bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine
Authors: Anthony Joshua C. , Lang Charles H. , Crozier Stephen J. , Anthony Tracy G. , MacLean David A. , Kimball Scot R. , Jefferson Leonard S.
Published: February 2015

Enhanced protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats is mimicked by oral leucine administration. To assess the contribution of insulin to the protein synthetic response to leucine, food-deprived (18 h) male rats (∼200 g) were intravenously administered a primed-constant infusion of somatostatin (60 μg + 3 μg · kg−1· h−1) or vehicle beginning 1 h before administration of leucine (1.35 gl-leucine/kg) or saline (control). Rats were killed 15, 30, 45, 60, or 120 min after leucine administration. Compared with controls, serum insulin concentrations were elevated between 15 and 45 min after leucine administration but returned to basal values by 60 min. Somatostatin maintained insulin concentrations at basal levels throughout the time course. Protein synthesis was increased between 30 and 60 min, and this effect was blocked by somatostatin. Enhanced assembly of the mRNA cap-binding complex (composed of eukaryotic initiation factors eIF4E and eIF4G) and hyperphosphorylation of the eIF4E-binding protein 1 (4E-BP1), the 70-kDa ribosomal protein S6 kinase (S6K1), and the ribosomal protein S6 (rp S6) were observed as early as 15 min and persisted for at least 60 min. Somatostatin attenuated the leucine-induced changes in 4E-BP1 and S6K1 phosphorylation and completely blocked the change in rp S6 phosphorylation but had no effect on eIF4G · eIF4E assembly. Overall, the results suggest that the leucine-induced enhancement of protein synthesis and the phosphorylation states of 4E-BP1 and S6K1 are facilitated by the transient increase in serum insulin. In contrast, assembly of the mRNA cap-binding complex occurs independently of increases in insulin and, by itself, is insufficient to stimulate rates of protein synthesis in skeletal muscle after leucine administration.
Protein-tyrosine-phosphatase 1B activation is regulated developmentally in muscle of neonatal pigs
Authors: Suryawan Agus , Davis Teresa A.
Published: February 2015

The high activity of the insulin-signaling pathway contributes to the enhanced feeding-induced stimulation of translation initiation in skeletal muscle of neonatal pigs. Protein-tyrosine-phosphatase 1B (PTP1B) is a negative regulator of the tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate 1 (IRS-1). The activity of PTP1B is determined mainly by its association with IR and Grb2. We examined the level of PTP1B activity, PTP1B protein abundance, PTP1B tyrosine phosphorylation, and the association of PTP1B with IR and Grb2 in skeletal muscle and liver of fasted and fed 7- and 26-day-old pigs. PTP1B activity in skeletal muscle was lower (P < 0.05) in 7- compared with 26-day-old pigs but in liver was similar in the two age groups. PTP1B abundances were similar in muscle but lower (P < 0.05) in liver of 7- compared with 26-day-old pigs. PTP1B tyrosine phosphorylation in muscle was lower (P < 0.05) in 7- than in 26-day-old pigs. The associations of PTP1B with IR and with Grb2 were lower (P < 0.05) at 7 than at 26 days of age in muscle, but there were no age effects in liver. Finally, in both age groups, fasting did not have any effect on these parameters. These results indicate that basal PTP1B activation is developmentally regulated in skeletal muscle of neonatal pigs, consistent with the developmental changes in the activation of the insulin-signaling pathway reported previously. Reduced PTP1B activation in neonatal muscle likely contributes to the enhanced insulin sensitivity of skeletal muscle in neonatal pigs.
Loss of regulation of lipogenesis in the Zucker diabetic rat. II. Changes in stearate and oleate synthesis
Authors: Bassilian Sara , Ahmed Syed , Lim Shu K. , Boros Laszlo G. , Mao Catherine S. , Lee W.-N. Paul
Published: February 2015

De novo lipogenesis and dietary fat uptake are two major sources of fatty acid deposits in fat of obese animals. To determine the relative contribution of fatty acids from these two sources in obesity, we have determined the distribution of c16 and c18 fatty acids of triglycerides in plasma, liver, and epididymal fat pad of Zucker diabetic fatty (ZDF) rats and their lean littermates (ZL) under two isocaloric dietary fat conditions. Lipogenesis was also determined using the deuterated water method. Conversion of palmitate to stearate and stearate to oleate was calculated from the deuterium incorporation by use of the tracer dilution principle. In the ZL rat, lipogenesis was suppressed from 70 to 24%, conversion of palmitate to stearate from 86 to 78%, and conversion of stearate to oleate from 56 to 7% in response to an increase in the dietary fat-to-carbohydrate ratio. The results suggest that suppression of fatty acid synthase and stearoyl-CoA desaturase activities is a normal adaptive mechanism to a high-fat diet. In contrast, de novo lipogenesis, chain elongation, and desaturation were not suppressed by dietary fat in the ZDF rat. The lack of ability to adapt to a high-fat diet resulted in a higher plasma triglyceride concentration and excessive fat accumulation from both diet and de novo synthesis in the ZDF rat.
Calorie restriction increases muscle insulin action but not IRS-1-, IRS-2-, or phosphotyrosine-PI 3-kinase
Authors: Davidson Robert T. , Arias Edward B. , Cartee Gregory D.
Published: February 2015

Skeletal muscle insulin sensitivity improves with a moderate reduction in caloric intake. We studied possible mechanisms in calorie-restricted [CR: 60% ad libitum (AL) intake] compared with AL rats, utilizing a time-matched feeding protocol (3, 5, 10, or 20 days). Visceral fat mass was lower for CR vs. AL at 10 and 20 days, but insulin-stimulated muscle 3- O-methylglucose transport was higher in CR vs. AL rats only at 20 days. Fructose 6-phosphate (precursor for the hexosamine biosynthetic pathway, which has inverse relationship with insulin sensitivity) was reduced only at 3 days of CR. Insulin stimulation of insulin receptor substrate (IRS)-1-, IRS-2-, and antiphosphotyrosine-associated phosphatidylinositol 3-kinase (PI3K) was similar for CR and AL. A PI3K inhibitor, wortmannin, reduced insulin-stimulated 3- O-methylglucose transport to basal levels, regardless of diet. With brief time-matched CR, reduced visceral fat mass precedes increased insulin sensitivity; transient reduction in fructose 6-phosphate may trigger more persistent changes but does not coincide with enhanced insulin action; and PI3K is essential for insulin-stimulated 3- O-methylglucose transport in CR as well as AL rats, although insulin-stimulated PI3K is not significantly greater in CR compared with AL animals.
Reduced activity of mtTFA decreases the transcription in mitochondria isolated from diabetic rat heart
Authors: Kanazawa Akio , Nishio Yoshihiko , Kashiwagi Atsunori , Inagaki Hidetoshi , Kikkawa Ryuichi , Horiike Kihachiro
Published: February 2015

To evaluate abnormalities in the mitochondrial transcription factor A (mtTFA) function as a cause of mitochondrial dysfunction in diabetes, we measured the mRNA contents of the proteins consisting of the mitochondrial respiratory chain as well as transcriptional and translational activities in the mitochondria isolated from controls and streptozotocin-induced diabetic rat hearts. Using Northern blot analysis, we found 40% reduced mRNA contents of mitochondrial-encoded cytochrome b and ATP synthase subunit 6 in diabetic rat hearts compared with control rats ( P< 0.05). These abnormalities were completely recovered by insulin treatment. Furthermore, the mitochondrial activities of transcription and translation were decreased significantly in mitochondria isolated from diabetic rats by 60% ( P < 0.01) and 71% ( P < 0.01), respectively, compared with control rats. The insulin treatment also completely normalized these abnormalities in diabetic rats. Consistently, gel retardation assay showed a reduced binding of mtTFA to the D-loop of mitochondrial DNA in diabetic rats, although there was no difference in the mtTFA mRNA and protein content between the two groups. On the basis of these findings, a reduced binding activity of mtTFA to the D-loop region in the hearts of diabetic rats may contribute to the decreased mitochondrial protein synthesis.
IL-6 and TNF-α expression in, and release from, contracting human skeletal muscle
Authors: Steensberg Adam , Keller Charlotte , Starkie Rebecca L. , Osada Takuya , Febbraio Mark A. , Pedersen Bente Klarlund
Published: February 2015

The aim of the present study was to examine whether IL-6 and TNF-α are expressed in, and released from, human skeletal muscle during exercise. We hypothesized that the skeletal muscle will release IL-6, but not TNF-α, during exercise because of previous observations that TNF-α negatively affects glucose uptake in skeletal muscle. Six healthy, male subjects performed 180 min of two-legged knee-extensor exercise. Muscle samples were obtained from the vastus lateralis of one limb. In addition, blood samples were obtained from a femoral artery and vein. Plasma was analyzed for IL-6 and TNF-α. We detected both IL-6 and TNF-α mRNA in resting muscle samples, and whereas IL-6 increased ( P < 0.05) ∼100-fold throughout exercise, no significant increase in TNF-α mRNA was observed. Arterial plasma TNF-α did not increase during exercise. Furthermore, there was no net release of TNF-α either before or during exercise. In contrast, IL-6 increased throughout exercise in arterial plasma, and a net IL-6 release from the contracting limb was observed after 120 min of exercise ( P < 0.05).
Corticosterone-dependent driving influence of the suprachiasmatic nucleus on adrenal sensitivity to ACTH
Authors: Sage Dominique , Maurel Daniel , Bosler Olivier
Published: February 2015

We investigated the effects of ablation of the suprachiasmatic nucleus (SCN) on corticosterone (CORT) responses to synthetic ACTH given in either the morning or evening. After dexamethasone treatment, evening ACTH injections in intact rats produced a significantly larger increase in plasma CORT compared with morning ones. In rats with SCN lesions, the ACTH-induced CORT secretion was independent of time of day, providing direct evidence for a driving influence of the SCN on the diurnal rhythm of adrenal sensitivity to ACTH. In the absence of dexamethasone treatment, the SCN-lesioned rats were selected for morning-like (ML) or evening-like (EL) basal levels of CORT. Responses to ACTH were not different in ML rats compared with sham-lesioned morning controls. In contrast, EL rats compared with sham-lesioned evening controls showed an ∼60% decrease in increment of CORT levels within the first 15 min postinjection. These results indicate that the SCN upregulates ACTH sensitivity of the adrenal cortex during the ascending phase of the daily CORT secretion and point to a critical role of glucocorticoids in determining SCN action.
Isotopomer spectral analysis of intermediates of cholesterol synthesis in human subjects and hepatic cells
Authors: Lindenthal B. , Aldaghlas T. A. , Holleran A. L. , Sudhop T. , Berthold H. K. , von Bergmann K. , Kelleher J. K.
Published: February 2015

Steroid intermediates of the cholesterol synthesis pathway are characterized by rapid turnover rates relative to cholesterol due to their small pool size. Because the small pools will label rapidly, these intermediates may provide valuable information about the incorporation of isotopes in de novo synthesis of cholesterol and related compounds. The labeling of cholesterol synthesis intermediates from [1-13C]acetate was investigated in human subjects and in liver cell models by means of isotopomer spectral analysis (ISA). In human subjects, infusing [1-13C]acetate into the duodenum for 12 h demonstrated that ∼50% of the plasma lathosterol pool was derived from de novo synthesis during this interval. The lipogenic acetyl-CoA precursor pool enrichment reached a constant value within 3 h of the start of the infusion. In vitro studies indicated that liver cell models decrease de novo lathosterol synthesis when cholesterol synthesis is inhibited by statins or cholesterol-containing serum. We propose a new calculation to increase the accuracy and precision of cholesterol synthesis estimates in vivo combining the ISA of lathosterol and cholesterol.
Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet
Authors: Burcelin Rémy , Crivelli Valérie , Dacosta Anabela , Roy-Tirelli Alexandra , Thorens Bernard
Published: February 2015

C57BL/6J mice were fed a high-fat, carbohydrate-free diet (HFD) for 9 mo. Approximately 50% of the mice became obese and diabetic (ObD), ∼10% lean and diabetic (LD), ∼10% lean and nondiabetic (LnD), and ∼30% displayed intermediate phenotype. All of the HFD mice were insulin resistant. In the fasted state, whole body glucose clearance was reduced in ObD mice, unchanged in the LD mice, and increased in the LnD mice compared with the normal-chow mice. Because fasted ObD mice were hyperinsulinemic and the lean mice slightly insulinopenic, there was no correlation between insulin levels and increased glucose utilization. In vivo, tissue glucose uptake assessed by 2-[14C]deoxyglucose accumulation was reduced in most muscles in the ObD mice but increased in the LnD mice compared with the values of the control mice. In the LD mice, the glucose uptake rates were reduced in extensor digitorum longus (EDL) and total hindlimb but increased in soleus, diaphragm, and heart. When assessed in vitro, glucose utilization rates in the absence and presence of insulin were similar in diaphragm, soleus, and EDL muscles isolated from all groups of mice. Thus, in genetically homogenous mice, HFD feeding lead to different metabolic adaptations. Whereas all of the mice became insulin resistant, this was associated, in obese mice, with decreased glucose clearance and hyperinsulinemia and, in lean mice, with increased glucose clearance in the presence of mild insulinopenia. Therefore, increased glucose clearance in lean mice could not be explained by increased insulin level, indicating that other in vivo mechanisms are triggered to control muscle glucose utilization. These adaptive mechanisms could participate in the protection against development of obesity.
TNF-α impairs heart and skeletal muscle protein synthesis by altering translation initiation
Authors: Lang Charles H. , Frost Robert A. , Nairn Angus C. , MacLean David A. , Vary Thomas C.
Published: February 2015

This study examined potential mechanisms contributing to the inhibition of protein synthesis in skeletal muscle and heart after administration of tumor necrosis factor (TNF)-α. Rats had vascular catheters implanted, and TNF-α was infused continuously for 24 h. TNF-α decreased in vivo-determined rates of global protein synthesis in gastrocnemius (39%) and heart (25%). The TNF-α-induced decrease in protein synthesis in the gastrocnemius involved a reduction in the synthesis of both myofibrillar and sarcoplasmic proteins. To identify potential mechanisms responsible for regulating mRNA translation, we examined several eukaryotic initiation factors (eIFs) and elongation factors (eEFs). TNF-α decreased the activity of eIF-2B in muscle (39%) but not in heart. This diminished activity was not caused by a reduction in the content of eIF-2Bε or the content and phosphorylation state of eIF-2α. Skeletal muscle and heart from TNF-α-treated rats demonstrated 1) an increased binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF-4E, 2) a decreased amount of eIF-4E associated with eIF-4G, and 3) a decreased content of the hyperphosphorylated γ-form of 4E-BP1. In contrast, the infusion of TNF-α did not alter the content of eEF-1α or eEF-2, or the phosphorylation state of eEF-2. In summary, these data suggest that TNF-α impairs skeletal muscle and heart protein synthesis, at least in part, by decreasing mRNA translational efficiency resulting from an impairment in translation initiation associated with alterations in eIF-4E availability.
Contrasting effects of fish oil and safflower oil on hepatic peroxisomal and tissue lipid content
Authors: Neschen Susanne , Moore Irene , Regittnig Werner , Yu Chun Li , Wang Yanlin , Pypaert Marc , Petersen Kitt Falk , Shulman Gerald I.
Published: February 2015

To examine the mechanism by which fish oil protects against fat-induced insulin resistance, we studied the effects of control, fish oil, and safflower oil diets on peroxisomal content, fatty acyl-CoA, diacylglycerol, and ceramide content in rat liver and muscle. We found that, in contrast to control and safflower oil-fed rats, fish oil feeding induced a 150% increase in the abundance of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase in liver but lacked similar effects in muscle. This was paralleled by an almost twofold increase in hepatic peroxisome content (both P < 0.002 vs. control and safflower). These changes in the fish oil-fed rats were associated with a more than twofold lower hepatic triglyceride/diacylglycerol, as well as intramuscular triglyceride/fatty acyl-CoA, content. In conclusion, these data strongly support the hypothesis that n-3 fatty acids protect against fat-induced insulin resistance by serving as peroxisome proliferator-activated receptor-α ligands and thereby induce hepatic, but not intramuscular, peroxisome proliferation. In turn, an increased hepatic β-oxidative capacity results in lower hepatic triglyceride/diacylglycerol and intramyocellular triglyceride/fatty acyl-CoA content.
Role of cytokines and testosterone in regulating lean body mass and resting energy expenditure in HIV-infected men
Authors: Roubenoff Ronenn , Grinspoon Steven , Skolnik Paul R. , Tchetgen Eric , Abad Leslie , Spiegelman Donna , Knox Tamsin , Gorbach Sherwood
Published: February 2015

Although catastrophic weight loss is no longer common in HIV-infected men, we hypothesized that a more gradual process of cachexia [loss of lean body mass (LBM) without severe weight loss, often accompanied by elevated resting energy expenditure (REE)] is still common and is driven by excessive production of the catabolic cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). We performed a longitudinal analysis of an ongoing cohort study of nutritional status in 172 men with HIV infection. LBM loss of >1 kg occurred in 35% of the cohort, and LBM loss of >5% occurred in 12.2% over 8 mo of observation, but classical wasting (loss of ≈10% of weight) was rare (2%). Both TNF-α (−150 g LBM · ng−1· ml−1, P < 0.02) and IL-1β production (−130 g LBM · ng−1· ml−1, P < 0.01) by peripheral blood mononuclear cells predicted loss of LBM. A rise in REE of >200 kcal/day was found in 17.7% of the subjects regardless of weight change. IL-1β (+9 kcal/day per ng/ml, P < 0.002) and TNF-α (+10 kcal/day per ng/ml, P < 0.02) production predicted ΔREE. Serum free testosterone was inversely associated with TNF-α production and was not an independent predictor of either ΔLBM or ΔREE after adjustment for cytokine production. Even though weight loss was rare in this cohort of patients treated with highly active antiretroviral therapy, loss of LBM was common and was driven by catabolic cytokines and not by inadequate dietary intake or hypogonadism.
New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle
Authors: Walzel Bernd , Speer Oliver , Boehm Ernie , Kristiansen Søren , Chan Sharon , Clarke Kierian , Magyar Joseph P. , Richter Erik A. , Wallimann Theo
Published: February 2015

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 ± 9.4 μM and maximal velocity value 17.3 ± 3.1 pmol · min−1· mg vesicle protein−1), which cotransports Cr into skeletal muscle together with Na+and Cl−ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH2-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of ∼58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [14C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.
Triiodothyronine is required for the stimulation of type II 5′-deiodinase mRNA in rat brown adipocytes
Authors: Martinez-deMena Raquel , Hernández Arturo , Obregón Maria-Jesús
Published: February 2015

Type II 5′-iodothyronine deiodinase (D2), produces triiodothyronine (T3) and is stimulated by cold exposure via norepinephrine (NE) release in brown adipose tissue. Cultured rat brown adipocytes require T3for the adrenergic stimulation of D2 activity. D2 mRNA expression in cultured brown adipocytes is undetectable with the use of basal conditions or NE without T3. Full D2 expression is achieved using NE + T3, especially after prolonged T3exposure. β3-Adrenergic agonists mimic the NE action, whereas cAMP analogs do not. Prolonged exposure to T3alone increases D2 mRNA. High T3doses (500 nM) inhibit the adrenergic stimulation of D2 activity while increasing D2 mRNA. The effects obtained with NE + T3or T3alone are suppressed by actinomycin, but not by cycloheximide, which leads to accumulation of short D2 mRNA transcripts. Prolonged or short exposure to T3did not change D2 mRNA half-life, but T3seemed to elongate it. In conclusion, T3is an absolute requirement for the adrenergic stimulation of D2 mRNA in brown adipocytes. T3upregulates D2 mRNA, an effect that might involve stimulation of factors required for transcription or for stabilization of D2 mRNA.
Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance
Authors: Nelson Bryce A. , Robinson Katherine A. , Buse Maria G.
Published: February 2015

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high glucose or glucosamine, provided insulin (0.6 nM) is present during preincubation. Insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity is unaffected (30). Total cellular IRS-1, PI 3-kinase, or Akt concentrations were unchanged. Akt activation in subcellular fractions was assessed by immunoblotting with two phospho-Akt-specific antibodies. Upon acute 100 nM insulin stimulation, plasma membrane (PM)-associated phospho-Akt was highest in cells preincubated in low glucose with no insulin, less in high glucose with no insulin, even less in low glucose+insulin, and lowest in high glucose+insulin. Only high glucose+insulin caused insulin-resistant glucose transport. Acute insulin stimulation increased total PM-Akt about twofold after preincubation without insulin in low or high glucose. Preincubation with 0.6 nM insulin decreased Akt PM translocation by ∼25% in low and ∼50% in high glucose. Preincubation with glucosamine did not affect Akt phosphorylation or translocation. Conclusions: chronic exposure to high glucose or insulin downregulates acute insulin-stimulated Akt activation, acting synergistically distal to PI 3-kinase. Maximal insulin activates more Akt than required for maximal glucose transport stimulation. Insulin resistance may ensue when PM-associated phospho-Akt decreases below a threshold. High glucose and glucosamine cause insulin resistance by different mechanisms in 3T3-L1 adipocytes.
Elevated endogenous cortisol reduces autonomic neuroendocrine and symptom responses to subsequent hypoglycemia
Authors: McGregor Veronica P. , Banarer Salomon , Cryer Philip E.
Published: February 2015

We tested the hypothesis that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia. Twelve healthy young adults were studied on two separate occasions, once after infusions of a pharmacological dose of α-(1–24)-ACTH (100 μg/h) from 0930 to 1200 and 1330 to 1600, which raised plasma cortisol levels to ∼45 μg/dl on day 1, and once after saline infusions on day 1. Hyperinsulinemic (2.0 mU · kg−1· min−1) stepped hypoglycemic clamps (90, 75, 65, 55, and 45 mg/dl glucose steps) were performed on the morning of day 2 on both occasions. These markedly elevated antecedent endogenous cortisol levels reduced the adrenomedullary ( P = 0.004, final plasma epinephrine levels of 489 ± 64 vs. 816 ± 113 pg/ml), sympathetic neural ( P = 0.0022, final plasma norepinephrine levels of 244 ± 15 vs. 342 ± 22 pg/ml), parasympathetic neural ( P = 0.0434, final plasma pancreatic polypeptide levels of 312 ± 37 vs. 424 ± 56 pg/ml), and neurogenic (autonomic) symptom ( P = 0.0097, final symptom score of 7.1 ± 1.5 vs. 10.6 ± 1.6) responses to subsequent hypoglycemia. Growth hormone, but not glucagon or cortisol, responses were also reduced. The findings that increased endogenous cortisol secretion reduces autonomic neuroendocrine and neurogenic symptom responses to subsequent hypoglycemia are potentially relevant to cortisol mediation of hypoglycemia-associated autonomic failure, and thus a vicious cycle of recurrent iatrogenic hypoglycemia, in people with diabetes mellitus.
Metabolic and anthropometric factors related to skeletal muscle UCP3 gene expression in healthy human adults
Authors: Calsbeek D. J. , Thompson T. L. , Dahl J. A. , Stob N. R. , Brozinick J. T. , Hill J. O. , Hickey M. S.
Published: February 2015

This cross-sectional investigation sought to determine the relationship between selected metabolic, endocrine, and anthropometric factors and skeletal muscle UCP3 mRNA in healthy adult humans. Twenty-four healthy adults (13 male and 11 female) across a range of aerobic capacity, age, and body composition were studied. Muscle biopsies were obtained from the vastus lateralis, from which UCP3 mRNA was quantified by Northern blot, and fiber type was determined by use of the myosin ATPase staining procedure. In addition, resting energy expenditure and maximum rate of oxygen consumption were determined by indirect calorimetry, body composition was determined by dual-energy X-ray absorptiometry, and fasting plasma leptin and insulin were determined by ELISA. UCP3 mRNA was correlated positively with the percent type I fibers ( r = 0.842, P < 0.001), plasma leptin ( r = 0.454, P = 0.026), and plasma insulin ( r = 0.615, P < 0.001) and inversely to age ( r = −0.411, P = 0.046). Stepwise multiple regression analysis determined that percent type I muscle fibers was the best predictor of vastus lateralis UCP3 mRNA, and no other variable entered the equation (model r 2 = 0.66). This study suggests that of the variables measured, UCP3 mRNA is primarily related to skeletal muscle fiber type in healthy adults. The factors that contribute to fiber-specific differences in UCP3 mRNA expression will need to be examined in future studies.
Increase in daily LH secretion in response to short-term calorie restriction in obese women with PCOS
Authors: Van Dam Eveline W. C. M. , Roelfsema Ferdinand , Veldhuis Johannes D. , Helmerhorst Frans M. , Frölich Marijke , Meinders A. Edo , Krans H. Michiel J. , Pijl Hanno
Published: February 2015

We hypothesized that short-term calorie restriction would blunt luteinizing hormone (LH) hypersecretion in obese women with polycystic ovary syndrome (PCOS) and thereby ameliorate the anovulatory endocrine milieu. To test this hypothesis, 15 obese patients with PCOS and nine age- and body mass index-matched healthy women underwent 24-h blood sampling to quantitate plasma LH, leptin, and insulin levels. PCOS subjects were prescribed a very low caloric liquid diet (4.2 MJ/day) for 7 days and were then resampled. Basal and pulsatile LH secretion was threefold higher in PCOS subjects, but plasma insulin and leptin levels were not different in the calorie-replete state. Contrary to expectation, calorie restriction enhanced basal and pulsatile LH secretion even further. As expected, plasma glucose, insulin, and leptin concentrations decreased by 18, 75, and 50%, respectively. Serum total testosterone concentration fell by 23%, whereas serum estrone, estradiol, sex hormone-binding globulin (SHBG), and androstenedione concentrations remained unchanged. Enhanced LH secretion in the presence of normal metabolic and hormonal adaptations to calorie restriction points to anomalous feedback control of pituitary LH release in PCOS.
Fatty acid transport: the roads taken
Authors: Schaffer Jean E.
Published: February 2015

Efficient uptake and channeling of long-chain fatty acids (LCFAs) are critical cellular functions. Although spontaneous flip-flop of nonionized LCFAs from one leaflet of a bilayer to the other is rapid, evidence is emerging that proteins are important mediators and/or regulators of trafficking of LCFAs into and within cells. Genetic screens have led to the identification of proteins that are required for fatty acid import and utilization in prokaryotic organisms. In addition, functional screens have elucidated proteins that facilitate fatty acid import into mammalian cells. Although the mechanisms by which these proteins mediate LCFA import are not well understood, studies in both prokaryotic and eukaryotic organisms provide compelling evidence that uptake of LCFAs across cellular membranes is coupled to esterification by acyl-CoA synthetases. This review will summarize results of studies of non-protein-mediated and protein-mediated LCFA transport and discuss how these different mechanisms may contribute to cellular metabolism.
Contributions of total and regional fat mass to risk for cardiovascular disease in older women
Authors: Van Pelt R. E. , Evans E. M. , Schechtman K. B. , Ehsani A. A. , Kohrt W. M.
Published: February 2015

The aim of this study was to determine whether trunk fat mass, measured by dual-energy X-ray absorptiometry (DEXA), is predictive of insulin resistance and dyslipidemia, independently of arm and leg fat mass, in postmenopausal women. Total and regional body composition was measured by DEXA in 166 healthy, postmenopausal women (66 ± 4 yr). Four primary markers of insulin resistance and dyslipidemia were assessed: 1) area under the curve for the insulin (INSAUC) response to an oral glucose tolerance test (OGTT), 2) product of the OGTT glucose and insulin areas (INSAUC×GLUAUC), 3) serum triglycerides (TG), and 4) high-density lipoprotein (HDL)-cholesterol. Trunk fat mass was the strongest independent predictor of each of the primary dependent variables. In multivariate regression models, trunk fat mass was associated with unfavorable levels of INSAUC, INSAUC×GLUAUC, TG, and HDL-C, whereas leg fat mass was favorably associated with each of these variables. Thus trunk fat is a strong independent predictor of insulin resistance and dyslipidemia in postmenopausal women, whereas leg fat appears to confer protective effects against metabolic dysfunction.
Accelerated rates of glycolysis in the hypertrophied heart: are they a methodological artifact?
Authors: Leong Hon Sing , Grist Mark , Parsons Hannah , Wambolt Richard B. , Lopaschuk Gary D. , Brownsey Roger , Allard Michael F.
Published: February 2015

Glycolysis, measured by3H2O production from [5-3H]glucose, is accelerated in isolated working hypertrophied rat hearts. However, nonglycolytic detritiation of [5-3H]glucose via the nonoxidative pentose phosphate pathway (PPP) could potentially lead to an overestimation of true glycolytic rates, especially in hypertrophied hearts where the PPP may be upregulated. To address this concern, we measured glycolysis using [5-3H]glucose and a second, independent method in isolated working hearts from halothane-anesthetized, sham-operated and aortic-constricted rats. Glycolysis was accelerated in hypertrophied hearts compared with control hearts regardless of the method used. There was also excellent concordance in glycolytic rates between the different methods. Moreover, activity of glucose-6-phosphate dehydrogenase and expression of transaldolase, enzymes controlling key steps in the oxidative and nonoxidative PPP, respectively, were not different between control and hypertrophied hearts. Thus nonglycolytic detritiation of [5-3H]glucose in the PPP is insignificant, and 3H2O production from [5-3H]glucose is an accurate means to measure glycolysis in isolated working normal and hypertrophied rat hearts. Furthermore, the PPP does not appear to be increased in cardiac hypertrophy induced by abdominal aortic constriction.
Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca2+
Authors: Ojuka Edward O. , Jones Terry E. , Nolte Lorraine A. , Chen May , Wamhoff Brian R. , Sturek Michael , Holloszy John O.
Published: February 2015

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+induces an increase in GLUT4. We found that raising cytosolic Ca2+by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.
Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine
Authors: Al Alam Denise , Danopoulos Soula , Schall Kathy , Sala Frederic G. , Almohazey Dana , Fernandez G. Esteban , Georgia Senta , Frey Mark R. , Ford Henri R. , Grikscheit Tracy , Bellusci Saverio
Published: February 2015

Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.
Heterogeneous patterns of vasoreactivity in the middle cerebral and internal carotid arteries
Authors: Coverdale Nicole S. , Lalande Sophie , Perrotta Amanda , Shoemaker J. Kevin
Published: February 2015

This study compared changes in cross-sectional area (CSA) and flow (Q) between the middle cerebral artery (MCA) and the internal carotid artery (ICA) at baseline and during 5 min of hypercapnia (HC; 6% CO2) and hypocapnia (HO; hyperventilation) and quantified how these changes contribute to estimates of cerebrovascular reactivity (CVR). Measures of MCA CSA were made using 3T magnetic resonance imaging. On a separate day, MCA flow velocity was measured with transcranial Doppler ultrasound and ICA diameters and flow velocity were measured with duplex ultrasound. Fourteen subjects (23 ± 3 yr, 7 females) participated, providing data for 11 subjects during HC and 9 subjects during HO. An increase in MCA CSA ( P < 0.05) was observed within the first minute of HC. During HO, the decrease in MCA CSA ( P < 0.05) was delayed until minute 4. No changes were observed in ICA CSA during HC or HO. The relative changes in QICA and QMCA were similar during HC and HO. Therefore, the MCA, but not ICA, dilates and constricts during 5 min of HC and HO, respectively. The consequent impact on QMCA significantly affects estimates of CVR, and reactivity cannot be attributed solely to changes in smaller arterioles.
Downhill running and exercise in hot environments increase leukocyte Hsp72 (HSPA1A) and Hsp90α (HSPC1) gene transcripts
Authors: Tuttle James A. , Castle Paul C. , Metcalfe Alan J. , Midgley Adrian W. , Taylor Lee , Lewis Mark P.
Published: February 2015

Stressors within humans and other species activate Hsp72 and Hsp90α mRNA transcription, although it is unclear which environmental temperature or treadmill gradient induces the largest increase. To determine the optimal stressor for priming the Hsp system, physically active but not heat-acclimated participants (19.8 ± 1.9 and 20.9 ± 3.6 yr) exercised at lactate threshold in either temperate (20°C, 50% relative humidity; RH) or hot (30°C, 50% RH) environmental conditions. Within each condition, participants completed a flat running (temperate flat or hot flat) and a downhill running (temperate downhill or hot downhill) experimental trial in a randomized counterbalanced order separated by at least 7 days. Venous blood samples were taken immediately before (basal), immediately after exercise, and 3 and 24 h postexercise. RNA was extracted from leukocytes and RT-quantitative PCR conducted to determine Hsp72 and Hsp90α mRNA relative expression. Leukocyte Hsp72 mRNA was increased immediately after exercise following downhill running (1.9 ± 0.9-fold) compared with flat running (1.3 ± 0.4-fold; P = 0.001) and in hot (1.9 ± 0.6-fold) compared with temperate conditions (1.1 ± 0.5-fold; P = 0.003). Leukocyte Hsp90α mRNA increased immediately after exercise following downhill running (1.4 ± 0.8-fold) compared with flat running (0.9 ± 0.6-fold; P = 0.002) and in hot (1.6 ± 1.0-fold) compared with temperate conditions (0.9 ± 0.6-fold; P = 0.003). Downhill running and exercise in hot conditions induced the largest stimuli for leukocyte Hsp72 and Hsp90α mRNA increases.
Atheroma Progression in Hyporesponders to Statin Therapy
Authors: Kataoka Yu , St. John Julie , Wolski Kathy , Uno Kiyoko , Puri Rishi , Tuzcu E. Murat , Nissen Steven E. , Nicholls Stephen J.
Published: February 2015

Objective—

Lowering low-density lipoprotein cholesterol (LDL-C) with statins has been demonstrated to slow plaque progression. This antiatherosclerotic effect in patients with minimal LDL-C lowering has not been investigated.

Approach and Results—

Six hundred forty-seven patients with angiographic coronary artery disease who were commenced on statin therapy underwent serial imaging with intravascular ultrasound. Responders were defined as a percentage reduction in LDL-C of <15%. Disease progression was compared in responders (n=517) and hyporesponders (n=130) to statin therapy. Twenty percentage of patients demonstrated minimal changes in LDL-C, despite commencement of statin therapy. Statin hyporesponders were younger (55 versus 57 years;
P
=0.01), more likely to be male (79% versus 66%;
P
=0.005), and obese (body mass index, 31.5±6.1 versus 30.3±5.9 kg/m
2
;
P
=0.04) and less likely to have a history of dyslipidemia (50% versus 66%;
P
<0.001). Baseline levels of systolic blood pressure (127±15 versus 132±17 mm Hg;
P
=0.01) and LDL-C (2.5±0.6 versus 3.4±0.8 mmol/L;
P
<0.001) were lower in statin hyporesponders. Baseline percent atheroma volume was similar between statin hyporesponders and responders (36.9±9.8% versus 38.3±9.2%;
P
=0.13). On serial evaluation, greater progression of percent atheroma volume (1.19±0.48% versus 0.09±0.43%;
P
=0.003) was observed in statin hyporesponders. After adjusting for baseline clinical characteristics and measures of plaque burden, statin hyporesponders still exhibited greater atheroma progression (+0.83±0.58% versus −0.21±0.52%;
P
=0.006).

Conclusions—

A substantial proportion of patients with coronary artery disease fail to achieve effective reductions in LDL-C, despite prescription of statin therapy. Greater progression of atherosclerosis is observed in these patients. Our current study underscores monitoring LDL-C level after the commencement of statin to ensure adequate response to statin therapy.
Etv2 and Fli1b Function Together as Key Regulators of Vasculogenesis and Angiogenesis
Authors: Craig Michael P. , Grajevskaja Viktorija , Liao Hsin-Kai , Balciuniene Jorune , Ekker Stephen C. , Park Joo-Seop , Essner Jeffrey J. , Balciunas Darius , Sumanas Saulius
Published: February 2015

Objective—

The E26 transformation-specific domain transcription factor
Etv2
/
Etsrp
/
ER71
is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis.

Approach and Results—

Zebrafish
fli1a
and
fli1b
mutants were obtained using transposon-mediated gene trap approach. Individual
fli1a
and
fli1b
homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older
etv2
mutant embryos does not occur in embryos lacking both
etv2
and
fli1b
.
Etv2
and
fli1b
double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast,
fli1a
mutation did not affect the recovery of
etv2
mutant phenotype. Overexpression analyses indicate that both
etv2
and
fli1b
, but not
fli1a
, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites.

Conclusions—

Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.
COX2 Inhibition Reduces Aortic Valve Calcification In Vivo
Authors: Wirrig Elaine E. , Gomez M. Victoria , Hinton Robert B. , Yutzey Katherine E.
Published: February 2015

Objective—

Calcific aortic valve disease (CAVD) is a significant cause of morbidity and mortality, which affects ≈1% of the US population and is characterized by calcific nodule formation and stenosis of the valve.
Klotho
-deficient mice were used to study the molecular mechanisms of CAVD as they develop robust aortic valve (AoV) calcification. Through microarray analysis of AoV tissues from
klotho
-deficient and wild-type mice, increased expression of the gene encoding cyclooxygenase 2 (COX2;
Ptgs2
) was found. COX2 activity contributes to bone differentiation and homeostasis, thus the contribution of COX2 activity to AoV calcification was assessed.

Approach and Results—

In
klotho
-deficient mice, COX2 expression is increased throughout regions of valve calcification and is induced in the valvular interstitial cells before calcification formation. Similarly, COX2 expression is increased in human diseased AoVs. Treatment of cultured porcine aortic valvular interstitial cells with osteogenic media induces bone marker gene expression and calcification in vitro, which is blocked by inhibition of COX2 activity. In vivo, genetic loss of function of COX2 cyclooxygenase activity partially rescues AoV calcification in
klotho
-deficient mice. Moreover, pharmacological inhibition of COX2 activity in
klotho
-deficient mice via celecoxib-containing diet reduces AoV calcification and blocks osteogenic gene expression.

Conclusions—

COX2 expression is upregulated in CAVD, and its activity contributes to osteogenic gene induction and valve calcification in vitro and in vivo.