Rat Soleus Muscle Strips Research Articles

A novel two-chain IGF-II-derived peptide from purified β-cell granules

Objective Insulin-like growth factor II (IGF-II) is a potent mitogen that regulates prenatal growth and development in both humans and rodents. Its role in post-natal life is less clear although immunohistochemical studies have observed IGF-II-like immunoreactivity (IGF-II-LI) associated with insulin-producing pancreatic β-cells. Here we isolated secretory granules from a β-cell line, βTC6-F7, and characterized the nature of the IGF-II-LI located therein. Design Secretory granules were isolated from cultured mouse βTC6-F7 cells by ultracentrifugation. Granule protein content was separated by reversed-phase HPLC, and assayed for IGF-II (radioimmunoassay) prior to identification by gas-phase NH 2-terminal sequencing and MALDI-TOF MS. Effects of glucose incorporation into muscle glycogen were determined by incubating with isolated rat soleus muscle strips. Results βTC6-F7 cells contained 60 ± 8 pmol of IGF-II-LI per 10 6 cells compared to 340 ± 44 pmol insulin-LI per 10 6 cells. IGF-II immunoreactive fractions were found to contain an IGF-II-like molecule with a molecular mass of 6847.6 Da. The protein was found to be a two-chain insulin-like product of Igf2 that corresponds to mouse des(37–40)IGF-II, which we termed ‘vesiculin’. This molecule was also detectable in βTC6-F7 cells by intact-cell mass spectrometry. Mouse vesiculin evoked concentration-dependent stimulation of muscle glycogen synthesis ex vivo with an EC 50 value of 131 nM ± 1.35. Conclusions Vesiculin, des(37–40)IGF-II, is a novel two-chain insulin-like hormone and the major “IGF-II-like” peptide found in purified mouse βTC6-F7 secretory granules. It stimulated ex vivo muscle glycogen synthesis with an efficacy greater than or equal to the intrinsic potency of IGF-II when compared to insulin derived from the same species.

AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus muscle

Muscle contraction increases glucose uptake and fatty acid (FA) metabolism in isolated rat skeletal muscle, due at least in part to an increase in AMP-activated kinase activity (AMPK). However, the extent to which AMPK plays a role in the regulation of substrate utilization during contraction is not fully understood. We examined the acute effects of 5-aminoimidazole-4-carboxamide riboside (AICAR; 2 mm), a pharmacological activator of AMPK, on FA metabolism and glucose oxidation during high intensity tetanic contraction in isolated rat soleus muscle strips. Muscle strips were exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mm; high fatty acid, HFA, 1 mm) to examine the role that FA availability may play in both exogenous and endogenous FA metabolism with contraction and AICAR. Synergistic increases in AMPK alpha2 activity (+45%; P<0.05) were observed after 30 min of contraction with AICAR, which further increased exogenous FA oxidation (LFA: +71%, P<0.05; HFA: +46%, P<0.05) regardless of FA availability. While there were no changes in triacylglycerol (TAG) esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +65%, P<0.05). AICAR significantly blunted endogenous TAG hydrolysis (LFA: -294%, P<0.001; HFA: -117%, P<0.05), but had no effect on endogenous oxidation rates, suggesting a better matching between TAG hydrolysis and subsequent oxidative needs of the muscle. There was no effect of AICAR on the already elevated rates of glucose oxidation during contraction. These results suggest that FA metabolism is very sensitive to AMPK alpha2 stimulation during contraction.

AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle

5-Amino-4-imidazolecarboxamide riboside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK), acutely stimulates glucose uptake and fatty acid (FA) oxidation in skeletal muscle. However, it is not fully understood whether AICAR-induced changes in glucose oxidation are secondary to changes in FA oxidation (i.e. glucose fatty acid cycle), or what role AMPK may be playing in the regulation of intramuscular triacylglycerol (TAG) esterification and hydrolysis. We examined the acute (60 min) effects of AICAR (2 mm) on FA metabolism, glucose oxidation and pyruvate dehydrogenase (PDH) activation in isolated resting rat soleus muscle strips exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mm; high fatty acid, HFA, 1 mm). AICAR significantly increased AMPK alpha2 activity (+192%; P<0.05) over 60 min, and simultaneously increased both FA (LFA: +33%, P<0.05; HFA: +36%, P<0.05) and glucose (LFA: +105%, P<0.05; HFA: +170, P<0.001) oxidation regardless of FA availability. While there were no changes in TAG esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +15%, P<0.05; HFA: +49%, P<0.05). AICAR had no effect on endogenous TAG hydrolysis and oxidation in resting soleus. The stimulation of glucose oxidation with AICAR was associated with an increase in PDH activation (+126%; P<0.05) but was without effect on pyruvate, an allosteric activator of the PDH complex, suggesting that AMPK may stimulate PDH directly. In conclusion, AMPK appears to be an important regulator of both FA metabolism and glucose oxidation in resting skeletal muscle.

Differences in troglitazone action on glucose metabolism in freshly isolated vs long-term incubated rat skeletal muscle.

1. Exposure of isolated skeletal muscle to troglitazone has resulted in inconsistent findings ranging from inhibition to stimulation of fuel oxidation and the glycogenic pathway. To better understand such variation in outcome, the present study used isolated rat soleus muscle strips to examine the interdependent influences of prolonged maintenance in vitro and of troglitazone exposure. 2. If freshly isolated muscle strips were exposed to troglitazone (1 micro mol l(-1)) for 24 h, glucose oxidation was markedly reduced (-26+/-1%, P<0.0001), whereas glycogen synthesis remained unaffected (+9+/-7%, n.s.). 3. In contrast, extended exposure to troglitazone for 72 h increased both glucose oxidation (+65+/-28%, P<0.05) and glycogen synthesis (+46+/-11%, P<0.005), and a similar stimulatory effect was also observed in muscles exposed to troglitazone only during the last 24 h of their 72 h preincubation period (glucose oxidation: +61+/-15%, P<0.001; glycogen synthesis: +43+/-15%, P<0.01). 4. Troglitazone thus stimulated glucose utilization in long-term incubated muscle independent of the duration of exposure (24 or 72 h), whereas it inhibited glucose utilization in freshly isolated muscle. 5. The observed differences in troglitazone action on freshly isolated vs long-term incubated muscle suggest that findings on muscle tissue subject to prolonged maintenance in vitro cannot be extrapolated to native muscle in vivo.

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.

Failure of leptin to affect basal and insulin-stimulated glucose metabolism of rat skeletal muscle in vitro.

Studies on different isolated tissues have provided evidence that leptin may directly modulate cellular glucose handling. The present study was performed to elucidate leptin's action on basal and insulin-stimulated glucose metabolism in native muscle tissue, which under physiological circumstances is the quantitatively most important target tissue of insulin. Isolated rat soleus muscle strips were incubated for 1 h in the absence or presence of leptin (0, 1, 10, or 100 nmol/l) under basal or insulin-stimulated conditions (10 nmol/l). No effects of leptin were found on the rates of 3H-2-deoxy-glucose transport (basal: control, 314+/-14; 1 nmol/l leptin, 320+/-17; 10 nmol/l leptin, 314+/-13; 100 nmol/l leptin, 322+/-16; insulin-stimulated: control, 690+/-33; 1 nmol/l leptin, 691+/-29; 10 nmol/l leptin, 665+/-26; 100 nmol/l leptin, 664+/-27; cpm x mg(-1) x h(-1); NS vs respective control) and on net glucose incorporation into glycogen (basal: control, 1.75+/-0.18; 1 nmol/l leptin, 2.01+/-0.13; 10 nmol/l leptin, 1.92+/-0.11; 100 nmol/l leptin, 1.81+/-0.13; insulin-stimulated: control, 5.98+/-0.40; 1 nmol/l leptin, 5.93+/-0.30; 10 nmol/l leptin, 5.46+/-0.25; 100 nmol/l leptin, 5.85+/-0.30; micromol x g(-1) x h(-1); NS vs respective control). In parallel, leptin failed to affect rates of aerobic and anaerobic glycolysis as well as muscle glycogen content. Further experiments revealed that the inability of leptin to directly affect muscle glucose handling prevailed independently of muscle fiber type (soleus and epitrochlearis muscle), of ambient insulin concentrations (0-30 nmol/l), and of leptin exposure time (1 h or 6 h). Thus, our findings fail to support speculations about a physiological role of direct insulin-mimetic or insulin-desensitizing effects of leptin on skeletal muscle tissue.

Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro.

1. The direct short-term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips. 2. In muscle strips from Sprague-Dawley rats, troglitazone (3.25 micromol l(-1)) increased basal and insulin-stimulated glucose transport by 24% and 41%, respectively (P<0.01 each). 3. In the presence of 5 nmol l(-1) insulin, stimulation of glucose transport by 3.25 micromol l(-1) troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling. 4. Whereas insulin retained its stimulant effect on [3H]-2-deoxy-glucose transport in hypoxia-stimulated muscle (by 44%; c.p.m. mg(-1) h(-1): 852+/-77 vs 1229+/-75, P<0.01), 3.25 micromol l(-1) troglitazone failed to increase glucose transport under hypoxic conditions (789+/-40 vs 815+/-28, NS) suggesting that hypoxia and troglitazone address a similar, non-insulin-like mechanism. 5. No differences between troglitazone and hypoxia were identified in respective interactions with insulin. 6. Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction-like manner, but it remains to be elucidated whether this contributes to the long-term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.

Acute and Chronic Exposure to Tumor Necrosis Factor- Fails to Affect Insulin-Stimulated Glucose Metabolism of Isolated Rat Soleus Muscle

To better understand the effects of tumor necrosis factor-α (TNFα) on insulin sensitivity, direct interaction of the peptide with freshly isolated rat soleus muscle strips was investigated. Muscles were exposed to TNFα at concentrations ranging from 0.01–5 nmol/liter. Rates of insulin-stimulated (5 or 100 nmol/liter) glucose metabolism were determined after periods of TNFα preexposure of 30 min, 6 h, and 24 h. Independent of exposure time, TNFα failed to exert any significant effect on rates of 3H-2-deoxy-glucose transport (stimulation by 100 nmol/liter insulin after preincubation without vs. with 5 nmol/liter TNFα, cpm/mg·h: 30 min, 779 ± 29 vs. 725 ± 29; 6 h, 652 ± 56 vs. 617 ± 60; 24 h, 911± 47 vs. 936 ± 31) or glucose incorporation into glycogen (μmol/g·h: 30 min, 5.19 ± 0.22 vs. 5.25 ± 0.41; 6 h, 2.08 ± 0.10 vs. 2.09 ± 0.17; 24 h, 2.51 ± 0.21 vs. 2.41 ± 0.26). In parallel, TNFα neither affected insulin-stimulated rates of glucose oxidation (CO2 production) and anaerobic glycolysis (lactate release), nor muscle glycogen content. In conclusion, these findings do not support the hypothesis of muscle insulin desensitization by TNFα via autocrine or paracrine mechanisms. The obtained data favor the concept that TNFα-dependent muscle insulin resistance in vivo depends on indirect effects rather than direct interaction of the peptide with skeletal muscle.

Acute and chronic exposure to tumor necrosis factor-alpha fails to affect insulin-stimulated glucose metabolism of isolated rat soleus muscle.

To better understand the effects of tumor necrosis factor-alpha (TNF alpha) on insulin sensitivity, direct interaction of the peptide with freshly isolated rat soleus muscle strips was investigated. Muscles were exposed to TNF alpha at concentrations ranging from 0.01-5 nmol/liter. Rates of insulin-stimulated (5 or 100 nmol/liter) glucose metabolism were determined after periods of TNF alpha preexposure of 30 min, 6 h, and 24 h. Independent of exposure time, TNF alpha failed to exert any significant effect on rates of 3H-2-deoxy-glucose transport (stimulation by 100 nmol/liter insulin after preincubation without vs. with 5 nmol/liter TNF alpha, cpm/mg x h: 30 min, 779 +/- 29 vs. 725 +/- 29; 6 h, 652 +/- 56 vs. 617 +/- 60; 24 h, 911 +/- 47 vs. 936 +/- 31) or glucose incorporation into glycogen (micromol/g x h: 30 min, 5.19 +/- 0.22 vs. 5.25 +/- 0.41; 6 h, 2.08 +/- 0.10 vs. 2.09 +/- 0.17; 24 h, 2.51 +/- 0.21 vs. 2.41 +/- 0.26). In parallel, TNF alpha neither affected insulin-stimulated rates of glucose oxidation (CO2 production) and anaerobic glycolysis (lactate release), nor muscle glycogen content. In conclusion, these findings do not support the hypothesis of muscle insulin desensitization by TNF alpha via autocrine or paracrine mechanisms. The obtained data favor the concept that TNF alpha-dependent muscle insulin resistance in vivo depends on indirect effects rather than direct interaction of the peptide with skeletal muscle.

In vitro effect of adenosine agonist GR79236 on the insulin sensitivity of glucose utilisation in rat soleus and human rectus abdominus muscle

The dose-response effects of a new adenosine agonist, GR79236, were examined in isolated rat soleus muscle strips and human rectus abdominus muscle strips. Effects on the insulin sensitivity of carbohydrate metabolism were examined, in particular upon insulin stimulated glycogen synthesis and glycolytic flux. In the presence of adenosine deaminase (ADA), GR79236 increased insulin sensitivity of pyruvate release from rat soleus muscle strips by 24% from 82.5 ± 10.0 to 102.5 ± 10.0 ( P < 0.01), by 27% to 105.0 ± 12.5 ( P < 0.01) and by 24% to 102.5 ± 10.0 ( P < 0.01) nmol/25 mg per h at 0.1, 1 and 10 μM GR79236, respectively. Rates of lactate release followed a similar but non-significant trend. Addition of GR79236 in the presence of ADA had no effect on rates of glycogen synthesis. Insulin stimulated rates of pyruvate or lactate release or of glycogen synthesis were unaffected by the addition of adenosine deaminase or GR79236 in human rectus abdominus muscle strips. Adenosine agonists may act indirectly to modulate insulin sensitivity of carbohydrate metabolism.

Long term culture of rat soleus muscle in vitro: Its effects on glucose utilization and insulin sensitivity

Rat soleus muscle strips cultured for 24 h in medium 199 were well preserved in terms of electron microscopy; ATP and creatine phosphate concentrations; rates of glucose utilization, glycogen and protein synthesis, and effects of insulin thereon. Culture led to modest changes in fluid spaces and intracellular (K+); increased basal glucose utilization up to two-fold; had no effect on the maximum response to insulin; and had no effect on sensitivity to insulin except in the presence of adenosine deaminase. Thus in vitro neither denervation nor absence of insulin had any marked effects in 24 h to decrease responses to insulin.

Total and myofibrillar protein degradation in isolated soleus muscles after exercise.

The effect of exercise on the rate of total and myofibrillar protein degradation was determined by measuring the rate of release of tyrosine and 3-methylhistidine, respectively, from isolated rat soleus muscle strips after exercise. The rate of tyrosine release was 30-50% greater from the muscles of the exercised rats, whereas the rate of 3-methylhistidine release was unchanged. Thus the exercise-induced increase in the rate of protein degradation is due to increased breakdown of nonmyofibrillar proteins. The rate of protein degradation increases as a function of exercise duration and rapidly returns to the preexercise level during recovery. The exercise-induced increase in the rate of protein degradation is not inhibited by chloroquine. Together these observations suggest that the increase in the rate of protein degradation observed immediately after exercise is due to the breakdown of nonmyofibrillar proteins and occurs via the nonlysosomal pathway of protein degradation.

Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle.

Diabetes-associated peptide has recently been isolated and characterized from the amyloid of the islets of Langerhans in type 2 (non-insulin-dependent) diabetics, and immunoreactivity with antibodies to the peptide has been demonstrated in islet B cells of both normal and type 2 diabetic subjects. In view of the evidence presented in this paper that this 37-amino acid peptide may be a hormone present in normal individuals, we now propose the name "amylin" to replace "diabetes-associated peptide." Because increased amylin, deposited as amyloid within the islets of Langerhans, is characteristic of type 2 diabetes, the study below was performed to examine the possible effects of amylin on peripheral glucose metabolism. Whole amylin was synthesized by using solid-phase techniques, with formation of the disulfide linkage by oxidation in dilute aqueous solution and recovery of the peptide by lyophilization. The effects of amylin on glucose metabolism were studied in two preparations in vitro, isolated rat soleus muscle strips and isolated rat adipocytes. In skeletal muscle exposed to 120 nM amylin for 1 hr, there was a marked decrease in both basal and submaximally insulin-stimulated rates of glycogen synthesis, which resulted in significant reduction in the rates of insulin-stimulated glucose uptake. In muscles treated with amylin there was no response at the concentration of insulin required to stimulate glucose uptake half-maximally in untreated (control) muscles. In marked contrast, amylin had no effect on either basal or insulin-stimulated rates of glucose incorporation into either CO2 or triacylglycerol in isolated adipocytes. Therefore, amylin may be a factor in the etiology of the insulin resistance in type 2 diabetes mellitus, as both deposition of the peptide in islet amyloid and decreased rates of glucose uptake and glycogen synthesis in skeletal muscle are characteristic of this condition.

Insulin sensitivity of glucose metabolism in obese rat soleus muscle strips

Insulin sensitivity of glucose metabolism in obese rat soleus muscle strips