5) methods. Weight changes were monitored on a daily basis and blood samples were collected on Days 0, 7 and 21 post surgery. A fecal sample was collected on Day 28 after surgery to evaluate fat content. One month post surgery, positive contrast radiography was conducted at 5, 10, 20, 40, 60 and 90 minutes after oral administration of barium sulfate, to evaluate the postoperative complications. There was a gradual decline in body weight in both experimental groups after surgery (P < 0.05). There was no difference in blood parameters at any time after surgery between the two methods (P > 0.05). Fecal fat content increased in the Roux-en-Y compared to the Jejunal loop interposition technique (P < 0.05). No major complications were found in radiographs and gastric emptying time was similar between the two groups (P > 0.05). CONCLUSION: Roux-en-Y and Jejunal loop interposition techniques might be considered as suitable approaches for reconstructing gastro-intestinal tract following gastrectomy in dogs. The results of this study warrant further investigation with a larger number of animals.

Evaluation of the correlation between serum biochemical values and liver ultrasonographic indices in periparturient cows with different body condition scores.

OBJECTIVE: To determine alterations of serum biochemical variables in relation to changes of near- and far-field mean grayscale histogram (MGSH) and attenuation rates in liver ultrasonograms of periparturient cows. ANIMALS: 67 Holstein cows. PROCEDURES: Cows were allocated on the basis of body condition score into underconditioned (n = 21), moderately conditioned (23), and overconditioned (23) groups. Serum samples (obtained every 10 days from 30 days before to 30 days after calving) were analyzed for aspartate aminotransferase, alanine aminotransferase, and γ-glutamyltransferase activities and BUN, albumin, calcium, and inorganic phosphorus concentrations along with digital estimation of near- and far-field MGSH values of liver ultrasonograms and deep attenuation. Values were compared among groups and within each group, and their correlations were determined in the pre- and postpartum periods. RESULTS: Serum biochemical variables did not differ significantly among groups. Aspartate aminotransferase and γ-glutamyltransferase activities increased in the postpartum period. Fluctuations of alanine aminotransferase activity were not significant; BUN decreased significantly in the peripartum period. Albumin concentration decreased prior to parturition and remained low, but significantly increased after parturition. Calcium concentration decreased on day 10 but subsequently increased. Phosphorus concentration decreased stepwise until day 10 after calving. Postpartum biochemical variables had weak correlations with near- and far-field MGSH values in overconditioned cows. The highest levels of sound attenuation were found in overconditioned cows on calving day. CONCLUSIONS AND CLINICAL RELEVANCE: Liver ultrasonographic features were poorly correlated with changes of serum biochemical variables. This suggests that liver ultrasonography is not a good technique for estimating functional liver abnormalities in periparturient cows.

Molecular considerations for development of phage antibody libraries.

Nowadays, phage display libraries are used as robust tools for discovery and evolution of peptide/protein based drugs as well as targeting molecules, in particular monoclonal antibodies (mAbs) and its fragments (i.e., scFvs, Fabs, or bivalent F(ab')2). Phage display technology, as a molecular diversity approach, enables selection of antibody fragments (e.g., scFv/Fab) with high affinity, specificity and effector functions against various targets. However, such selection process itself is largely dependent upon various molecular factors such as methods for construction of phage library, phage/phagemid vectors, helper phage, host cells and biopanning processes. The current review article provides important molecular considerations for successful development of phage antibody libraries that may be used as a platform for translation of antibody fragments into viable diagnostic/therapeutic reagents.

Palmitate-activated macrophages confer insulin resistance to muscle cells by a mechanism involving protein kinase C θ and ε.

BACKGROUND: Macrophage-derived factors contribute to whole-body insulin resistance, partly by impinging on metabolically active tissues. As proof of principle for this interaction, conditioned medium from macrophages treated with palmitate (CM-PA) reduces insulin action and glucose uptake in muscle cells. However, the mechanism whereby CM-PA confers this negative response onto muscle cells remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: L6-GLUT4myc myoblasts were exposed for 24 h to palmitate-free conditioned medium from RAW 264.7 macrophages pre-treated with 0.5 mM palmitate for 6 h. This palmitate-free CM-PA, containing selective cytokines and chemokines, inhibited myoblast insulin-stimulated insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, AS160 phosphorylation, GLUT4 translocation and glucose uptake. These effects were accompanied by a rise in c-Jun N-terminal kinase (JNK) activation, degradation of Inhibitor of κBα (IκBα), and elevated expression of proinflammatory cytokines in myoblasts. Notably, CM-PA caused IRS1 phosphorylation on Ser1101, and phosphorylation of novel PKCθ and ε. Co-incubation of myoblasts with CM-PA and the novel and conventional PKC inhibitor Gö6983 (but not with the conventional PKC inhibitor Gö6976) prevented PKCθ and ε activation, JNK phosphorylation, restored IκBα mass and reduced proinflammatory cytokine production. Gö6983 also restored insulin signalling and glucose uptake in myoblasts. Moreover, co-silencing both novel PKC θ and ε isoforms in myoblasts by RNA interference, but not their individual silencing, prevented the inflammatory response and restored insulin sensitivity to CM-PA-treated myoblasts. CONCLUSIONS/CLINICAL SIGNIFICANCE: The results suggest that the block in muscle insulin action caused by CM-PA is mediated by novel PKCθ and PKCε. This study re-establishes the participation of macrophages as a relay in the action of fatty acids on muscle cells, and further identifies PKCθ and PKCε as key elements in the inflammatory and insulin resistance responses of muscle cells to macrophage products. Furthermore, it portrays these PKC isoforms as potential targets for the treatment of fatty acid-induced, inflammation-linked insulin resistance.

Periparturition alterations to liver ultrasonographic echo-texture and fat mobilization parameters in clinically healthy Holstein cows.

The objective of present study was to record the sequential alterations in liver echo-texture through digital analyzing of the B-mode ultrasonography in three groups of under-conditioned (UC), moderate-conditioned (MC), and over-conditioned (OC) clinically healthy Holstein cows from 30 days to calving until 30 days in milk. Furthermore, to compare their changes in association with the changes of fat mobilization parameters of non-esterified fatty acids (NEFA), betahydroxybutyric acid (BHBA), and body condition score (BCS). Although the cows lost significant (P < 0.05) BCS from calving and the NEFA values showed an increasing trend near calving and the BHBA values significantly inclined postparturition, the mean grey scale histogram (MGSH) of liver images did not reveal significant fluctuations unless a significant decrease on calving day (P < 0.05). The MGSH drop was predicted to be the result of anatomical changes in abdominal cavity, related to delivery and liver's blood flow. OC cows had higher NEFA on day +20 than UC and MC cows (P < 0.05). UC cows showed higher MGSH values on day -30 than MC cows and again on day -10 comparing to MC and OC cows (P < 0.05). MGSH values correlated with BCS values (rUC = -0.186; rMC = -0.283; rOC = -0.158). It was concluded that the studied cows did not show significant alterations in textural changes in their liver ultrasound whilst going through fat mobilization. As quantitative ultrasonography has shown the potential to detect cases of fatty liver, it could gain the attention to become a feasible device for liver health monitoring on a herd basis.

Blood parameters in female Zandi lambs as affected by liver biopsy methodology.

The aim of the present study was to investigate the normal blood parameters of Iranian fat-tailed sheep (Zandi) and their changes due to rapid liver biopsy technique with a tru-cut biopsy needle. In ten ewe lambs, blood samples were collected from jugular vein and biopsy needle was inserted through the dorsal one third of the 11th intercostal space, on the right hand side of the lambs and liver specimen was collected. Physical examinations were performed on alternate days during the experiment. Blood collection was done on both before (day 1) and after (day 17) the biopsy. All animals were slaughtered at day 17. Values were compared using paired t test. While biopsy did not make any significant changes in mean values of body temperature, heart rate, respiratory rate, PCV, WBC, neutrophil, lymphocyte, eosinophil, monocyte, total serum protein, AST, ALT, and serum calcium (p > 0.05), it made a significant difference on the values of ALP (p < 0.001), serum inorganic phosphate (p = 0.035), and magnesium (p = 0.013). Necropsy examination revealed the points of hitting the biopsy needles on the diaphragmatic surface of the livers, surrounded by a zone of intense hyperemia. Peritoneal adhesions accompanying with typical strands of fibrous connective tissue between diaphragmatic surface of the liver and adjacent abdominal wall were found in two cases.

Airway inflammatory events in diabetic-antigen sensitized guinea pigs.

Experimental evidence indicates that the relative lack of insulin in an organism results in an overall reduction in inflammatory reactions. This study was planned to determine the inflammatory events in antigen sensitized diabetic guinea pigs. Twenty-five male guinea pigs were categorized into five groups of five each as follows: diabetic, antigen sensitized, antigen sensitized diabetic, insulin-treated antigen sensitized diabetic and control animals. Induction of experimental diabetes and antigen sensitization was performed by injection of streptozotocin and ovalbumin, respectively. Animals were killed by exsanguination and bronchoalveolar lavage was performed. Bronchoalveolar lavage fluid cellular and protein contents were determined. Airway responsiveness to acetylcholine was assessed using isolated tracheal triple-ring. Histopathological examinations were performed on the lungs. Decreases in the airway reactivity in diabetic and antigen sensitized diabetic animals were found compared with antigen sensitized animals. Experimental diabetes also decreased antigen-induced protein leakage into the airspace as well as the accumulation of inflammatory cells (eosinophils, neutrophils, lymphocytes and macrophages) in bronchoalveolar lavage fluid of antigen sensitized animals. Insulin treatment prevented these decreases in protein content and inflammatory cells infiltration in bronchoalveolar lavage fluid observed in the antigen sensitized guinea pigs with diabetes. Histopathological results showed that coinduction of experimental diabetes significantly reduces the number of eosinophils in the lungs of antigen sensitized animals. Again, treatment with insulin increased the number of eosinophils in the antigen sensitized diabetic animals. Experimental diabetes causes were found to decrease the airway reactivity and inflammatory responsiveness induced by antigen sensitization due to a reduction in the insulin levels.