Thiamine deficiency induces anorexia by inhibiting hypothalamic AMPK.

Obesity and eating disorders are prevailing health concerns worldwide. It is important to understand the regulation of food intake and energy metabolism. Thiamine (vitamin B1) is an essential nutrient. Thiamine deficiency (TD) can cause a number of disorders in humans, such as Beriberi and Wernicke-Korsakoff syndrome. We demonstrated here that TD caused anorexia in C57BL/6 mice. After feeding a TD diet for 16days, the mice displayed a significant decrease in food intake and an increase in resting energy expenditure (REE), which resulted in a severe weight loss. At the 22nd day, the food intake was reduced by 69% and 74% for male and female mice, respectively in TD group. The REE increased by ninefolds in TD group. The loss of body weight (17-24%) was similar between male and female animals and mainly resulted from the reduction of fat mass (49% decrease). Re-supplementation of thiamine (benfotiamine) restored animal's appetite, leading to a total recovery of body weight. The hypothalamic adenosine monophosphate-activated protein kinase (AMPK) is a critical regulator of food intake. TD inhibited the phosphorylation of AMPK in the arcuate nucleus (ARN) and paraventricular nucleus (PVN) of the hypothalamus without affecting its expression. TD-induced inhibition of AMPK phosphorylation was reversed once thiamine was re-supplemented. In contrast, TD increased AMPK phosphorylation in the skeletal muscle and upregulated the uncoupling protein (UCP)-1 in brown adipose tissues which was consistent with increased basal energy expenditure. Re-administration of thiamine stabilized AMPK phosphorylation in the skeletal muscle as well as energy expenditure. Taken together, TD may induce anorexia by inhibiting hypothalamic AMPK activity. With a simultaneous increase in energy expenditure, TD caused an overall body weight loss. The results suggest that the status of thiamine levels in the body may affect food intake and body weight.

Contrasting mammalian PTH promoters: identification of transcription factors controlling PTH gene expression.

Parathyroid hormone (PTH) is a key component in the maintenance of calcium and phosphate homeostasis. The steady-state expression of the PTH gene can be modeled as a balance between transcriptional activators and repressors. During renal failure, the gradual loss of kidney function is often accompanied by increased circulating concentrations of PTH and decreased synthesis of 1,25-di-hydroxyvitamin D3 (1,25(OH)2D3). The latter finding results in impaired calcium absorption and the removal of a known repressor of PTH gene transcription. Current regimens for treating secondary hyperparathyroidism associated with renal insufficiency are focused on boosting activities that repress PTH gene transcription or secretion of the hormone, and involve the use of vitamin D and its analogues or calcimimetic agents. However, in recent years, concerns have arisen over the use of the steroid hormone and alternative treatments are being sought. Here, we present new information regarding transcription factors controlling PTH gene expression, which include the specificity proteins (Sp) and the nuclear factor Y (NF-Y) complex. A highly conserved DNA response element for the Sp proteins has been identified in mammalian promoters, while an NF-Y binding site is uniquely positioned in the human promoter. Both of these factors are expressed in the parathyroid gland and their DNA elements appear to be functioning as activators of PTH gene expression. Further elucidation of such pathways may offer novel approaches for treating hyperparathyroidism associated with renal failure via suppression of transcriptional activators. That work is currently in progress.

Cell-free synthesis and affinity isolation of proteins on a nanomole scale.

The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action. The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus. They can be used in translation, transcription-translation or replication-translation reactions. Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein. The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day. The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.