Diaphragm Glycogen Research Articles

Minimum Effective Dose to Achieve Biochemical Correction with Adeno-Associated Virus Vector-Mediated Gene Therapy in Mice with Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy. Muscle weakness progresses despite currently available therapy, which has prompted the development of gene therapy with adeno-associated virus (AAV) type 2 vectors cross-packaged as AAV8 (2/8). Preclinical studies of gene therapy demonstrated that the minimum effective dose (MED) for biochemical correction with AAV2/8-LSPhGAA was ∼2 × 1011 vector genomes (vg)/kg body weight. The current study examined the transduction of AAV2/8-LSPeGFP vector in adult GAA-KO mice with Pompe disease, and correlated that degree of transduction with the biochemical correction achieved by the same dose of AAV2/8-LSPhGAA. The MED was found to be ∼2 × 1011 vg/kg, with all hepatocytes variably transducing at this dose. At this dose, liver GAA significantly increased, while liver glycogen significantly decreased. The 2 × 1011 vg/kg dose was sufficient to significantly decrease diaphragm glycogen. However, the heart, diaphragm, and quadriceps all required a fourfold higher dose to achieve correction of GAA deficiency in association with significant clearance of stored glycogen, which correlated with increased serum GAA activity. These data indicate that AAV2/8-LSPeGFP transduced all hepatocytes when the 2 × 1011 vg/kg dose was administered, which correlated with partial biochemical correction from the equivalent dose of AAV2/8-LSPhGAA. Altogether, these data support the conclusion that substantial transduction of the liver is required to achieve biochemical correction from AAV2/8-LSPhGAA.

NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.20Results from ATB200-02: first-in-human, open-label, phase 1/2 study of ATB200 co-administered with AT2221 for Pompe disease

Late-onset Pompe disease (LOPD) is caused by impaired lysosomal glycogen clearance due to acid alpha-glucosidase (GAA) deficiency. Accumulation of glycogen in skeletal muscle and diaphragm leads to loss of muscle and respiratory function. Recombinant human GAA enzyme replacement therapy (rhGAA ERT; alglucosidase alfa) is standard treatment for LOPD. ATB200-02 (NCT02675465) is a first-in-human, open-label, Phase 1/2 trial to evaluate ATB200, a next-generation rhGAA ERT, co-administered with AT2221, an oral pharmacological chaperone, in adults with LOPD. Twenty patients (pts) were enrolled: ERT-switch ambulatory (Cohort 1, n=11), ERT-switch nonambulatory (Cohort 2, n=4), and ERT-naive ambulatory (Cohort 3, n=5). Mean±SD 6-minute walk test increased for ambulatory ERT-switch (Month [Mo] 6 [n=10], +23.9±52.2; Mo 9 [n=10], +24.5±40.8; Mo 12 [n=8], +57.4±34.4 m) and ERT-naive pts (Mo 6 [n=5], +41.8±29.4; Mo 9 [n=5], +63.5±23.1; Mo 12 [n=2], +86.8±11.1 m). Other motor function tests were generally consistent with these results. Increases were observed in upper extremity strength in nonambulatory ERT-switch pts at Mo 6 and 9. Forced vital capacity (% predicted) increased in ERT-naive pts (Mo 6 [n=5], +4.2; Mo 9 [n=5], +6.2; Mo 12 [n=2], +6.0) and was generally stable in ambulatory ERT-switch pts (Mo 6 [n=9], -1.3; Mo 9 [n=9], -1.7; Mo 12 [n=7], -3.1). All cohorts demonstrated improvements in the Fatigue Severity Scale. ATB200/AT2221 was associated with reductions in creatine kinase and urine hexose tetrasaccharide. The most common treatment-emergent adverse events were upper and lower abdominal pain (n=8), diarrhea (n=8), and nasopharyngitis (n=6). Three events of infusion-associated reactions occurred in 550+ infusions. Data from this interim analysis show a clinical benefit of ATB200/AT2221 in ERT-naive pts and in pts who have been on ERT for ∼5 years. ATB200/AT2221 has the potential to be a significant treatment alternative for patients with Pompe disease.

Acid alpha‐glucosidase gene replacement therapy to the diaphragm remodels ventilatory function in severe Pompe disease (1091.8)

Pompe disease is a neuromuscular disorder due to mutations in the gene that encodes the acid alpha‐glucosidase (GAA) enzyme, which results in lysosomal glycogen accumulation in striated muscle and motor neurons. The severe infantile form leads to cardiac hypertrophy, respiratory failure and early mortality. An approved enzyme replacement therapy reduces the cardiomyopathy and increases survival, yet many patients eventually require mechanical ventilation (MV). Our objective is to investigate an alternative treatment, by correcting the defective gene in an open‐label, Phase I/II clinical study of AAV‐GAA therapy to the diaphragm. We hypothesized that retrograde transport of AAV‐GAA would promote diaphragm glycogen clearance to restore inspiratory motor function. To date, five children (ages 2‐15) with full‐time MV dependence have received intramuscular rAAV1‐CMV‐GAA delivery into the diaphragm and completed follow‐up ventilatory testing. Maximal pressure inspiratory generation did not improve one year after dosing, but maximum unassisted tidal volume increased significantly (median: 22% gain, range: 11‐77%). Moreover, all subjects generated improved spontaneous breathing without MV assistance (median: 586%, range: 192‐2901% improvement over baseline levels). We conclude these findings may be related to vector transduction properties, fiber atrophy from chronic MV, and properties of the postoperative muscle conditioning prescription.Grant Funding Source: K12 HD055929; CRB‐HLBI1‐S‐10‐00223

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype

AbstractPompe disease (acid α-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector–mediated expression of human acid α-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid α-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid α-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

Locomotor and peripheral effects of sibutramine modulated by 5-HT2 receptors

Sibutramine has been described as an anti-obesity drug with the ability to inhibit serotonin (5-HT), noradrenaline, and dopamine re-uptake, but without affinity to histamine and muscarinic receptors. On the other hand, cyproheptadine antagonizes serotonin 5-HT(2A), 5-HT(2B), and 5-HT(2C), histamine H1, and muscarinic (M) receptors. There are many reports concerning the influence of sibutramine on central serotoninergic pathways. In this study, we suggest that peripheral pathways may also be involved in the serotoninergic effects of sibutramine. In vivo experiments were undertaken to investigate the serotoninergic effects of sibutramine on body mass, the glycogen concentration in the diaphragm of rats, and locomotor behaviour. Rats were submitted to oral treatment with sibutramine, cyproheptadine, or sibutramine applied in combination with cyproheptadine, for a period of 2 months to investigate the 5-HT2 effects of sibutramine on these parameters. As the results demonstrated, the lower increase in body mass and the increased glycogen levels in the diaphragm muscle of rats treated with sibutramine seem to be modulated by 5-HT2 receptors, since these effects were completely antagonized by cyproheptadine in the group treated with the 2 drugs co-applied. Furthermore, the behavioural results also suggest that mechanisms modulated by 5-HT2 receptors are involved in the increase of locomotion in the rats treated with sibutramine, since the effect did not occur in the rats treated with sibutramine co-applied with the 5-HT2 receptor antagonist, cyproheptadine. The results suggest that sibutramine modifies energy-related parameters such as body mass, diaphragm glycogen, and locomotor behaviour in rats via 5-HT2 serotoninergic pathways.

1124. Correction of Glycogen Storage Disease Type II by an AAV Vector Encoding Highly-Secreted, Chimeric Acid α-glucosidase

Top of pageAbstract GSD-II (Pompe disease; MIM 232300) is an autosomal recessive disorder caused by the deficiency in the activity of the lysosomal enzyme acid |[alpha]|-glucosidase (GAA; acid maltase; EC 3.2.1.20). The disease is characterized by the massive accumulation of glycogen in lysosomes and in the cytoplasm of striated muscle, with an accompanying disruption of cellular functions. The efficacy of therapy with lysosomal enzymes, such as GAA, could be markedly improved if secretion of these proteins from producer cells were increased. Secreted proteins are directed extracellularly by the presence of an N-terminal signal peptide. We have investigated the hypothesis that a chimeric lysosomal enzyme containing an alternative signal peptide will achieve much higher secretion of the recombinant lysosomal enzyme from transduced cells. The coding sequence of human GAA was modified by substituting alternative signal peptides from highly secreted proteins for the native GAA signal peptide (denoted chimeric GAA). The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold, compared to the secretion of unmodified GAA. The efficacy of this strategy for gene therapy in Pompe disease was analyzed in GAA-knockout (GAA-KO) mice. An adeno-associated virus (AAV) vector containing a liver-specific promoter to drive the expression of GAA was constructed (AAV-LSPSPhGAApA) and pseudotyped as AAV8. The long-term efficacy of SP-hGAA was evaluated by the quantitation of GAA activity and glycogen content following AAV-LSPSPhGAApA administration. A dose-response analysis revealed sustained, high GAA activity in plasma following administration of at least 3 |[times]| 1010 particles; however, an equivalent number of particles for a vector encoding unmodified GAA did not elevate GAA activity in plasma. Receptor-mediated uptake of secreted GAA can be demonstrated only if plasma levels exceed a therapeutic threshold, and the GAA activity of the heart and diaphragm were significantly elevated 18 weeks following administration of as few as 1 |[times]| 1010 particles of AAV-LSPSPhGAApA. GAA activity in the quadriceps muscle was increased with as few as 3 |[times]| 1010 particles of AAV-LSPSPhGAApA. The lower threshold for significant reduction of heart, diaphragm, and quadriceps glycogen content with AAV-LSPSPhGAApA was 3 |[times]| 1010 particles. Neither fewer particles for that vector, nor an equivalent number of particles of the analogous vector encoding unmodified GAA affected glycogen content in the quadriceps. Thus, an AAV vector encoding chimeric GAA increased the secretion of hGAA from the liver, and reduced the number of vector particles required to achieve a therapeutic effect in GSD-II mice.

Metabolism of d-Glucose and Its Pentaacetate Ester in Muscles and Pancreatic Islets of GLUT4 Null Mice

The metabolism of d-glucose and its pentaacetate ester was investigated in GLUT4 null mice and control C57Bl6/CBA mice. The incorporation of d-[U-14C]glucose (1.7 mM) into glycogen of diaphragm, soleus, and extensor digitorium longus muscles averaged, in the GLUT4 null mice, only 34 ± 7% of the mean corresponding control values. The utilization of d-[5-3H]glucose and conversion of d-[U-14C]glucose to 14CO2 and radioactive acidic metabolites or amino acids were little affected, however, in the muscles from GLUT4 null mice. Likewise, under steady-state conditions, the intracellular pool of 6-deoxy-6-iodo-d-glucose (10 μM) was not significantly different in muscles from GLUT4 null and control mice. The incorporation of d-[U-14C]glucose pentaacetate (1.7 mM) into glycogen and utilization of d-[5-3H]glucose pentaacetate were also not significantly different in muscles from GLUT4 null and control animals. They were about 10–30 times lower than the corresponding values found with the unesterified hexose. In pancreatic islets, however, the metabolism of d-glucose pentaacetate was not lower than that of unesterified d-glucose. Moreover, the utilization of d-[5-3H]glucose and catabolism of d-[U-14C]glucose were significantly higher in the islets from GLUT4 null mice than in those from control animals. These findings indicate that the defect of d-glucose metabolism in GLUT4 null mice occurs in muscles but not in pancreatic islets, affects preferentially glycogen synthesis rather than glycolysis, and can be bypassed by using the pentaacetate ester of the hexose. The present data also reveal a striking difference between muscles and islets when comparing the metabolism of d-glucose to that of its ester.

Chloroquine Extends the Lifetime of the Activated Insulin Receptor Complex in Endosomes

Insulin signal transduction, initiated by binding of insulin to its receptor at the plasma membrane, activates the intrinsic receptor tyrosine kinase and leads to internalization of the activated ligand-receptor complex into endosomes. This study addresses the role played by the activated insulin receptor within hepatic endosomes and provides evidence for its central role in insulin-stimulated events in vivo. Rats were treated with chloroquine, an acidotrophic agent that has been shown previously to inhibit endosomal insulin degradation, and then with insulin. Livers were removed and fractionated by density gradient centrifugation to obtain endosomal and plasma membrane preparations. Chloroquine treatment increased the amount of receptor-bound insulin in endosomes at 2 min after insulin injection by 93% as determined by exclusion from G-50 columns and by 90% as determined by polyethylene glycol precipitation (p < 0.02). Chloroquine treatment also increased the insulin receptor content of endosomes after insulin injection (integrated over 0-45 min) by 31% when compared with controls (p < 0.05). Similarly, chloroquine increased both insulin receptor phosphotyrosine content and its exogenous tyrosine kinase activity after insulin injection (64%; p < 0.01 and 96% and p < 0. 001, respectively). In vivo chloroquine treatment was without any observable effect on insulin binding to plasma membrane insulin receptors, nor did it augment insulin-stimulated receptor autophosphorylation or kinase activity in the plasma membrane. Concomitant with its effects on endosomal insulin receptors, chloroquine treatment augmented insulin-stimulated incorporation of glucose into glycogen in diaphragm (p < 0.001). These observations are consistent with the hypothesis that chloroquine-dependent inhibition of endosomal insulin receptor dissociation and subsequent degradation prolongs the half-life of the active endosomal receptor and potentiates insulin signaling from this compartment.

Effects of cortisone and testosterone on diaphragmatic function and biochemistry in the rabbit.

The effects of cortisone and testosterone on in vivo diaphragmatic function and biochemistry were assessed in rabbits to determine whether androgens can prevent the effects of corticosteroids on diaphragmatic function and biochemistry and what mechanisms of muscle function may be altered. Cortisone altered respiratory muscle endurance and diaphragmatic glycogen and lactate levels but had little effect on diaphragmatic strength. Testosterone had no significant impact on diaphragmatic strength, endurance, or biochemistry. However, concomitant administration of testosterone with cortisone blunted the effect of cortisone on respiratory muscle endurance, even though it failed to block the effects of cortisone on diaphragmatic glycogen and lactate levels. Alterations in respiratory muscle endurance were not due to low- or high-frequency contractile fatigue of the diaphragm or diaphragmatic glycogen depletion or lactate accumulation in any of the treatment conditions. On the other hand, alterations in central drive played a dominant role, as evidenced by alterations in inspiratory duty cycle and a falloff in the intensity of central drive during loading. Although the exact mechanisms underlying the alterations in central drive remain unknown, concomitant administration of androgens with corticosteroids blunted the impact of corticosteroids on respiratory muscle function.

In vitro [U-14C]glucose utilization by tissues of weanling rats with lateral hypothalamic area lesions one month after lesion production.

The role of the lateral hypothalamic area (LHA) in intermediary metabolism was investigated by quantitation of [U-14C]glucose oxidation to 14CO2 and 14C incorporation into the glycogen and lipid fraction of the liver, epididymal fat pad, and diaphragm. Weanling male Sprague-Dawley rats received bilateral electrolytic lesions in the LHA (LHAL rats). Sham operated rats were either fed ad libitum (CON-ADLIB) or pair-gained to the LHAL rats (CON-PG). The experiment was terminated 1 month after lesion production. LHAL rats were significantly (SIG) lighter and shorter and ate less than CON-ADLIB; LHAL rats were also SIG shorter than CON-PG, pointing to a food intake-independent lesion effect. Both LHAL and CON-PG rats had SIG less percent carcass fat than CON-ADLIB, but there was no SIG difference between LHAL and CON-PG rats. Also, LHAL rats had a SIG higher percentage of carcass protein than both CON-ADLIB and CON-PG. Furthermore, LHAL rats incorporated SIG less glucose into liver glycogen than CON-ADLIB but SIG more into CON-PG, whereas CON-PG rats incorporated SIG less into liver glycogen than CON-ADLIB, again suggesting a food intake-independent effect. There was no difference among the groups in glucose oxidation and incorporation into lipids and glycogen in both diaphragm and epididymal fat pads and liver total lipid. However, livers of CON-PG metabolized SIG more [U-14C]glucose to CO2 than did livers of CON-ADLIB, suggesting a food intake-dependent effect. There was no difference between LHAL and CON-PG rats in this parameter.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo insulin mimetic effects of pV compounds: role for tissue targeting in determining potency.

Peroxovanadium (pV) compounds activate the insulin receptor kinase in hepatocytes and inhibit the dephosphorylation of insulin receptors in hepatic endosomes with highly correlated potencies (Posner, B. I., R. Faure, J. W. Burgess, A. P. Bevan, D. Lachance, G. Zhang-Sun, J. B. Ng, D. A. Hall, B. S. Lum, and A. Shaver J. Biol. Chem. 269: 4596-4604, 1994). After intravenous administration, K2[VO(O2)2(picolinato)].2H2O [bpV(pic)], VO(O2) (picolinato) (H2O)2 [mpV(pic)], K[VO(O2)2(picolinato)].3H2O [bpV(phen)], and K[VO(O2)2(4,7-dimethyl-1,10-phenanthroline)].1/2H2O [bpV(Me2phen)] produced 50% of their maximal hypoglycemic effect at doses of 0.04, 0.04, 0.32, and 0.65 mumol/100 g body wt, respectively. In contrast, their potencies as inhibitors of dephosphorylation were bpV(pic) = bpV(phen) > mpV(pic) = bpV(Me2phen). bpV(pic) stimulated [14C]glucose incorporation into rat diaphragm glycogen in vivo, and its effect was dose dependent, synergistic with insulin, and evident in other skeletal muscles. In contrast, bpV(phen) displayed no effect on glycogen synthesis in skeletal muscle. mpV(pic) stimulated and bpV(Me2phen) had no effect on glycogen synthesis in the diaphragm. bpV(pic) augmented rat diaphragm insulin receptor kinase 2.2-fold with a time-integrated response 70% that of insulin. In contrast, the effect of bpV(phen) was delayed and much reduced. Thus, the in vivo potencies of pV compounds reflect differing capacities to act on skeletal muscle. The ancillary ligand within the pV complex may target one tissue in preference to another.

Circadian rhythm of glycogen in the hamster diaphragm.

The purpose of this study was to determine the diurnal fluctuation of glycogen stores for the whole hemidiaphragm and within a specific myofibrillar ATPase (M-ATPase) fibre type and diaphragmatic region. Fifty-six golden Syrian hamsters were randomly divided into six groups according to the time of sampling biopsies from the diaphragm: 03:00, 07:00, 11:00, 15:00, 19:00, and 23:00. The right hemidiaphragm was quick frozen and biochemically assayed for glycogen levels. Biopsies from the left hemidiaphragm of the same animal were cut from the anterior costal and crural regions, and stained with periodic acid--Schiff (PAS) and for M-ATPase. Optical density measures of PAS-stained fibres were determined to quantitate glycogen in different M-ATPase fibre types and diaphragmatic regions. Biochemical assay of the entire hemidiaphragm showed slightly greater glycogen content of biopsies taken at 11:00 and 15:00 than at 03:00, 19:00, and 23:00 (range of differences: 6.4-10.0%). However, glycogen levels within a specific M-ATPase fibre type and diaphragm region were not different in biopsies sampled at different times. Because the hamster has a small diurnal variation of glycogen in the diaphragm, which is similar to the small diurnal variation of glycogen in human skeletal muscle, this species may be a good animal model for metabolic studies of the diaphragm that could be affected by diurnal glycogen variability.

Effects of endotoxic shock on diaphragmatic function in mechanically ventilated rats.

Diaphragmatic function was investigated in mechanically ventilated rats during endotoxic shock (group E, n = 18) and after saline solution injection (group C, n = 8). Endotoxic shock was produced by a 1-min injection of Escherichia coli endotoxin (10 mg/kg iv) suspended in saline. Diaphragmatic strength was assessed before (T0) and 15 (T15) and 60 (T60) min after injection by measuring transdiaphragmatic pressure (Pdi) generated during bilateral phrenic stimulation at 0.5, 10, 20, 30, 50, and 100 Hz. Diaphragmatic neuromuscular transmission was assessed by measuring the integrated electrical activity of the diaphragm. Diaphragmatic endurance was assessed 75 min after injection from the rate of Pdi decline after a 30-s continuous 10-Hz phrenic stimulation. In 16 additional animals, diaphragmatic glycogen content was determined 60 min after inoculation with endotoxin (n = 8) or 0.9% sodium chloride solution (n = 8). Diaphragmatic resting membrane potential (Em) was measured in 16 additional animals 60 min after endotoxin (n = 8) or saline injection (n = 8). Mean blood pressure decreased from 74 +/- 3 to 53 +/- 6 mmHg at T60 in group E, whereas it was maintained in group C. At T60 Pdi was decreased in group E for frequencies of 50 and 100 Hz and was associated with a decreased diaphragmatic electromyographic activity of 25.3 +/- 2.5 and 26.5 +/- 5.2% for 50- and 100-Hz stimulations, respectively, in comparison with T0 values.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Acute Fasting on Diaphragm Strength and Endurance

The effects of short periods of fasting on diaphragm contractile function remain unclear. The purpose of the present study was (1) to examine the relationship between duration of acute fasting and diaphragm contractile performance, and (2) to assess the effects of fasting on diaphragm glycogen stores and the relationship between changes in diaphragm function and alterations in muscle glycogen stores. Studies were performed on four groups of Syrian hamsters (nine animals in each group). One group served as a control and was allowed to feed normally, whereas the other three groups were fasted for either 1, 2, or 3 days. Diaphragm strips from animals were studied in vitro by measuring tension during electrically induced contractions. Two strips from each animal were studied; one strip was examined with a bath glucose equal to the prevailing blood glucose, and the second was preincubated in a high glucose solution (170 mg/dl) for 20 min. Fasting resulted in reductions in body weight, blood glucose concentrations, diaphragm strength, and diaphragm endurance in strips tested at the prevailing blood glucose levels. These effects were pronounced in animals fasted for 3 days, with little or no change in diaphragm contractility observed in animals fasted for shorter periods. Diaphragm weight, thickness, and glycogen content were unchanged in the fasted animals, as was the weight of the soleus muscle. Preincubation of strips from 3-day-fasted animals in a high glucose medium resulted in a significant increase in diaphragm strip strength and endurance.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of Diaphragm Glycogen, Lactate, and Function to Respiratory Failure

Diaphragmatic function and biochemical changes were studied during respiratory failure induced by incremental inspiratory threshold loading in anesthetized rabbits (1) who were unbound and spontaneously breathing, (2) during lower thoracic and abdominal binding, and (3) while bound and undergoing transvenous phrenic nerve pacing of the diaphragm. There was no evidence of contractile fatigue or alterations in glycogen or lactate concentrations in the diaphragm of unbound spontaneously breathing animals. With thoracoabdominal binding, there was a fall in maximal transdiaphragmatic pressure (Pdimax) and the ratio of diaphragmatic force divided by neural input (Pdi/Edi ratio); there was no change in diaphragm glycogen, but there was a significant rise in lactate. In the bound and phrenic-paced animals Pdimax and Pdi/Edi ratio fell, and there was significant glycogen depletion and lactate accumulation. There was a significant correlation between diaphragm function and the levels of diaphragm glycogen and lactate at the point of respiratory failure. We conclude that (1) respiratory failure induced by incremental inspiratory threshold loading was not associated with either contractile fatigue of the diaphragm or diaphragmatic biochemical changes in unbound spontaneously breathing animals, (2) when mechanisms that preserve diaphragmatic function are circumvented by phrenic pacing and/or thoracoabdominal binding, diaphragm fatigue and biochemical changes occur, and (3) there is a significant relationship between in vivo evidence of contractile fatigue of the diaphragm and diaphragmatic glycogen depletion and lactate accumulation.

Effect of Corticosteroids on Diaphragm Function and Biochemistry in the Rabbit

The effects of corticosteroid administration for 2 weeks on diaphragm histology, biochemistry, and function were assessed in the rabbit at rest and during inspiratory loading to respiratory failure, and compared to the extensor digitorum longus and untreated control animals. There were marked pathologic changes in the diaphragm after corticosteroid administration and significant alterations in diaphragm glycogen level (118.7 +/- 2.8 versus 56.2 +/- 1.0 mmol/kg wet weight, p less than 0.01) and lactate level (5.3 +/- 0.3 versus 3.5 +/- 0.4 mmol/kg wet weight, p less than 0.01) with only minimal changes in the skeletal muscle. Although respiratory muscle endurance in the control group increased from 142 +/- 17 min at baseline to 155 +/- 20 at Week 1 and 183 +/- 13 at Week 2 (p less than 0.05), it fell progressively from 135 +/- 19 min at baseline to 99 +/- 5 at Week 1 and 88 +/- 9 at Week 2 (p less than 0.01) in the corticosteroid-treated animals. On the other hand, there was no significant change in resting Pdimax after corticosteroid administration. In addition, there was no significant change in Pdimax, Pdi/Edi ratio, or glycogen level at the point of respiratory failure, but diaphragm lactate levels were increased (8.5 +/- 0.8 versus 5.3 +/- 0.3, p less than 0.05). When variable degrees of diaphragm dysfunction were induced by phrenic nerve pacing and/or thoracoabdominal binding, the degree of biochemical change associated with diaphragm dysfunction was significantly greater in corticosteroid-treated animals. We conclude that corticosteroid administration in the rabbit alters diaphragm histopathology and biochemistry as well as respiratory muscle endurance, but not strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum Half-Life and Biological Activity of Mutants of Human Insulin-Like Growth Factor I which Do Not Bind to Serum Binding Proteins

We have characterized the biological properties of two mutants of human insulin-like growth factor I (IGF-I) which, as we have shown previously, have normal affinity for the type I IGF receptor, but drastically reduced affinity for the acid-stable components of human serum binding proteins. [Phe-1,Val1,Asn2,Gln3,His4,Ser8,His9,Glu12 ,Tyr15,Leu16]IGF I (B-chain mutant) and [Gln3,Ala4,Tyr15,Leu16]IGF I have 1000 and 500 times lower affinity than IGF-I for the native 150K binding protein in adult rat serum. Like IGF-I, these two peptides migrate as monomers during size exclusion chromatography on TSK 125. [125I]IGF-I, [125I]B-chain mutant, and [125I] [Gln3,Ala4,Tyr15,Leu16]IGF-I have in vivo serum half-lives of 100, 27.5, and 26.9 min, respectively, after iv injection. These data suggest that serum binding protein-bound peptide is cleared from the serum more slowly than free peptide. The tissue distributions of [125I]IGF-I and [125I]B-chain mutant are similar 10 min after dosing, with more than 80% of the tissue-sequestered intact radioactive peptides in the kidney. Despite decreased serum half-lives, the B-chain mutant and [Gln3,Ala4,Tyr15,Leu16]IGF-I are, respectively, 4 times and twice as active as IGF-I in stimulating the incorporation of [14C]glucose into glycogen in rat diaphragm in vivo. This effect of IGF-I is thought to be mediated by the type 1 IGF receptor in muscle, since the same doses of peptide that stimulated glycogen synthesis more than 30-fold did not stimulate the incorporation of [14C]glucose into total lipid in adipose tissue, an effect known to be mediated by the insulin receptor. These data support the hypothesis that serum- or tissue-derived binding proteins impair the ability of IGF-I to exert its effects through the type 1 IGF receptor in vivo.

Exercise-induced glycogen utilization by the respiratory muscles.

Some controversy exists in the literature as to whether or not diaphragmatic glycogen is utilized during exercise. In this study male Sprague-Dawley rats were used to determine whether prolonged treadmill exercise would result in a significant reduction of glycogen concentration in the respiratory muscles. Untrained rats were run to exhaustion at a speed of 24 m/min, up a 10% grade. Run time averaged 48:30 min. After exercise a significant reduction in glycogen was observed in the diaphragm (43% of control), intercostals (43%), heart (39%), and plantaris (76%). In the diaphragm a significant reduction was shown in both types I and II fibers using the periodic acid-Schiff (PAS) stain for glycogen. These findings show that muscles with vastly different aerobic capacities utilize endogenous glycogen during moderately intense submaximal endurance exercise and that the costal diaphragm muscle is not an exception as has recently been suggested.

Effect of Copper on Insulin Release by the Intact Rat Pancreas and the Perfused Rat Pancreas

In previous studies, we have shown that rats fed a copper-poor diet have an impaired glucose tolerance and insulin response to an oral glucose load. Furthermore, CuCl2 X 2H2O added to the incubation medium stimulated glucose incorporation into the diaphragm glycogen and epididimal fat of rats. The purpose of the present study was to examine the effect of copper on insulin release in vivo and in the perfused pancreas. Copper chloride stimulated the insulin release by the perfused pancreas of rats fed laboratory chow (copper content, 6.7 ppm). Stimulation with 0.5 mg/dl CuCl2 X 2H2O for 20 min resulted in release of 14.7 +/- 3.1 compared with 18.9 +/- 4.3 mu insulin following stimulation with 16.7 mM glucose. The CuCl2 X 2H2O effect depended on the presence of calcium in the medium--as with other insulinotropic agents--but not on the presence of magnesium. Animals fed a copper-poor (1.2 ppm) sucrose diet have a selective impairment to i.v. glucose-induced insulin stimulation, but not to i.v. arginine or aminophylline stimulation when compared with controls fed a sucrose copper-supplemented diet or a laboratory chow diet. This selective beta cell defect is not necessarily genetically inherited and may be acquired. The role of copper on insulin release is discussed.

Acute metabolic effects and half-lives of intravenously administered insulinlike growth factors I and II in normal and hypophysectomized rats.

Insulinlike growth factors (IGF) act qualitatively like insulin on insulin target tissues in vitro. In the circulation in vivo they are bound to specific carrier proteins. In this form or when continuously infused into hypophysectomized (hypox) rats they do not exert acute insulinlike effects on glucose homeostasis. This study definitively shows that intravenous bolus injections of pure IGF I or II act acutely on glucose homeostasis: they lower the blood sugar, enhance the disappearance of U-[14C]glucose from serum and increase its incorporation into diaphragm glycogen in normal and hypox rats in the presence of antiinsulin serum. The same effects were obtained with recombinant human IGF I injected intravenously either with or without antiinsulin serum into normal rats. Free fatty acid levels decreased transiently only in normal animals. Lipid synthesis from glucose in adipose tissue was not stimulated in hypox and barely stimulated in normal rats. The half-life of injected IGF I or II in normal rats (approximately 4 h) is strikingly different from that in hypophysectomized rats (20-30 min) and appears to depend on the growth hormone-induced 150,000-200,000-mol wt IGF carrier protein that is lacking in hypophysectomized rats. 15 min after the bolus serum IGF I and II concentrations were similar to steady state levels during long-term infusion in hypox rats. Free IGF was barely detectable, however, in the infused animals, whereas 40-100% was found free 15 min after the bolus. These observations for the first time confirm the hypothesis that only free IGF, but not the IGF carrier protein complex, is bioavailable to insulin target tissues.