Increasing Dietary Fat Elicits Similar Changes in Fat Oxidation and Markers of Muscle Oxidative Capacity in Lean and Obese Humans

In lean humans, increasing dietary fat intake causes an increase in whole-body fat oxidation and changes in genes that regulate fat oxidation in skeletal muscle, but whether this occurs in obese humans is not known. We compared changes in whole-body fat oxidation and markers of muscle oxidative capacity differ in lean (LN) and obese (OB) adults exposed to a 2-day high-fat (HF) diet. Ten LN (BMI = 22.5±2.5 kg/m2, age = 30±8 yrs) and nine OB (BMI = 35.9±4.93 kg/m2, 38±5 yrs, Mean±SD) were studied in a room calorimeter for 24hr while consuming isocaloric low-fat (LF, 20% of energy) and HF (50% of energy) diets. A muscle biopsy was obtained the next morning following an overnight fast. 24h respiratory quotient (RQ) did not significantly differ between groups (LN: 0.91±0.01; OB: 0.92±0.01) during LF, and similarly decreased during HF in LN (0.86±0.01) and OB (0.85±0.01). The expression of pyruvate dehydrogenase kinase 4 (PDK4) and the fatty acid transporter CD36 increased in both LN and OB during HF. No other changes in mRNA or protein were observed. However, in both LN and OB, the amounts of acetylated peroxisome proliferator-activated receptor γ coactivator-1-α (PGC1-α) significantly decreased and phosphorylated 5-AMP-activated protein kinase (AMPK) significantly increased. In response to an isoenergetic increase in dietary fat, whole-body fat oxidation similarly increases in LN and OB, in association with a shift towards oxidative metabolism in skeletal muscle, suggesting that the ability to adapt to an acute increase in dietary fat is not impaired in obesity

The effects of glucose on the energy metabolism of Novikoff ascites hepatoma cells.

Glucose addition to Novikoff ascites hepatoma cells causes a rapid depletion of ATP and a decrease in total adenine nucleotides. Reestablishment of the steady state concentrations of ATP requires 50 to 60 minutes. Decreased rates of protein synthesis, glycogen synthesis, ion transport, PRPP synthesis, and incorporation of adenine and hypoxanthine into the acid soluble fraction are associated with the decrease in ATP concentration. During the first 6 minutes following glucose addition fructose diphosphate accumulates and the concentration of inorganic phosphate decreases. Glycolysis and glucose utilization decrease in the interval between 1 and 6 minutes after the addition of glucose. After preincubation of the cells, addition of glucose does not cause large changes in ATP levels and the period of decreased metabolic activity is eliminated. The maximum rates of PRPP synthesis, glycolysis, and adenine incorporation are greater in non-preincubated than in preincubated cells. Partial replacement of sodium by potassium ions in the incubation medium causes a prolongation of the period of ATP recovery and of decreased metabolic activity. Possible factors involved in the regulation of the above events are discussed

Properties of a 5′-nucleotidase purified from mouse liver plasma membranes

1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate–Tris buffer (sarcosyl–Tris buffer), solubilized 90% of the protein and 70% of the 5′-nucleotidase activity. 2. The proteins of the sarcosyl–Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose–detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5′-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5′-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl–Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl–Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000–150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5′-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported

Relative rates of degradation of mouse-liver surface-membrane proteins

1. The relative rates of degradation of the proteins of liver membranes and soluble fractions were examined by using a double-isotope labelling technique. 2. The proteins of plasma membrane and smooth microsomal fraction were more rapidly degraded than those of the mitochondrial and soluble fractions. 3. No great variation in the rates of degradation of the proteins of plasma membrane and microsomal subfractions was detected. The proteins of intercellular gap-junctions (nexuses) were degraded slowly. 4. Extraction of plasma membranes with aqueous and organic solvents and detergents indicated that fractions known to be enriched in glycoproteins underwent more rapid degradation.5The high molecular weight components of liver plasma membranes and derivative fractions were more rapidly degraded than those of low molecular weight. Since glycoproteins predominated amongst the high molecular weight components of plasma membranes, the functional significance of this finding is discussed

The distribution of surface antigens during fractionation of mouse liver plasma membranes

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified γ-globulin antibody fraction was iodinated with (125)I. The reaction of the (125)I-labelled γ-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The γglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. `Light' and `heavy' mouse liver plasma-membrane subfractions bound similar amounts of γ-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO(3)–Na(2)CO(3) buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane

Immunochemical characterization of proteins from mouse liver plasma membranes

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an (125)I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate–1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5′-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate–Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate–Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose–detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5′-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000–130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed