The cellular alterations associated with skeletal muscle differentiation share a high degree of similarity with key phenotypic changes usually ascribed to apoptosis. For example, actin fiber disassembly͞reorga-nization is a conserved feature of both apoptosis and differentiating myoblasts and the conserved muscle contractile protein, myosin light chain kinase, is required for the apoptotic feature of membrane blebbing. As such, these observations suggest that the induction of differentiation and apoptosis in the myogenic lineage may use overlapping cellular mechanisms. Here, we report that skeletal muscle differentiation depends on the activity of the key apoptotic protease, caspase 3. Peptide inhibition of caspase 3 activity or homologous deletion of caspase 3 leads to dramatic reduction in both myotube͞ myofiber formation and expression of muscle-specific proteins. Subsequently, we have identified Mammalian Sterile Twenty-like kinase as a crucial caspase 3 effector in this cellular process. Mammalian Sterile Twenty-like kinase is cleavage-activated by caspase 3, and restoration of this truncated kinase in caspase 3 null myoblasts restores the differentiation phenotype. Taken together, these results confirm a unique and unanticipated role for a caspase 3-mediated signal cascade in the promotion of myogenesis.

SUMMARY Brown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3′UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.

Caspase proteases have become the focal point for the development and application of anti-apoptotic therapies in a variety of central nervous system diseases. However, this approach is based on the premise that caspase function is limited to invoking cell death signals. Here, we show that caspase-3 activity is elevated in nonapoptotic differentiating neuronal cell populations. Moreover, peptide inhibition of protease activity effectively inhibits the differentiation process in a cultured neurosphere model. These results implicate caspase-3 activation as a conserved feature of neuronal differentiation and suggest that targeted inhibition of this protease in neural cell populations may have unintended consequences.

Ga radioisotopes, including the generator-produced positron-emitting isotope (68)Ga (t1/2 = 68 min), are of increasing interest for the development of new radiopharmaceuticals. Bifunctional chelates (BFCs) that can be efficiently radiolabeled with Ga to yield complexes with good in vivo stability are needed. To this end, we undertook a systematic comparison of four BFCs containing different chelating moieties: two novel BFCs, p-NO2-Bn-Oxo (1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) and p-NO2-Bn-PCTA (3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and two more commonly used BFCs, p-NO2-Bn-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and p-NO2-Bn-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Each BFC was compared with respect to radiolabeling conditions, radiochemical yield, stability, and in vivo clearance properties. p-NO2-Bn-PCTA, p-NO2-Bn-Oxo, and p-NO2-Bn-NOTA were all more efficiently radiolabeled with Ga compared to p-NO2-Bn-DOTA. p-NO2-Bn-DOTA required longer reaction time, higher concentrations of BFC, or heating to obtain equivalent radiochemical yields. Better stability was observed for p-NO2-Bn-NOTA and p-NO2-Bn-PCTA compared to p-NO2-Bn-DOTA and p-NO2-Bn-Oxo, especially with respect to transmetalation to transferrin. Ga-radiolabled p-NO2-Bn-Oxo was found to be kinetically labile and therefore unstable in vivo. Ga-radiolabeled p-NO2-Bn-NOTA and p-NO2-Bn-PCTA were relatively inert, while Ga-radiolabeled p-NO2-Bn-DOTA had intermediate stability, losing >20% of Ga in less than one hour when incubated with apo-transferrin. Similar stability differences were seen when incubating at pH 2. In vivo PET imaging and biodistribution studies in mice showed that (68)Ga-radiolabeled p-NO2-Bn-PCTA, p-NO2-Bn-NOTA, and p-NO2-Bn-DOTA all cleared through the kidneys. While there was no statistical difference in the biodistribution results of (68)Ga-radiolabeled p-NO2-Bn-PCTA and p-NO2-Bn-DOTA, (68)Ga-radiolabeled p-NO2-Bn-NOTA cleared more rapidly from blood and muscle tissue but retained at up to 5 times higher activity in the kidneys.