Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated β-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

The genetic defect in producing the adipose hormone leptin results among others in a drastic increase of bone mass. The current understanding is that under normal circumstances, osteoblast activity is indirectly suppressed by a hypothalamic relay induced by leptin-signalling in the brain. To investigate whether leptin might also regulate osteoblast activity in a direct manner, expression of leptin receptors in rat osteoblasts was determined and their functionality was analyzed upon recombinant leptin treatment. Reverse transcription-PCR con¢rmed the expression of four among the six currently described receptor isoforms, which were also able to transduce cell signalling as shown by STAT3 phosphorylation after activation. ß

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a b s t r a c tThe Drosophila sNPF gene regulates growth through the ERK-insulin pathway. sNPF encodes a precursor protein that is processed and produces biologically active sNPF peptides. However, the functions of these peptides are not known. In Drosophila neuronal cells in culture and in flies in vivo, sNPF1 and sNPF2 activated the ERK-insulin pathway and regulated body growth. In addition, the sNPF precursor and the processed sNPF peptide were co-localized in the neurons of the central nervous system. These results indicate that sNPF1 and sNPF2 peptides processed from the sNPF precursor are sufficient for regulating body growth through the ERK-insulin pathway in Drosophila.

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.