bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitive methods to study human apolipoprotein B metabolism using stable isotope-labeled amino acids
Authors: Demant T. , Packard C. J. , Demmelmair H. , Stewart P. , Bedynek A. , Bedford D. , Seidel D. , Shepherd J.
Published: December 2017

The objective of the study was to develop a sensitive method using stable isotope-labeled tracers that would permit the determination of apolipoprotein B (apoB) metabolism in very low-density lipoprotein subfractions (VLDL1, Sf 60-400; VLDL2, Sf 20-60), intermediate-density lipoprotein (IDL, Sf 12-20), and low-density lipoprotein (LDL, Sf 0-12). Six normolipidemic subjects were given trideuterated leucine, and its clearance from plasma and appearance in the four apoB-containing lipoprotein fractions were followed by use of a highly sensitive gas chromatography-mass spectrometry technique in which the m + 3-to-m + 2 ion ratio was selectively monitored. This analytic approach permitted the precise measurement of low enrichments in IDL and LDL and extension of the turnover out to 250-300 h. A compartmental model was developed to derive rate constants from the plasma and apoB enrichment curves. The model was uniquely identifiable once parameter dependencies had been introduced to reduce the number of unknowns. Values were obtained for apoB input into all lipoprotein density intervals, together with rates of interconversion and catabolism; these agreed well with results from radioiodinated tracer experiments. An alternative model structure was also explored in which input occurred only into VLDL1. Altering the protocol of tracer administration (bolus vs. primed constant infusion) and dose (over a 10-fold range) had no influence on the results obtained. The analytic and modeling approach described will permit stable isotopes to be used to elucidate key features of apoB metabolism in normal and pathological states.
Effects of somatostatin on pulsatile insulin secretion: elective inhibition of insulin burst mass
Authors: Porksen N. , Munn S. R. , Steers J. L. , Veldhuis J. D. , Butler P. C.
Published: December 2017

Although it is well known that somatostatin inhibits net insulin secretion, it is unknown whether this is achieved by regulation of the basal or pulsatile components of insulin secretion and, if the latter, whether this is through modulation of pulse mass or frequency. We addressed these questions with a canine model. Portal vein blood was sampled at 1-min intervals in five dogs for 60 min before (basal) and 90 min after ingestion of 30 g glucose on two different occasions, during a saline (SAL) or a somatostatin (SMS, 175 ng/min) infusion. Plasma glucose concentrations were similar during SAL and SMS. SMS had no effect on pulse frequency before (8.4 +/- 0.7 vs. 9.2 +/- 1.0 pulses/h, SMS vs. SAL, P = 0.54) or after glucose (13.3 +/- 1.1 vs. 11.6 +/- 0.9 pulses/h, SMS vs. SAL, P = 0.22). In contrast, SMS decreased insulin pulse mass in the postabsorptive (84 +/- 28 vs. 214 +/- 73 pmol/pulse, SMS vs. SAL, P < 0.05) and fed states (676 +/- 143 vs. 913 +/- 183 pmol/pulse, SMS vs. SAL, P < 0.05). In the postabsorptive state, SMS decreased insulin clearance by approximately 50% (0.32 +/- 0.04 vs. 0.60 +/- 0.09 l/min, P < 0.05), but after glucose ingestion, insulin clearance was comparable during SMS or SAL (0.72 +/- 0.04 vs. 0.80 +/- 0.08 l/min, P = 0.4). SMS appeared to alter insulin clearance through modulation of insulin pulse amplitude, because in the postabsorptive state clearance was closely correlated to the pulse amplitude (r = + 0.87, P < 0.0001). In conclusion, somatostatin regulates the rate of insulin secretion by selective inhibition of pulsatile insulin secretion. Regulation of secretory burst mass (and amplitude) may secondarily influence transhepatic and thus total body clearance of endogenously secreted insulin and thereby serve as a novel mechanism to dictate the systemic insulin concentration.
Age-related changes in body composition are associated with hepatic insulin resistance in conscious rats
Authors: Barzilai N. , Rossetti L.
Published: December 2017

Age-dependent changes in body composition and hepatic ([3H]glucose) glucose metabolism were examined in 2-, 4-, and 14-mo-old (n = 26) conscious Sprague-Dawley rats. Hepatic glucose production (HGP) and hepatic glucose-6-phosphatase maximum velocity were decreased 18 and 30%, respectively, between 2 and 4 mo but were unchanged with further aging. However, between 4 and 14 mo, twofold higher plasma insulin levels were required to maintain similar HGP, suggesting that hepatic insulin resistance develops with age. Utilizing hepatic-pancreatic clamp technique, we showed that a much higher rate of insulin infusion (1.6 +/- 0.1 vs. 0.8 +/- 0.1 mU.kg-1.min-1) was needed to achieve similar plasma glucose levels and HGP. Furthermore, when 4-mo-old rats were infused with insulin at similar rates as the 14-mo-old rats, HGP was decreased by approximately 30%. Because hepatic insulin sensitivity was inversely related to the increase in body weight (r2 = 0.876) and free fatty acid levels (r2 = 0.843), we suggest that age-related changes in body composition may lead to the impairment of hepatic glucose metabolism.
Compartmentalization of protein traffic in insulin-sensitive cells
Authors: Kandror K. V. , Pilch P. F.
Published: December 2017

Insulin-sensitive cells, adipocytes and myocytes, translocate a number of intracellular proteins to the cell surface in response to insulin. Among these proteins are glucose transporters 1 and 4 (GLUT-1 and GLUT-4, respectively), receptors for insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) and transferrin, the aminopeptidase gp 160, caveolin, and a few others. In the case of insulin-activated glucose transport, this translocation has been proven to be the major, if not the only regulatory mechanism of this process. It seems likely that the cell surface recruitment of the IGF-II/Man-6-P and transferrin receptors also serves the nutritional needs of cells, whereas the physiological role of the aminopeptidase gp160 remains uncertain. Analysis of the compartmentalization and trafficking pathways of translocatable proteins in fat cells identified more than one population of recycling vesicles, although all have identical sedimentation coefficients and buoyant densities in vitro. GLUT-4-containing vesicles include essentially all the intracellular GLUT-4, gp160, and the acutely recycling populations of receptors for IGF-II/Man-6-P and transferrin. Besides these proteins, which can be considered as vesicle “cargo”, GLUT-4-containing vesicles have other components, like secretory carrier-associated membrane proteins (SCAMP), Rab(s), and vesicle-associated membrane protein (VAMP)/cellubrevin, which are ubiquitous to secretory vesicles and granules from different tissues. GLUT-1 and caveolin are excluded from GLUT-4-containing vesicles and form different vesicular populations of unknown polypeptide composition. In skeletal muscle, two independent populations of GLUT-4-containing vesicles are found, insulin sensitive and exercise sensitive, which explains the additive effect of insulin and exercise on glucose uptake. Both vesicular populations are similar to each other and to analogous vesicles in fat cells.
Microdialysis measurements in skin: evidence for significant lactate release in healthy humans
Authors: Jansson P. A. , Krogstad A. L. , Lonnroth P.
Published: December 2017

To assess net lactate release from dermal skin, seven lean, healthy men were studied after overnight fasting. Two microdialysis catheters were inserted in the upper dermal tissue, as ensured by ultrasound scanning, in the periumbilical area. Each catheter was calibrated in situ to get an estimate of the lactate concentration in interstitial fluid (1,001 +/- 24 mumol/l), which in turn enabled calculation of the local capillary-venous lactate concentration (963 +/- 25 mumol/l). Concomitantly, arterialized venous plasma lactate (673 +/- 32 mumol/l), blood hematocrit (43 +/- 1%), and skin blood flow (3.8 +/- 0.9 ml.100 g-1.min-1) as measured by 133Xe clearance were determined, and dermal apparent lactate release (570 +/- 89 nmol.100 g-1.min-1) was estimated according to the Fick principle. During an oral glucose tolerance test (OGTT, 75 g), the dermal interstitial-arterial lactate difference decreased significantly to reach a nadir at 60 min. Moreover, no significant increase in skin blood flow was seen during the OGTT. In conclusion, we found a significant net lactate release from abdominal dermal skin after overnight fasting, whereas no significant increase was observed during an OGTT.
Dexamethasone rapidly increases hypothalamic neuropeptide Y secretion in adrenalectomized ob/ob mice
Authors: Chen H. L. , Romsos D. R.
Published: December 2017

A single intracerebroventricular injection of dexamethasone (DEX) rapidly (within 30 min) suppresses brown adipose tissue thermogenesis and increases plasma insulin concentrations in adrenal-ectomized (ADX) ob/ob mice but not in ADX lean mice. Intracerebroventricular neuropeptide Y (NPY) administered intracerebroventricularly causes these same metabolic changes within 30 min in both ob/ob and lean ADX mice. We therefore hypothesized that DEX exerts these rapid-onset metabolic actions in ob/ob mice via a phenotype-specific enhancement of NPY secretion within the central nervous system. In support of this hypothesis, DEX (a type II glucocorticoid receptor agonist) administered intracerebroventricularly selectively lowered NPY concentrations in the whole hypothalamus of ADX ob/ob mice by 35% and in the arcuate nucleus region by approximately 70% within 30 min but not in the brain stem or hippocampus or in any of these regions of lean mice. DEX also functioned in vitro to enhance depolarization-dependent release of NPY from hypothalamic blocks of ADX ob/ob mice but not of ADX lean mice. Thus DEX acts in the hypothalamus of ob/ob mice in a phenotype-specific manner to evoke rapid transport of NPY from cell bodies within the arcuate nucleus to terminal regions including the dorsomedial and ventromedial hypothalamic regions for release.
Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts
Authors: Johnson D. L. , McAllister T. N. , Frangos J. A.
Published: December 2017

Interstitial fluid flow may mediate skeletal remodeling in response to mechanical loading. Because nitric oxide (NO) has been shown to be an osteoblast mitogen and inhibitor of osteoclastic resorption, we investigated and characterized the role of fluid shear on the release of NO in osteoblasts. Rat calvarial cells in a stationary culture produced undetectable levels of NO. Fluid shear stress (6 dyn/cm2) rapidly increased NO release rate to 9.8 nmol.h-1.mg protein-1 and sustained this production for 12 h of exposure to flow. Cytokine treatment also induced NO synthesis after a 12-h lag phase of zero production, followed by a production rate of 0.6 nmol.h-1.mg protein-1. Flow-induced NO production was blocked by the NO synthase (NOS) inhibitor NG-amino-L-arginine, but not by dexamethasone, which suggests that the flow stimulated a constitutive NOS isoform. This is the first time that a functional constitutively present NOS isoform has been identified in osteoblasts. Moreover, fluid flow represents the most potent stimulus of NO release in osteoblasts reported to date. Fluid flow-induced NO production may therefore play a primary role in bone maintenance and remodeling.
Kinetic analysis of receptor-mediated endocytosis of G-CSF derivative, nartograstim, in rat bone marrow cells
Authors: Kuwabara T. , Kobayashi S. , Sugiyama Y.
Published: December 2017

To elucidate the mechanism of the receptor-mediated clearance of granulocyte colony-stimulating factor (G-CSF), we performed kinetic analyses of the receptor-mediated endocytosis (RME) processes using a human G-CSF derivative, nartograstim (NTG), and isolated rat bone marrow cells. The first-order rate constants involved in RME processes were obtained by computerized model fitting of the time courses of the ligand-receptor complex on both the cell surface and in the cell interior and the degradation products in the medium in the pulse-chase experiment. They were also calculated based on a kinetic model involving the ligand concentration dependence of the initial binding rate, the steady-state degradation rate, and the steady-state amounts of ligand on both the cell surface and in the interior. The rate constants for the RME processes after receptor binding determined in the different experiments were similar, that is, the half-times for the dissociation, internalization, and degradation of the ligand-receptor complex were 770, 10-30, and 20 min, respectively. However, the association constant obtained by measuring the initial binding was fivefold greater than that calculated under steady-state conditions. These kinetic analyses support the hypothesis that the internalization of the receptor may be accelerated by ligand binding, causing downregulation of the receptor on the cell surface. These overall kinetic analyses based on steady-state and non-steady-state data of the RME processes clarify the dynamics of the interaction between NTG and its receptor.
Gastrointestinal tract protein synthesis and mRNA levels for proteolytic systems in adult fasted rats
Authors: Samuels S. E. , Taillandier D. , Aurousseau E. , Cherel Y. , Le Maho Y. , Arnal M. , Attaix D.
Published: December 2017

We studied protein turnover in the gastrointestinal tract of adult fasted rats, since the mechanisms responsible for protein wasting in these tissues are poorly understood. Protein mass of stomach, small intestine, and colon decreased by 14-29 and 21-49% after 1 and 5 days of fasting, respectively. The fractional rate of in vivo protein synthesis (ks) was approximately 34% lower in the stomach after 1 and 5 days of fasting due to decreased capacity for protein synthesis (Cs). In small intestine and colon, ks was not different after 1 day, but was approximately 26% lower on day 5, mainly because of a reduction in Cs. Thus protein wasting in the stomach is primarily mediated by decreased protein synthesis but not in small intestine and colon during short-term fasting. To determine which proteolytic systems may be activated in the gut, we measured mRNA levels for critical components of the lysosomal (cathepsins B and D), Ca(2+)-activated (m-calpain), and ubiquitin-dependent (ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C8, and C9 proteasome subunits) proteolytic pathways. mRNA levels for most of these components increased during fasting, suggesting that a coordinated activation of multiple proteolytic systems contributed to intestinal protein wasting.
Dissociation of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation
Authors: Yoshihara T. , Honma S. , Katsuno Y. , Honma K.
Published: December 2017

Extracellular neuropeptide Y (NPY) in the vicinity of the paraventricular nucleus (PVN) as well as NPY concentrations in the PVN were measured in rats under ad libitum feeding and 2-day and 10-day food deprivation. Plasma corticosterone levels were not changed by 2-day food deprivation but were increased by subsequent refeeding. In contrast, the extracellular NPY levels were increased by 2-day food deprivation and were decreased rapidly by refeeding. The NPY concentrations were also increased and increased further by refeeding. On the other hand, plasma corticosterone levels were elevated by 10-day food deprivation and were decreased by subsequent refeeding. The extracellular NPY levels were also increased by food deprivation and decreased gradually after refeeding. However, the postprandial levels were still elevated when plasma corticosterone levels were returned to the basal levels. The NPY concentrations were also increased and increased further by refeeding. The amount of food intake after refeeding was positively correlated with the extracellular NPY levels. It is concluded that extracellular NPY levels in the PVN do not necessarily covariate with plasma corticosterone levels in rats under food deprivation.
Effects of chronic renal failure on plasma clearance of insulin-like growth factor I, des-(1-3)IGF-I, and LR3IGF-I
Authors: Gillespie C. M. , Hazel S. J. , Walton P. E. , Martin A. A.
Published: December 2017

Using a rat model of chronic renal failure (CRF), we examined insulin-like growth factor I (IGF-I) clearance, degradation, organ distribution, and IGF binding profiles in plasma. The effects of IGF-binding proteins (IGFBP) on IGF clearance and degradation in CRF were studied using the IGF-I analogues des-(1-3)IGF-I and LR3IGF-I, which bind poorly to IGFBP. Although total clearance of IGF-I was not significantly altered in CRF, half-life and area under the curve were increased in the rapid distribution phase and were reduced in the slow elimination phase. Total clearance of LR3IGF-I was significantly increased. Reduced binding of IGF-I in the 150-kDa complex and increased binding to smaller-molecular-weight IGFBP were observed in CRF. Increased degradation of both IGF-I and LR3IGF-I was associated with reduced IGF binding in the 150-kDa complex. The results suggest that the accumulation of lower-molecular-weight IGFBP with reduced IGF binding in the 150-kDa complex, associated with increased degradation of peptide, may explain, at least in part, the reduced bioactivity of IGF-I observed in CRF.
Insulin sensitivity and hemodynamic responses to insulin in Wistar-Kyoto and spontaneously hypertensive rats
Authors: Pitre M. , Nadeau A. , Bachelard H.
Published: December 2017

The insulin-mediated vasodilator effect has been proposed as an important physiological determinant of insulin action on glucose disposal in normotensive humans. The present study was designed to further examine the acute regional hemodynamic effects of insulin in different vascular beds and to explore the relationships between insulin vascular effects and insulin sensitivity during euglycemic hyperinsulinemic clamps in conscious normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The rats were instrumented with intravascular catheters and pulsed Doppler flow probes to measure blood pressure, heart rate, and regional blood flows. In WKY rats, the euglycemic infusion of insulin (4 and 16 mU.kg-1.min-1) causes vasodilations in renal and hindquarter vascular beds but no changes in mean blood pressure, heart rate, or superior mesenteric vascular conductance. In contrast, in SHR, the same doses of insulin produce vasoconstrictions in superior mesenteric and hindquarter vascular beds and, at high doses, increase blood pressure. Moreover, at the lower dose of insulin tested, we found a reduction in the insulin sensitivity index in the SHR compared with the WKY rats. The present findings provide further evidence for an association between insulin sensitivity and insulin-mediated hemodynamic responses.
A cross-sectional study on body composition and energy expenditure in women athletes during aging
Authors: Ryan A. S. , Nicklas B. J. , Elahi D.
Published: December 2017

The relationships between total and regional body composition, intra-abdominal adipose tissue (IAAT), resting metabolic rate (RMR), and substrate oxidation were examined in 43 highly trained women athletes and 14 sedentary women aged 18-69 yr. Athletes were divided into four groups (18-29, 30-39, 40-49, and 50-69 yr) and controls into two groups (18-29 and 40-50 yr). Maximal oxygen consumption declined with age (r = -0.52, P < 0.0005) in the athletes and was higher in all groups of athletes than in controls (P < 0.0001). No differences in percent fat and fat-free mass (FFM) were found between the youngest and oldest athletes. Although body mass index was < 25 kg/m2 in all subjects, percent body fat and total fat mass were higher in controls than in athletes for both young and older women (all P < 0.05). FFM was higher in young athletes than in young controls (P < 0.0001). Despite similar percent fat among athletes, IAAT increased with age (r = 0.75, P < 0.0001), but subcutaneous abdominal fat and sagittal diameter did not. IAAT and subcutaneous abdominal fat were also higher in young controls than in young athletes and in older controls than in older athletes (all P < 0.005). Age and FFM were independent predictors of the decline in RMR in the athletes. Fat oxidation (g/day) was highest in the youngest athletes and declined with age (r = -0.47, P < 0.005). We conclude that intense chronic exercise in women athletes prevented the decline in FFM with age. Endurance-trained women have low IAAT stores, which may potentially reduce subsequent risk associated with the metabolic syndrome.
Calcium content and respiratory control index of skeletal muscle mitochondria during exercise and recovery
Authors: Madsen K. , Ertbjerg P. , Djurhuus M. S. , Pedersen P. K.
Published: December 2017

The purpose of this study was to evaluate the relationship between mitochondrial Ca2+ concentration and the respiratory control index (RCI; state III/state IV) in isolated mitochondria before and after exhaustive exercise at 75% of maximal O2 consumption. Muscle biopsies of 100-150 mg from 12 moderately trained men were sampled at rest, immediately after exercise, and 30 or 60 min after exercise. The mitochondrial Ca2+ content after exhaustive exercise was significantly higher than the preexercise level [15.1 (range 39.4) vs. 11.6 (range 6.5) nmol/mg protein, respectively; P < 0.05], and RCI increased from 11.6 (range 14.4) at rest to 13.7 (range 15.0) at exhaustion (P < 0.05). After 60 min of recovery, the mitochondrial Ca2+ content was still high [18.8 (range 29.9) nmol/mg protein], but the RCI value was significantly depressed because of the increased state IV value and, in fact, was lower than the preexercise value [8.6 (range 5.1); P < 0.05]. Our results show that the mitochondrial Ca2+ content is increased in human skeletal muscle after prolonged exhaustive exercise and that this is followed by an elevated RCI value, with slightly increased state III and decreased state IV respiration. The restoration of the elevated mitochondrial Ca2+ level is slow and could be related to an increased state IV respiration, which together indicate uncoupled Ca2+ respiration during recovery.
Contributions of liver and kidneys to glycerol production and utilization in the dog
Authors: Previs S. F. , Martin S. K. , Hazey J. W. , Soloviev M. V. , Keating A. P. , Lucas D. , David F. , Koshy J. , Kirschenbaum D. W. , Tserng K. Y. , Brunengraber H.
Published: December 2017

The classical concept holds that liver and kidneys are the main sinks of glycerol released by adipose tissue. However, rates of glycerol appearance (Ra) exceed the rate of glycerol delivery to liver and kidneys. We measured the hepatic and renal contributions to glycerol production and utilization in anesthetized dogs that were fasted either overnight or for 24 h after 3 days on a carbohydrate-free diet. Dogs were infused with [2H5]glycerol, and the concentration and 2H enrichment of glycerol were measured across liver and kidney. After a baseline period, either norepinephrine or glucose plus insulin was infused to alter the rate of glycerol production. Our study shows that the production of glycerol by liver and kidneys amounted to 4-9% and 4-7% of the Ra of glycerol, respectively. Uptake of glycerol by liver and kidneys amounted to 26-30 and 10-19% of the Ra of glycerol, respectively. Thus, contrary to the classical concept, the bulk of glycerol utilization occurs in nonhepatic, nonrenal tissues that have very low glycerol kinase activity per gram.
Fat and carbohydrate metabolism during exercise in elderly and young subjects
Authors: Sial S. , Coggan A. R. , Carroll R. , Goodwin J. , Klein S.
Published: December 2017

We evaluated the effect of aging on fat and carbohydrate metabolism during moderate intensity exercise. Glycerol, free fatty acid (FFA), and glucose rate of appearance (Ra) in plasma and substrate oxidation were determined during 60 min of cycle ergometer exercise in six elderly (73 +/- 2 yr) and six young adults (26 +/- 2 yr) matched by gender and lean body mass. The elderly group was studied during exercise performed at 56 +/- 3% of maximum oxygen uptake, whereas the young adults were studied during exercise performed at the same absolute and at a similar relative intensity as the elderly subjects. Mean fat oxidation during exercise was 25-35% lower in the elderly subjects than in the young adults exercising at either the same absolute or similar relative intensities (P < 0.05). Mean carbohydrate oxidation in the elderly group was 35% higher than the young adults exercising at the same absolute intensity (P < 0.001) but 40% lower than the young adults exercising at the same relative intensity (P < 0.001). Average FFA Ra in the elderly subjects was 85% higher than in the young adults exercising at the same absolute intensity (P < 0.05) but 35% lower than the young adults exercising at a similar relative intensity (P < 0.05). We conclude that fat oxidation is decreased while carbohydrate oxidation is increased during moderate intensity exercise in elderly men and women. The shift in substrate oxidation was caused by age-related changes in skeletal muscle respiratory capacity because lipolytic rates and FFA availability were not rate limiting in the older subjects.
Maintaining muscle protein anabolism after a metabolic stress: role of dextrose vs. amino acid availability
Authors: Borel M. J. , Williams P. E. , Jabbour K. , Hibbard J. C. , Flakoll P. J.
Published: December 2017

The effect of chronic hypocaloric parenteral infusions of amino acids (AA) vs. dextrose (D) on protein homeostasis after a generalized metabolic stress was examined. Multicatheterized mongrel dogs were metabolically challenged by a 4-day fast and then administered a 4-day intravenous infusion of saline (S, n = 8), D (n = 8), or isocaloric AA (n = 7). Although nitrogen balance (g.kg.1.day-1) was similarly negative with S (-0.37 +/- 0.05), D (-0.28 +/- 0.03), and AA (-0.37 +/- 0.04) during the fasting period, it was less negative (P < or = 0.05) with AA (-0.06 +/- 0.04) than with D (-0.20 +/- 0.03) or S (-0.23 +/- 0.04) during nutrient infusion. AA resulted in net hindlimb uptake and D in net hindlimb release of essential AA (570 +/- 261 vs. -248 +/- 59 nmol.kg-1.min-1). Whereas S and D infusions led to net hindlimb muscle protein loss (-37 +/- 24 and -89 +/- 33 micrograms.kg-1.min-1, respectively, P < or = 0.05 vs. AA), parenteral AA resulted in net deposition (169 +/- 62 micrograms.kg-1.min-1) as measured using L-[ring-2H5]phenylalanine. Thus hypocaloric parenteral D infusion after a metabolic stress does not favor nitrogen conservation, because net whole body nitrogen loss, skeletal muscle protein catabolism, and hindlimb AA release were not blunted compared with S infusion. Conversely, hypocaloric AA infusion preserves whole body and muscle protein stores.
Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3-L1 adipocytes
Authors: Katagiri H. , Asano T. , Inukai K. , Ogihara T. , Ishihara H. , Shibasaki Y. , Murata T. , Terasaki J. , Kikuchi M. , Yazaki Y. , Oka Y.
Published: December 2017

The dominant negative p85alpha regulatory subunit (delta p85alpha) of phosphatidylinositol (PI) 3-kinase or dominant negative Ras (N17Ras) was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Functional expression of delta p85alpha and N17Ras was confirmed by marked inhibition of insulin-stimulated PI 3-kinase activity and mitogen-activated protein kinase activity, respectively. N17Ras expression did not affect glucose transport activity, whereas delta p85alpha expression inhibited insulin-stimulated glucose transport with impairment of GLUT-4 translocation, although inhibition of glucose transport activity was less remarkable than that of PI 3-kinase activity in delta p85alpha-expressing cells. Thus the Ras signaling pathway does not play a major part in either translocation or intrinsic activity of glucose transporters, but PI 3-kinase activation, via phosphotyrosyl proteins and heterodimeric PI 3-kinase, plays a pivotal role in insulin-stimulated glucose transport. However, a discrepancy was observed between PI 3-kinase activity and glucose transport activity, suggesting a possibility that a different pathway(s) is involved in insulin-stimulated intrinsic activity of glucose transporters.
Agouti regulation of intracellular calcium: role of melanocortin receptors
Authors: Kim J. H. , Kiefer L. L. , Woychik R. P. , Wilkison W. O. , Truesdale A. , Ittoop O. , Willard D. , Nichols J. , Zemel M. B.
Published: December 2017

Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+]i) is elevated in viable yellow mice. Because [Ca2+]i has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+]i was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+]i in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+]i with an apparent concentration eliciting 50% of the maximal response (EC50) of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+]i, we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+]i, whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC50 of 18 nM, which is comparable to that for agouti antagonism of (125)I-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+]i suggests a potential mechanism for agouti-induced insulin resistance.
Glucose production, recycling, Cori cycle, and gluconeogenesis in humans: relationship to serum cortisol
Authors: Tayek J. A. , Katz J.
Published: December 2017

Six normal subjects (NL group) and 13 cancer patients (CAI and CAII groups) were fasted overnight and infused with [U-(13)C]glucose (0.016-0.058 mg x min(-1) x kg(-1)). Plasma glucose and lactate were isolated, and mass isotopomer distributions were determined by gas chromatography-mass spectroscopy. Applying equations modified from those previously described [J. A. Tayek and J. Katz. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E709-E717, 1996], we determined glucose production (GP), recycling of glucose carbons, fraction of recycled molecules in blood glucose (Cori cycle), formation of pyruvate from unlabeled carbons, dilution of pyruvate via the tricarboxylic acid cycle and other reactions, and rate of gluconeogenesis. Glucose production was similar in all groups: 2.4 +/- 0.2 mg x min(-1) x kg (-1). The fraction of recycled carbon and of the Cori cycle were elevated in the CAI group vs. the CAII and NL groups: 15 and 33% vs. 7.8 and 19%, respectively (P = 0.01). Gluconeogenesis was 1.9 +/- 0.1, 1.0 +/- 0.1, and 0.83 +/- 0.11 mg x min(-1) x kg(-1) in the CAI, CAII, and NL groups, respectively. In the NL and CAII groups, 20% of GP is via recycling, 20% from unlabeled carbon sources (muscle glycogen, amino acids), and 60% from hepatic glycogenolysis; in the CAI group, 30% is from recycling, 50% from unlabeled carbon, and 20% from glycogen and other sources. Serum cortisol was elevated in the CAI group vs. the CAII group: 11.2 +/- 1.2 vs. 7.7 +/- 1.2 microg/dl (P < 0.05). There was a strong correlation between plasma cortisol and Cori cycle in the NL (r = 0.963) and CAI and CAII groups (r = 0.771). Serum cortisol was directly, and insulin was inversely, correlated with gluconeogenesis in the NL (r2 = 0.967) and CAI and CAII groups (r2 = 0.727). We conclude that whereas the cancer population is heterogeneous with respect to gluconeogenesis, many cancer patients derive their GP predominantly from gluconeogenesis compared with healthy controls, who derive less than one-half of their GP from gluconeogenesis.
Effects of exercise and feeding on the hexosamine biosynthetic pathway in rat skeletal muscle
Authors: Nelson B. A. , Robinson K. A. , Koning J. S. , Buse M. G.
Published: December 2017

Products of the hexosamine biosynthesis pathway (HSNP) have been implicated in glucose-induced insulin resistance. We measured the major products of HSNP, UDP-N-acetyl hexosamines (UDP-HexNAc), and the activity of L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT, rate-limiting enzyme) in rat hindlimb muscles immediately after exercise and 1, 3, and 16 h postexercise (swimming) in fed and fasted rats and sedentary controls. Muscle glycogen decreased by 50-75% postexercise. In sedentary rats, muscle GFAT activity decreased by approximately 30% (P < 0.002) after an 18-h fast. GFAT activity was not affected by exercise under any condition. Muscle UDP-HexNAc increased approximately 30% postexercise (P < 0.01) in ad libitum-fed but not in fasted rats. UDP-HexNAc remained elevated (approximately 30%, P < 0.002) for 16 h if animals were fed postexercise. Concentrations of UDP-hexoses, GDP-mannose, and UDP were unchanged postexercise. Conclusions are that muscle GFAT activity is regulated by the nutritional state but not by acute exercise. Glucose flux via HNSP may be increased postexercise in muscles of ad libitum-fed rats. Increased HSNP products may serve as negative feedback regulators to limit excessive muscle glycogen deposition postexercise.
Effects of diabetes on rodent cardiac thyroid hormone receptor and isomyosin expression
Authors: Haddad F. , Bodell P. W. , McCue S. A. , Baldwin K. M.
Published: December 2017

Previous studies show that diabetes induces marked transformations in cardiac myosin heavy chain (MHC) gene expression that are somehow linked to the cellular action of thyroid hormone 3,5,3'-triiodothyronine (T3). In this study, we tested the hypothesis that diabetes induces a reduced expression of thyroid hormone receptors (TRs), which are known to be important transcription factors interacting with thyroid response elements (TREs) in the promoter region of both alpha- and beta-MHC genes. Adult female rats were randomly assigned to either a normal control (NC) or diabetic (D) group. Three and/or six weeks after induction of diabetes via streptozotocin injection, the hearts of the animals were analyzed for MHC and TR mRNA isoforms expression, nuclear T3 binding, and nuclear extract interaction with a palindromic TRE. Results showed that diabetes induced significant alteration in alpha- and beta-MHC expression. Northern blot analyses indicated no diabetes-associated differences in TR isoform mRNA signals. Cardiac nuclear T3 binding studies suggested no differences in either the binding capacity or the equilibrium binding constant among the two groups, indicating no changes in either the number of nuclear TRs or their affinity for T3. Furthermore, gel mobility shift assays detected no difference between NC and D groups for cardiac nuclear extract binding to palindromic TRE. Collectively, these findings suggest that, whereas diabetes exerts a profound effect on cardiac isomyosin gene expression, the underlying mechanism, although dependent on factors linked to T3 function, does not involve alterations in TR expression.
Exercise training reverses insulin resistance in muscle by enhanced recruitment of GLUT-4 to the cell surface
Authors: Etgen G. J. , Jensen J. , Wilson C. M. , Hunt D. G. , Cushman S. W. , Ivy J. L.
Published: December 2017

The effects of exercise training on cell surface GLUT-4 in skeletal muscle of the obese (fa/fa) Zucker rat were investigated using the impermeant glucose transporter photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis- (D-mannos-4-yloxy)-2-propylamine (ATB-BMPA). In the absence of insulin, 3-O-methyl-D-glucose transport activity was no different in either fast-twitch (epitrochlearis) or slow-twitch (soleus) muscles of trained and sedentary obese rats. Likewise, basal ATB-BMPA-labeled GLUT-4 was not altered in these muscles with training. In contrast, the trained group exhibited significantly greater insulin-stimulated (2 mU/ml) glucose transport activity in epitrochlearis muscles than the sedentary group (0.53 +/- 0.03 vs. 0.18 +/- 0.03 mumol.g-1 x 10 min-1 for trained and sedentary, respectively), which was paralleled by a significant enhancement of insulin-stimulated cell surface GLUT-4 (5.33 +/- 0.20 vs. 1.57 +/- 0.14 disintegrations.min-1.mg-1 for trained and sedentary, respectively). Exercise training, however, did not alter insulin-stimulated glucose transport activity or cell surface GLUT-4 in soleus muscles. Finally, exercise training did not alter the ability of muscle contraction to elevate glucose transport activity or cell surface GLUT-4 in either epitrochlearis or soleus muscles of the obese rat. These results indicate that training improves insulin-stimulated glucose transport in muscle of the obese Zucker rat by increasing GLUT-4 content and by altering the normal intracellular distribution of these transporters such that they are now capable of migrating to the cell surface in response to the insulin stimulus.
Extracellular ATP as an autocrine/paracrine regulator of prolactin release
Authors: Nunez L. , Villalobos C. , Frawley L. S.
Published: December 2017

Recent evidence demonstrates that ATP, costored with a number of hormones and neurotransmitters in secretory granules, is coreleased during exocytosis of these agents. Here, we explored the possibility that extracellular ATP subserves an autocrine and/or paracrine role in the regulation of prolactin (PRL) release by subjecting rat pituitary cells to various experimental manipulations aimed at evaluating putative interactions between ATP and mammotropes. Our results strongly support the view that ATP functions as a local regulator of PRL secretion. To be more specific, we observed that ATP is released in a predictable manner by physiologically relevant secretagogues that are reasonably targeted to mammotropes. Moreover, we found that ATP can act directly on pituitary cells to stimulate the release of PRL from most (if not all) mammotropes. Finally, we determined that antagonism or removal of ATP leads to a diminution of PRL export from pituitary cells cultured under basal or thyrotropin-releasing hormone-stimulated conditions. On the basis of these results, we propose that ATP acts locally to amplify and prolong the PRL secretory response elicited by a more traditional hypophysiotropic signal.
Osteogenic protein-1 downregulates endothelin A receptors in primary rat osteoblasts
Authors: Kitten A. M. , Harvey S. A. , Criscimagna N. , Asher M. , Lee J. C. , Olson M. S.
Published: December 2017

Osteogenesis is a complex process whereby growth factors and mediators from both local and systemic sources modulate the bone-forming activities of osteoblasts. In the present study we utilized primary cultures of fetal rat calvarial cells to characterize osteoblast responsiveness to the vascular mediator endothelin-1 (ET-1) and to investigate whether ET-1 responses are regulated by osteogenic protein-1 (OP-1). We found that a 1- to 2-day exposure to OP-1 diminished ET-1 receptor ligand binding and signal transduction by downregulating ET-1 receptor mRNA expression. ET-1-mediated calcium signaling and ligand binding were completely abolished by the ETA receptor antagonist BQ-123, suggesting that ET-1 effects are mediated by this receptor. Northern analysis of total RNA revealed that ETA mRNA expression was inhibited approximately 50% by OP-1 treatment, whereas ETB receptor mRNA was not detected by this method of analysis. In OP-1-treated cultures, the magnitude and duration of ET-1 calcium signals varied among individual cells. This finding may be related to a heterogeneous OP-1 response, indicated by alkaline phosphatase induction in only a subpopulation of cells. These results suggest that modulation of osteoblast function by ET-1 occurs during distinct periods of phenotypic development and imply that downregulation of ET-1 responsiveness may be necessary for optimal bone formation in vivo.
Regional lipolytic responses to isoproterenol in women
Authors: Guo Z. , Johnson C. M. , Jensen M. D.
Published: December 2017

We previously found that epinephrine, a mixed beta- and alpha-adrenoreceptor agonist, stimulates systemic and nonsplanchnic upper body free fatty acid (FFA) release but not lower body FFA release in healthy nonobese women. To evaluate the role of beta-adrenergic-mediated effects on this regional difference in lipolysis, we measured systemic, leg, and splanchnic FFA kinetics ([3H]palmitate) in seven healthy nonobese women before and during an intravenous isoproterenol infusion. Isoproterenol increased systemic palmitate flux (87 +/- 12 vs. 100 +/- 10 mumol/min, P < 0.05) but failed to affect leg [10.8 +/- 1.2 vs. 11.4 +/- 2.3 mumol/min, P = not significant (NS)] or splanchnic (10.8 +/- 3.2 vs. 10.0 +/- 1.8 mumol/min, P = NS) palmitate release. Upper body nonsplanchnic palmitate release increased from 56 +/- 14 to 71 +/- 10 mumol/min. Systemic O2 consumption increased (227 +/- 11 to 241 +/- 10 ml/min, P = 0.006) during isoproterenol infusion, as did leg (318 +/- 42 vs. 404 +/- 53 ml/min, P < 0.01) and splanchnic (827 +/- 104 vs. 970 +/- 108 ml/min, P < 0.05) plasma flow. These results suggest that lower body adipose tissue lipolysis in women is less sensitive or responsive than nonsplanchnic upper body adipose tissue to beta-adrenergic stimulation and that regional differences in alpha 2-adrenergic-receptor responses were not responsible for the similar regional differences we observed previously with epinephrine.
The somatotropic axis in neonatal calves can be modulated by nutrition, growth hormone, and Long-R3-IGF-I
Authors: Hammon H. , Blum J. W.
Published: December 2017

Effects on the somatotropic axis [plasma levels of insulin-like growth factors (IGFs) I and II, IGF-binding proteins (IGFBPs), and growth hormone (GH)] of feeding different amounts of colostrum or milk replacer, of Long-R3-IGF-I (administered subcutaneously or orally; 50 micrograms.kg body wt-1.day-1 for 7 days), and of subcutaneously injected recombinant bovine GH (rbGH; 1 mg.kg body wt-1.day-1 for 7 days) were evaluated in calves during the 1st wk of life. Plasma Long-R3-IGF-I increased after subcutaneous application but not with the oral dose. Endogenous IGF-I was higher in calves fed colostrum six times compared with those fed only milk replacer. Native IGF-I was highest in rbGH-injected calves but was lowered by the subcutaneous injection of Long-R3-IGF-I. IGF-II concentrations were not modified by any of the treatments. IGFBP-2 increased in calves fed only milk replacer and those receiving subcutaneous Long-R3-IGF-I. GH was not modulated by differences in nutrition but increased after rbGH administration and similarly in all groups after intravenous injection of GH-releasing factor analog GRF-(1-29). Parenteral administration of Long-R3-IGF-I decreased GH concentration but did not affect the secretory pattern. The data demonstrate that the somatotrophic axis is basically functioning in neonatal calves and is influenced by nutrition, GH, and Long-R3-IGF-I.
In vivo NMR investigation of intramuscular glucose metabolism in conscious rats
Authors: Jucker B. M. , Rennings A. J. , Cline G. W. , Petersen K. F. , Shulman G. I.
Published: December 2017

In vivo 13C nuclear magnetic resonance (NMR) spectroscopy was used to determine quantitatively the flux of muscle glycolysis, glycogen synthesis, pyruvate dehydrogenase, and pyruvate carboxylation in the hindlimb of conscious rats. 13C NMR spectroscopy was used to observe [1-13C]glucose label precursor incorporation into intramuscular [1-13C]glycogen, [3-13C]lactate, and [3-13C]alanine during a hyperglycemic (approximately 11 mM)-hyperinsulinemic (10 mU.kg-1.min-1) clamp. The glycogen synthesis rate was calculated to be 224 +/- 23 nmol.g-1.min-1. The kinetic data obtained from the label turnover in the intramuscular C-3 lactate and C-3 alanine metabolite pools, as well as in plasma C-3 lactate and C-3 alanine, were combined with a steady-state rate analysis to determine the glycolytic flux (67.4 +/- 10.1 nmol.g-1.min-1). Steady-state isotopomer analysis of glutamate and pyruvate in skeletal muscle tissue extracts was used to determine the anaplerotic contribution of substrate via pyruvate carboxylation (Vpc). The pyruvate dehydrogenase flux (Vpdh) was calculated after a steady-state flux correction for Vpc. Calculated values of Vpc and Vpdh were 24.8 +/- 4.3 and 110.0 +/- 18.7 nmol.g-1.min-1, respectively. In addition, [2-13C]acetate was used in a separate study to determine that pyruvate carboxylation was the major pathway for anaplerosis in skeletal muscle under conditions of hyperglycemia-hyperinsulinemia.
Constant infusion and bolus injection of stable-label tracer give reproducible and comparable fasting HGO
Authors: Hovorka R. , Eckland D. J. , Halliday D. , Lettis S. , Robinson C. E. , Bannister P. , Young M. A. , Bye A.
Published: December 2017

We have investigated the reproducibility of fasting hepatic glucose output (HGO) estimates by use of isotope dilution methodology of stable-label tracers. Six normal subjects were studied on two occasions 1 wk apart. After an overnight fast, the subjects received a bolus injection of 7 mg/kg of [U-13C]glucose and, simultaneously, a primed constant infusion of 0.05 mg.kg-1.min-1 of [6,6(-2)H]glucose. The bolus injection provided one estimate of HGO (HGOBOL), and the constant infusion provided two estimates of HGO, namely, HGO at 2 h (HGOINF2) and HGO at 4 h (HGOINF4), both with the assumption of steady-state conditions. All estimates were similar in value; HGOBOL was highest, followed by HGOINF2 and HGOINF4 [2.30 +/- 0.11 (SE), 2.17 +/- 0.12, and 2.01 +/- 0.13 mg.kg-1.min-1]. The constant infusion gave highly reproducible results. In the case of HGOINF2, the within-subject coefficient of variation (CV) was only 3% compared with 5% of HGOINF4. The reproducibility of HGOBOL was comparable with the within-subject CV of 7%. We conclude that a constant infusion and a bolus injection of stable-label tracer give reproducible and comparable estimates of HGO.
Molecular analysis of insulin resistance in isolated ventricular cardiomyocytes of obese Zucker rats
Authors: Kolter T. , Uphues I. , Eckel J.
Published: December 2017

Isolated ventricular cardiomyocytes obtained from lean and genetically (fa/fa) obese Zucker rats were used to correlate alterations of insulin-induced glucose transport activation and GLUT-4 translocation to possible defects of the insulin signaling cascade. Maximal stimulation with insulin was found to produce an unaltered translocation of GLUT-4 to the plasma membrane (4.2- and 3.7-fold increase for lean and obese rats, respectively). However, a largely reduced sensitivity of 3-O-methylglucose transport could be detected in obese rats at physiological doses of insulin (completely unresponsive at 8 x 10(-11) M compared with 3-fold stimulation of glucose transport in lean controls). Tyrosine phosphorylation of the insulin receptor beta-subunit and the insulin receptor substrate 1 (IRS-1) was stimulated identically in cardiomyocytes from both lean and obese rats. Labeling of cells with [33P]orthophosphate revealed a marked increase in the serine and/or threonine phosphorylation of IRS-1 in the obese group (370% of lean controls), with a concomitant reduction in IRS-1 abundance (30-40%). The reduced sensitivity of glucose transport at 8 x 10(-11) M insulin was then found to correlate to a completely blunted response of IRS-1-associated phosphatidylinositol 3-kinase activity in cardiomyocytes from obese rats. Those data show that cardiac insulin resistance of obesity involves defective insulin signaling at low concentrations of the hormone, whereas GLUT-4 translocation is fully operative in the isolated cell. It is suggested that hyperphosphorylation of IRS-1 may significantly contribute to the pathogenesis of insulin resistance in the heart.