Increased Leucine Incorporation Research Articles

Activation of the hypoxia response pathway protects against age-induced cardiac hypertrophy

AimsWe have previously demonstrated protection against obesity, metabolic dysfunction, atherosclerosis and cardiac ischemia in a hypoxia-inducible factor (HIF) prolyl 4-hydroxylase-2 (Hif-p4h-2) deficient mouse line, attributing these protective effects to activation of the hypoxia response pathway in a normoxic environment. We intended here to find out whether the Hif-p4h-2 deficiency affects the cardiac health of these mice upon aging. Methods and resultsWhen the Hif-p4h-2 deficient mice and their wild-type littermates were monitored during normal aging, the Hif-p4h-2 deficient mice had better preserved diastolic function than the wild type at one year of age and less cardiomyocyte hypertrophy at two years. On the mRNA level, downregulation of hypertrophy-associated genes was detected and shown to be associated with upregulation of Notch signaling, and especially of the Notch target gene and transcriptional repressor Hairy and enhancer-of-split-related basic helix-loop-helix (Hey2). Blocking of Notch signaling in cardiomyocytes isolated from Hif-p4h-2 deficient mice with a gamma-secretase inhibitor led to upregulation of the hypertrophy-associated genes. Also, targeting Hey2 in isolated wild-type rat neonatal cardiomyocytes with siRNA led to upregulation of hypertrophic genes and increased leucine incorporation indicative of increased protein synthesis and hypertrophy. Finally, oral treatment of wild-type mice with a small molecule inhibitor of HIF-P4Hs phenocopied the effects of Hif-p4h-2 deficiency with less cardiomyocyte hypertrophy, upregulation of Hey2 and downregulation of the hypertrophy-associated genes. ConclusionsThese results indicate that activation of the hypoxia response pathway upregulates Notch signaling and its target Hey2 resulting in transcriptional repression of hypertrophy-associated genes and less cardiomyocyte hypertrophy. This is eventually associated with better preserved cardiac function upon aging. Activation of the hypoxia response pathway thus has therapeutic potential for combating age-induced cardiac hypertrophy.

Blastocyst—endometrial interactions in early pregnancy in the sheep

Summary. This review analyses the endocrine recognition of pregnancy in the sheep. Particular emphasis is placed on the effect of a blastocyst on prostaglandin and protein metabolism in the endometrium. The preimplantation period is associated with increased prostaglandin content in the endometrium and uterine lumen, increased leucine incorporation into endometrial protein and increased blood flow to the uterus. It is concluded that there are substantial alterations in endometrial function during the protracted preimplantation period and that these alterations constitute an endocrine form of maternal recognition of pregnancy. However, it is not yet possible to attribute these alterations to specific embryonic signals.

Megestrol acetate treatment influences tissue amino acid uptake and incorporation during cancer cachexia

Summary Background & aims Cachexia is a multi-organic syndrome associated with cancer, characterized by body weight loss, muscle and adipose tissue wasting and inflammation, being often associated with anorexia. The aim of the present investigation was to study the effects of megestrol acetate (MA) on the rate of leucine incorporation into muscle tissue and on the uptake of AIB (a non-metabolizable analogue of alanine) in both skeletal muscle and liver tissue. Methods The effects of MA (100 mg/kg) were tested in cachectic tumour-bearing rats (Yoshida AH-130 ascites hepatoma). Tissue amino acid uptake was assessed by means of the alanine analogue α-amino-3H-isobutyrate (AIB). 14C-leucine oxidation and tissue protein incorporation were also determined. Results Administration of MA to tumour-bearing rats resulted in an important reversal of the muscle wasting process, as reflected by individual muscle weights. Treatment with the progestogen resulted in an increased AIB uptake together with an increased leucine incorporation in skeletal muscle. MA treatment significantly decreased AIB uptake by the liver. Conclusions MA treatment results in both a significant increased uptake of neutral amino acids (AIB) by skeletal muscle and a significant incorporation of the branched-chain amino acid leucine into muscle protein, indicating a clear anabolic action of the drug on muscle tissue.

Effects of High Glucose on AVP-Induced Hyperplasia, Hypertrophy, and Type IV Collagen Synthesis in Cultured Rat Mesangial Cells

Introduction. Hyperglycemia is a principal characteristic of diabetes and influences many cellular functions. Diabetic nephropathy is characterized by glomerular mesangial expansion which could result from increased mesangial cell extracellular matrix synthesis induced by hyperglycemia. Methods. To investigate whether the physiological functions of mesangial cells are altered in a diabetic environment, we evaluated the effect of high extracellular glucose concentration on thymidine/leucine incorporation, hyperplasia/hypertrophy, and type IV collagen synthesis, induced by vasopressin (AVP), in cultured rat mesangial cells. Results. The exposure of mesangial cells to a high glucose concentration (30 mM) significantly reduced AVP-induced thymidine incorporation and hyperplasia compared with normal glucose (10 mM). By contrast, treatment of mesangial cells with AVP in the presence of high extracellular glucose significantly increased leucine incorporation, hypertrophy, and type IV collagen synthesis compared with those at normal glucose levels. The administration of staurosporine, a protein kinase C inhibitor, reversed these effects of high-glucose conditions. Furthermore, the nonpeptide AVP V1A receptor-selective antagonists potently inhibited these AVP-induced physiological responses in mesangial cells cultured in high-glucose conditions. Conclusions. These results demonstrate that high glucose suppresses mesangial cell proliferation but enhances hypertrophy and type IV collagen synthesis induced by AVP. This increased mesangial cell hypertrophy and extracellular matrix synthesis may play a crucial role in the glomerular mesangial expansion common to diabetic nephropathy.

Resistin Promotes Cardiac Hypertrophy via the AMP-activated Protein Kinase/Mammalian Target of Rapamycin (AMPK/mTOR) and c-Jun N-terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Impact of Growth Hormone and Dehydroepiandrosterone on Protein Metabolism in Glucocorticoid-Treated Patients

Chronic pharmacological glucocorticoid (GC) use causes substantial morbidity from protein wasting. GH and androgens are anabolic agents that may potentially reverse GC-induced protein loss. Our objective was to assess the effect of GH and dehydroepiandrosterone (DHEA) on protein metabolism in subjects on long-term GC therapy. This was an open, stepwise GH dose-finding study (study 1), followed by a randomized cross-over intervention study (study 2). The studies were performed at a clinical research facility. In study 1, six subjects (age 69+/-4 yr) treated with long-term (>6 months) GCs (prednisone dose 8.3+/-0.8 mg/d) were studied before and after two sequential GH doses (0.8 and 1.6 mg/d) for 2 wk each. In study 2, 10 women (age 71+/-3 yr) treated with long-term GCs (prednisone dose 5.4+/-0.5 mg/d) were studied at baseline and after 2-wk treatment with GH 0.8 mg/d, DHEA 50 mg/d, or GH and DHEA (combination treatment). Changes in whole body protein metabolism were assessed using a 3-h primed constant infusion of 1-[13C]leucine, from which rates of leucine appearance, leucine oxidation, and leucine incorporation into protein were estimated. In study 1, GH 0.8 and 1.6 mg/d significantly reduced leucine oxidation by 19% (P=0.03) and 31% (P=0.02), and increased leucine incorporation into protein by 10% (P=0.13) and 19% (P=0.04), respectively. The lower GH dose did not cause hyperglycemia, whereas GH 1.6 mg/d resulted in fasting hyperglycemia in two of six subjects. In study 2, DHEA did not significantly change leucine metabolism alone or when combined with GH. Blood glucose was not affected by DHEA. GH, at a modest supraphysiological dose of 0.8 mg/d, induces protein anabolism in chronic GC users without causing diabetes. DHEA 50 mg/d does not enhance the effect of GH. GH may safely prevent or reverse protein loss induced by chronic GC therapy.

Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts

Effects of tanshinone VI, a diterpene from Tan-Shen, on humoral factor-induced phosphorylation of ERK and Akt during hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts isolated from neonatal rats were examined. Treatment of cultured cardiomyocytes with 10 nM insulin-like growth factor-1 (IGF-1) or 10 nM endothelin-1 resulted in an increase in leucine incorporation into acid-insoluble fraction. Treatment of cultured cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II increased incorporation of proline. IGF-1 increased phosphorylated extracellular signal-regulated kinase (pERK) and protein kinase B (pAkt) of cardiomyocytes, whereas endothelin-1 increased pERK, but not pAkt. Treatment of cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II also increased pERK, whereas pAkt was increased by treatment with IGF-1 alone. When the cardiomyocytes were incubated in the presence of 10 μM tanshinone VI, IGF-1- and endothelin-1-induced increases in pERK, but not pAkt, were partially attenuated. Treatment of cardiac fibroblasts with 10 μM tanshinone VI also attenuated IGF-1-induced increases in pERK and pAkt. Tanshinone VI also partially attenuated angiotensin II-induced increase in proline incorporation into cardiac fibroblasts. PD98059, an inhibitor for phosphorylation of extracellular signal-regulated kinase (ERK), but not wortmannin, that for protein kinase B phosphorylation, attenuated an increase in leucine incorporation into cardiomyocytes in the presence of either IGF-1 or endothelin-1. These results suggest that tanshinone VI is a possible agent that can attenuate the humoral factor-induced hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts via an attenuation of ERK phosphorylation in these cells.

Abstract 143: Caveolae Regulate PI3K/Akt/RhoA-Mediated p38 MAPK Nuclear Translocation in Leptin-Induced Cardiomyocyte Hypertrophy

Background : Obesity is associated with increased leptin production which may contribute to cardiac hypertrophy. However, the mechanism of leptin-induced cardiac hypertrophy remains incompletely understood. Previous studies have shown that the RhoA/ROCK/cofilin pathway and p38 MAPK but not ERK1/2 activation are major contributors to leptin-induced cardiac hypertrophy. In this study we explored the roles of caveolae and the PI3K/Akt pathway in regulating RhoA and p38 MAPK activation during leptin-induced cardiomyocyte hypertrophy. Methods and Results : Neonatal rat ventricular myocytes were cultured with 3.1 nmol/L leptin for 24 hours. Caveolae number and expression of caveolin-3 were significantly increased after leptin treatment (2 and 3 fold, respectively; p<0.01). These effects were associated with a 29% (p<0.05) increase in cell surface area and a 40% (p<0.05) increase in leucine incorporation, indicating cardiomyocyte hypertrophy. Disruption of cardiomyocyte caveolae with 5 mM methyl-beta-cyclodextrin (MβCD) significantly inhibited leptin-induced hypertrophy. RhoA was detected in caveolae fractions of a sucrose gradient after cardiomyocytes were treated with leptin for 5 min, demonstrating subcellular translocation of RhoA. Treatment with MβCD, 50 ng/ml C3 exoenzyme (a RhoA inhibitor) or 50 nM latrunculin B (actin filaments depolymerization agent) significantly attenuated leptin-induced RhoA translocation into caveolae fractions. Moreover, Western blot analysis showed that leptin-dependent activation of PI3K (116%; p<0.05), Akt (115%; p<0.05) and RhoA (330%; p<0.05) were significantly inhibited by 50 μM LY294002, a specific PI3K inhibitor. In addition, LY294002 significantly attenuated leptin-induced increases in cell surface area and in leucine incorporation. Furthermore, we found that leptin-induced activation of p38 (189%; p<0.05) and ERK1/2 (220%; p<0.05) was associated with p38 MAPK but not ERK1/2 nuclear translocation. MβCD, C3 exoenzyme and LY294002 potently attenuated leptin-induced p38 MAPK nuclear translocation. Conclusions : Our results demonstrate that caveolae, the PI3K/Akt/RhoA pathway and p38 MAPK nuclear translocation play a pivotal role in leptin-induced cardiomyocyte hypertrophy.

Elevated glucose activates protein synthesis in cultured cardiac myocytes

Diabetes mellitus results in chronic hyperglycemia, a serious metabolic disorder associated with a markedly increased risk of cardiovascular disease. However, the effects of high glucose (HG) on cardiac myocyte growth have not been fully clarified. In this study, the effect of glucose on cardiac myocyte growth was examined using leucine incorporation as an index of protein synthesis. High glucose (HG, 25 mmol/L) increased leucine incorporation (167% ± 0.2% over normal glucose, n = 4, P < .01) compared with a physiological glucose concentration (5.5 mmol/L, normal glucose). The HG-induced increase in leucine incorporation was time- and dose-dependent and was not due to osmotic changes because 25 mmol/L mannitol did not change leucine incorporation. High glucose also significantly reduced elongation factor 2 phosphorylation, an effect known to result in increased protein synthesis at the elongation step. Western blot analysis showed that HG-activated protein kinase B (PKB), also called Akt (PKB/Akt), at 18 hours. High glucose–induced leucine incorporation was attenuated with phosphatidylinositol 3-kinase (PI3K) inhibition using wortmannin and LY294002 and by rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, 72%, 64%, and 65% ( P < .05), respectively. High glucose also activated extracellular signal–regulated kinase 1/2 activity with peak stimulation at 5 minutes. In addition, PD98059, an inhibitor of mitogen-activated protein kinase kinase, attenuated HG-induced leucine incorporation. These data show for the first time that elevated glucose increases protein synthesis in cardiac myocytes. The increase appears to be mediated by activation of PI3K-PKB/Akt and/or PI3K-mTOR as well as extracellular signal–regulated kinase 1/2. These results provide new evidence for a direct effect of glucose independent of insulin on cardiac myocyte growth.

Leptin induces rat glomerular mesangial cell hypertrophy, but does not regulate hyperplasia or apoptosis

There is an increased incidence of renal glomerulosclerosis in obese individuals. One of the major structural changes observed in nephropathy is the increase in kidney size, which may occur due to hypertrophy or changes in the rate of hyperplasia or apoptosis. Here we investigated whether leptin, the product of the obese (ob) gene which is found at high plasma levels in obese and diabetic individuals, alters any of these parameters. We show that leptin increased hypertrophy of these cells. This was indicated by an approximately 33% increase in cell size and 40% increase in leucine incorporation. Furthermore, we show that the hypertrophic effect of leptin was mediated via PI 3-kinase and ERK1/2 by using the inhibitors LY294002 and PD98059, respectively. We also confirm that leptin activates both PI 3-kinase and ERK1/2 in these cells. We show that hyperplasia was not affected by leptin by measuring rat glomerular mesangial cell number and by assessing bromodeoxyuridine uptake. Leptin also did not alter caspase 3-like activity under control conditions or upon induction of apoptosis by ultraviolet light, suggesting that apoptosis was not regulated by leptin in these cells. Our results show that leptin induced glomerular mesangial cell hypertrophy via PI 3-kinase and ERK1/2, and that hyperplasia and apoptosis were not altered by leptin. The hypertrophic effect of leptin may play a role in the pathophysiology of nephropathy associated with obesity.

Mitogen-activated Protein Kinases and Mitogen-activated Protein Kinase Phosphatases Mediate the Inhibitory Effects of All-trans Retinoic Acid on the Hypertrophic Growth of Cardiomyocytes

All-trans retinoic acid (RA) has been implicated in mediation of cardiac growth inhibition in neonatal cardiomyocytes. However, the associated signaling mechanisms remain unclear. Utilizing neonatal cardiomyocytes, we demonstrated that RA suppressed the hypertrophic features induced by cyclic stretch or angiotensin II (Ang II). Cyclic stretch- or Ang II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAP kinase) was dose- and time-dependently inhibited by RA. Significant inhibition was observed by 5 microm RA, from 8 to 24 h of pretreatment. This inhibitory effect was not mediated at the level of mitogen-activated protein kinase kinases (MKKs), because RA had no effect on stretch- or Ang II-induced phosphorylation of MEK1/2, MKK4, and MKK3/6. However, the phosphatase inhibitor vanadate reversed the inhibitory effect of RA on MAP kinases and protein synthesis. RA up-regulated the expression level of MAP kinase phosphatase-1 (MKP-1) and MKP-2, and the time course was correlated with the inhibitory effect of RA on activation of MAP kinases. Overexpression of wild-type MKP-1 inhibited the phosphorylation of JNK and p38 in cardiomyocytes. These data indicated that MKPs were involved in the inhibitory effect of RA on MAP kinases. Using specific RAR and RXR antagonists, we demonstrated that both RARs and RXRs were involved in regulating stretch- or Ang II-induced activation of MAP kinases. Our findings provide the first evidence that the anti-hypertrophic effect of RA is mediated by up-regulation of MKPs and inhibition of MAP kinase signaling pathways.

Identification of multiple proteins whose synthetic rates are enhanced by high amino acid levels in rat hepatocytes.

Amino acids are key regulators of protein synthesis in liver. However, it remains to be determined whether amino acids stimulate synthesis of all or certain specific liver proteins. No techniques are currently available to simultaneously measure synthetic rates of several individual proteins. Here we report studies performed on rat hepatocyte primary cultures in which we used metabolic labeling with [(14)C]leucine, two-dimensional gel electrophoresis (2DGE), and tandem mass spectrometry to identify proteins that showed increased leucine incorporation when high amino acid levels were present in the media. Rat hepatocytes were isolated by in situ collagenase perfusion, cultured in serum-free medium containing insulin, and incubated for 2, 4, and 8 h in media of standard and high amino acid concentrations. SDS-PAGE and 2DGE were performed to separate proteins from cell lysates. Proteins that consistently showed increased synthesis on triplicate cultures, as detected by phosphorimaging of gels, were identified by tandem mass spectrometry. The combination of these approaches enabled the detection of 16 specific liver proteins whose synthetic rates were enhanced by increased amino acid concentration. These proteins are involved in specific functions such as translation initiation, protein folding and modification, oxidative phosphorylation, antioxidant defense, signal transduction, and transport, as well as cell motility and tissue integrity. No quantitative changes for any of these proteins were detected by gel staining, indicating that no detectable changes in protein concentration occurred. In contrast, measurable changes in synthetic rates occurred in 16 proteins. In conclusion, amino acids stimulate the synthesis of several liver proteins with important cellular functions.

Stimulation of DNA and RNA synthesis in cultured rabbit cardiac fibroblasts by angiotensin IV.

1. Although angiotensin II (ANG II) has been identified as a key factor in the development of cardiac hypertrophy and remodelling, the role of its degradation fragment ANG II (3-8), angiotensin IV (ANG IV), is unknown. The presence of ANG IV in the blood circulation as well as the identification of ANG IV receptors in the heart and other organs indicates that ANG IV may act as a peptide hormone. 2. ANG IV receptors were characterized by binding of 125I-ANG IV to membranes of cultured rabbit cardiac fibroblasts. Incorporation of [3H]thymidine, [3H]uridine and [3H]leucine into DNA, RNA and proteins, respectively, was determined to analyse the growth effects of ANG IV, ANG II and the combination of both peptides. 3. ANG IV displaces 125I-ANG IV bound to membranes of rabbit cardiac fibroblasts with high affinity, whereas ANG II receptor-specific ligands ([Sar1,Ile8]ANG II, losartan, CGP 42 112 A) do not. 125I-ANG IV binds to a single class of binding site with a dissociation constant (Kd) of 4.87 +/- 0.11 nmol/l. The density of ANG IV receptors (Bmax) is 371 +/- 8.3 fmol/mg of protein. 125I-ANG IV binding is not markedly affected in the presence of the non-hydrolysable GTP analogue GTP gamma S, whereas binding of 125I-ANG II is reduced. 4. In quiescent cells, a 24 h exposure of ANG IV (100 nmol/l) increased rates of thymidine and uridine incorporation by 127% and 246%, respectively. A small but statistically insignificant increase in leucine incorporation was observed under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein and energy balance following femoral neck fracture in geriatric patients

To elucidate the effect of total peripheral parenteral nutrition (TPPN) on protein kinetics following injury, we compared the whole-body leucine kinetic response using a primed-constant infusion of l-[1- 14C]leucine in 33 elderly patients (aged 82 ± 1.0 years) following hip fracture and 33 healthy elderly control subjects (aged 75 ± 0.7 years). Following a 36-hour fast, leucine release from protein breakdown was 1.2 ± 0.10 μmol · kg −1 · min −1 and leucine incorporation into protein was 0.94 ± 0.095 μmol · kg −1 · min −1 in control subjects, and in injured subjects leucine release from protein breakdown was 1.3 ± 0.14 μmol · kg −1 · min −1 and leucine incorporation into protein was 0.97 ± 0.092 μmol · kg −1 · min −1. Control and injured subjects were then administered TPPN (protein, 1.5 g amino acids · kg −1; carbohydrate, 10.0 kcal · kg −1; lipid, 15.0 kcal · kg −1) for 24 hours, and leucine kinetics were redetermined. Compared with protein kinetics in the fasting state, leucine release from protein decreased to 1.0 ± 0.14 μmol · kg −1 · min −1 and leucine incorporation into protein increased to 1.16 ± 0.097 μmol · kg −1 · min −1 in control subjects. Injured patients also responded to TPPN with a decrease in leucine release from protein breakdown (1.12 ± 0.156 μmol · kg −1 · min −1) and an increase in leucine incorporation into protein (1.29 ± 0.164 μmol · kg −1 · min −1). These results indicate that in a geriatric population, whole-body leucine kinetics following hip fracture and the anabolic response to TPPN are not significantly altered from those of uninjured subjects. The overall metabolic response of energy expenditures and efficiency of nitrogen retention does suggest a mild catabolic response that may be reversed by nutritional intervention.

Effect of myo-inositol on cell proliferation and collagen transcription and secretion in proximal tubule cells cultured in elevated glucose.

Tubulointerstitial lesions often develop in the kidneys of patients and experimental animals with diabetes mellitus. In an in vitro model of diabetic renal disease, it has been previously demonstrated in this laboratory that elevated glucose levels stimulate procollagen transcription and secretion in proximal tubule cells in culture while inducing cellular hypertrophy and reducing cellular proliferation. Previous experiments in other tissues have suggested that myo-inositol supplementation, probably by reversing a disturbance in cell myo-inositol metabolism related to increased activity of the polyol pathway, reverses the effects of glucose on cell function. We tested the effect of myo-inositol supplementation on proximal tubule cells in culture in the presence of elevated medium glucose level. Incubation in 450 mg/dL of glucose media reduced cell proliferation; 450 mg/dL of glucose plus myo-inositol (800 microM) increased proliferation, returning the value to that seen in cells incubated in 100 mg/dL of glucose. Incubation in 450 mg/dL of glucose media increased type IV and type I procollagen mRNA levels and peptide secretion rates compared with those seen in cells incubated in medium containing 100 mg/dL of glucose. This glucose-induced stimulation of procollagen mRNA levels and procollagen secretion was not observed when the elevated glucose medium was supplemented with 800 microM myo-inositol. On the other hand, myo-inositol supplementation did not prevent the glucose-induced cellular hypertrophy: there was no reduction in the increased leucine incorporation and cellular protein content. Cell incubation in 450 mg/dL of glucose media did not lead to a measurable decrease in total cellular myo-inositol.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Myoblast Membrane Protein Synthesis by 25-Hydroxy-Vitamin D 3

The effects of 25-hydroxy-vitamin D3 (25 OHD3) on myoblast protein synthesis were studied in connection with its role on muscle cell phosphate metabolism. The sterol markedly increased leucine incorporation into total cell proteins in cultured chick embryo myoblasts. This enhancement was greater than that produced by 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) and occurred prior to a significant stimulation of cell phosphate accumulation. Maximum effects of 25 OHD3 (8 h) on myoblast phosphate uptake were suppressed by cycloheximide indicating that they are mediated by de novo protein synthesis. At a similar treatment period, labelling of myoblasts with [3H]leucine (control) and [14C]leucine (+25 OHD3) followed by co-electrophoresis of total protein extracts on SDS-PAGE and isoelectrofocusing gels revealed that the sterol selectively affects the synthesis of proteins of 20 kDa and 50 kDa. These macromolecules were recovered in the microsomal fraction after differential centrifugation of homogenates. Further fractionation of myoblast microsomes on sucrose density gradients showed co-localization of the 50 kDa and 20 kDa proteins with microsomal subfractions which preferentially bind [3H-alpha]bungarotoxin, suggesting that the proteins induced by 25 OHD3 are associated to plasma membranes and may play a role in the effects of the sterol on cell phosphate uptake.

Culture of normal and inflamed rabbit colonic explants. Medium-term prostaglandin profile.

Rabbit colonic biopsy specimens cultured under well-standardized conditions showed a linear uptake of leucine, thymidine and arachidonic acid (AA) for at least 48 h. Prostaglandin E2 (PGE2) production displayed a time-dependent profile. Initial high and fluctuating levels were seen as a result of the preparation trauma. Within 5-7 h a semi-steady state was reached. Addition of the Ca++ ionophore A23187 significantly increased PGE2 production throughout the 48-h time course, but stimulation was optimal (fivefold) around 6 h, as compared with two- to three-fold at 24 and 48 h. Phorbol 12-myristate 13-acetate stimulation of biopsy specimens labelled for 24 h with 14C-AA showed significantly higher proportional release of 14C-PGE2 than after A23187 or a combination of both, indicating stimulus utilization of different AA pools, with variable turnover. Mucosa from rabbits with acetic acid-induced colitis demonstrated a significantly increased leucine incorporation during the first 24-h period and normal incorporation during the second period. PGE2 production reached a high A23187-insensitive maximum at 24 h. This study shows the importance of sample timing, the existence of different AA pools, and the feasibility of studying inflamed mucosa in medium-term tissue culture.

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis.

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.

Studies on the effect of inflammation on the acute phase response using rat liver slices.

Liver slices from control and inflamed rats were incubated in McCoy's medium and incorporation of [3H]leucine into liver and medium proteins and into albumin and alpha 1-acid glycoprotein was monitored over 48 hr. The release of the new acute phase reactant, sialyltransferase was also monitored in this system. Earlier observations in which liver slices were incubated for 6 hr showed that increased leucine incorporation into liver and medium proteins and alpha 1-acid glycoprotein, coupled with decreased incorporation into albumin, correlated with the acute phase response of these proteins. Increased incorporation of leucine into these proteins was found following 48 hr incubation in McCoy's medium showing that slices were able to express the changes characteristic of the acute phase response over this longer time period of incubation. Sialyltransferase was released into medium in a linear fashion up to 15 hr and continued to increase for 30 hr in this system; there was a substantial increase in release of enzyme activity from slices from inflamed rats when compared to controls. Monokine-conditioned medium prepared from peritoneal exudate cells isolated from rats at various times after lipopolysaccharide administration was used to induce the acute phase response by intraperitoneal injection. Slices were prepared from these rats and sialyltransferase release from slices was monitored. Monokines prepared from peritoneal exudate cells isolated from rats at about 30 hr were most effective in stimulating sialyltransferase release from liver slices.

Whole body leucine metabolism in adolescents with Crohn's disease and growth failure during nutritional supplementation

The adaptive response of whole body leucine metabolism to nutritional supplementation was determined in 6 adolescents with Crohn's disease and growth failure. Five healthy adolescents served as controls for body composition studies. In the first study period, all subjects received a constant diet comparable to usual intakes. In the second period, the patients were given overnight intragastric supplemental feedings that increased dietary protein and energy intakes approximately 40% (to 3.2 g/kg · day and 96 kcal/kg · day, respectively). During each dietary period, O2 consumption, nitrogen balance, whole body potassium (40K), and urinary creatinine measurements were obtained on all adolescents, and the patients received a primed, constant, intravenous infusion of [13C]leucine for 4 h in the fed state. Plasma leucine and expired carbon dioxide 13C-enrichments were determined by mass spectrometric techniques. In the first study, O2 consumption and nitrogen balances were similar between groups; in patients, 40K and urinary creatinine were reduced by 30% and 36%, respectively. With nutritional supplementation, nitrogen balance increased fourfold; O2 consumption and 40K increased by 32% and 10%, respectively. Similarly, whole body leucine flux increased from 166.9 ± 5.9 to 201.3 ± 11.2 μmol/kg · h (p < 0.05) due to a 66% increase in leucine incorporation into body protein (p < 0.01) and a 41% decrease in leucine oxidation (p < 0.05). Thus, these studies demonstrate that the mechanisms responsible for lean body mass accretion during nutritional supplementation in adolescents with Crohn's disease and growth failure are increased rates of amino acid incorporation into body protein (via protein synthesis] and decreased rates of amino acid oxidation.