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Peroxisome proliferatoractivated receptor-[delta] (PPAR[delta]) activation results in upregulation of genes associated with skeletal muscle fatty acid oxidation and mitochondrial uncoupling. However, direct, noninvasive assessment of lipid metabolism and mitochondrial energy coupling in skeletal muscle following PPAR[delta] stimulation has not been examined. Therefore, in this study we examined the response of a selective PPAR[delta] agonist (GW610742X at 5 or 100 mg x [kg.sup.-1] x [day.sup.-1] for 8 days) on skeletal-muscle lipid metabolism and mitochondrial coupling efficiency in rats by using in vivo magnetic resonance spectroscopy (MRS). There was a decrease in the intramyocellular lipid-to-total creatine ratio as assessed by in vivo [1.sup.H]-MRS in soleus and tibialis anterior muscles by day 7 (reduced by 49 and 46%, respectively; P < 0.01) at the high dose. Following the [1.sup.H]-MRS experiment (day 8), [1-[sup.13]C]glucose was administered to conscious rats to assess metabolism in the soleus muscle. The relative fat-vs.-carbohydrate oxidation rate increased in a dose-dependent manner (increased by 52 and 93% in the 5 and 100 mg x [kg.sup.-1] x [day.sup.-1] groups, respectively; P < 0.05). In separate experiments where mitochondrial coupling was assessed in vivo (day 7), [sup.31]P-MRS was used to measure hindlimb ATP synthesis and [sup.13]C-MRS was used to measure the hindlimb tricarboxylic acid cycle flux ([V.sub.tca]), There was no alteration, at either dose, in mitochondrial coupling efficiency measured as the ratio of unidirectional ATP synthesis flux to [V.sub.tca]. Soleus muscle GLUT4 expression was decreased by twofold, whereas pyruvate dehydrogenase kinase 4, carnitine palmitoyl transferase 1a, and uncoupling protein 2 and 3 expression was increased by two- to threefold at the high dose (P < 0.05). In summary, these are the first noninvasive measurements illustrating a selective PPAR[delta]-mediated decrease in muscle lipid content that was consistent with a shift in metabolic substrate utilization from carbohydrate to lipid. However, the mitochondrial-energy coupling efficiency was not altered in the presence of increased uncoupling protein expression. peroxisome proliferator-activated receptor-[delta]; intramyocellular lipid; mitochondrial coupling; muscle

Aim: Cabotegravir long‐acting (LA) intramuscular (IM) injection is being investigated for HIV preexposure prophylaxis due to its potent antiretroviral activity and infrequent dosing requirement. A subset of healthy adult volunteers participating in a Phase I study assessing cabotegravir tissue pharmacokinetics underwent serial magnetic resonance imaging (MRI) to assess drug depot localization and kinetics following a single cabotegravir LA IM targeted injection. Methods: Eight participants (four men, four women) were administered cabotegravir LA 600 mg under ultrasonographic‐guided injection targeting the gluteal muscles. MRI was performed to determine injection‐site location in gluteal muscle (IM), subcutaneous (SC) adipose tissue and combined IM/SC compartments, and to quantify drug depot characteristics, including volume and surface area, on Days 1 (≤2 hours postinjection), 3 and 8. Linear regression analysis examined correlations between MRI‐derived parameters and plasma cabotegravir exposure metrics, including maximum observed concentration (Cmax) and partial area under the concentration–time curve (AUC) through Weeks 4 and 8. Results: Cabotegravir LA depot locations varied by participant and were identified in the IM compartment (n = 2), combined IM/SC compartments (n = 4), SC compartment (n = 1) and retroperitoneal cavity (n = 1). Although several MRI parameter and exposure metric correlations were determined, total depot surface area on Day 1 strongly correlated with plasma cabotegravir concentration at Days 3 and 8, Cmax and partial AUC through Weeks 4 and 8. Conclusion: MRI clearly delineated cabotegravir LA injection‐site location and depot kinetics in healthy adults. Although injection‐site variability was observed, drug depot surface area correlated with both plasma Cmax and partial AUC independently of anatomical distribution. [ABSTRACT FROM AUTHOR]

A noninvasive procedure to determine the amount of intracellular glucose is presented. The method makes use of isotopic carbon nuclear magnetic resonance spectroscopy. Intracellular glucose concentration is derived from the measure of the total tissue glucose and the amount of glucose in the extracellular space. A number of corrections for the extracellular glucose concentration must be taken into consideration. The applications of intracellular glucose determination are discussed.

A nuclear magnetic resonance imaging technique has been developed to identify and measure intracellular glucose metabolism in the skeletal muscle of conscious rats. The method can also be used to measure glucose metabolism in humans. Specific measurements were taken for muscle glycolysis, glycogen synthesis, pyruvate dehydrogenase and pyruvate carboxylation. The anaplerotic contribution of substrate via pyruvate carboxylation was established for glutamate and pyruvate using steady-state isotopomer analysis.

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining [sup.13]C NMR to measure rates of mitochondrial substrate oxidation with [sup.31]P NMR to assess unidirectional ATP synthesis flux. These combined [sup.31]P/[sup.13]C NMR measurements were performed in control, 10-day triiodo-L-thyronine ([T.sub.3])-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold ([+ or -] 1.1) and 2.8-fold ([+ or -] 0.8), respectively, in the [T.sub.3]-treated vs. control rat gastrocnemius muscle. [sup.13]C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 [+ or -] 21,148 [+ or -] 25, and 310 [+ or -] 48 nmol/g per min in the control, [T.sub.3], and DNP groups, respectively. [sup.31]P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 [+ or -] 14, 84 [+ or -] 14, and 73 [+ or -] 7 nmol/g per s in the control, [T.sub.3], and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of [T.sub.3] decrease energy coupling by [approximately equals] 80% and [approximately equals] 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.

Jucker, Beat M., Nicole Barucci, and Gerald I. Shulman. Metabolic control analysis of insulin-stimulated glucose disposal in rat skeletal muscle. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E505-E512, 1999.--Metabolic control analysis was used to calculate the distributed control of insulin-stimulated skeletal muscle glucose disposal in awake rats. Three separate hyperinsulinemic infusion protocols were performed: 1) protocol I was a euglycemic (~6 mM)-hyperinsulinemic (10 mU [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) clamp, 2) protocol II was a hyperglycemic (~11 mM)-hyperinsulinemic (10 mU [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1]) clamp, and 3) protocol III was a euglycemic (~6 mM)-hyperinsulinemic (10 mU [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1])-lipid/heparin increased plasma free fatty acid) clamp. [1-[sup.13]C]glucose was administered in all three protocols for a 3-h period, during which time [1-[sup.13]C]glucose label incorporation into [1-[sup.13]C]glycogen, [3-[sup.13]C]lactate, and [3-[sup.13]C]alanine was detected in the hindlimb of awake rats via [sup.13]C-NMR. Combined steady-state and kinetic data were used to calculate rates of glycogen synthesis and glycolysis. Additionally, glucose 6-phosphate (G-6-P) was measured in the hindlimb muscles with the use of in vivo [sup.31]P-NMR during the three infusion protocols. The clamped glucose infusion rates were 31.6 [+ or -] 2.9, 49.7 [+ or -] 1.0, and 24.0 [+ or -] 1.5 mg [multiplied by] [kg.sup.-1] [multiplied by] [min.sup.-1] at 120 min in protocols I-III, respectively. Rates of glycolysis were 62.1 [+ or -] 10.3, 71.6 [+ or -] 11.8, and 19.5 [+ or -] 3.6 nmol [multiplied by] [g.sup.-1] [multiplied by] [min.sup.-1] and rates of glycogen synthesis were 125 [+ or -] 15, 224 [+ or -] 23, and 104 [+ or -] 17 nmol [multiplied by] [g.sup.-1] [multiplied by] [min.sup.-1] in protocols I-III, respectively. Insulin-stimulated G-6-P concentrations were 217 [+ or -] 8, 265 [+ or -] 12, and 251 [+ or -] 9 nmol/g in protocols I-III, respectively. A top-down approach to metabolic control analysis was used to calculate the distributed control among glucose transport/phosphorylation [GLUT-4/hexokinase (HK)], glycogen synthesis, and glycolysis from the metabolic flux and G-6-P data. The calculated values for the control coefficients (C) of these three metabolic steps ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] = 0.55 [+ or -] 0.10, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]= 0.30 [+ or -] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]=0.15 [+ or -] 0.02; where J is glucose disposal flux, and glycogen syn is glycogen synthesis) indicate that there is shared control of glucose disposal and that glucose transport/phosphorylation is responsible for the majority of control of insulin-stimulated glucose disposal in skeletal muscle. nuclear magnetic resonance; flux control; glycogen synthesis; glycolysis; glucose 6-phosphate

Objectives: In our laboratory we evaluated the use of micro‐computed tomography (micro‐CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). Methods: To show concordance between traditional Alizarin Red S stain and micro‐CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro‐CT, then stained and re‐examined using traditional methods. Results: A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low‐density skeletal bones may be visible by Alizarin Red stain but not by micro‐CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro‐CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro‐CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro‐CT scanned and traditional Alizarin Red S stained skeletons. Conclusion: Micro‐CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies. [ABSTRACT FROM AUTHOR]