Pancreatic β cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive β cells into more pliable states with reduced cellular insulin levels, increased β cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, β cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner.

The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis.

The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk of age-related disease in mammals. We compared Ins2 mice to Ins2 littermate controls, on a genetically stable Ins1 null background. Proteomic and transcriptomic analyses of livers from 25-week-old mice suggested potential for healthier aging and altered insulin sensitivity in Ins2 mice. Halving Ins2 lowered circulating insulin by 25%-34% in aged female mice, without altering Igf1 or circulating Igf1. Remarkably, decreased insulin led to lower fasting glucose and improved insulin sensitivity in aged mice. Moreover, lowered insulin caused significant lifespan extension, observed across two diverse diets. Our study indicates that elevated insulin contributes to age-dependent insulin resistance and that limiting basal insulin levels can extend lifespan.

Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM.

A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.Wolfe-Simon et al. isolated strain GFAJ-1 from the arsenic-rich sediments of California's Mono Lake by its ability to grow through multiple subculturings in artificial Mono Lake medium AML60 that lacked added phosphate but had high concentrations of arsenate (+As/ −P condition) (1). Because GFAJ-1 grew in −P medium only when arsenate was provided, and because significant amounts of arsenate were detected in subcellular fractions, growth was attributed to the use of arsenate in place of phosphate. However, the basal level of phosphate contaminating the −P medium was reported to be 3-4 M (1), which previous studies of low-phosphate microbial communities suggest is sufficient to support moderate growth (2). GFAJ-1 grew well on medium supplemented with ample phosphate but no arsenate (1500 M PO 4 , +P/-As condition), indicating that GFAJ-1 is not obligately arsenate-dependent. Wolfe-Simon et al.(1) further reported that arsenic was incorporated into the DNA backbone of GFAJ-1 in place of phosphorus, with an estimated 4% replacement of P by As based on the As:P ratio measured in agarose gel slices containing DNA samples. This † Corresponding author: redfield@zoology.ubc.ca. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript finding was surprising because arsenate is predicted to reduce rapidly to arsenite in physiological conditions (3, 4), and because arsenate esters in aqueous solution are known to be rapidly hydrolyzed (5). We have now tested this report by culturing GFAJ-1 cells supplied by the authors (1) and by analyzing highly purified DNA from phosphate-limited cells grown with and without arsenate.Wolfe-Simon et al. reported that GFAJ-1 cells grew very slowly in AML60 medium (doubling time ~12 hours), and that, when phosphate was not added to the medium, cells failed to grow unless arsenate (40 mM) was provided (1). However, although we obtained strain GFAJ-1 from these authors, in our hands GFAJ-1 was unable to grow at all in AML60 medium containing the specified trace elements and vitamins, even with 1500 M sodium phosphate added as specified in (1). We confirmed the strain's identity using RT-PCR and sequencing of 16S rRNA, using primers specified by Wolfe-Simon et al.(1); this gave a sequence identical to that reported for strain GFAJ-1. We then found that addition of small amounts of yeast extract, tryptone or individual amino acids to basal AML60 medium allowed growth, with do...