The role of Plasmodium V-ATPase in vacuolar physiology and antimalarial drug uptake.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Human primary cells can tell body time: Dedicated to Steven A. Brown.

The field of chronobiology has advanced significantly since ancient observations of natural rhythms. The intricate molecular architecture of circadian clocks, their hierarchical organization within the mammalian body, and their pivotal roles in organ physiology highlight the complexity and significance of these internal timekeeping mechanisms. In humans, circadian phenotypes exhibit considerable variability among individuals and throughout the individual's lifespan. A fundamental challenge in mechanistic studies of human chronobiology arises from the difficulty of conducting serial sampling from most organs. The concept of studying circadian clocks in vitro relies on the groundbreaking discovery by Ueli Schibler and colleagues that nearly every cell in the body harbours autonomous molecular oscillators. The advent of circadian bioluminescent reporters has provided a new perspective for this approach, enabling high-resolution continuous measurements of cell-autonomous clocks in cultured cells, following in vitro synchronization pulse. The work by Steven A. Brown has provided compelling evidence that clock characteristics assessed in primary mouse and human skin fibroblasts cultured in vitro represent a reliable estimation of internal clock properties in vivo. The in vitro approach for studying molecular human clocks in cultured explants and primary cells, pioneered by Steve Brown, represents an invaluable tool for assessing inter-individual differences in circadian characteristics alongside comprehensive genetic, biochemical and functional analyses. In a broader context, this reliable and minimally invasive approach offers a unique perspective for unravelling the functional inputs and outputs of oscillators operative in nearly any human tissue in physiological contexts and across various pathologies.

Advances in understanding bat infection dynamics across biological scales.

Over the past two decades, research on bat-associated microbes such as viruses, bacteria and fungi has dramatically increased. Here, we synthesize themes from a conference symposium focused on advances in the research of bats and their microbes, including physiological, immunological, ecological and epidemiological research that has improved our understanding of bat infection dynamics at multiple biological scales. We first present metrics for measuring individual bat responses to infection and challenges associated with using these metrics. We next discuss infection dynamics within bat populations of the same species, before introducing complexities that arise in multi-species communities of bats, humans and/or livestock. Finally, we outline critical gaps and opportunities for future interdisciplinary work on topics involving bats and their microbes.

Adipose Tissue in Breast Cancer Microphysiological Models to Capture Human Diversity in Preclinical Models.

Female breast cancer accounts for 15.2% of all new cancer cases in the United States, with a continuing increase in incidence despite efforts to discover new targeted therapies. With an approximate failure rate of 85% for therapies in the early phases of clinical trials, there is a need for more translatable, new preclinical in vitro models that include cellular heterogeneity, extracellular matrix, and human-derived biomaterials. Specifically, adipose tissue and its resident cell populations have been identified as necessary attributes for current preclinical models. Adipose-derived stromal/stem cells (ASCs) and mature adipocytes are a normal part of the breast tissue composition and not only contribute to normal breast physiology but also play a significant role in breast cancer pathophysiology. Given the recognized pro-tumorigenic role of adipocytes in tumor progression, there remains a need to enhance the complexity of current models and account for the contribution of the components that exist within the adipose stromal environment to breast tumorigenesis. This review article captures the current landscape of preclinical breast cancer models with a focus on breast cancer microphysiological system (MPS) models and their counterpart patient-derived xenograft (PDX) models to capture patient diversity as they relate to adipose tissue.

KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum-infected erythrocytes.

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence.

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

A particle-based computational model to analyse remodelling of the red blood cell cytoskeleton during malaria infections.

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Glucolipotoxicity promotes the capacity of the glycerolipid/NEFA cycle supporting the secretory response of pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to high glucose and fatty acids has been proposed to induce glucolipotoxicity. However, contradictory results suggest adaptations of the beta cells, which might be instrumental for partial preservation of the secretory response. In this context, we delineated the expression pattern of genes related to lipid pathways along with fat storage/mobilisation during glucose-stimulated insulin secretion. METHODS: Insulin-secreting cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mmol/l) without or with BSA-complexed 0.4 mmol/l palmitate and oleate. Then, transcriptomic analyses of lipid pathways were performed in human islets by RNA-Seq and in INS-1E cells and rat islets by quantitative RT-PCR. Storage of fat was assessed in INS-1E cells by electron microscopy and Bodipy staining, which was also used for measuring lipid mobilisation rate. The secretory response was monitored during acute 15 mmol/l glucose stimulation using online luminescence assay for INS-1E cells and by radioimmunoassay for rat islets. RESULTS: In human islets, chronic exposure to palmitate and oleate modified expression of a panel of genes involved in lipid handling. Culture at 25 mmol/l glucose upregulated genes encoding for enzymes of the glycerolipid/NEFA cycle and downregulated receptors implicated in fatty acid signalling. Similar results were obtained in INS-1E cells, indicating enhanced capacity of the glycerolipid/NEFA cycle under glucotoxic conditions. Exposure to unsaturated C18:1 fatty acid favoured intracellular lipid accumulation in a glucose-dependent way, an effect also observed with saturated C16:0 fatty acid when combined with the panlipase inhibitor Orlistat. After the glucolipotoxic culture, intracellular fat mobilisation was required for acute glucose-stimulated secretion, particularly in oleate-treated cells under glucotoxic culture conditions. The lipid mobilisation rate was governed chiefly by the levels of stored fat as a direct consequence of the culture conditions rather than energetic demands, except in palmitate-loaded cells. CONCLUSIONS/INTERPRETATION: Glucolipotoxic conditions promote the capacity of the glycerolipid/NEFA cycle thereby preserving part of the secretory response. The cycle of fat storage/mobilisation emerges as a mechanism helping the beta cell to cope with glucotoxic conditions.

Functionalized supported membranes for quantifying adhesion of P. falciparum-infected erythrocytes.

The pathology of Plasmodium falciparum malaria is largely defined by the cytoadhesion of infected erythrocytes to the microvascular endothelial lining. The complexity of the endothelial surface and the large range of interactions available for the infected erythrocyte via parasite-encoded adhesins make analysis of critical contributions during cytoadherence challenging to define. Here, we have explored supported membranes functionalized with two important adhesion receptors, ICAM1 or CD36, as a quantitative biomimetic surface to help understand the processes involved in cytoadherence. Parasitized erythrocytes bound to the receptor-functionalized membranes with high efficiency and selectivity under both static and flow conditions, with infected wild-type erythrocytes displaying a higher binding capacity than do parasitized heterozygous sickle cells. We further show that the binding efficiency decreased with increasing intermolecular receptor distance and that the cell-surface contacts were highly dynamic and increased with rising wall shear stress as the cell underwent a shape transition. Computer simulations using a deformable cell model explained the wall-shear-stress-induced dynamic changes in cell shape and contact area via the specific physical properties of erythrocytes, the density of adhesins presenting knobs, and the lateral movement of receptors in the supported membrane.

Urban rat exposure to anticoagulant rodenticides and zoonotic infection risk.

Anticoagulant rodenticides (ARs) deployed to control rodent pest populations can increase the risk of pathogen infection for some wildlife. However, it is unknown whether ARs also increase infection risk for target rodents, which are common hosts for zoonotic (animal-to-human transmitted) pathogens. In this study, we tested whether rats exposed to ARs were more likely to be infected with zoonotic pathogens, specifically Leptospira spp. or Escherichia coli, after controlling for known predictors of infection (i.e. sex, age, body condition). We collected biological samples from 99 rats trapped in Chicago alleys and tested these for Leptospira infection, E. coli shedding and AR exposure. We found that rats that had been exposed to ARs and survived until the time of trapping, as well as older rats, were significantly more likely to be infected with Leptospira spp. than other rats. We found no significant association between E. coli shedding and any predictors. Our results show that human actions to manage rats can affect rat disease ecology and public health risks in unintended ways, and more broadly, contribute to a growing awareness of bidirectional relationships between humans and natural systems in cities.

Thermal tolerance and environmental persistence of a protozoan parasite in monarch butterflies.

Many parasites have external transmission stages that persist in the environment prior to infecting a new host. Understanding how long these stages can persist, and how abiotic conditions such as temperature affect parasite persistence, is important for predicting infection dynamics and parasite responses to future environmental change. In this study, we explored environmental persistence and thermal tolerance of a debilitating protozoan parasite that infects monarch butterflies. Parasite transmission occurs when dormant spores, shed by adult butterflies onto host plants and other surfaces, are later consumed by caterpillars. We exposed parasite spores to a gradient of ecologically-relevant temperatures for 2, 35, or 93 weeks. We tested spore viability by feeding controlled spore doses to susceptible monarch larvae, and examined relationships between temperature, time, and resulting infection metrics. We also examined whether distinct parasite genotypes derived from replicate migratory and resident monarch populations differed in their thermal tolerance. Finally, we examined evidence for a trade-off between short-term within-host replication and long-term persistence ability. Parasite viability decreased in response to warmer temperatures over moderate-to-long time scales. Individual parasite genotypes showed high heterogeneity in viability, but differences did not cluster by migratory vs. resident monarch populations. We found no support for a negative relationship between environmental persistence and within-host replication, as might be expected if parasites invest in short-term reproduction at the cost of longer-term survival. Findings here indicate that dormant spores can survive for many months under cooler conditions, and that heat dramatically shortens the window of transmission for this widespread and virulent butterfly parasite.

Identification of Bioactive Compounds of Asparagus officinalis L.: Permutation Test Allows Differentiation among "Triguero" and Hybrid Green Varieties.

In this study, we determined the phytochemical profile of the Spanish "triguero" asparagus landrace "verde-morado" (Asparagus officinalis L.), a wild traditional landrace, and the improved "triguero" HT-801, together with two commercial green asparagus varieties. For comparison, we used reverse-phase high-performance liquid chromatography coupled with diode array electrospray time-of-flight mass spectrometry (RP-HPLC-DAD-ESI-TOF/MS) followed by a permutation test applied using a resampling methodology valid under a relaxed set of assumptions, such as i.i.d. errors (not necessarily normal) that are exchangeable under the null hypothesis. As a result, we postulate that "triguero" varieties (the improved HT-801 followed by its parent "verde-morado") have a significantly different phytochemical profile from that of the other two commercial hybrid green varieties. In particular, we found compounds specific to the "triguero" varieties, such as feruloylhexosylhexose isomers, or isorhamnetin-3-O-glucoside, which was found only in the "triguero" variety HT-801. Although studies relating the phytochemical content of "triguero" asparagus varieties to its health-promoting effects are required, this characteristic phytochemical profile can be used for differentiating and revalorizating these asparagus cultivars.

Phosphomimetic substitution at Ser-33 of the chloroquine resistance transporter PfCRT reconstitutes drug responses in Plasmodium falciparum.

The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance-conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ∼50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT.

In vivo stabilization of OPA1 in hepatocytes potentiates mitochondrial respiration and gluconeogenesis in a prohibitin-dependent way.

Patients with fatty liver diseases present altered mitochondrial morphology and impaired metabolic function. Mitochondrial dynamics and related cell function require the uncleaved form of the dynamin-like GTPase OPA1. Stabilization of OPA1 might then confer a protective mechanism against stress-induced tissue damages. To study the putative role of hepatic mitochondrial morphology in a sick liver, we expressed a cleavage-resistant long form of OPA1 (L-OPA1Δ) in the liver of a mouse model with mitochondrial liver dysfunction (i.e. the hepatocyte-specific prohibitin-2 knockout (Hep-Phb2-/-) mice). Liver prohibitin-2 deficiency caused excessive proteolytic cleavage of L-OPA1, mitochondrial fragmentation, and increased apoptosis. These molecular alterations were associated with lipid accumulation, abolished gluconeogenesis, and extensive liver damage. Such liver dysfunction was associated with severe hypoglycemia. In prohibitin-2 knockout mice, expression of L-OPA1Δ by in vivo adenovirus delivery restored the morphology but not the function of mitochondria in hepatocytes. In prohibitin-competent mice, elongation of liver mitochondria by expression of L-OPA1Δ resulted in excessive glucose production associated with increased mitochondrial respiration. In conclusion, mitochondrial dynamics participates in the control of hepatic glucose production.

Nanofocused Scanning X-ray Fluorescence Microscopy Revealing an Effect of Heterozygous Hemoglobin S and C on Biochemical Activities in Plasmodium falciparum-Infected Erythrocytes.

While there is ample evidence suggesting that carriers of heterozygous hemoglobin S and C are protected from life-threatening malaria, little is known about the underlying biochemical mechanisms at the single cell level. Using nanofocused scanning X-ray fluorescence microscopy, we quantify the spatial distribution of individual elements in subcellular compartments, including Fe, S, P, Zn, and Cu, in Plasmodium falciparum-infected (P. falciparum-infected) erythrocytes carrying the wild type or variant hemoglobins. Our data indicate that heterozygous hemoglobin S and C significantly modulate biochemical reactions in parasitized erythrocytes, such as aberrant hemozoin mineralization and a delay in hemoglobin degradation. The label-free scanning X-ray fluorescence imaging has great potential to quantify the spatial distribution of elements in subcellular compartments of P. falciparum-infected erythrocytes and unravel the biochemical mechanisms underpinning disease and protective traits.

Landscape-level toxicant exposure mediates infection impacts on wildlife populations.

Anthropogenic landscape modification such as urbanization can expose wildlife to toxicants, with profound behavioural and health effects. Toxicant exposure can alter the local transmission of wildlife diseases by reducing survival or altering immune defence. However, predicting the impacts of pathogens on wildlife across their ranges is complicated by heterogeneity in toxicant exposure across the landscape, especially if toxicants alter wildlife movement from toxicant-contaminated to uncontaminated habitats. We developed a mechanistic model to explore how toxicant effects on host health and movement propensity influence range-wide pathogen transmission, and zoonotic exposure risk, as an increasing fraction of the landscape is toxicant-contaminated. When toxicant-contaminated habitat is scarce on the landscape, costs to movement and survival from toxicant exposure can trap infected animals in contaminated habitat and reduce landscape-level transmission. Increasing the proportion of contaminated habitat causes host population declines from combined effects of toxicants and infection. The onset of host declines precedes an increase in the density of infected hosts in contaminated habitat and thus may serve as an early warning of increasing potential for zoonotic spillover in urbanizing landscapes. These results highlight how sublethal effects of toxicants can determine pathogen impacts on wildlife populations that may not manifest until landscape contamination is widespread.

Understanding suicidal behavior and its prevention among youth and young adults in Mexico.

Suicide rates in Mexico have increased and have more than doubled in the state of Aguascalientes over the past 10 years. Few studies have been able to control for family, neighborhood, and occupational environment factors that may confound the association between psychosocial characteristics and suicidal behavior. We study suicidal behavior among adolescents and young adults in Mexico utilizing epidemiologic research strategies to overcome prior research deficiencies. In a case-control study with youth and adults 14-42 years of age, recent cases of severe suicidal behavior (n = 150) were individually matched with up to three controls who had never had a suicidal attempt by age and sex, as well as within familial, neighborhood, and occupational contexts (n = 353). Data were collected through standardized face-to-face interviews to measure suicidal behavior and several covariates, including family relations, psychological resources, hopelessness, depression, self-esteem, stress, impulsivity, anxiety, and substance use. All measures demonstrated good to excellent precision and accuracy. Compared with their matched controls, cases perceived life events as more stressful and had worse depression and familial relationships; poorer development of affective, religious, and social resources; higher levels of hopelessness and impulsive behavior; and lower self-esteem. Evidence from multivariate analysis suggests highly probable MDE combined with low self-esteem and the use of two or more drugs in the past month more clearly differentiate cases and controls and, therefore, may best predict suicidal attempt among adolescents and young adults in Aguascalientes, Mexico.

AMPK Profiling in Rodent and Human Pancreatic Beta-Cells under Nutrient-Rich Metabolic Stress.

Chronic exposure of pancreatic ß-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in ß-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in ß-cells. Then, we investigated the expression profile of AMPK pathways in ß-cells following metabolic stresses. INS-1E ß-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E ß-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the ß-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.