Angiopoietin-1 (Ang-1) is essential for the maturation of blood vessels during vasculogenesis. Besides angiogenesis, recent publications indicate that Ang-1 is also a potent survival factor for endothelial cells; however, the mechanisms by which pathways remain elusive. Doxorubicin (DOX) is a powerful anticancer drug, but its use is severely restricted by its cardiotoxicity. The authors report here that Ang-1 inhibits DOX-induced cell death in human umbilical vein endothelial cells (HUVECs). Interestingly, the DOX-induced up-regulation in Fas (CD95/APO-1) and Fas ligand expression could be blocked by Ang-1, indicating a pivotal role of Ang-1 in DOX-induced Fas and Fas ligand expression. In addition, the prevention of cell death in this model system seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K)/Akt, as Ang-1 fails to inhibit DOX-induced cell death while PI3K/Akt pathway was blocked by the PI3K inhibitor LY294002. Moreover, Ang-1 inhibits DOX-induced up-regulation of p53 through PI3K/Akt. Therefore, Ang-1 is a potent inhibitor for DOX-induced cell death through Fas and PI3K/Akt-mediated pathways.

Tumor necrosis factor-alpha (TNF-alpha) and hyperglycemia both impair insulin sensitivity in vivo. This may be secondary to stimulation of adipose tissue lipolysis and consequent increased circulating free fatty acids (FFAs). Here we report that neither TNF-alpha nor glucose alone has a pronounced effect on lipolysis in 3T3-L1 adipocytes. However, the combination of TNF-alpha plus glucose markedly stimulates lipolysis. Glucose does not affect the ability of isoproterenol to stimulate lipolysis. Alternative substrates such as acetate, pyruvate, and lactate do not allow the TNF-alpha effect. Mannose was almost as effective as glucose; fructose was marginally effective, but galactose was ineffective. The effectiveness of the sugars corresponded with production of lactate, i.e., the cells readily produced lactate from glucose or mannose, slightly from fructose, and not at all from galactose. The ability of TNF-alpha to phosphorylate extracellular signal-regulated kinase 1 (ERK1) and ERK2 and to downregulate perilipin (which has been implicated in the lipolytic effect of TNF-alpha) was not affected by glucose. We conclude that the lipolytic action of TNF-alpha is influenced by glucose in 3T3-L1 adipocytes. The findings suggest that glucose metabolism is required for the lipolytic response to TNF-alpha but not for early signaling events. These findings suggest novel mechanisms by which TNF-alpha and hyperglycemia raise FFA levels and induce insulin resistance.

Multiple isoforms of glucose transporters are found in muscle, the tissue that normally accounts for 85% of insulin-stimulated glucose uptake. Glucose uptake into muscle cells in the fasting state is mediated primarily by GLUT1 and GLUT3 glucose transporters, whereas postprandial (insulin-stimulated) and exercise-related increments in muscle glucose uptake are mediated primarily by GLUT4. To determine if glucose transporters are abnormally expressed in muscle from insulin-resistant subjects, muscle samples were obtained from 10 normal subjects and 6 obese, nondiabetic subjects with severe insulin resistance and acanthosis nigricans. Both GLUT4 total protein and mRNA were normal in the insulin-resistant subjects. Muscle GLUT3 protein and mRNA were lower than controls by 62% and 71%, respectively. GLUT1 mRNA was twice normal, whereas GLUT1 protein content was not significantly increased. GLUT4 protein was markedly redistributed to the muscle plasma membrane in subjects with severe insulin resistance compared with normals (92% v 40% GLUT4 in plasma membrane-enriched fractions, P <.001), whereas the percentage of GLUT1 and GLUT3 protein found in the plasma membrane-enriched fractions was not different from controls. These data document differences in the expression of genes for GLUT1 and GLUT3 in muscle from normal and insulin-resistant subjects. Further, insulin resistance with fasting hyperinsulinemia was associated with a redistribution of GLUT4 to the muscle cell surface with no change in total GLUT4 protein. These data suggest that glucose transporter gene expression and their basal distribution in human muscle are related to insulin resistance and could be determinants of whole body insulin responsiveness.

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Basal, “insulin-independent” glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 ± 10, 46 ± 4, and 156 ± 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P < 0.05). The augmented negative amino acid balance was the result of an increased muscle protein breakdown (P < 0.05) without a concomitant change in muscle protein synthesis. Muscle efflux of glutamine and alanine increased significantly after bed rest due to a significant increase in de novo synthesis (P < 0.05). Thus, inactivity sensitizes skeletal muscle to the catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

A method has been developed in which the concentrations of insulin in dried blood spots on filter paper can be estimated. The technique involves elution of blood from the paper, evaporation, and rehydration in assay buffer. The resuspended samples are then assayed by radioimmunoassay. The ratio of control plasma to control dried blood spot insulin values is used as a conversion factor for unknown samples on paper to express the dried blood spot insulin concentration in plasma equivalents. Intra-assay variability was 14%, and interassay variability was 25%, but the elution conversion factor had a variability of about 40% among individuals. This technique has substantial advantages for field studies, but elevated values should be confirmed by a venous plasma sample measurement in a conventional radioimmunoassay.

OBJECTIVE: Insulin is known to be mitogenic to a variety of cells in culture. The purpose of this study was to investigate the possible role of insulin in the growth and development of endometrial cancers.STUDY DESIGN: Specific binding and growth effects of insulin were studied in 5 different human endometrial cancer cell lines derived from cancers with different degrees of differentiation: HEC-1-A and HEC-1-B (from a moderately well-differentiated adenocarcinoma), RL95-2 (from a moderately well-differentiated adenosquamous carcinoma), KLE (from poorly differentiated carcinoma), and AN3 CA (from a metastatic undifferentiated endometrial carcinoma). The receptors were further characterized by competitive binding and chemical cross-linking studies.RESULTS: Binding studies with I-125-insulin revealed the presence of high-affinity binding sites for insulin on all the 5 cell lines. Binding of insulin was found to be highly specific. Competitive binding studies with I-125-insulin revealed that insulin was most effective in displacing the labeled hormone, whereas insulin-like growth factor-I and insulin-like growth factor-II competed for binding only at very high concentrations. Scatchard analysis of the binding data revealed that the association constant for the high-affinity binding sites ranged from 0.72 to 1.91 x 10(9) L/mol. Estrogen-receptor-negative cell lines HEC-1-A and HEC-1-B had the highest number of insulin receptors, whereas the estrogen-receptor-positive cell line RL95-2 had the least number of receptors. The effect of insulin on cell proliferation was studied by monitoring cell number and incorporating [H-3]thymidine into deoxyribonucleic acid of the cells. Insulin stimulated cell growth of all the cell lines.CONCLUSIONS: The results of this study indicate the potential role of hyperinsulinemia in the growth and development of endometrial cancer.

The prevalences of obesity and of non-insulin dependent diabetes mellitus (NIDDM) have increased in the United States population over the past two decades, and thus diabetes preven tion has become a major concern of public health agencies such as the National Institutes of Health. Identification of individuals at risk for diabetes is an essential first step in designing and implementing intervention programs. Insulin resistance is the hallmark of the pathophysiology of NIDDM. Subjects with hyperinsulinemia, impaired glucose tolerance, or gestational diabetes are well accepted as being at high risk for diabetes. We propose that the easily identifiable skin lesion, acanthosis nigricans, is common in the major minority groups in the United States and that its presence is a surrogate for laboratory-determined hyperinsulinemia. Clin Pediatr. 1998;37:73-80

Compared with the US white, non-Hispanic population, the African-American population has a nearly two-fold higher prevalence of noninsulin-dependent diabetes mellitus (NIDDM). Obesity, which usually precedes NIDDM, is associated with the skin lesion acanthosis nigricans in African Americans. This study was undertaken to determine what the relationship of acanthosis nigricans was to hyperinsulinemia, a major risk factor for NIDDM. Eighty-nine African-American subjects with acanthosis nigricans and 25 others without the skin lesion were evaluated using oral glucose tolerance testing and responsiveness to insulin. Noninsulin-dependent diabetes mellitus was present in 19 of the subjects with acanthosis nigricans. The prevalence of NIDDM in this group increased with increasing age, reaching 50% among those in their 40s. Fasting plasma insulin concentration was in direct proportion to the severity of the acanthosis nigricans involvement of the neck. These data suggest that among African Americans, this skin lesion is a marker for hyperinsulinemia and insulin resistance. Furthermore, the presence of acanthosis nigricans identifies a subset with a much higher prevalence of NIDDM than is present in African Americans in the general population.

We sought to determine the extent to which the loss of lean body mass and nitrogen during inactivity was due to alterations in skeletal muscle protein metabolism. Six male subjects were studied during 7 days of diet stabilization and after 14 days of stimulated microgravity (-6 degrees bed rest). Nitrogen balance became more negative (P < 0.03) during the 2nd wk of bed rest. Leg and whole body lean mass decreased after bed rest (P < 0.05). Serum cortisol, insulin, insulin-like growth factor I, and testosterone values did not change. Arteriovenous model calculations based on the infusion of L-[ring-13C6]-phenylalanine in five subjects revealed a 50% decrease in muscle protein synthesis (PS; P < 0.03). Fractional PS by tracer incorporation into muscle protein also decreased by 46% (P < 0.05). The decrease in PS was related to a corresponding decrease in the sum of intracellular amino acid appearance from protein breakdown and inward transport. Whole body protein synthesis determined by [15N]alanine ingestion on six subjects also revealed a 14% decrease (P < 0.01). Neither model-derived nor whole body values for protein breakdown change significantly. These results indicate that the loss of body protein with inactivity is predominantly due to a decrease in muscle PS and that this decrease is reflected in both whole body and skeletal muscle measures.

We tested the hypothesis that glucose plus insulin determine the rate of fat oxidation in humans by controlling the rate of fatty acid entrance into the mitochondria. We gave constant infusions of [1-13C]oleate, a long-chain fatty acid, and [1-14C]octanoate, a medium-chain fatty acid, for 3 h in seven volunteers (basal). Immediately after the basal period, a hyperinsulinemic (insulin infusion = 120 mU x m(-2) min(-1)), hyperglycemic (plasma glucose = 140 mg/dl) clamp was started and continued for 5 h. During the last 3 h of the clamp, the infusions of [1-13C]oleate and [1-14C]octanoate were repeated. Intracellular acylcarnitine concentrations were measured in muscle biopsies obtained before and after the clamp. Plasma oleate enrichment and FFA concentration were kept constant by means of variable infusions of lipids and heparin. Oleate, but not octanoate, requires carnitine binding to gain access to the mitochondrial matrix; hence, if glucose and/or insulin limit long-chain fatty acid entrance into the mitochondria, then, during the clamp, long-chain acylcarnitine formation should be decreased, causing a decrease in oleate, but not octanoate, oxidation. Oleate oxidation decreased from the basal value of 0.7+/-0.1 to 0.4+/-0.1 micromol x kg(-1) x min(-1) (P < 0.05). In contrast, octanoate oxidation remained unchanged. Long-chain acylcarnitine concentration decreased from 855+/-271 in the basal state to 376+/-83 nmol/gram dry weight during the clamp (P < 0.05). We conclude that glucose and/or insulin determine fatty acid oxidation by controlling the rate of long-chain fatty acid entrance into the mitochondria.