bluuebunny/crossref_metadata_2025 huggingface dataset:
Isomer-specific actions of conjugated linoleic acid on muscle glucose transport in the obese Zucker rat
Authors: Henriksen Erik J. , Teachey Mary K. , Taylor Zachary C. , Jacob Stephan , Ptock Arne , Krämer Klaus , Hasselwander Oliver
Published: March 2015

The fatty acid-conjugated linoleic acid (CLA) enhances glucose tolerance and insulin action on skeletal muscle glucose transport in rodent models of insulin resistance. However, no study has directly compared the metabolic effects of the two primary CLA isomers, cis-9, trans-11-CLA (c9,t11-CLA) and trans-10, cis-12-CLA (t10,c12-CLA). Therefore, we assessed the effects of a 50:50 mixture of these two CLA isomers (M-CLA) and of preparations enriched in either c9,t11-CLA (76% enriched) or t10,c12-CLA (90% enriched) on glucose tolerance and insulin-stimulated glucose transport in skeletal muscle of the insulin-resistant obese Zucker ( fa/ fa) rat. Animals were treated daily by gavage with either vehicle (corn oil), M-CLA, c9,t11-CLA, or t10,c12-CLA (all CLA treatments at 1.5 g total CLA/kg body wt) for 21 consecutive days. During an oral glucose tolerance test, glucose responses were reduced ( P < 0.05) by 10 and 16%, respectively, in the M-CLA and t10,c12-CLA animals, respectively, whereas insulin responses were diminished by 21 and 19% in these same groups. There were no significant alterations in these responses in the c9,t11-CLA group. Insulin-mediated glucose transport activity was enhanced by M-CLA treatment in both type I soleus (32%) and type IIb epitrochlearis (58%) muscles and by 36 and 48%, respectively, with t10,c12-CLA. In the soleus, these increases were associated with decreases in protein carbonyls (index of oxidative stress, r = -0.616, P = 0.0038) and intramuscular triglycerides ( r = -0.631, P = 0.0028). Treatment with c9,t11-CLA was without effect on these variables. These results suggest that the ability of CLA treatment to improve glucose tolerance and insulin-stimulated glucose transport activity in insulin-resistant skeletal muscle of the obese Zucker rat are associated with a reduction in oxidative stress and muscle lipid levels and can be specifically ascribed to the actions of the t10,c12 isomer. In the obese Zucker rat, the c9,t11 isomer of CLA is metabolically neutral.
Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells
Authors: Suárez Cecilia , Díaz-Torga Graciela , Gonzalez-Iglesias Arturo , Vela Jorge , Mladovan Alejandro , Baldi Alberto , Becu-Villalobos Damasia
Published: March 2015

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.
Insulin action on protein metabolism in acromegalic patients
Authors: Battezzati Alberto , Benedini Stefano , Fattorini Annalisa , Losa Marco , Mortini Pietro , Bertoli Simona , Lanzi Roberto , Testolin Giulio , Biolo Gianni , Luzi Livio
Published: March 2015

Insulin resistance in acromegaly causes glucose intolerance and diabetes, but it is unknown whether it involves protein metabolism, since both insulin and growth hormone promote protein accretion. The effects of acromegaly and of its surgical cure on the insulin sensitivity of glucose and amino acid/protein metabolism were evaluated by infusing [6,6-2H2]glucose, [1-13C]leucine, and [2-15N]glutamine during a euglycemic insulin (1 mU · kg−1 · min−1) clamp in 12 acromegalic patients, six studied again 6 mo after successful adenomectomy, and eight healthy controls. Acromegalic patients, compared with postsurgical and control subjects, had higher postabsorptive glucose concentration (5.5 ± 0.3 vs. 4.9 ± 0.2 μmol/l, P < 0.05, and 5.1 ± 0.1 μmol/l) and flux (2.7 ± 0.1 vs. 2.0 ± 0.2 μmol · kg−1 · min−1, P < 0.01, and 2.2 ± 0.1 μmol · kg−1 · min−1, P < 0.05) and reduced insulin-stimulated glucose disposal (+15 ± 9 vs. +151 ± 18%, P < 0.01, and 219 ± 58%, P < 0.001 from basal). Postabsorptive leucine metabolism was similar among groups. In acromegalic and postsurgical subjects, insulin suppressed less than in controls the endogenous leucine flux (−9 ± 1 and −12 ± 2 vs. −18 ± 2%, P < 0.001 and P< 0.05), the nonoxidative leucine disposal (−4 ± 3 and −1 ± 3 vs. −18 ± 2%, P < 0.01 and P < 0.05), respectively, indexes of proteolysis and protein synthesis, and leucine oxidation (−17 ± 6% in postsurgical patients vs. −26 ± 6% in controls, P < 0.05). Within 6 mo, surgery reverses insulin resistance for glucose but not for protein metabolism. After adenomectomy, more leucine is oxidized during hyperinsulinemia.
Inhibiting NOS blocks microvascular recruitment and blunts muscle glucose uptake in response to insulin
Authors: Vincent M. A. , Barrett E. J. , Lindner J. R. , Clark M. G. , Rattigan S.
Published: March 2015

We examined the effects of inhibiting nitric oxide synthase with Nω-nitro-l-arginine-methyl ester (l-NAME) on total hindlimb blood flow, muscle microvascular recruitment, and hindlimb glucose uptake during euglycemic hyperinsulinemia in vivo in the rat. We used two independent methods to measure microvascular perfusion. In one group of animals, microvascular recruitment was measured using the metabolism of exogenously infused 1-methylxanthine (1-MX), and in a second group contrast-enhanced ultrasound (CEU) was used. Limb glucose uptake was measured by arterial-venous concentration differences after 2 h of insulin infusion. Saline alone did not alter femoral artery flow, glucose uptake, or 1-MX metabolism. Insulin (10 mU·min-1·kg-1) significantly increased hindlimb total blood flow (0.69 ± 0.02 to 1.22 ± 0.11 ml/min, P < 0.05), glucose uptake (0.27 ± 0.05 to 0.95 ± 0.08 μmol/min, P < 0.05), 1-MX uptake (5.0 ± 0.5 to 8.5 ± 1.0 nmol/min, P < 0.05), and skeletal muscle microvascular volume measured by CEU (10.0 ± 1.6 to 15.0 ± 1.2 video intensity units, P < 0.05). Addition of l-NAME to insulin completely blocked the effect of insulin on both total limb flow and microvascular recruitment (measured using either 1-MX or CEU) and blunted glucose uptake by 40% ( P < 0.05). We conclude that insulin specifically recruits flow to the microvasculture in skeletal muscle via a nitric oxide-dependent pathway and that this may be important to insulin's overall action to regulate glucose disposal.
Hormone-sensitive lipase activity and triacylglycerol hydrolysis are decreased in rat soleus muscle by cyclopiazonic acid
Authors: Watt Matthew J. , Steinberg Gregory R. , Heigenhauser G. J. F. , Spriet Lawrence L. , Dyck David J.
Published: March 2015

Cyclopiazonic acid (CPA) is a sarcoplasmic reticulum Ca2+-ATPase inhibitor that increases intracellular calcium. The role of CPA in regulating the oxidation and esterification of palmitate, the hydrolysis of intramuscular lipids, and the activation of hormone-sensitive lipase (HSL) was examined in isolated rat soleus muscles at rest. CPA (40 μM) was added to the incubation medium to levels that resulted in subcontraction increases in muscle tension, and lipid metabolism was monitored using the previously described pulse-chase procedure. CPA did not alter the cellular energy state, as reflected by similar muscle contents of ATP, phosphocreatine, free AMP, and free ADP. CPA increased total palmitate uptake into soleus muscle (11%, P < 0.05) and was without effect on palmitate oxidation. This resulted in greater esterification of exogenous palmitate into the triacylglycerol (18%, P < 0.05) and phospholipid (89%, P < 0.05) pools. CPA decreased ( P < 0.05) intramuscular lipid hydrolysis, and this occurred as a result of reduced HSL activity (20%, P < 0.05). Incubation of muscles with 3 mM caffeine, which is also known to increase Ca2+without affecting the cellular energy state, reduced HSL activity (24%, P < 0.05). KN-93, a calcium/calmodulin-dependent kinase inhibitor (CaMKII), blocked the effects of CPA and caffeine, and HSL activity returned to preincubation values. The results of the present study demonstrate that CPA simultaneously decreases intramuscular triacylglycerol (IMTG) hydrolysis and promotes lipid storage in isolated, intact soleus muscle. The decreased IMTG hydrolysis is likely mediated by reduced HSL activity, possibly via the CaMKII pathway. These responses are not consistent with the increased hydrolysis and decreased esterification observed in contracting muscle when substrate availability and the hormonal milieu are tightly controlled. It is possible that more powerful signals or a higher [Ca2+] may override the lipid-storage effect of the CPA-mediated effects during muscular contractions.
Sex-specific p38 MAP kinase activation following trauma-hemorrhage: involvement of testosterone and estradiol
Authors: Angele Martin K. , Nitsch Stefan , Knöferl Markus W. , Ayala Alfred , Angele Peter , Schildberg Friedrich W. , Jauch Karl W. , Chaudry Irshad H.
Published: March 2015

Although immune functions are markedly depressed in males and not in proestrous females following trauma-hemorrhage (T-H), the mechanisms responsible for the divergent responses remain unknown. Because sex steroids modulate the activation of p38, our aim was to determine whether differences in the activation of p38 by phosphorylation (p38-P) might contribute to the sex-dimorphic immune response following T-H. The effects of testosterone and estradiol on the activation of p38 were also examined. Intact male mice (C3H/HeN), castrated males treated with vehicle, 5α-dihydrotestosterone (DHT), or 17β-estradiol, and proestrous females were subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (35 ± 5 mmHg for 90 min and resuscitation) or sham operation. At 2 h thereafter, splenic (SMφ) and peritoneal macrophages (PMφ) were harvested and cultured (with 10 μg/ml LPS), and Western blot analysis was carried out for quantification of p38 and p38-P. Sex, testosterone and estradiol plasma levels, and T-H did not alter the constitutive expression of p38 in SMφ and PMφ. In contrast, the activated form of p38 (p38-P) was markedly increased in SMφ and PMφ from female shams compared with male shams. Moreover, the phosphorylation of p38-P increased in males after T-H, whereas it decreased in females under those conditions. Castration before T-H prevented the increase in p38-P in males. Castrated animals treated with DHT displayed increased p38-P following T-H, whereas 17β-estradiol had no effect on p38-P in castrated mice. Thus 1) sex influences the activation of p38 MAP kinase, 2) DHT is responsible for the increased activation of p38 in male mice, and 3) this sex-specific activation of p38 might be responsible for the sexually dimorphic immune response following T-H.
Visceral adiposity, C-peptide levels, and low lipase activities predict HIV-dyslipidemia
Authors: Yarasheski Kevin E. , Tebas Pablo , Claxton Sherry , Marin Donna , Coleman Trey , Powderly William G. , Semenkovich Clay F.
Published: March 2015

Protease inhibitor-based highly active antiretroviral therapy (PI-HAART) has been implicated in dyslipidemia, peripheral insulin resistance, and abnormal adipose tissue deposition in human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome, or AIDS. In vitro evidence indicates that some PIs reduce adipocyte lipoprotein (LPL) and hepatic lipase (HL) expression and activities. We examined whether LPL and HL activities are reduced in HIV-infected patients with dyslipidemia. Fasting serum lipids, glucoregulatory hormones, and postheparin LPL and HL activities, as well as whole body and regional adiposity, were measured in 19 HIV-seronegative controls, 9 HIV+ patients naive to all anti-HIV medications, 9 HIV+ patients naive to PIs, 9 HIV+ patients with prior PI experience but not currently receiving PIs, and 47 HIV+ patients receiving PI-HAART. The PI-HAART group had low LPL and HL activities. However, multiple linear regression analysis indicated that low postheparin LPL activity contributed only partially to HIV-dyslipidemia. Central adiposity and high C-peptide levels (an indicator of high insulin secretion) were stronger predictors of HIV-dyslipidemia. Low LPL and HL activities, by themselves, were insufficient to explain HIV-dyslipidemia because the PI-naive group had low LPL and HL activities but had normal adiposity, C-peptide levels, and serum lipid and lipoprotein levels. HDL-cholesterol was lower in PI-HAART and PI-naive groups than seronegative controls and was directly associated with LPL activity. These findings suggest that HIV-dyslipidemia is mediated primarily by factors that influence triglyceride and lipoprotein synthesis (e.g., central adiposity and hyperinsulinemia) and mediated only partially by factors that influence triglyceride clearance (e.g., lipase activity).
Characterization of rat iodothyronine sulfotransferases
Authors: Kester Monique H. A. , Kaptein Ellen , Roest Thirza J. , van Dijk Caren H. , Tibboel Dick , Meinl Walter , Glatt Hansruedi , Coughtrie Michael W. H. , Visser Theo J.
Published: March 2015

Sulfation appears to be an important pathway for the reversible inactivation of thyroid hormone during fetal development. The rat is an often used animal model to study the regulation of fetal thyroid hormone status. The present study was done to determine which sulfotransferases (SULTs) are important for iodothyronine sulfation in the rat, using radioactive T4, T3, rT3, and 3,3′-T2as substrates, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as cofactor, and rat liver, kidney and brain cytosol, and recombinant rat SULT1A1, -1B1, -1C1, -1E1, -2A1, -2A2, and -2A3 as enzymes. Recombinant rat SULT1A1, -1E1, -2A1, -2A2, and -2A3 failed to catalyze iodothyronine sulfation. For all tissue SULTs and for rSULT1B1 and rSULT1C1, 3,3′-T2was by far the preferred substrate. Apparent Kmvalues for 3,3′-T2amounted to 1.9 μM in male liver, 4.4 μM in female liver, 0.76 μM in male kidney, 0.23 μM in male brain, 7.7 μM for SULT1B1, and 0.62 μM for SULT1C1, whereas apparent Kmvalues for PAPS showed less variation (2.0-6.9 μM). Sulfation of 3,3′-T2was inhibited dose dependently by other iodothyronines, with similar structure-activity relationships for most enzymes except for the SULT activity in rat brain. The apparent Kmvalues of 3,3′-T2in liver cytosol were between those determined for SULT1B1 and -1C1, supporting the importance of these enzymes for the sulfation of iodothyronines in rat liver, with a greater contribution of SULT1C1 in male than in female rat liver. The results further suggest that rSULT1C1 also contributes to iodothyronine sulfation in rat kidney, whereas other, yet-unidentified forms appear more important for the sulfation of thyroid hormone in rat brain.
Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity, leading to PKC-dependent insulin exocytosis
Authors: Lehtihet Mikael , Welsh Nils , Berggren Per-Olof , Cook George A. , Sjöholm Åke
Published: March 2015

Hypoglycemic sulfonylureas such as glibenclamide have been widely used to treat type 2 diabetic patients for 40 yr, but controversy remains about their mode of action. The widely held view is that they promote rapid insulin exocytosis by binding to and blocking pancreatic β-cell ATP-dependent K+(KATP) channels in the plasma membrane. This event stimulates Ca2+influx and sets in motion the exocytotic release of insulin. However, recent reports show that >90% of glibenclamide-binding sites are localized intracellularly and that the drug can stimulate insulin release independently of changes in KATPchannels and cytoplasmic free Ca2+. Also, glibenclamide specifically and progressively accumulates in islets in association with secretory granules and mitochondria and causes long-lasting insulin secretion. It has been proposed that nutrient insulin secretagogues stimulate insulin release by increasing formation of malonyl-CoA, which, by blocking carnitine palmitoyltransferase 1 (CPT-1), switches fatty acid (FA) catabolism to synthesis of PKC-activating lipids. We show that glibenclamide dose-dependently inhibits β-cell CPT-1 activity, consequently suppressing FA oxidation to the same extent as glucose in cultured fetal rat islets. This is associated with enhanced diacylglycerol (DAG) formation, PKC activation, and KATP-independent glibenclamide-stimulated insulin exocytosis. The fat oxidation inhibitor etomoxir stimulated KATP-independent insulin secretion to the same extent as glibenclamide, and the action of both drugs was not additive. We propose a mechanism in which inhibition of CPT-1 activity by glibenclamide switches β-cell FA metabolism to DAG synthesis and subsequent PKC-dependent and KATP-independent insulin exocytosis. We suggest that chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may explain the prolonged stimulation of insulin secretion in some diabetic patients even after drug removal that contributes to the sustained hypoglycemia of the sulfonylurea.
Model to assess muscle protein turnover: domain of validity using amino acyl-tRNA vs. surrogate measures of precursor pool
Authors: Toffolo Gianna , Albright Robert , Joyner Michael , Dietz Niki , Cobelli Claudio , Nair K. Sreekumaran
Published: March 2015

Current models to measure protein turnover across muscle bed are based on many surrogate measures of amino acyl-tRNA. We measured muscle protein turnover based on tracer-to-tracee ratios of the stable isotopes of leucine, phenylalanine, and ketoisocaproate (KIC) in artery and vein and muscle amino acyl-tRNA and muscle tissue fluid (TF) in 26 healthy subjects. A three-compartment model calculation based on arteriovenous and tRNA measurements was first performed and its domain of validity assessed. The results were then compared with those using simpler approaches based on surrogate measures of tRNA such as those of TF and KIC and a one-compartment model based on arteriovenous amino acids. In 96% of cases, the model using tRNA was applicable, but only in a lower percentage of cases were the results using surrogate measures applicable. Protein breakdown, protein synthesis, and shunting of amino acids from artery to vein were consistently underestimated, and fluxes of amino acid from artery to intracellular compartment and from intracellular compartment to vein were overestimated, when surrogate measures were used. The one-compartment model also underestimated protein breakdown and synthesis. Measurements using tissue fluid gave results closer to those based on tRNA. In conclusion, a three-compartment model using arteriovenous samples and amino acyl-tRNA provides measurements of muscle protein turnover of acceptable precision in 96% of cases. The precision was unacceptable in a substantial percentage of cases, and the accuracy of the estimation of protein fluxes was significantly affected when surrogate measures were used.
Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin
Authors: Reisz-Porszasz Suzanne , Bhasin Shalender , Artaza Jorge N. , Shen Ruoqing , Sinha-Hikim Indrani , Hogue Aimee , Fielder Thomas J. , Gonzalez-Cadavid Nestor F.
Published: March 2015

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18–24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.
Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans
Authors: Bruun Jens M. , Lihn Aina S. , Verdich Camilla , Pedersen Steen B. , Toubro Søren , Astrup Arne , Richelsen Bjørn
Published: March 2015

Adiponectin is an adipose tissue-specific protein that is abundantly present in the circulation and suggested to be involved in insulin sensitivity and development of atherosclerosis. Because cytokines are suggested to regulate adiponectin, the aim of the present study was to investigate the interaction between adiponectin and three adipose tissue-derived cytokines (IL-6, IL-8, and TNF-α). The study was divided into three substudies as follows: 1) plasma adiponectin and mRNA levels in adipose tissue biopsies from obese subjects [mean body mass index (BMI): 39.7 kg/m2, n = 6] before and after weight loss; 2) plasma adiponectin in obese men (mean BMI: 38.7 kg/m2, n = 19) compared with lean men (mean BMI: 23.4 kg/m2, n = 10) before and after weight loss; and 3) in vitro direct effects of IL-6, IL-8, and TNF-α on adiponectin mRNA levels in adipose tissue cultures. The results were that 1) weight loss resulted in a 51% ( P < 0.05) increase in plasma adiponectin and a 45% ( P < 0.05) increase in adipose tissue mRNA levels; 2) plasma adiponectin was 53% ( P < 0.01) higher in lean compared with obese men, and plasma adiponectin was inversely correlated with adiposity, insulin sensitivity, and IL-6; and 3) TNF-α ( P < 0.01) and IL-6 plus its soluble receptor ( P < 0.05) decreased adiponectin mRNA levels in vitro. The inverse relationship between plasma adiponectin and cytokines in vivo and the cytokine-induced reduction in adiponectin mRNA in vitro suggests that endogenous cytokines may inhibit adiponectin. This could be of importance for the association between cytokines (e.g., IL-6) and insulin resistance and atherosclerosis.
Influence of prolonged endurance cycling and recovery diet on intramuscular triglyceride content in trained males
Authors: van Loon Luc J. C. , Schrauwen-Hinderling Vera B. , Koopman René , Wagenmakers Anton J. M. , Hesselink Matthijs K. C. , Schaart Gert , Kooi M. Eline , Saris Wim H. M.
Published: March 2015

Intramuscular triglycerides (IMTG) are assumed to form an important substrate source during prolonged endurance exercise in trained males. This study investigated the effects of endurance exercise and recovery diet on IMTG content in vastus lateralis muscle. Nine male cyclists were provided with a standardized diet for 3 days, after which they performed a 3-h exercise trial at a 55% maximum workload. Before and immediately after exercise and after 24 and 48 h of recovery, magnetic resonance spectroscopy (MRS) was performed to quantitate IMTG content. Muscle biopsies were taken after 48 h of recovery to determine IMTG content by using quantitative fluorescence microscopy. The entire procedure was performed two times; in one trial, a normal diet containing 39% energy (En%) as fat was provided (NF) and in the other a typical carbohydrate-rich athlete's diet (LF: 24 En% fat) was provided. During exercise, IMTG content decreased by 21.4 ± 3.1%. During recovery, IMTG content increased significantly in the NF trial only, reaching preexercise levels within 48 h. In accord with MRS, fluorescence microscopy showed significantly higher IMTG content in the NF compared with the LF trial, with differences restricted to the type I muscle fibers (2.1 ± 0.2 vs. 1.4 ± 0.2% area lipid staining, respectively). In conclusion, IMTG content in the vastus lateralis muscle declines significantly during prolonged endurance exercise in male cyclists. When a normal diet is used, IMTG contents are subsequently repleted within 48 h of postexercise recovery. In contrast, IMTG repletion is impaired substantially when a typical, carbohydrate-rich athlete's diet is used. Data obtained by quantitative fluorescence microscopy correspond well with MRS results, implying that both are valid methods to quantify IMTG content.
Control of LH secretory-burst frequency and interpulse-interval regularity in women
Authors: Keenan Daniel M. , Evans William S. , Veldhuis Johannes D.
Published: March 2015

Hypothalamic neurons generate discrete bursts of gonadotropin-releasing hormone (GnRH) and thereby pulses of luteinizing hormone (LH) at randomly timed intervals centered on a probabilistic mean frequency. We tested the hypothesis that physiological mechanisms govern not only the number but also the stochastic dispersion of the GnRH/LH pulse-renewal process in humans; for example, in young women in the early (EF) and late (LF) follicular and midluteal (ML) phases of the menstrual cycle ( n = 18) and in postmenopausal individuals (PM, n = 16). To this end, we quantify stochastic interpulse variability by way of the order-independent, two-parameter Weibull renewal process (Keenan DM and Veldhuis J. Am J Physiol Regul Integr Comp Physiol 281: R1917–R1924, 2001) and the sequence-specific, model-free approximate-entropy statistic (ApEn) (Pincus SM. Proc Natl Acad Sci USA 88: 2297–2301, 1991). Statistical testing unveiled 1) reduced probabilistic mean LH secretory-burst frequency (lower λ of the Weibull distribution) in ML compared with each of EF, LF, and PM ( P < 0.001); 2) quantifiably more regular LH interburst-interval sets (elevated γ of the Weibull density) in PM than in each of EF, LF, and ML ( P < 0.01); 3) uniquely prolonged latency to maximal LH secretion within individual secretory bursts in ML ( P < 0.01); and 4) comparably mean random, sequential LH interburst-interval and mass values (normalized ApEn) among the distinct hormonal milieus. From these data, we postulate that sex steroids and age determine daily LH secretory-burst number, quantifiable pulse-renewal variability, and secretory-waveform evolution.
Serine metabolism in human pregnancy
Authors: Kalhan Satish C. , Gruca Lourdes L. , Parimi Prabhu S. , O'Brien Alicia , Dierker Leroy , Burkett Ed
Published: March 2015

Serine plays an important role in intermediary metabolism as a source of one carbon pool for nucleotide biosynthesis, as a precursor for glycine and glucose, and as a contributor to cysteine biosynthesis. A unique serine-glycine cycling between the liver and the placenta has been demonstrated in the sheep fetus. We hypothesized that, because of serine's role in growth and development, significant changes in serine metabolism will occur in pregnancy with advancing gestation. The rate of appearance (Ra) of serine and its metabolism were quantified in healthy women longitudinally through pregnancy with a [2-15N13C]serine tracer. The contribution of serine N to urea and the rate of oxidation of serine were measured using the precursor-product relation. Plasma serine concentrations and serine Ra were lower in pregnant (P) women, in both early and late gestation, compared with nonpregnant (NP) women [plasma serine: NP, 113 ± 24.5; P early, 71.9 ± 6.2; P late, 68.5 ± 9.6 μmol/l; serine Ra: NP ( n = 7), 152.9 ± 42.8; P early ( n = 12), 123.7 ± 21.5; P late ( n = 8), 102.8 ± 18.2 μmol · kg−1 · h−1]. Serine contributed ∼6% to urea N and 15–20% to the plasma glycine pool, and oxidation of serine represented ∼8% of Ra. There was no significant difference between P and NP subjects. Glucose infusion, at 3 mg · kg−1 · min−1in P subjects, resulted in a decrease in serine Ra and an increase in oxidation. The decrease in serine turnover in pregnancy may represent a decrease in α-amino nitrogen turnover related to a decreased rate of branched-chain amino acid transamination and caused by pregnancy-related hormones aimed at nitrogen conservation and accretion.
Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise
Authors: Coven David L. , Hu Xiaoyue , Cong Lin , Bergeron Raynald , Shulman Gerald I. , Hardie D. Grahame , Young Lawrence H.
Published: March 2015

AMP-activated protein kinase (AMPK) is emerging as a key signaling pathway that modulates cellular metabolic processes. In skeletal muscle, AMPK is activated during exercise. Increased myocardial substrate metabolism during exercise could be explained by AMPK activation. Although AMPK is known to be activated during myocardial ischemia, it remains uncertain whether AMPK is activated in response to the physiological increases in cardiac work associated with exercise. Therefore, we evaluated cardiac AMPK activity in rats at rest and after 10 min of treadmill running at moderate (15% grade, 16 m/min) or high (15% grade, 32 m/min) intensity. Total AMPK activity in the heart increased in proportion to exercise intensity ( P < 0.05). AMPK activity associated with the α2-catalytic subunit increased 2.8 ± 0.4-fold ( P < 0.02 vs. rest) and 4.5 ± 0.6-fold ( P < 0.001 vs. rest) with moderate- and high-intensity exercise, respectively. AMPK activity associated with the α1-subunit increased to a lesser extent. Phosphorylation of the Thr172-regulatory site on AMPK α-catalytic subunits increased during exercise ( P < 0.001). There was no increase in Akt phosphorylation during exercise. The changes in AMPK activity during exercise were associated with physiological AMPK effects (GLUT4 translocation to the sarcolemma and ACC phosphorylation). Thus cardiac AMPK activity increases progressively with exercise intensity, supporting the hypothesis that AMPK has a physiological role in the heart.
Human intestinal monoacylglycerol acyltransferase: differential features in tissue expression and activity
Authors: Lockwood John F. , Cao Jingsong , Burn Paul , Shi Yuguang
Published: March 2015

Acyl CoA-monoacylglycerol acyltransferase (MGAT) catalyzes the first step in triacyglycerol resynthesis involved in dietary absorption in enterocytes. Despite its potentially important role in dietary fat absorption, a gene encoding a human intestinal MGAT has not been identified. In this study, we report the identification and functional characterization of a human intestinal MGAT ( hMGAT2) and its splice variant (hMGAT2V). The hMGAT2 gene encodes a peptide of 334 amino acids with a molecular mass of 38.2 kDa that shares 81 and 47% amino acid identities with the mouse MGAT2 and the human diacylglycerol acyltransferase (DGAT2) enzymes, respectively. The hMGAT2 gene is localized on chromosome 11q13.5, adjacent to the DGAT2 gene, suggesting gene duplication. Transient expression of hMGAT2, but not an alternatively spliced variant, hMGAT2V, in COS-7 cells led to a ninefold increase in the synthesis of DAG. The human and mouse differ significantly in tissue distribution of MGAT2.In addition to a predominant expression in the small intestine in both species, distinct levels were also found in the human liver, contrasting with higher levels in the mouse kidney. In comparison with a single 1.8-kb transcript in mouse, the hMGAT2 gene expressed two transcripts of 3.0 and 6.0 kb in size that encode MGAT2 and an inactive peptide with unknown functions, respectively. Despite a significant level of hMGAT2 mRNA in the human liver, little MGAT activity was detected in liver microsomes when tested against monoacyglcerols with different unsaturated side chains, suggesting possible posttranscriptional regulation.
Ghrelin promotes slow-wave sleep in humans
Authors: Weikel J. C. , Wichniak A. , Ising M. , Brunner H. , Friess E. , Held K. , Mathias S. , Schmid D. A. , Uhr M. , Steiger A.
Published: March 2015

Ghrelin, an endogenous ligand of the growth hormone (GH) secretagogue (GHS) receptor, stimulates GH release, appetite, and weight gain in humans and rodents. Synthetic GHSs modulate sleep electroencephalogram (EEG) and nocturnal hormone secretion. We studied the effect of 4 × 50 μg of ghrelin administered hourly as intravenous boluses between 2200 and 0100 on sleep EEG and the secretion of plasma GH, ACTH, cortisol, prolactin, and leptin in humans ( n = 7). After ghrelin administration, slow-wave sleep was increased during the total night and accumulated δ-wave activity was enhanced during the second half of the night. Rapid-eye-movement (REM) sleep was reduced during the second third of the night, whereas all other sleep EEG variables remained unchanged. Furthermore, GH and prolactin plasma levels were enhanced throughout the night, and cortisol levels increased during the first part of the night (2200–0300). The response of GH to ghrelin was most distinct after the first injection and lowest after the fourth injection. In contrast, cortisol showed an inverse pattern of response. Leptin levels did not differ between groups. Our data show a distinct action of exogenous ghrelin on sleep EEG and nocturnal hormone secretion. We suggest that ghrelin is an endogenous sleep-promoting factor. This role appears to be complementary to the already described effects of the peptide in the regulation of energy balance. Furthermore, ghrelin appears to be a common stimulus of the somatotropic and hypothalamo-pituitary-adrenocortical systems. It appears that ghrelin is a sleep-promoting factor in humans.
Developmental switch in brain nutrient transporter expression in the rat
Authors: Vannucci Susan J. , Simpson Ian A.
Published: March 2015

Normal development of both human and rat brain is associated with a switch in metabolic fuel from a combination of glucose and ketone bodies in the immature brain to a nearly total reliance on glucose in the adult. The delivery of glucose, lactate, and ketone bodies from the blood to the brain requires specific transporter proteins, glucose and monocarboxylic acid transporter proteins (GLUTs and MCTs), respectively. Developmental expression of the GLUTs in rat brain, i.e., 55-kDa GLUT1 in the blood-brain barrier (BBB), 45-kDa GLUT1 and GLUT3 in vascular-free brain, corresponds to maturational increases in cerebral glucose uptake and utilization. It has been suggested that MCT expression peaks during suckling and sharply declines thereafter, although a comparable detailed study has not been done. This study investigated the temporal and regional expression of MCT1 and MCT2 mRNA and protein in the BBB and the nonvascular brain during postnatal development in the rat. The results confirmed maximal MCT1 mRNA and protein expression in the BBB during suckling and a decline with maturation, coincident with the switch to glucose as the predominant cerebral fuel. However, nonvascular MCT1 and MCT2 levels do not reflect changes in cerebral energy metabolism, suggesting a more complex regulation. Although MCT1 assumes a predominantly glial expression in postweanling brain, the concentration remains fairly constant, as does that of MCT2 in neurons. The maintenance of nonvascular MCT levels in the adult brain implies a major role for these proteins, in concert with the GLUTs in both neurons and astrocytes, to transfer glycolytic intermediates during cerebral energy metabolism.
Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes
Authors: Ferone Diego , Pivonello Rosario , van Hagen P. Martin , Dalm Virgil A. S. H. , Lichtenauer-Kaligis Elgin G. R. , Waaijers Marlijn , van Koetsveld Peter M. , Mooy Diana M. , Colao Annamaria , Minuto Francesco , Lamberts Steven W. J. , Hofland Leo J.
Published: March 2015

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3in normal human thymic tissue and of sst1and sst2Aon isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity125I-Tyr11-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2Aand sst3mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2Aand sst3are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3mRNA was higher in the intermediate/mature CD3+fraction compared with the immature CD2+CD3−one, whereas sst2AmRNA was less abundant in the intermediate/mature CD3+thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3−thymocytes. Subtype sst3is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3is involved in this process. Moreover, sst3has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2AmRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.
Influence of growth hormone on bone marrow adipogenesis in hypophysectomized rats
Authors: Appiagyei-Dankah Yaw , Tapiador Carmen D. , Evans Jodi F. , Castro-Magana Mariano , Aloia John F. , Yeh James K.
Published: March 2015

The hypophysectomized rat has been used as a model to study the effects of growth hormone deficiency on bone. Here, we have investigated the influence of growth hormone administration to hypophysectomized rats (HX) for 6 wk on accumulation of triglycerides in bone marrow and on the differentiation of primary marrow stromal cells into adipocytes under in vitro conditions. We found that hypophysectomy significantly increased triglyceride concentration in bone marrow, which was attenuated by growth hormone administration. Primary bone marrow stromal cells derived from HX rats also had more adipocytes at confluence compared with growth hormone-treated hypophysectomized (GH) rats. When stimulated with 3-isobutyl-1-methylxanthine plus dexamethasone (IBMX-Dex), preadipocyte colony counts increased more significantly in GH rats. Markers of adipocyte differentiation were higher in HX than in control or GH rats at confluence. However, after stimulation with IBMX-Dex, increased expression of markers was seen in GH compared with HX rats. In conclusion, growth hormone administration to hypophysectomized rats attenuated triglyceride accumulation in bone marrow and inhibited the differentiation of stromal cells into adipocytes in vitro.
Endotoxemia reduces skeletal muscle protein synthesis in neonates
Authors: Orellana Renan A. , O'Connor Pamela M. J. , Nguyen Hanh V. , Bush Jill A. , Suryawan Agus , Thivierge M. Carole , Fiorotto Marta L. , Davis Teresa A.
Published: March 2015

Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets ( n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 μg · kg−1 · h−1]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [3H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-α, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis.
Akt promotes increased mammalian cell size by stimulating protein synthesis and inhibiting protein degradation
Authors: Faridi Jesika , Fawcett Janet , Wang Lihong , Roth Richard A.
Published: March 2015

Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.
Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA
Authors: Duan Jinhong , Zhang Hai-Ying , Adkins Steven D. , Ren Bonnie H. , Norby Faye L. , Zhang Xiaochun , Benoit Joseph N. , Epstein Paul N. , Ren Jun
Published: March 2015

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90) and maximal velocity of shortening/relengthening (±d L/d t). Intracellular Ca2+was evaluated as Ca2+-induced Ca2+release [difference in fura 2 fluorescent intensity (ΔFFI)] and fluorescence decay rate (τ). Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a, phospholamban (PLB), Na+-Ca2+exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, ±d L/d t, and ΔFFI associated with prolonged TPS, TR90, and τ. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and ΔFFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA.
Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs
Authors: Bertolo Robert F. P. , Brunton Janet A. , Pencharz Paul B. , Ball Ronald O.
Published: March 2015

We have previously shown that arginine deficiency is exacerbated by the removal of dietary proline in orally, but not parenterally, fed piglets. Therefore, we hypothesized that the net interconversions of proline, ornithine, and arginine primarily occur in the small intestine of neonatal piglets. Ten intragastrically fed piglets received either intraportal (IP) or intragastric (IG) primed, constant infusions of [guanido-14C]arginine and [U-14C]ornithine + [2,3-3H]proline. By infusing amino acid isotopes via the stomach compared with the portal vein, we isolated small intestinal first-pass metabolism in vivo. During IP infusion, fractional net conversions (%) from proline to ornithine (0), ornithine to arginine (11 ± 6), and ornithine to proline (5 ± 1) were lower ( P < 0.05) than during IG infusion (39 ± 8, 18 ± 6, and 42 ± 12, respectively); we speculate that these data are due to the localization of ornithine aminotransferase to the gut. The balance of these conversions indicated a large synthesis of arginine (70.0 μmol · kg−1 · h−1) by the gut, with a corresponding degradation of ornithine (70.8 μmol · kg−1 · h−1) and no change in proline balance. Gut synthesis of arginine from proline (48.1 μmol · kg−1 · h−1) was 50% of its requirement, whereas proline synthesis from arginine (33.0 μmol · kg−1 · h−1) amounted to 10% of its requirement. Overall, arginine synthesis is more dependent on the gut than proline synthesis. In situations in which gut metabolism is compromised, such as during parenteral nutrition or gastrointestinal disease, arginine and proline are individually indispensable because their biosyntheses are negligible.