Acquisition of peak bone mass in a Norwegian youth cohort: longitudinal findings from the Fit Futures study 2010-2022.

In a Norwegian youth cohort followed from adolescence to young adulthood, bone mineral density (BMD) levels declined at the femoral neck and total hip from 16 to 27 years but continued to increase at the total body indicating a site-specific attainment of peak bone mass. PURPOSE: To examine longitudinal trends in bone mineral density (BMD) levels in Norwegian adolescents into young adulthood. METHOD: In a prospective cohort design, we followed 980 adolescents (473 (48%) females) aged 16-19 years into adulthood (age of 26-29) on three occasions: 2010-2011 (Fit Futures 1 (FF1)), 2012-2013 (FF2), and 2021-2022 (FF3), measuring BMD (g/cm2) at the femoral neck, total hip, and total body with dual x-ray absorptiometry (DXA). We used linear mixed models to examine longitudinal BMD changes from FF1 to FF3. RESULTS: From the median age of 16 years (FF1), femoral neck BMD (mean g/cm2 (95% CI)) slightly increased in females from 1.070 (1.059-1.082) to 1.076 (1.065-1.088, p = 0.015) at the median age of 18 years (FF2) but declined to 1.041 (1.029-1.053, p < 0.001) at the median age of 27 years (FF3). Similar patterns were observed in males: 16 years, 1.104 (1.091-1.116); 27 years, 1.063 (1.050-1.077, p < 0.001); and for the total hip in both sexes (both p < 0.001). Total body BMD increased from age 16 to 27 years in both sexes (females: 16 years, 1.141 (1.133-1.148); 27 years, 1.204 (1.196-1.212), p < 0.001; males: 16 years, 1.179 (1.170-1.188); 27 years, 1.310 (1.296-1.315), p < 0.001). CONCLUSION: BMD levels increased from 16 to 18 years at the femoral and total hip sites in young Norwegian females and males, and a small decline was observed at the femoral sites when the participants were followed up to 27 years. Total body BMD continued to increase from adolescence to young adulthood.

Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/ß-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Electrostatic sheathing of lipoprotein lipase is essential for its movement across capillary endothelial cells.

GPIHBP1, an endothelial cell (EC) protein, captures lipoprotein lipase (LPL) within the interstitial spaces (where it is secreted by myocytes and adipocytes) and transports it across ECs to its site of action in the capillary lumen. GPIHBP1's 3-fingered LU domain is required for LPL binding, but the function of its acidic domain (AD) has remained unclear. We created mutant mice lacking the AD and found severe hypertriglyceridemia. As expected, the mutant GPIHBP1 retained the capacity to bind LPL. Unexpectedly, however, most of the GPIHBP1 and LPL in the mutant mice was located on the abluminal surface of ECs (explaining the hypertriglyceridemia). The GPIHBP1-bound LPL was trapped on the abluminal surface of ECs by electrostatic interactions between the large basic patch on the surface of LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the surface of ECs. GPIHBP1 trafficking across ECs in the mutant mice was normalized by disrupting LPL-HSPG electrostatic interactions with either heparin or an AD peptide. Thus, GPIHBP1's AD plays a crucial function in plasma triglyceride metabolism; it sheathes LPL's basic patch on the abluminal surface of ECs, thereby preventing LPL-HSPG interactions and freeing GPIHBP1-LPL complexes to move across ECs to the capillary lumen.

100 YEARS OF VITAMIN D: Combined hormonal contraceptives and vitamin D metabolism in adolescent girls.

Objective: Combined hormonal contraceptive (CHC) use has been associated with higher total 25-hydroxyvitamin D (25(OH)D) levels. Here, we investigate the relation between CHC use and vitamin D metabolism to elucidate its clinical interpretation. Methods: The cross-sectional Fit Futures 1 included 1038 adolescents. Here, a subgroup of 182 girls with available 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)2D), 24,25-dihydroxyvitamin D (24,25(OH)2D), vitamin D-binding protein (DBP) and measured free 25(OH)D levels, in addition to parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), was investigated. Vitamin D metabolites were compared between girls using (CHC+) and not using CHC (CHC-). Further, the predictability of CHC on 25(OH)D levels was assessed in a multiple regression model including lifestyle factors. The ratios 1,25(OH)2D/25(OH)D and 24,25(OH)2D/25(OH)D (vitamin D metabolite ratio (VMR)) in relation to 25(OH)D were presented in scatterplots. Results: CHC+ (n = 64; 35% of the girls) had higher 25(OH)D levels (mean ± s.d., 60.3 ± 22.2) nmol/L) than CHC- (n = 118; 41.8 ± 19.3 nmol/L), P -values <0.01. The differences in 25(OH)D levels between CHC+ and CHC- were attenuated but remained significant after the adjustment of lifestyle factors. CHC+ also had higher levels of 1,25(OH)2D, 24,25(OH)2D, DBP and calcium than CHC-, whereas 1,25(OH)2D/25(OH)D, PTH, FGF23 and albumin were significantly lower. Free 25(OH)D and VMR did not statistically differ, and both ratios appeared similar in relation to 25(OH)D, irrespective of CHC status. Conclusion: This confirms a clinical impact of CHC on vitamin D levels in adolescents. Our observations are likely due to an increased DBP-concentration, whereas the free 25(OH)D appears unaltered.

Expression and one-step purification of active LPL contemplated by biophysical considerations.

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (Tm is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (Tm increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies.

The influence of snuff and smoking on bone accretion in late adolescence. The Tromsø study, Fit Futures.

Areal bone mineral density (aBMD) predicts future fracture risk. This study explores associations between use of tobacco and bone accretion in Norwegian adolescents. Our results indicate that use of snuff is negatively associated with accretion of aBMD in adolescence and may be a signal of increased future fracture risk. PURPOSE: Bone mineral accrual in childhood and adolescence is a long-term primary preventive strategy of osteoporosis. Areal bone mineral density (aBMD) is a surrogate measure of bone strength and a predictor of fracture risk. The aim of this population-based 2-year follow-up cohort study was to explore associations between use of snuff and smoking and changes (∆) in aBMD in Norwegian girls and boys aged 15-17 years at baseline. METHODS: The first wave of the Tromsø study, Fit Futures was conducted from 2010 to 2011. Femoral neck (FN), total hip (TH), and total body (TB) bone mineral content (BMC) and aBMD were measured by dual-energy X-ray absorptiometry. Information on use of snuff, smoking habits, and other lifestyle related variables were collected through self-administered questionnaires. Two years later, during 2012-2013, the measurements were repeated in the second wave. The present study included 349 girls and 281 boys and compared "non-users" (n = 243 girls, 184 boys) with "users" (n = 105 girls, 96 boys) of snuff and "non-smokers" (n = 327 girls, 249 boys) with "smokers" (n = 21 girls, 31 boys) using linear regression adjusted for age, baseline height and weight, change in height and weight, pubertal maturation, physical activity, ethnicity, alcohol consumption, diagnosis known to affect bone, and medication known to affect bone. The influence of "double use" on bone accretion was also explored. RESULTS: In girls, no associations between use of snuff and ∆aBMD were found. In boys, use of snuff was associated with reduced bone accretion in all ∆aBMD models. Sensitivity analysis with exclusion of "sometimes" users of snuff strengthened associations at femoral sites in girls and attenuated all associations in boys. In girls, no associations between smoking and ∆aBMD were found. In boys, only the association with TB ∆aBMD was significant in the fully adjusted models. In girls, "double users" analyses showed similar association to smoking. In boys, nearly all models showed statistically significant associations with a difference of ~ 1-2% in ∆aBMD between "non-users" and "double users" during 2 years of follow-up. CONCLUSIONS: Our results indicate that tobacco use in late adolescence could be detrimental to bone accretion and may be a signal of increased fracture risk in adult life.

GPIHBP1 and ANGPTL4 Utilize Protein Disorder to Orchestrate Order in Plasma Triglyceride Metabolism and Regulate Compartmentalization of LPL Activity.

Intravascular processing of triglyceride-rich lipoproteins (TRLs) is crucial for delivery of dietary lipids fueling energy metabolism in heart and skeletal muscle and for storage in white adipose tissue. During the last decade, mechanisms underlying focal lipolytic processing of TRLs along the luminal surface of capillaries have been clarified by fresh insights into the functions of lipoprotein lipase (LPL); LPL's dedicated transporter protein, glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1); and its endogenous inhibitors, angiopoietin-like (ANGPTL) proteins 3, 4, and 8. Key discoveries in LPL biology include solving the crystal structure of LPL, showing LPL is catalytically active as a monomer rather than as a homodimer, and that the borderline stability of LPL's hydrolase domain is crucial for the regulation of LPL activity. Another key discovery was understanding how ANGPTL4 regulates LPL activity. The binding of ANGPTL4 to LPL sequences adjacent to the catalytic cavity triggers cooperative and sequential unfolding of LPL's hydrolase domain resulting in irreversible collapse of the catalytic cavity and loss of LPL activity. Recent studies have highlighted the importance of the ANGPTL3-ANGPTL8 complex for endocrine regulation of LPL activity in oxidative organs (e.g., heart, skeletal muscle, brown adipose tissue), but the molecular mechanisms have not been fully defined. New insights have also been gained into LPL-GPIHBP1 interactions and how GPIHBP1 moves LPL to its site of action in the capillary lumen. GPIHBP1 is an atypical member of the LU (Ly6/uPAR) domain protein superfamily, containing an intrinsically disordered and highly acidic N-terminal extension and a disulfide bond-rich three-fingered LU domain. Both the disordered acidic domain and the folded LU domain are crucial for the stability and transport of LPL, and for modulating its susceptibility to ANGPTL4-mediated unfolding. This review focuses on recent advances in the biology and biochemistry of crucial proteins for intravascular lipolysis.

The Importance of Lipoprotein Lipase Regulation in Atherosclerosis.

Lipoprotein lipase (LPL) plays a major role in the lipid homeostasis mainly by mediating the intravascular lipolysis of triglyceride rich lipoproteins. Impaired LPL activity leads to the accumulation of chylomicrons and very low-density lipoproteins (VLDL) in plasma, resulting in hypertriglyceridemia. While low-density lipoprotein cholesterol (LDL-C) is recognized as a primary risk factor for atherosclerosis, hypertriglyceridemia has been shown to be an independent risk factor for cardiovascular disease (CVD) and a residual risk factor in atherosclerosis development. In this review, we focus on the lipolysis machinery and discuss the potential role of triglycerides, remnant particles, and lipolysis mediators in the onset and progression of atherosclerotic cardiovascular disease (ASCVD). This review details a number of important factors involved in the maturation and transportation of LPL to the capillaries, where the triglycerides are hydrolyzed, generating remnant lipoproteins. Moreover, LPL and other factors involved in intravascular lipolysis are also reported to impact the clearance of remnant lipoproteins from plasma and promote lipoprotein retention in capillaries. Apolipoproteins (Apo) and angiopoietin-like proteins (ANGPTLs) play a crucial role in regulating LPL activity and recent insights into LPL regulation may elucidate new pharmacological means to address the challenge of hypertriglyceridemia in atherosclerosis development.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding.

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/ß-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including ß2, ß3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on ß3-α3 and progress to ß5 and ß4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/ß-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

Blockade of Oncogenic NOTCH1 with the SERCA Inhibitor CAD204520 in T Cell Acute Lymphoblastic Leukemia.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).

Body Weight and Body Mass Index Influence Bone Mineral Density in Late Adolescence in a Two-Year Follow-Up Study. The Tromsø Study: Fit Futures.

Determinants of bone acquisition in late adolescence and early adulthood are not well-described. This 2-year follow-up study explored the associations of body weight (BW), body mass index (BMI), and changes in weight status with adolescent bone accretion in a sample of 651 adolescents (355 girls and 296 boys) between 15 and 19 years of age from The Tromsø Study: Fit Futures. This Norwegian population-based cohort study was conducted from 2010 to 2011 and was repeated from 2012 to 2013. We measured femoral neck, total hip, and total body bone mineral content and areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry. We measured height, BW, calculated BMI (kg/m 2), and collected information on lifestyle at both surveys. Mean BMI (SD) at baseline was 22.17 (3.76) and 22.18 (3.93) in girls and boys, respectively. Through multiple linear regression, baseline BW and BMI were positively associated with ∆aBMD over 2 years of follow-up at all skeletal sites in boys ( p < 0.05), but not in girls. ∆BW and ∆BMI predicted ∆aBMD and ∆BMC in both sexes, but the strength of the associations was moderate. Individuals who lost weight during follow-up demonstrated a slowed progression of aBMD accretion compared with those gaining weight, but loss of BW or reduction of BMI during 2 years was not associated with net loss of aBMD. In conclusion, our results confirm that adequate BW for height in late adolescence is important for bone health. Associations between change in weight status and bone accretion during follow-up were moderate and unlikely to have any clinical implication on adolescents of normal weight. Underweight individuals, particularly boys, are at risk of not reaching optimal peak bone mass and could benefit from an increase in BMI. © 2019 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Evolution and Medical Significance of LU Domain-Containing Proteins.

Proteins containing Ly6/uPAR (LU) domains exhibit very diverse biological functions and have broad taxonomic distributions in eukaryotes. In general, they adopt a characteristic three-fingered folding topology with three long loops projecting from a disulfide-rich globular core. The majority of the members of this protein domain family contain only a single LU domain, which can be secreted, glycolipid anchored, or constitute the extracellular ligand binding domain of type-I membrane proteins. Nonetheless, a few proteins contain multiple LU domains, for example, the urokinase receptor uPAR, C4.4A, and Haldisin. In the current review, we will discuss evolutionary aspects of this protein domain family with special emphasis on variations in their consensus disulfide bond patterns. Furthermore, we will present selected cases where missense mutations in LU domain-containing proteins leads to dysfunctional proteins that are causally linked to genesis of human disease.

Adolescent body composition and associations with body size and growth from birth to late adolescence. The Tromsø study: Fit Futures-A Norwegian longitudinal cohort study.

BACKGROUND: Fat and fat-free masses and fat distribution are related to cardiometabolic risk. OBJECTIVES: to explore how birth weight, childhood body mass index (BMI) and BMI gain were related to adolescent body composition and central obesity. METHODS: In a population-based longitudinal study, body composition was measured by dual-energy X-ray absorptiometry in 907 Norwegian adolescents (48% girls). Associations between birth weight, BMI categories, and BMI gain were evaluated by fitting linear mixed models and conditional growth models with fat mass index (FMI, kg/m2 ), fat-free mass index (FFMI, kg/m2 ) standard deviation scores (SDS), and central obesity at 15 to 20 years, as well as change in FMI SDS and FFMI SDS between ages 15 to 17 and 18 to 20 as outcomes. RESULTS: Birth weight was associated with FFMI in adolescence. Greater BMI gain in childhood, conditioned on prior body size, was associated with higher FMI, FFMI, and central overweight/obesity with the strongest associations seen at age 6 to 16.5 years: FMI SDS: ß = 0.67, 95% CI (0.63-0.71), FFMI SDS: 0.46 (0.39, 0.52), in girls, FMI SDS: 0.80 (0.75, 0.86), FFMI SDS: 0.49 (0.43, 0.55), in boys. CONCLUSIONS: Compared with birth and early childhood, high BMI and greater BMI gain at later ages are strong predictors of higher fat mass and central overweight/obesity at 15 to 20 years of age.

How Is Adolescent Bone Mass and Density Influenced by Early Life Body Size and Growth? The Tromsø Study: Fit Futures-A Longitudinal Cohort Study From Norway.

The effect of birth weight and childhood body mass index (BMI) on adolescents' bone parameters is not established. The aim of this longitudinal, population-based study was to investigate the association of birth weight, childhood BMI, and growth, with adolescent bone mass and bone density in a sample of 633 adolescents (48% girls) from The Tromsø Study: Fit Futures. This population-based cohort study was conducted in 2010-2011 and 2012-2013 in Tromsø, Norway. Bone mineral content (BMC) and areal BMD (aBMD) were measured at total hip (TH) and total body (TB) by dual-energy X-ray absorptiometry (DXA) and converted to internal Z-scores. Birth weight and childhood anthropometric measurements were retrospectively obtained from the Medical Birth Registry of Norway and childhood health records. Associations between birth weight, BMI, and growth were evaluated by fitting linear mixed models with repeated measures of BMC and aBMD at ages 15 to 17 and 18 to 20 years as the outcome. In crude analysis, a significant positive association (p < 0.05) with TB BMC was observed per 1 SD score increase in birth weight, observed in both sexes. Higher rate of length growth, conditioned on earlier size, from birth to age 2.5 years, and higher rate of weight gain from ages 6.0 to 16.5 years, conditioned on earlier size and concurrent height growth, revealed stronger associations with bone accrual at ages 15 to 20 years compared with other ages. Compared with being normal weight, overweight/obesity at age 16.5 years was associated with higher aBMD Z-scores: ß coefficient (95% confidence interval [CI]) of 0.78 (0.53, 1.03) and 1.08 (0.85, 1.31) in girls, 0.63 (0.42, 0.85) and 0.74 (0.54, 0.95) in boys at TH and TB, respectively. Similar associations were found for BMC. Being underweight was consistently negatively associated with bone parameters in adolescence. In conclusion, birth weight influences adolescent bone mass but less than later growth and BMI in childhood and adolescence. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans.

Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this "zinc flux" could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen.

Novel Zinc-Attenuating Compounds as Potent Broad-Spectrum Antifungal Agents with In Vitro and In Vivo Efficacy.

An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here we report the identification and characterization of two novel zinc-attenuating compounds, ZAC307 and ZAC989, which exhibit broad-spectrum in vitro antifungal activity and in vivo efficacy in a fungal kidney burden candidiasis model. The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc-attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects on Candida albicans and Aspergillus fumigatus could be inactivated by the addition of exogenous zinc to fungal growth media. We determined the ZACs to be fungistatic, with a low propensity for resistance development. Gene expression analysis suggested that the ZACs interfere negatively with the expression of genes encoding the major components of the A. fumigatus zinc uptake system, thus supporting perturbance of zinc homeostasis as the likely mode of action. With demonstrated in vitro and in vivo antifungal activity, low propensity for resistance development, and a novel mode of action, the ZACs represent a promising new class of antifungal compounds, and their advancement in a drug development program is therefore warranted.

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity.

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H+-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H+-ATPase inhibitors as a new class of antifungal agents.

Bone mineral density at the hip and its relation to fat mass and lean mass in adolescents: the Tromsø Study, Fit Futures.

BACKGROUND: Positive association between body weight and bone mass is well established, and the concept of body mass index (BMI) is associated with higher areal bone mineral density (aBMD) and reduced fracture risk. BMI, that comprises both fat mass (FM) and lean mass (LM) may contribute to peak bone mass achievement in different ways. This study explored the influence of body composition in terms of total body LM and FM on hip aBMD-values in adolescence. METHODS: In 2010/2011, 93% of the region's first-year upper-secondary school students (15-17 years old) in Tromsø, Norway attended the Tromsø Study, Fit Futures. Areal BMD at femoral neck (aBMDFN) and total hip (aBMDTH) (g/cm2), total body LM and FM (g) were measured by dual energy X-ray absorptiometry (DXA). Height and weight were measured, and BMI calculated. Lifestyle variables were collected by self-administered questionnaires and interviews, including questions on time spent on leisure time physical activity. Stratified analyses of covariance and regression models included 395 girls and 363 boys. Crude results were adjusted for age, height, sexual maturation, physical activity levels, vitamin D levels, calcium intake, alcohol consumption and smoking habits. RESULTS: Unadjusted distribution indicated higher aBMD-levels at higher LM-levels in both genders (p < 0.001), but higher aBMD at higher FM-levels were found only in girls (p < 0.018). After multiple adjustments, aBMDFN-levels in girls were associated by 0.053 g/cm2 and 0.032 g/cm2 per standard deviation (SD) change in LM and FM (p < 0.001). Corresponding values in boys were 0.072 and 0.025 (p < 0.001). The high LM groups accounted for the highest aBMD-levels, while aBMD-levels at the LM/FM-combinations indicated different patterns in girls compared to boys. The adjusted odds ratio (95% CI) for low levels of aBMDFN was 6.6 (3.4,13.0) in boys, compared to 2.8 (1.6,4.9) in girls per SD lower LM. CONCLUSIONS: LM and FM should be regarded as strong predictors for bone mass and hence bone strength in adolescents. A gender specific difference indicated that high lean mass is of crucial importance prominently in boys. In adolescents with low lean mass, especially in girls, high fat mass may partially ameliorate the effect of deficient lean mass levels.

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives.

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H+-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H+-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H+-ATPase within minutes, leading to fungal death after 1-3h of compound exposure in vitro. The tested compounds are not selective for the fungal H+-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca2+-ATPase) and the sodium potassium ATPase (Na+,K+-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H+-ATPase indirectly, but Candida albicans cells exposed to potent H+-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H+-ATPase.