Impaired Macrophage and Satellite Cell Infiltration Occurs in a Muscle-Specific Fashion Following Injury in Diabetic Skeletal Muscle

Background

Systemic elevations in PAI-1 suppress the fibrinolytic pathway leading to poor collagen remodelling and delayed regeneration of tibialis anterior (TA) muscles in type-1 diabetic Akita mice. However, how impaired collagen remodelling was specifically attenuating regeneration in Akita mice remained unknown. Furthermore, given intrinsic differences between muscle groups, it was unclear if the reparative responses between muscle groups were different.

Principal Findings

Here we reveal that diabetic Akita muscles display differential regenerative responses with the TA and gastrocnemius muscles exhibiting reduced regenerating myofiber area compared to wild-type mice, while soleus muscles displayed no difference between animal groups following injury. Collagen levels in TA and gastrocnemius, but not soleus, were significantly increased post-injury versus controls. At 5 days post-injury, when degenerating/necrotic regions were present in both animal groups, Akita TA and gastrocnemius muscles displayed reduced macrophage and satellite cell infiltration and poor myofiber formation. By 10 days post-injury, necrotic regions were absent in wild-type TA but persisted in Akita TA. In contrast, Akita soleus exhibited no impairment in any of these measures compared to wild-type soleus. In an effort to define how impaired collagen turnover was attenuating regeneration in Akita TA, a PAI-1 inhibitor (PAI-039) was orally administered to Akita mice following cardiotoxin injury. PAI-039 administration promoted macrophage and satellite cell infiltration into necrotic areas of the TA and gastrocnemius. Importantly, soleus muscles exhibit the highest inducible expression of MMP-9 following injury, providing a mechanism for normative collagen degradation and injury recovery in this muscle despite systemically elevated PAI-1.

Conclusions

Our findings suggest the mechanism underlying how impaired collagen remodelling in type-1 diabetes results in delayed regeneration is an impairment in macrophage infiltration and satellite cell recruitment to degenerating areas; a phenomena that occurs differentially between muscle groups.     

Adipogenic and lipolytic effects of chronic glucocorticoid exposure

Glucocorticoids have been proposed to be both adipogenic and lipolytic in action within adipose tissue, although it is unknown whether these actions can occur simultaneously. Here we investigate both the in vitro and in vivo effects of corticosterone (Cort) on adipose tissue metabolism. Cort increased 3T3-L1 preadipocyte differentiation in a concentration-dependent manner, but did not increase lipogenesis in adipocytes. Cort increased lipolysis within adipocytes in a concentration-dependent manner (maximum effect at 1-10 μM). Surprisingly, removal of Cort further increased lipolytic rates (～320% above control, P < 0.05), indicating a residual effect on basal lipolysis. mRNA and protein expression of adipose triglyceride lipase and phosphorylated status of hormone sensitive lipase (Ser563/Ser660) were increased with 48 h of Cort treatment. To test these responses in vivo, Sprague-Dawley rats were subcutaneously implanted with wax pellets with/without Cort (300 mg). After 10 days, adipose depots were removed and cultured ex vivo. Both free fatty acids and glycerol concentrations were elevated in fed and fasting conditions in Cort-treated rats. Despite increased lipolysis, Cort rats had more visceral adiposity than sham rats (10.2 vs. 6.9 g/kg body wt, P < 0.05). Visceral adipocytes from Cort rats were smaller and more numerous than those in sham rats, suggesting that adipogenesis occurred through preadipocyte differentiation rather than adipocyte hypertrophy. Visceral, but not subcutaneous, adipocyte cultures from Cort-treated rats displayed a 1.5-fold increase in basal lipolytic rates compared with sham rats (P < 0.05). Taken together, our findings demonstrate that chronic glucocorticoid exposure stimulates both lipolysis and adipogenesis in visceral adipose tissue but favors adipogenesis primarily through preadipocyte differentiation.     

Inward Flux of Lactate- through Monocarboxylate Transporters Contributes to Regulatory Volume Increase in Mouse Muscle Fibres

Mouse and rat skeletal muscles are capable of a regulatory volume increase (RVI) after they shrink (volume loss resultant from exposure to solutions of increased osmolarity) and that this RVI occurs mainly by a Na-K-Cl-Cotransporter (NKCC) - dependent mechanism. With high-intensity exercise, increased extracellular osmolarity is accompanied by large increases in extracellular [lactate^-]. We hypothesized that large increases in [lactate^-] and osmolarity augment the NKCC-dependent RVI response observed with a NaCl (or sucrose) - induced increase in osmolarity alone; a response that is dependent on lactate^- influx through monocarboxylate transporters (MCTs). Single mouse muscle fibres were isolated and visualized under light microscopy under varying osmolar conditions. When solution osmolarity was increased by adding NaLac by 30 or 60 mM, fibres lost significantly less volume and regained volume sooner compared to when NaCl was used. Phloretin (MCT1 inhibitor) accentuated the volume loss compared to both NaLac controls, supporting a role for MCT1 in the RVI response in the presence of elevated [lactate^-]. Inhibition of MCT4 (with pCMBS) resulted in a volume loss, intermediate to that seen with phloretin and NaLac controls. Bumetanide (NKCC inhibitor), in combination with pCMBS, reduced the magnitude of volume loss, but volume recovery was complete. While combined phloretin-bumetanide also reduced the magnitude of the volume loss, it also largely abolished the cell volume recovery. In conclusion, RVI in skeletal muscle exposed to raised tonicity and [lactate^-] is facilitated by inward flux of solute by NKCC- and MCT1-dependent mechanisms. This work demonstrates evidence of a RVI response in skeletal muscle that is facilitated by inward flux of solute by MCT-dependent mechanisms. These findings further expand our understanding of the capacities for skeletal muscle to volume regulate, particularly in instances of raised tonicity and lactate^- concentrations, as occurs with high intensity exercise.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Microsequence analysis of peptides and proteins : II. Separation of amino acid phenylthiohydantoin derivatives by high-performance liquid chromatography on octadecylsilane supports

A comparison of the separation of the common phenylthiohydantoin derivatives of amino acids on DuPont octadecylsilane with that obtained with Ultrasphere octadecylsilane supports is given together with the effect of acetate, phosphate, and trifluoroacetate buffers in the elution solvents. An important change in performance for two different batches of DuPont Zorbax octadecylsilane was noted. The use of combined trifluoroacetate/acetate buffer with Ultrasphere octadecylsilane gives optimal separations and peak sharpness. Practical examples of the performance of this system in low-nanomole NH₂-terminal sequence analysis are discussed with emphasis on identification of unusual amino acid derivatives and interfering background peaks.     

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH₂-terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.     

Biosynthesis of monogalactosyl diglyceride by chloroplasts from spinacia oleracea and from some gramineae

Synthetic diglycerides which differed in unsaturation of fatty acids gave the same incorporation of [¹⁴C]galactose from UDP-[¹⁴C]galactose when added to acetone powders of spinach chloroplasts up to about 0·6 mg diglyceride/20 mg acetone powder. Diolein and the endogenous diglyceride isolated from the acetone extract of chloroplasts stimulated galactolipid biosynthesis to a similar extent. With all diglycerides used, monogalactosyl diglyceride was the main product with little accompanying synthesis of digalactosyl diglyceride. The radioactivity in the monogalactosyl diglyceride synthesized from UDP-[¹⁴C]galactose by whole chloroplasts was distributed widely among the monogalactosyl diglycerides with different fatty acid composition. It is concluded that the enzyme which catalyses the transfer of galactose from UDP-galactose to diglyceride is not specific for polyunsaturated diglycerides and that the polyunsaturated monogalactosyl diglycerides arise either by desaturation of the fatty acyl residues after monogalactolipid synthesis or by transacylation. Acetone powders of chloroplasts prepared from several Gramineae did not exhibit transferase activity although whole chloroplasts were active.     

Homer modulates NFAT-dependent signaling during muscle differentiation

While changes in intracellular calcium are well known to influence muscle contraction through excitation contraction coupling, little is understood of the calcium signaling events regulating gene expression through the calcineurin/NFAT pathway in muscle. Here, we demonstrate that Ca(+2) released via the inositol trisphosphate receptor (IP3R) increases nuclear entry of NFAT in undifferentiated skeletal myoblasts, but the IP3R Ca(+2) pool in differentiated myotubes promotes nuclear exit of NFAT despite a comparable quantitative change in [Ca(+2)]i. In contrast, Ca(+2) released via ryanodine receptors (RYR) increases NFAT nuclear entry in myotubes. The scaffolding protein Homer, known to interact with both IP3R and RYR, is expressed as part of the myogenic differentiation program and enhances NFAT-dependent signaling by increasing RYR Ca(+2) release. These results demonstrate that differentiated skeletal myotubes employ discrete pools of intracellular calcium to restrain (IP3R pool) or activate (RYR pool) NFAT-dependent signaling, in a manner distinct from undifferentiated myoblasts. The selective expression of Homer proteins contributes to these differentiation-dependent features of calcium signaling.