Brief food restriction increases FA oxidation and glycogen synthesis under insulin-stimulated conditions

To determine the effects of brief food restriction on fatty acid (FA) metabolism, hindlimbs of F344/BN rats fed either ad libitum (AL) or food restricted (FR) to 60% of baseline food intake for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions (20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [3-(3)H]glucose, and [1-(14)C]palmitate). Basal glucose and insulin levels were significantly lower (P < 0.05) in FR vs. AL rats. Palmitate uptake (34.3 +/- 2.7 vs. 24.5 +/- 3.1 nmol/g/min) and oxidation (3.8 +/- 0.2 vs. 2.7 +/- 0.3 nmol.g(-1).min(-1)) were significantly higher (P < 0.05) in FR vs. AL rats, respectively. Glucose uptake was increased in FR rats and was accompanied by significant increases in red and white gastrocnemius glycogen synthesis, indicating an improvement in insulin sensitivity. Although muscle triglyceride (TG) levels were not significantly different between groups, glucose uptake and total preperfusion TG concentration were negatively correlated (r(2) = 0.27, P < 0.05). In conclusion, our results show that under hyperglycemic-hyperinsulinemic conditions, brief FR resulted in an increase in FA oxidative disposal that may contribute to the improvement in insulin sensitivity.     

Impaired fatty acid oxidation in muscle of aging rats perfused under basal conditions

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under conditions of euglycemia with low insulin levels. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 8 mM glucose, 1 mM palmitate, 25 microU/ml insulin, [1-(14)C]palmitate, and [3-(3)H]glucose. Glucose and palmitate uptake were similar among age groups. The percent and total palmitate oxidized (nmol.min(-1).g(-1)) were 30-36 and 41-49% lower (P < 0.05) in 15-mo- and 24-mo-old than in 5-mo-old animals. Compared with 5-mo- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 91-305% in red and 118-219% in white muscles of 24-mo-old animals. Fatty acid-binding protein content was 40-64% higher (P < 0.05) in 24-mo- than in 5-mo- or 15-mo-old animals. In red muscle, hormone-sensitive lipase (HSL) content was 28% lower (P < 0.05) in 24-mo- than in 5-mo-old animals. These results indicate that, under euglycemic conditions in the presence of low insulin levels, the reduction in FA disposal to oxidation and the decrease in HSL content may contribute to the accumulation of TG in muscle of old animals.     

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.     

Immunoprophylaxis with attenuated ehrlich ascites tumor cells in admixture with BCG

Summary During propagation in tissue culture, the Ehrlich ascites carcinoma was found to lose some of its tumor-producing capacity (oncogenicity) when implanted IP or SC into CF-1 mice. On the other hand, attenuated cells retained their immunoprotective capacity; immunization of mice with a single dose (1×10⁴) of these cells induced a high degree of resistance against a challenge 1 month later with virulent Ehrlich cells maintained by IP transplantation. The admixture of BCG (1×10⁶ viable units) with attenuated cells further improved their immunogenicity. The immunogenicity of attenuated cells was almost completely abolished by gamma-irradiation (2,500 rads), but this property was significantly restored by the addition of BCG. Some evidence is presented that suggests that attenuated cells have a higher immunoprotective capacity than the corresponding virulent cells.     

Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.     

Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, β-lactoglobulin and α-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of α-lactalbumin was determined using the IDRing^® assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 ± 0.97 vs. 2.24 ± 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 ± 0.46 vs. 0.90 ± 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the α-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.