Genotoxic effects induced by iprodione and tebuconazole in meristematic cells of Allium cepa: responses dependent on concentration and exposure time.

The present work explores the genotoxicity of the fungicides iprodione (IP) and tebuconazole (TB) using the Allium cepa assay as an in vivo biological model. Both short-term and long-term exposures were studied, revealing concentration- and time-dependent cytological and genotoxic effects. IP exhibited genotoxicity over a wider concentration range (5-50 µg/ml) and required 30 h of exposure, while TB showed genotoxicity at higher concentrations (10 and 30 µg/ml) within a 4-h exposure period. The study highlights the importance of assessing potential risks associated with fungicide exposure, including handling, disposal practices, and concerns regarding food residue. Moreover, the research underscores the genotoxic effects of IP and TB on plant cells and provides valuable insights into their concentration and time-response patterns.

Medium & long-chain acylcarnitine's relation to lipid metabolism as potential predictors for diabetic cardiomyopathy: a metabolomic study.

BACKGROUND: Acylcarnitine is an intermediate product of fatty acid oxidation. It is reported to be closely associated with the occurrence of diabetic cardiomyopathy (DCM). However, the mechanism of acylcarnitine affecting myocardial disorders is yet to be explored. This current research explores the different chain lengths of acylcarnitines as biomarkers for the early diagnosis of DCM and the mechanism of acylcarnitines for the development of DCM in-vitro. METHODS: In a retrospective non-interventional study, 50 simple type 2 diabetes mellitus patients and 50 DCM patients were recruited. Plasma samples from both groups were analyzed by high throughput metabolomics and cluster heat map using mass spectrometry. Principal component analysis was used to compare the changes occurring in the studied 25 acylcarnitines. Multivariable binary logistic regression was used to analyze the odds ratio of each group for factors and the 95% confidence interval in DCM. Myristoylcarnitine (C14) exogenous intervention was given to H9c2 cells to verify the expression of lipid metabolism-related protein, inflammation-related protein expression, apoptosis-related protein expression, and cardiomyocyte hypertrophy and fibrosis-related protein expression. RESULTS: Factor 1 (C14, lauroylcarnitine, tetradecanoyldiacylcarnitine, 3-hydroxyl-tetradecanoylcarnitine, arachidic carnitine, octadecanoylcarnitine, 3-hydroxypalmitoleylcarnitine) and factor 4 (octanoylcarnitine, hexanoylcarnitine, decanoylcarnitine) were positively correlated with the risk of DCM. Exogenous C14 supplementation to cardiomyocytes led to increased lipid deposition in cardiomyocytes along with the obstacles in adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways and affecting fatty acid oxidation. This further caused myocardial lipotoxicity, ultimately leading to cardiomyocyte hypertrophy, fibrotic remodeling, and increased apoptosis. However, this effect was mitigated by the AMPK agonist acadesine. CONCLUSIONS: The increased plasma levels in medium and long-chain acylcarnitine extracted from factors 1 and 4 are closely related to the risk of DCM, indicating that these factors can be an important tool for DCM risk assessment. C14 supplementation associated lipid accumulation by inhibiting the AMPK/ACC/CPT1 signaling pathway, aggravated myocardial lipotoxicity, increased apoptosis apart from cardiomyocyte hypertrophy and fibrosis were alleviated by the acadesine.

Bioactive compounds from Artemisia dracunculus L. activate AMPK signaling in skeletal muscle.

An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a "DMC-2 knock out extract" (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.

Cytogenotoxic activity of the pesticides imidacloprid and iprodione on Allium cepa root meristem.

Effects of imidacloprid and iprodione, isolated and in mixture, were assessed by using seed germination and root growth test, flow cytometry, and chromosomal aberrations test on Allium cepa root meristem. The highest concentrations of imidacloprid, including field concentration, increased the frequency of sub-G1 particles, decreased the frequency of nuclei in G2/M, increased the coefficient of variation of G1 (CVG1) and the frequency of aberrant cells, and inhibited the mitotic index culminating in the reduction in root length. All doses of iprodione also presented cytogenotoxic action. The highest concentration of the fungicide affected the growth of A. cepa roots. In response to exposure to pesticide mixtures, the cell cycle of A. cepa was blocked in the G1 phase. The mixtures with low doses of the pesticides significantly decreased the mitotic index, and as a consequence, the genotoxicity was reduced. In the mixtures with the highest doses of the agrochemicals, the blockage of the cell cycle was insufficient for damage repair, resulting in a significant increase of chromosomal aberrations. The results suggest caution in the use of pesticides doses that induce cytological abnormalities in non-target organisms.

Residue behavior and removal of iprodione in garlic, green garlic, and garlic shoot.

BACKGROUND: Iprodione is considered to be an endocrine-disturbing pesticide, which could harm consumers. The garlic crop has three edible parts: the garlic, the green garlic, and the garlic shoot, which correspond to different stages of its growth. In this study, iprodione residue dissipation and distribution in these three edible parts were investigated, and dietary risk was evaluated. RESULTS: Iprodione residues were present in these samples in the following order: green garlic > garlic shoot > > garlic. The dissipation of iprodione in green garlic was slow with a half-life of 5.82-19.25 days. A very high RQchronic value of 207.35-407.30% suggested that the residual iprodione in green garlic had an unacceptable level of risk. Iprodione residue was significantly eliminated (59-90%) by an alkaline solution. The order for removing iprodione by soaking was the alkaline solutions (0.5% and 2% NaHCO3 ) > the acidic solutions (5% and 10% of vinegar) ≈ the neutral solutions (the 1% and 2% of table salt) > tap water. Processing factors (PFs) were <1, indicating that processing could decrease the iprodione residue level. CONCLUSION: This work could contribute to establishing maximum residue limits (MRLs) for iprodione in garlic, green garlic, and garlic shoots, and could provide guidance on the safe and appropriate use of iprodione in the garlic crop. © 2020 Society of Chemical Industry.

Methotrexate and its mechanisms of action in inflammatory arthritis.

Despite the introduction of numerous biologic agents for the treatment of rheumatoid arthritis (RA) and other forms of inflammatory arthritis, low-dose methotrexate therapy remains the gold standard in RA therapy. Methotrexate is generally the first-line drug for the treatment of RA, psoriatic arthritis and other forms of inflammatory arthritis, and it enhances the effect of most biologic agents in RA. Understanding the mechanism of action of methotrexate could be instructive in the appropriate use of the drug and in the design of new regimens for the treatment of RA. Although methotrexate is one of the first examples of intelligent drug design, multiple mechanisms potentially contribute to the anti-inflammatory actions of methotrexate, including the inhibition of purine and pyrimidine synthesis, transmethylation reactions, translocation of nuclear factor-κB (NF-κB) to the nucleus, signalling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and nitric oxide production, as well as the promotion of adenosine release and expression of certain long non-coding RNAs.

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1). METHODS: Human adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were grown in either normal medium alone (control group), the medium supplemented with AICAR (1 mM) and NAM (5 mM), or in the presence of both for 5 weeks to P10. Cell proliferation, differentiation capacity, level of apoptosis and autophagy, morphological changes, total cellular reactive oxygen species (ROS), and activity of mTORC1 and AMPK were compared among different treatment groups. RESULTS: MSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of ß-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy. CONCLUSIONS: Selective inhibition of mTORC1 by AICAR and NAM boosts autophagy, retains MSCs' self-renewal and multi-lineage differentiation capacity, and postpones senescence-associated changes after prolonged in vitro culture. Additionally, co-administration of AICAR and NAM shows an additive or probably a synergistic effect on cellular senescence.

The fungicide iprodione affects midgut cells of non-target honey bee Apis mellifera workers.

The honey bee Apis mellifera is an important pollinator of agricultural crops and natural forests. Honey bee populations have declined over the years, as a result of diseases, pesticides, and management problems. Fungicides are the main pesticides found in pollen grains, which are the major source of protein for bees. The objective of this study was to evaluate the cytotoxic effects of the fungicide iprodione on midgut cells of adult A. mellifera workers. Bees were fed on iprodione (LD50, determined by the manufacturer) for 12 or 24 h, and the midgut was examined using light and transmission electron microscopies. The expression level of the autophagy gene atg1 was assessed in midgut digestive cells. Cells of treated bees had signs of apoptosis: cytoplasmic vacuolization, apical cell protrusions, nuclear fragmentation, and chromatin condensation. Ultrastructural analysis revealed some cells undergoing autophagy and necrosis. Expression of atg1 was similar between treated and control bees, which can be explained by the facts that digestive cells had autolysosomes, whereas ATG-1 is found in the initial phases of autophagy. Iprodione acts by inhibiting the synthesis of glutathione, leading to the generation of reactive oxygen species, which in turn can induce different types of cell death. The results indicate that iprodione must be used with caution because it has side effects on non-target organisms, such as pollinator bees.

Toxicological effects of comercial formulations of fungicides based on procymidone and iprodione in seedlings and root tip cells of Allium cepa.

In this study the phytotoxic, cytotoxic, genotoxic and mutagenic effects of two commercial fungicide-active compounds, procymidone (PR) and iprodione (IP), were determined. The parameters evaluated were germination and root growth, mitotic index, chromosomal and nuclear aberrations, and molecular analyses were also performed in the model plant Allium cepa L. The results demonstrated that the active compounds PR and IP were phytotoxic, delaying germination and slowing the development of A. cepa seedlings. Moreover, PR and IP showed cytogenotoxicity towards A. cepa meristematic cells, inducing chromosomal changes and cell death. The mutagenic activity of the active compounds was demonstrated by the detection of DNA changes in simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers in the treated cells compared to the negative control. Together, these results contribute to a better understanding of the damage caused by these substances in living organisms and reveal a promising strategy for prospective studies of the toxic effects of environmental pollutants.

Cascade therapy with doxorubicin and survivin-targeted tailored nanoparticles: An effective alternative for sensitization of cancer cells to chemotherapy.

Chemotherapy frequently involves combination treatment protocols to maximize tumor cell killing. Unfortunately these intensive chemotherapeutic regimes, often show disappointing results due to the development of drug resistance and higher nonspecific toxicity on normal tissues. In cancer treatment, it is critically important to minimize toxicity while preserving efficacy. We have previously addressed this issue and proposed a nanoparticle-based combination therapy involving both a molecularly targeted therapy and chemotherapeutic agent for neutralizing antiapoptotic survivin (BIRC5) to potentiate the efficacy of doxorubicin (DOX). Although the particles exhibited strong anticancer effect on the lung carcinoma A549 and the cervical carcinoma HeLa cells, there were lower-level therapeutic outcomes on the colon carcinoma HCT-116, the leukemia Jurkat and the pancreatic carcinoma MIA PaCa-2 cells. Since targeted therapies are one of the key approaches for overcoming drug resistance, tailoring the treatment of cancer cells with distinct characteristics is necessary to improve the therapeutic outcome of cancer therapy and to minimize potential pharmacokinetic interactions of drugs. In the light of this issue, this study examined whether a cascade therapy with low-dose DOX and survivin-targeted tailored nanoparticles is more effective at sensitizing HCT-116, Jurkat and MIA PaCa-2 cancer cells to DOX-chemotherapy than simultaneous combination therapy. The results demonstrated that the sequential therapy with the protocol comprising addition of the nanoparticles after incubation of cells with DOX clearly advanced the therapeutic outcome of related cancer cells, whereas the reverse protocol resulted in a reduction or delay in apoptosis, emphasizing the critical importance of formulating synergistic drug combinations in cancer therapy.

The Chinese medicine Chai Hu Li Zhong Tang protects against non-alcoholic fatty liver disease by activating AMPKα.

An effective treatment for non-alcoholic fatty liver disease (NAFLD) is urgently needed. In the present study, we investigated whether the Chinese medicine Chai Hu Li Zhong Tang (CHLZT) could protect against the development of NAFLD. Rats in an animal model of NAFLD were treated with CHLZT, and their serum levels of cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were detected with an automatic biochemical analyzer. A cellular model of NAFLD was also established by culturing HepG2 cells in a medium that contained a long chain fat emulsion. Those cells were treated with CHLZT that contained serum from rats. After treatment, the levels of adenylate-activated protein kinase (AMPK) α (AMPKα), p-AMPKα, acetyl coenzyme A carboxylase (ACC) α (ACCα), pACCα, PPARγ, and SREBP-2 were detected. The AMPK agonist, acadesine (AICAR), was used as a positive control compound. Our results showed that CHLZT or AICAR significantly decreased the serum levels of TG, TC, LDL-C, AST, ALT, and insulin in NAFLD rats, and significantly increased their serum HDL-C levels. Treatments with CHLZT or AICAR significantly decreased the numbers of lipid droplets in NAFLD liver tissues and HepG2 cells. CHLZT and AICAR increased the levels of p-AMPKα and PPARγ in the NAFLD liver tissues and HepG2 cells, but decreased the levels of ACC-α, p-ACC-α, SREBP-2, and 3-hydroxyl-3-methylglutaryl-coenzyme A reductase (HMGR). CHLZT protects against NAFLD by activating AMPKα, and also by inhibiting ACC activity, down-regulating SREBP2 and HMGR, and up-regulating PPAR-γ. Our results suggest that CHLZT might be useful for treating NAFLD in the clinic.

The effects of iprodione fungicide on survival, behavior, and brood development of honeybees (Apis mellifera L.) after one foliar application during flowering on mustard.

A semifield study to assess the effects of iprodione on honeybees at label use rates was conducted on a bloom mustard crop. The present study followed the Organisation for Economic Co-operation and Development guideline 75 tunnel test and consisted of 3 groups: the iprodione-treated group, the untreated control group, and the toxic reference item group. In addition to the tunnels used for biological assessments, a tunnel was set up in the treatment and control groups to determine the level of residues in flowers, nectar, and pollen. The major endpoints to assess the effects of the application of iprodione were mortality, flight intensity, behavior, condition of the colonies, and development of the brood. Residue analysis showed that honeybees were exposed to significant residues of iprodione. However, no adverse effects were observed on overall mortality, flight intensity, behavior, or brood development of honeybees compared to control. It is concluded that iprodione does not adversely affect the health of honeybees when applied in agriculture at commercially relevant rates in a worst-case exposure scenario. Environ Toxicol Chem 2018;37:3086-3094. © 2018 SETAC.

Activation of AMPK by metformin improves withdrawal signs precipitated by nicotine withdrawal.

Cigarette smoking is the leading cause of preventable disease and death in the United States, with more persons dying from nicotine addiction than any other preventable cause of death. Even though smoking cessation incurs multiple health benefits, the abstinence rate remains low with current medications. Here we show that the AMP-activated protein kinase (AMPK) pathway in the hippocampus is activated following chronic nicotine use, an effect that is rapidly reversed by nicotine withdrawal. Increasing pAMPK levels and, consequently, downstream AMPK signaling pharmacologically attenuate anxiety-like behavior following nicotine withdrawal. We show that metformin, a known AMPK activator in the periphery, reduces withdrawal symptoms through a mechanism dependent on the presence of the AMPKα subunits within the hippocampus. This study provides evidence of a direct effect of AMPK modulation on nicotine withdrawal symptoms and suggests central AMPK activation as a therapeutic target for smoking cessation.

Combination therapy of lovastatin and AMP-activated protein kinase activator improves mitochondrial and peroxisomal functions and clinical disease in experimental autoimmune encephalomyelitis model.

Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/ß, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.

Variation in Fungicide Sensitivity Among Rhizoctonia Isolates Recovered from Potatoes in South Africa.

Rhizoctonia is a major pathogen of potato causing substantial yield losses worldwide. Control of Rhizoctonia diseases is based predominantly on the application of fungicides. However, little is known about the fungicide response variability of different Rhizoctonia anastomosis groups associated with potato diseases in South Africa. A total of 131 Rhizoctonia isolates were obtained from potato growing regions of South Africa from 2012 to 2014 and evaluated for sensitivity to fungicides in vitro and in vivo. The fungicides comprised six chemical formulations and one bio-fungicide representing seven Fungicide Resistance Action Committee groups. All Rhizoctonia anastomosis groups were sensitive to tolclofos-methyl (EC50: 0.001 to 0.098 µg a.i. ml-1) and fludioxonil (EC50: 0.06 to 0.09 µg a.i. ml-1) and showed variation in sensitivity to pencycuron, iprodione, benomyl, and Bacillus subtilis QST 713. However, for azoxystrobin, Rhizoctonia isolates exhibited variable sensitivity ranging from sensitivity (EC50: <0.09 µg a.i. ml-1) to insensitivity with EC50 values exceeding 5 µg a.i. ml-1. In greenhouse and field trials, tolclofos-methyl and fludioxonil exhibited significantly greater control of stem and black scurf whereas azoxystrobin was the least effective. This work demonstrated variable sensitivity within and among anastomosis groups of R. solani and binucleate Rhizoctonia to different fungicides. Information on fungicide sensitivity of Rhizoctonia isolates is crucial in the development of effective Rhizoctonia control strategies and facilitates monitoring of fungicide insensitive isolates in the pathogen population.

Autophagy induction enhances tetrandrine-induced apoptosis via the AMPK/mTOR pathway in human bladder cancer cells.

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the roots of Stephania tetrandra is a traditional Chinese medicine and exerts anticancer capacity in various types of cancers. Previous studies have shown that tetrandrine induces apoptosis in bladder cancer cells via activation of the caspase cascade. However, the underlying mechanism has not yet been reported. Autophagy is a cellular process involved in the degradation of broken proteins and aging organelles to maintain homeostasis. Recent studies indicate that autophagy is implicated in cancer therapy. Thus, we focused on the correlation between autophagy and apoptosis upon tetrandrine treatment in human bladder cancer cells. Firstly, our results observed a marked increase in autophagic double-membrane vacuoles and fluorescent puncta of red fluorescence protein-green fluorescence protein-LC3 (GRP-RFP-LC3) upon tetrandrine treatment, as evidenced by transmission electron microscopy and confocal fluorescence microscopy. Secondly, the expression of LC3-II was increased in tetrandrine-treated T24 and 5637 cells in a time- and concentration-dependent manner. Subsequently, downregulation of p62 and LC3 turnover assay further confirmed that tetrandrine induced autophagic flux in bladder cancer T24 and 5637 cells. Thirdly, the protein levels of phosphorylated-AMP-activated protein kinase (AMPK) and phosphorylated-acetyl-coenzyme A carboxylase (ACC) were upregulated in the tetrandrine-treated cells, while the mammalian target of rapamycin (mTOR)-related proteins were downregulated. Moreover, AICAR, a common AMPK activator, further increased the expression the LC3-II, while AMPK inhibitor compound C partially reversed the LC3-II protein levels in bladder cancer T24 cells. Finally, AICAR significantly reinforced the growth inhibition and apoptosis induction of tetrandrine in T24 and 5637 cells, while compound C had an opposite effect, suggesting that AMPK-mediated autophagy enhanced the cytotoxic and pro-apoptosis effect of tetrandrine in human bladder cancer cells. Taken together, the present study showed that tetrandrine induced autophagy in human bladder cancer cells by regulating the AMPK/mTOR signaling pathway, which contributed to the apoptosis induction by tetrandrine, indicating that tetrandrine may be a potential anticancer candidate for the treatment of bladder cancer, and autophagy may be a possible mechanism for cancer therapy.

5-Aminoimidazole-4-carboxamide ribonucleotide prevents fat gain following the cessation of voluntary physical activity.

NEW FINDINGS: What is the central question of this study? We investigated whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) could prevent acute increases in body fat and changes in omental and subcutaneous adipose tissue following the sudden transition from physical activity to physical inactivity. What is the main finding and its importance? AICAR prevented fat gains following the transition from physical activity to inactivity to levels comparable to rats that remained physically active. AICAR and continuous physical activity produced depot-specific changes in cyclin A1 mRNA and protein that were associated with the prevention of fat gain. These findings suggest that targeting AMP-activated protein kinase signalling could oppose rapid adipose mass growth. The transition from physical activity to inactivity is associated with drastic increases in 'catch-up' fat that in turn foster the development of many obesity-associated maladies. We tested whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) treatment would prevent gains in body fat following the sudden transition from a physically active state to an inactive state by locking a voluntary running wheel. Male Wistar rats were either sedentary (SED) or given wheel access for 4 weeks, at which time rats with wheels continued running (RUN), had their wheel locked (WL) or had WL with daily AICAR injection (WL + AICAR) for 1 week. RUN and WL + AICAR prevented gains in body fat compared with SED and WL (P < 0.001). Cyclin A1 mRNA, a marker of cell proliferation, was decreased in omental, but not subcutaneous adipose tissue, in RUN and WL + AICAR compared with SED and WL groups (P < 0.05). Both cyclin A1 mRNA and protein were positively associated with gains in fat mass (P < 0.05). Cyclin A1 mRNA in omental, but not subcutaneous, adipose tissue was negatively correlated with p-AMPK levels (P < 0.05). Differences in fat gain and omental mRNA and protein levels were independent of changes in food intake and in differences in select hypothalamic mRNAs. These findings suggest that AICAR treatment prevents acute gains in adipose tissue following physical inactivity to levels of rats that continuously run, and that together, continuous physical activity and AICAR could, at least initially in these conditions, exert similar inhibitory effects on adipogenesis in a depot-specific manner.

Glucocorticoids induced high fat diet preference via activating hypothalamic AMPK signaling in chicks.

Glucocorticoids (GCs) stimulate appetite, contributing to enhanced fat deposition. Our present study was conducted to determine whether GCs could evoke an appetite specifically for fat-rich diets in chicks. Chicks were subjected to a subcutaneous injection of corticosterone (CORT, 2mg/kg body weight/day) or corn oil (control), and food preference was tested. The results showed that CORT-chicks consumed more high-fat diet (HFD) compared with controls. In HFD-fed chicks, hypothalamic phosphorylated AMP-activated protein kinase α (AMPKα) and neuropeptide Y (NPY) mRNA levels were increased by CORT treatment. Activating AMPK with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, an AMPK activator, via intracerebroventricular injection further enhanced the CORT-induced HFD consumption and concurrently up-regulated NPY mRNA levels and phosphorylated AMPKα and acetyl-coenzyme A carboxylase levels. The dramatic increase in HFD consumption and upregulation of NPY mRNA levels and phospho-AMPKα levels induced by peripheral CORT injection was not altered by intracerebroventricular infusion of compound C (4-16µg), an AMPK inhibitor. In conclusion, CORT challenge caused a HFD preference by enhancing the AMPK pathway in the hypothalamus.

Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 µM)], inhibit mitochondrial function [sodium azide (75 µM), rotenone (1 µM), berberine (100 µM), DNP (500 µM)], or directly activate AMPK [AICAR (250 µM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 µM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 µM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.