Endocytosis in mouse blastocysts: Characterization and quantification of the fluid phase component

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, ³H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4°C. Insulin stimulated uptake of ³H-dextran by 30% (P<0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more ³H-dextran (38%; P<0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased ³H-dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 ± 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/I and 10 g BSA/I, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells. An analysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant contributions of both phases of endocytosis to blastocyst metabolism in vivo. © 1995 Wiley-Liss, Inc.     

Studies on the inhibitory effect of bacitracin on 125I-labelled insulin internalization in the rat hepatocyte

Previous studies have suggested that transglutaminase has a role in the internalization of some polypeptide hormones and is inhibited by the antibiotic, bacitracin. Bacitracin has been used in insulin-receptor studies to inhibit extracellular degradation of ¹²⁵I-labelled insulin. The aim of this study was to investigate bacitracin's effect on ¹²⁵I-labelled insulin-receptor interactions in isolated rat hepatocytes. 1 g/l bacitracin increased cell-associated ¹²⁵I-labelled insulin at 20, 30 and 37°C (P < 0.001, 0.0005 and 0.0005, respectively). At 5 and 15°C (internalization does not occur), bacitracin did not affect cell-associated ¹²⁵I-labelled insulin. The bacitracin effect was concentration dependent, increasing to 2 g/l. Scatchard analysis showed that bacitracin did not alter insulin receptor affinity or number. 1 g/l bacitracin abolished the effect of chloroquine. The increased cell-associated radioactivity with bacitracin was surface-bound in nature. 0.5 g/l bacitracin decreased ¹²⁵I-labelled insulin degradation in hepatocyte suspensions (P < 0.001) and in buffer previously incubated with hepatocytes (P < 0.0005). More ¹²⁵I-labelled insulin remained associated with cells during dissociation studies at 37°C when the buffer contained 1 g/l bacitracin. Label that appeared in the buffer after 60 min was significantly more intact in the presence of bacitracin (P < 0.025). These results suggest that bacitracin retards the internalization of ¹²⁵I-labelled insulin in isolated rat hepatocytes.     

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

• 1.1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture.
• 2.2. Specific ¹²⁵I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K_d~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing ¹²⁵I-labelled insulin.
• 3.3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of ¹²⁵I-labelled insulin and ¹²⁵I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types.
• 4.4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

Insulin inhibition of calcium binding by human placental membrane

Insulin and its analogues displaced membrane-bound calcium within a physiological range of insulin concentration, in proportion to both biological potency and ability to displace porcine ¹²⁵I-labelled insulin from the insulin receptor. Mild tryptic digestion of the membrane reduced insulin binding but did not reduce specific calcium binding. Displacement of membrane-bound calcium by insulin was dependent on insulin binding to its intact receptor. These studies suggest that Ca²⁺ may exert a controlling influence on insulin-receptor binding in vivo.     

Timing of the first cleavage post‐insemination affects cryosurvival of in vitro–produced bovine blastocysts

The time of the first cleavage of bovine zygotes during in vitro culture can affect the rate of development and cell number of the blastocysts. The aim of this work was to study the effect of the timing of first cleavage on the cryosurvival of the resulting blastocysts. Following standard IVM and IVF, zygotes were cultured in modified synthetic oviduct fluid (SOF), with 10% fetal calf serum (FCS) added 48 hr post insemination, in a humidified atmosphere of 5% CO_2, 5% O₂ and 90% N₂. Embryos which cleaved by 24, 27 30, 33, or 36 hr after insemination (IVF) were harvested and further cultured to the blastocyst stage (day 7 or day 8 post IVF). All developing blastocysts on days 7 and 8 were classified into three groups and were cryopreserved by vitrification. Group A consisted of blastocysts (<150 μm, small blastocysts); group B consisted of expanded or hatching blastocysts (>150 μm, large blastocysts); and group C consisted of morphologically poor quality blastocysts. The vitrification solution consisted of 6.5 M glycerol and 6% bovine serum albumin in PBS (VS3a). Thawed embryos were cultured further and survival was defined as the re‐expansion and maintenance of the blastocoel over 24, 48, and 72 hr, respectively. Overall survival and hatching at 72 hr post‐thawing was higher in blastocysts formed by day 7 than those formed by day 8 (60% vs. 40% survival; 63% vs. 45% hatching). Large blastocysts from day‐7 and day‐8 groups survived significantly better than small or poor quality blastocysts (76% vs. 63% and 31%; 72% vs. 30% and 26%, respectively; P < 0.05). Day‐7 blastocysts from the 27‐ and 30‐hr cleavage groups survived significantly better than those from the 36‐hr group (63% and 66% vs. 25%, P < 0.05). Day‐8 blastocysts from later cleaved (30 hr) zygotes had a higher survival than the 27‐hr cleavage groups (52% vs. 26%, P < 0.05). These results indicate that the day of blastocyst appearance, developmental stage, and timing of the first cleavage post‐insemination can influence the cryosurvival of bovine blastocysts following vitrification. Mol. Reprod. Dev. 53:318–324, 1999. © 1999 Wiley‐Liss, Inc.     

Receptor of Insulin-Like Growth Factor 1 in Tissues of the Mollusc Anodonta cygnea

To identify insulin-like receptors in the mollusc Anodonta cygnea, specific binding of ¹²⁵I-insulin and ¹²⁵I-IGF-1 by WGA-purified glycoprotein fractions of foot muscles and neural ganglia is studied. The binding sites for IGF-1 are detected for the first time in invertebrates, both in the muscles, and in the neural tissue of the mollusc. The level of ¹²⁵I-IGF-1 binding in the muscle tissue was equal to 2.8 ± 0.1, in the neural tissue, to 4.0 ± 0.2% per 5 µg of protein. The equilibrium dissociation constant (K _d) was equal to 4.8 ± 0.3 and 4.3 ± 0.2 nM, respectively. The relative affinity of the binding sites to insulin did not exceed 1% of their affinity to IGF-1. Binding of ¹²⁵I-insulin in the muscle tissue was not detected; the level of labeled insulin binding in the neural tissue was equal to 0.5% per 5 µg of protein. In the sarcolemmal fraction of the mollusc foot, IGF-1 and, to a lesser degree, insulin at a dose of 100 nM initiated phosphorylation of tyrosine in a protein with mol. mass of 70 kDa. The minor band of the phosphorylation was also detected in the zone of protein of 80 kDa. The conclusion is made about the existence in molluscan tissues of high-conserved receptors-tyrosine kinases identical by functional parameters to the mammalian receptor of IGF-1. From this, it is suggested that the peptides close by structure to vertebrate IGF-1 may be involved in physiological processes in A. cygnea. The problem of the nature of the insulin-binding sites in the molluscan neural tissue is discussed.     

Neural and hormonal stimulation of DNA and protein synthesis in cultured regeneration blastemata in the newt Notophthalmus viridescens

Distal portions of cone-stage newt forelimb blastemata were cultured transfilter to spinal ganglia for 36 or 72 hr. Addition of insulin to the medium consistently resulted in a significant increase (250% in ganglionated and 238% in nonganglionated blastemata) in the incorporation of [³H]thymidine into DNA, as compared to nontreated controls. When blastemata were cultured without ganglia for 36 or 72 hr, DNA synthesis decreased to 73 and 71%, respectively, of that achieved by ganglionated explants. When insulin was excluded from the medium, DNA synthesis decreased to 40% of insulin-treated explants, and, in the absence of both nerves and insulin, it declined to 31% of insulin-treated, innervated explants. The presence of insulin in the medium also resulted in an augmentation of (¹⁴C)-labeled amino acid incorporation into proteins; the average increase was 168%, as compared to untreated controls. l-thyroxine, growth hormone and hydrocortisone in combination with insulin, did not enhance the effects on DNA or protein synthesis of insulin alone. Also, exogenous cyclic nucleotides (cAMP, cGMP) and alterations of their endogenous levels with acetylcholine, sodium azide, theophylline or prostaglandins failed to elicit significant changes in DNA or protein synthesis. The existence of a synergistic action on DNA synthesis between nerves and insulin is suggested.     

Total protein content and protein synthesis within pre-elongation stage bovine embryos

Protein content was measured in zona-free bovine oocytes and pre-elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml^-1 bovine serum albumin (BSA). Values (ng embryo^-1) of 122 ± 7.8, 137 ± 8.6, 111 ± 8.8, 115 ± 10.4, 139 ± 9.0 and 152 ± 10.1 were obtained for zona-free mature oocytes, 2-cell (day 2), 8-cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona-enclosed blastocysts was 337 ± 58.0 ng embryo^-1. These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml^-1 BSA, 8 mg ml^-1 BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS-D1), 8 mg ml^-1 BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo-derived day 7 blastocysts. Protein content was significantly (P< 0.05) lower in PVA-cultured embryos than other treatments. To determine if this difference in PVA-cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA-culture and either PVA-culture, FCS-D1 culture or in vivo-derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L-[2,3,4,5,6-³H]-phenylalanine into the TCA-precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC-labelled BSA or β-casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra-embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of embryos in protein-free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo-derived embryos. Mol. Reprod. Dev. 50:139–145, 1998. © 1998 Wiley-Liss, Inc.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Low IGF-1 levels are associated with cardiovascular risk factors in haemodialysis patients

Cardiovascular diseases (CVD) constitute a significant risk and may, in part, explain the high morbidity and mortality rates among haemodialysis (HD) patients. Several studies have implicated reduced insulin like growth factor (IGF-1) levels in the development of CVD. However, it is not clear whether IGF-1, and its relationship with other hormones such as leptin, insulin, and growth hormone (GH), as well as anthropometric variables may explain the high incidence of vascular complications in chronic kidney disease (CKD) patients. This study was designed to measure total serum IGF-1, leptin, insulin and GH levels in CKD patients and in age-matched control subjects and to elucidate the relationship between IGF-1 and GH, leptin, and insulin as well as other known aetiological risk factors for CVD including blood pressure, body mass index (BMI), and age. The study consisted of 50 CKD patients [36 M and 14 F; mean age; 41.8 ± 10.3 years) on maintenance haemodialysis and 50 healthy control subjects (36 M and 14 F; mean age 41.6 ± 10.2 years) matched for age and sex. None of the subject among patients and controls reported either smoking or history of diabetes mellitus. The circulating levels of IGF-1 were significantly lower (P < 0.001) in both male and female patients compared to the control subjects. Moreover, IGF-1 was strongly and inversely correlated with both systolic blood pressure (SBP) (r = −0.360; P < 0.01) and diastolic blood pressure (DBP) (r = −0.512; P < 0.001) in the CKD group, and when the two groups were combined SBP (r = −0.396; P < 0.001) and DBP (r = −0.296; P < 0.01). When adjusted for age, the correlation was more significant, however, when adjusted for BMI no significant correlation was observed between IGF-1 and blood pressure. IGF-1 was inversely correlated with age (r = −0.367; P < 0.01) and BMI (r = −0.310; P < 0.05) in the control group, but not the patient group. In controls and patients, respectively, a positive correlation between leptin and BMI (r = 0.358; P < 0.01; r = 0.640, P < 0.001) was observed. The results show that circulating levels of IGF-1 were significantly lower in CKD patients as compared to healthy normal subjects and were inversely correlated with SBP and DBP independent of age, but not BMI indicative of a strong relationship between cardiovascular risk factors and low IGF-1 levels. Although, the data do not clearly indicate low IGF-1 levels as a cause or an effect of these cardiovascular risk factors, they do point to an interesting relationship between low IGF-1 levels and increased cardiovascular risk factors among CKD patients as compared to age-matched healthy control subjects.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

Cerebral cortical microvessels: An insulin-sensitive tissue

Cerebral cortical microvessels were isolated from bovine brains and incubated in the presence or absence of porcine insulin. The conversion of D-glucose to CO₂ and lipids was increased significantly in insulin-treated microvessels. Insulin also caused a significant increase in the activity of cyclic-AMP phosphodiesterase in these microvessels. Specific binding of ¹²⁵I-insulin to isolated brain microvessels, displaceable by cold insulin was also observed. The effects of insulin reported in this communication mark the first direct evidence that insulin is capable of regulating the metabolic activity of cerebral cortical microvascular tissue. The possible physiological significance of this finding is discussed.     

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake

The purification of a phosphate-binding protein (P_iBP₂) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit P_i uptake by whole cells. The inhibition is a mixed type of inhibition (V _m and K _m are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in P_i uptake. The binding of ¹²⁵I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.Abbreviations P_i inorganic phosphate - P_iBP phosphate-binding protein - Tris Tris (hydroxymethyl)-aminoethane - MES (2(N-Morpholino) ethanesulfonic acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid, disodium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - Fab fragments, fragment antigen binding     

The Cellular Regulation of Vesicle Exocytosis by Entamoeba histolytica1,2

ABSTRACT. We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed ¹²⁵iodine (¹²⁵I) labeled tyrosine conjugated dextran; ¹²⁵I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of ¹²⁵I-dextran released after 120 min at 37°C, 31°C, 25°C, and 4°C, respectively (P < 0.01 for each). Exocytosis at 37°C was inhibited by cytochalasin D (10 μg/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 μM) (P < 0.01 for each at ≥ 60 min). Calcium ionophore A23187 (1 μM) enhanced exocytosis at 5 and 15 min (P < 0.01). Elevation of vesicle pH with NH₄Cl (10 mM) had no effect on release of ¹²⁵I-dextran; phorbol myristate acetate (10^?6 M) increased exocytosis by 46% at 30 min (P < 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased ¹²⁵I-dextran release into the cell supernatant after 30 and 60 min at 37°C (by 40% and 42%, respectively, P < 0.01); release of ¹²⁵I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

A 12‐Week Aerobic Exercise Program Reduces Hepatic Fat Accumulation and Insulin Resistance in Obese,Hispanic Adolescents

The rise in obesity‐related morbidity in children and adolescents requires urgent prevention and treatment strategies. Currently, only limited data are available on the effects of exercise programs on insulin resistance, and visceral, hepatic, and intramyocellular fat accumulation. We hypothesized that a 12‐week controlled aerobic exercise program without weight loss reduces visceral, hepatic, and intramyocellular fat content and decreases insulin resistance in sedentary Hispanic adolescents. Twenty‐nine postpubertal (Tanner stage IV and V), Hispanic adolescents, 15 obese (7 boys, 8 girls; 15.6 ± 0.4 years; 33.7 ± 1.1 kg/m²; 38.3 ± 1.5% body fat) and 14 lean (10 boys, 4 girls; 15.1 ± 0.3 years; 20.6 ± 0.8 kg/m²; 18.9 ± 1.5% body fat), completed a 12‐week aerobic exercise program (4 × 30 min/week at ≥70% of peak oxygen consumption (VO₂peak)). Measurements of cardiovascular fitness, visceral, hepatic, and intramyocellular fat content (magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS)), and insulin resistance were obtained at baseline and postexercise. In both groups, fitness increased (obese: 13 ± 2%, lean: 16 ± 4%; both P < 0.01). In obese participants, intramyocellular fat remained unchanged, whereas hepatic fat content decreased from 8.9 ± 3.2 to 5.6 ± 1.8%; P < 0.05 and visceral fat content from 54.7 ± 6.0 to 49.6 ± 5.5 cm²; P < 0.05. Insulin resistance decreased indicated by decreased fasting insulin (21.8 ± 2.7 to 18.2 ± 2.4 µU/ml; P < 0.01) and homeostasis model assessment of insulin resistance (HOMA_IR) (4.9 ± 0.7 to 4.1 ± 0.6; P < 0.01). The decrease in visceral fat correlated with the decrease in fasting insulin (R² = 0.40; P < 0.05). No significant changes were observed in any parameter in lean participants except a small increase in lean body mass (LBM). Thus, a controlled aerobic exercise program, without weight loss, reduced hepatic and visceral fat accumulation, and decreased insulin resistance in obese adolescents.     

Insulin Sensitivity,Body Fat Distribution,and Family Diabetes History: The IRAS Family Study

Objectives : Markers of insulin resistance are often apparent in nondiabetic relatives of subjects with type 2 diabetes. Whether diabetes family history (FH) also predicts visceral fat accumulation and, if so, whether the increased insulin resistance in relatives of diabetic subjects occurs independently of visceral fat accumulation are not known. Research Methods and Procedures : To examine this issue, we studied the relationship of diabetes FH with insulin sensitivity and fat measures, measured by minimal model analysis and computed tomography, respectively, in families participating in the Insulin Resistance Atherosclerosis (IRAS) Family Study. FH scores were based on the diabetes status of the participants’ parents and older siblings. Results : FH scores were significantly correlated with reduced insulin sensitivity (p < 0.05) and increased subcutaneous (p < 0.05) and visceral (p < 0.05, San Antonio only) fat in families from San Antonio and Los Angeles but not in the leaner Hispanic families from San Luis Valley. There was no evidence for a stronger association of FH score with visceral fat accumulation than with subcutaneous fat or insulin resistance. Discussion : The absence of an association between FH score and insulin resistance/fat accumulation in San Luis Valley is consistent with the idea that the expression of transmitted diabetes genes may be suppressed in leaner, more physically active populations.     

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation,protein synthesis and protein breakdown in mouse C2C12 myotubes

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor ¹⁴C-glucose oxidation or to measure protein synthesis by incorporation of ³H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P<0.01, n=3 trials), whereas bovine insulin (1 µM) stimulated glucose oxidation (P<0.05, n=3 trials). Treatment with AGP in combination with insulin reduced ¹⁴C-glucose oxidation (P<0.05, n=3 trials), similar to the effect of AGP alone. Glucose transport, as measured by ³H-deoxyglucose uptake, was increased by 38% with 1 µM insulin (P<0.05, n=3 trials), whereas AGP alone increased glucose uptake by 36% (P<0.05, n=3 trials). The combination of insulin and AGP in the medium resulted in an 88% increase in glucose uptake (P<0.01, n=3 trials). Protein synthesis was measured by ³H-tyrosine incorporation into C2C12 myotubes. Insulin stimulated a 18% increase in ³H-tyrosine incorporation (P<0.05, n=6 trials). The incorporation of ³H-tyrosine into myotubes was reduced by 20% with AGP incubation (P<0.01, n=6 trials), like the 20% decrease in ³H-tyrosine incorporation in response to the combination of AGP and insulin (P<0.01, n=6 trials). Protein breakdown, as measured by the release of ³H-tyrosine from C2C12 myotubes, was reduced 27% by insulin (P<0.01, n=6 trials). Treatment with AGP had no effect on protein breakdown (P>0.05, n=6 trials), whereas incubation with both AGP and insulin reduced ³H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.