To ascertain if IGF-1 is a regulator of local muscle growth, total RNA was extracted from rabbit muscle induced to undergo rapid hypertrophy using active stretch and from control muscles. This was analysed by Northern hybridization with a 280 base pair probe containing sequences derived from exons 3 and 4 of the insulin-like growth factor 1 gene. Two types of insulin-like growth factor 1 mRNA were shown to be strong expressed in the stretched muscles. In situ hybridization using the same probe (280 base pair) showed that IGF-1 is strongly expressed in muscle that is induced to grow rapidly and is expressed in the muscle fibres themselves. Using RT-PCR a single insulin-like growth factor 1 isoform cDNA (IGF-1Ea) could be cloned from the normal resting muscles. However, an additional isoform of insulin-like growth factor 1 (insulin-like growth factor 1Eb) was found to be expressed in stretched muscle undergoing hypertrophy. The E domain sequence of the additional isoform differs from the liver insulin-like growth factor 1Ea by the presence a 52 base pair insert. This changes the reading frame of the derived carboxyl-terminal resulting in a different precursor insulin-like growth factor 1 isoform. This insulin-like growth factor 1 mRNA probably encodes the precursor insulin-like growth factor 1 isoform that is responsible for local muscle growth regulation in response to mechanical stimulation. To confirm that alternative splicing of the insulin-like growth factor 1 gene occurs in muscle in response to physical activity, oligonucleotide primers were made which specifically amplify the cDNAs of two isoforms (insulin-like growth factors 1Ea and Eb) in the human as well as the rabbit. Following altered physical activity for 2 h to 6 days, appreciable levels of insulin-like growth factor 1Eb (in human the Ec) isoform were detected in skeletal muscle by using RT-PCR. In contrast very little if any of this splice variant could be detected in control muscle not subjected to stretch or extra physical activity.

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na+ overload was found to accompany the cell death. Depletion of Na+ in culture medium or pretreatment of cells with the Na+/K+-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na+/K+-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na+/K+-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na+/K+-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.

The relationship between IGF-I and changes in muscle fibre phenotype in response to 6 d of stretch or disuse of the lower limb muscles of the rabbit was studied by combining in situ hybridisation and immunohistochemistry procedures. Passive stretch by plaster cast immobilisation of the muscle in its lengthened position not only induced an increase in IGF-I mRNA expression within the individual muscle fibres but also an increase in the percentage of fibres expressing neonatal and slow myosin. This change in phenotype was also found to be accompanied by a rapid and marked increase of muscle mass, total RNA content as well as IGF-I gene expression. In contrast, IGF-I appears not to be involved in muscle atrophy induced by immobilisation in the shortened position and the inactivity which results from this procedure. The level of increase in expression of IGF-I mRNA varied from fibre to fibre. By using adjacent serial sections, the fibres which expressed IGF-I mRNA at the highest levels were identified as expressing neonatal and the slow type 1 myosin. These data suggest that the expression of IGF-I within individual muscle fibres is correlated not only with hypertrophy but also with the muscle phenotypic adaptation that results from stretch and overload.

Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM.

No abstract