Intracellular glucose concentration in skeletal muscle of awake rats was determined under conditions of hyperglycemic (10.2 ± 0.6 mM) hyperinsulinemia (∼1,200 pM) and hyperglycemic (20.8 ± 1.5 mM) hypoinsulinemia (<12 pM) by use of13C nuclear magnetic resonance (NMR) spectroscopy during a prime-constant infusion of [1-13C]glucose and [1-13C]mannitol with either insulin (10 mU ⋅ kg−1 ⋅ min−1) or somatostatin (1.0 μg ⋅ kg−1 ⋅ min−1). Intracellular glucose was calculated as the difference between the concentrations of total tissue glucose (calculated from the in vivo13C NMR spectrum with mannitol as an internal concentration standard) and extracellular glucose, corrected by the ratio of intra- and extracellular water space. Extracellular concentration was corrected for an interstitial fluid-to-plasma glucose concentration gradient of 0.83 ± 0.07, determined by open-flow microperfusion. The mean ratio of intra- to extracellular glucose space, determined from the relative NMR signal intensities and concentrations of mannitol and total creatine, was 9.2 ± 1.1 (hyperglycemic hyperinsulinemia, n = 10), and 9.0 ± 1.7 (hyperglycemic hypoinsulinemia, n= 7). Mean muscle intracellular glucose concentration was <0.07 mM under hyperglycemic-hyperinsulinemic conditions ( n = 10) and 0.32 ± 0.06 mM under hyperglycemic-hypoinsulinemic conditions ( n = 7). This method is noninvasive and should prove useful for resolving the question of whether glucose transport or phosphorylation is responsible for the reduced rate of muscle glycogen synthesis observed in diabetic subjects.
The effects of increased plasma free fatty acids (FFA) on insulin-dependent whole body glucose disposal, skeletal muscle glycolysis, glycogen synthesis, pyruvate versus FFA/ketone oxidation, and glucose 6-phosphate (Glu-6-P) were investigated in the awake rat. A control group (glycerol-infused) and high plasma FFA group (Liposyn-infused) were clamped at euglycemia (ϳ6 mM)-hyperinsulinemia (10 milliunits/kg/min) throughout the experiment (180 -240 min). In the initial experiment, 13 C NMR was used to observe [1-13 C]glucose incorporation into [1-13 C]glycogen in the rat hindlimb for glycogen synthesis calculations and into [3-13 C]lactate and [3-13 C]alanine for glycolytic flux calculations. These experiments were followed by 31 P NMR measurements of Glu-6-P changes under identical conditions of the initial experiment. Plasma FFA concentrations were 2.25 ؎ 0.36 and 0.20 ؎ 0.03 mM in the high plasma FFA and control groups respectively (p < 0.0005). Glucose infusion rates (G inf ) decreased significantly in the Liposyninfused rats (29.5 ؎ 0.7 and 27.2 ؎ 1.2 mg/kg/min for control and high plasma FFA group, respectively, at 15 min to 30.7 ؎ 2.3 and 17.7 ؎ 1.3 mg/kg/min, respectively, at the end of the experiment, p < 0.002). Glycogen synthesis rates were 163 ؎ 32 and 104 ؎ 17 nmol/g/min, and glycolytic rates were 57.9 ؎ 8.0 and 19.5 ؎ 3.6 nmol/g/min (p < 0.002) in the control and high plasma FFA groups, respectively. The relative flux of pyruvate versus free fatty acids and ketones entering the tricarboxylic acid cycle was greater in the control (57 ؎ 9%) versus high plasma FFA group (25 ؎ 4%) (p < 0.005) as assessed by [4-13 C]glutamate/[3-13 C]lactate steady state isotopic enrichment measurements. Finally, Glu-6-P concentrations increased by 29.8 ؎ 7.0 and 52.8 ؎ 12.3% (p < 0.05) in the control and high plasma FFA groups, respectively, above their basal concentrations by 180 min.In conclusion, we have demonstrated the ability to use in vivo NMR to elucidate the metabolic fate of glucose within skeletal muscle of an awake rat during a euglycemic-hyperinsulinemic clamp and increased levels of plasma FFA. These data suggest that increased concentrations of plasma FFA inhibit insulin-stimulated muscle glucose metabolism in the rat through inhibition of glycolysis.Increased levels of plasma free fatty acids (FFA) 1 are prevalent in people with non-insulin-dependent diabetes mellitus and may play an important role in mediating the insulin resistance associated with this disease. A decrease in insulin-dependent glucose uptake by increased FFA was originally observed in vitro in heart and diaphragm muscle by Randle et al.(1). This phenomenon was postulated to be a result of pyruvate dehydrogenase (PDH) enzyme allosteric control by increased intramitochondrial acetyl-CoA/CoA and NADH/NAD ϩ ratios that activates pyruvate dehydrogenase kinase enzyme which subsequently deactivates the PDH enzyme complex (2). Intramitochondrial citrate also increases under conditions of increased FFA oxidation and decreased flux through isoci...
TMPRSS6 variants that affect protein function result in impaired matriptase-2 function and consequently uninhibited hepcidin production, leading to iron refractory iron deficiency anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. Much is still unknown about its pathophysiology, genotype-phenotype correlation, and optimal clinical management. We describe 14 different TMPRSS6 variants, of which 9 are novel, in 21 phenotypically affected IRIDA patients from 20 families living in the Netherlands; 16 out of 21 patients were female. In 7 out of 21 cases DNA sequencing and multiplex ligation dependent probe amplification demonstrated only heterozygous TMPRSS6 variants. The age at presentation, disease severity, and response to iron supplementation were highly variable, even for patients and relatives with similar TMPRSS6 genotypes. Mono-allelic IRIDA patients had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia than bi-allelic patients. Transferrin saturation (TSAT)/hepcidin ratios were lower in IRIDA probands than in healthy relatives. Most patients required parenteral iron. Genotype alone was not predictive for the response to oral iron. We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. The high proportion of female patients and the discrepancy between phenotypes of probands and relatives with the same genotype, suggest a complex interplay between genetic and acquired factors in the pathogenesis of IRIDA. In the absence of inflammation, the TSAT/hepcidin ratio is a promising diagnostic tool, even after iron supplementation has been given.Am. J. Hematol. 91:E482-E490,
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