Impact of Acutely Increased Endogenous- and Exogenous Ketone Bodies on FGF21 Levels in Humans.

PURPOSE: Fibroblast growth factor (FGF) 21 is a circulating hormone with metabolic regulatory importance. In mice, FGF21 increases in response to a ketogenic diet and fasting. In humans, a similar increase is only observed after prolonged starvation. We aim to study the acute effects of ketone bodies on circulating FGF21 levels in humans. METHODS: Participants from three randomized, placebo-controlled crossover studies, with increased endogenous or exogenous ketone bodies, were included. Study 1: patients with type 1 diabetes (T1D) (n = 9) were investigated after a) insulin deprivation and lipopolysaccharide (LPS) injection and b) insulin-controlled euglycemia. Study 2: patients with T1D (n = 9) were investigated after a) total insulin deprivation for 9 hours and b) insulin-controlled euglycemia. Study 3: Healthy adults (n = 9) were examined during a) 3-hydroxybutyrate (OHB) infusion and b) saline infusion. Plasma FGF21 was measured with immunoassay in serial samples. RESULTS: Circulating OHB levels were significantly increased to 1.3, 1.5, and 5.5 mmol/l in the three studies, but no correlations with FGF21 levels were found. Also, no correlations between FGF21, insulin, or glucagon were found. Insulin deprivation and LPS injection resulted in increased plasma FGF21 levels at t = 120 min (p = .005) which normalized at t = 240 min. CONCLUSION: We found no correlation between circulating FGF21 levels and levels of ketone bodies. This suggests that it is not ketosis per se which controls FGF21 production, but instead a rather more complex regulatory mechanism. TRIAL REGISTRATION: clinicaltrials.gov ID number: Study 1: NCT02157155 (5/6-2014), study 2: NCT02077348 (4/3-2014), and study 3: NCT02357550 (6/2-2015).

Substrate metabolism, hormone and cytokine levels and adipose tissue signalling in individuals with type 1 diabetes after insulin withdrawal and subsequent insulin therapy to model the initiating steps of ketoacidosis.

AIMS/HYPOTHESIS: Lack of insulin and infection/inflammation are the two most common causes of diabetic ketoacidosis (DKA). We used insulin withdrawal followed by insulin administration as a clinical model to define effects on substrate metabolism and to test whether increased levels of counter-regulatory hormones and cytokines and altered adipose tissue signalling participate in the early phases of DKA. METHODS: Nine individuals with type 1 diabetes, without complications, were randomly studied twice, in a crossover design, for 5 h followed by 2.5 h high-dose insulin clamp: (1) insulin-controlled euglycaemia (control) and (2) after 14 h of insulin withdrawal in a university hospital setting. RESULTS: Insulin withdrawal increased levels of glucose (6.1 ± 0.5 vs 18.6 ± 0.5 mmol/l), NEFA, 3-OHB (127 ± 18 vs 1837 ± 298 µmol/l), glucagon, cortisol and growth hormone and decreased HCO3- and pH, without affecting catecholamine or cytokine levels. Whole-body energy expenditure, endogenous glucose production (1.55 ± 0.13 vs 2.70 ± 0.31 mg kg-1 min-1), glucose turnover, non-oxidative glucose disposal, lipid oxidation, palmitate flux (73 [range 39-104] vs 239 [151-474] µmol/min), protein oxidation and phenylalanine flux all increased, whereas glucose oxidation decreased. In adipose tissue, Ser473 phosphorylation of Akt and mRNA levels of G0S2 decreased, whereas CGI-58 (also known as ABHD5) mRNA increased. Protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase phosphorylations were unaltered. Insulin therapy decreased plasma glucose concentrations dramatically after insulin withdrawal, without any detectable effect on net forearm glucose uptake. CONCLUSIONS/INTERPRETATION: Release of counter-regulatory hormones and overall increased catabolism, including lipolysis, are prominent features of preacidotic ketosis induced by insulin withdrawal, and dampening of Akt insulin signalling and transcriptional modulation of ATGL activity are involved. The lack of any increase in net forearm glucose uptake during insulin therapy after insulin withdrawal indicates muscle insulin resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT02077348 FUNDING: This study was supported by Aarhus University and the KETO Study Group/Danish Agency for Science Technology and Innovation.

Metabolic effects of insulin in a human model of ketoacidosis combining exposure to lipopolysaccharide and insulin deficiency: a randomised, controlled, crossover study in individuals with type 1 diabetes.

AIMS/HYPOTHESIS: Diabetic ketoacidosis (DKA) is often caused by concomitant systemic inflammation and lack of insulin. Here we used an experimental human model to test whether and how metabolic responses to insulin are impaired in the early phases of DKA with a specific focus on skeletal muscle metabolism. METHODS: Nine individuals with type 1 diabetes from a previously published cohort were investigated twice at Aarhus University Hospital using a 120 min infusion of insulin (3.0/1.5 mU kg-1 min-1) after an overnight fast under: (1) euglycaemic conditions (CTR) or (2) hyperglycaemic ketotic conditions (KET) induced by an i.v. bolus of lipopolysaccharide and 85% reduction in insulin dosage. The primary outcome was insulin resistance in skeletal muscle. Participants were randomly assigned to one of the two arms at the time of screening using www.randomizer.org . The study was not blinded. RESULTS: All nine volunteers completed the 2 days and are included in the analysis. Circulating concentrations of glucose and 3-hydroxybutyrate increased during KET (mean ± SEM 17.7 ± 0.6 mmol/l and 1.6 ± 0.2 mmol/l, respectively), then decreased after insulin treatment (6.6 ± 0.7 mmol/l and 0.1 ± 0.07 mmol/l, respectively). Prior to insulin infusion (KET vs CTR) isotopically determined endogenous glucose production rates were 17 ± 1.7 µmol kg-1 min-1 vs 8 ± 1.3 µmol kg-1 min-1 (p = 0.003), whole body phenylalanine fluxes were 2.9 ± 0.5 µmol kg-1 min-1 vs 3.1 ± 0.4 µmol kg-1 min-1 (p = 0.77) and urea excretion rates were 16.9 ± 2.4 g/day vs 7.3 ± 1.7 g/day (p = 0.01). Insulin failed to stimulate forearm glucose uptake and glucose oxidation in KET compared with CTR (p < 0.05). Glycogen synthase phosphorylation was impaired in skeletal muscle. CONCLUSIONS/INTERPRETATION: In KET, hyperglycaemia is primarily driven by increased endogenous glucose production. Insulin stimulation during early phases of DKA is associated with reduced glucose disposal in skeletal muscle, impaired glycogen synthase function and lower glucose oxidation. This underscores the presence of muscle insulin resistance in the pathogenesis of DKA. Trial registration www.clinicaltrials.gov (ID number: NCT02157155). Funding This work was funded by the Danish Council for Strategic Research (grant no. 0603-00479B).

Effects of insulin-induced hypoglycaemia on lipolysis rate, lipid oxidation and adipose tissue signalling in human volunteers: a randomised clinical study.

AIMS/HYPOTHESIS: The aims of this study were to determine the role of lipolysis in hypoglycaemia and define the underlying intracellular mechanisms. METHODS: Nine healthy volunteers were randomised to treatment order of three different treatments (crossover design). Treatments were: (1) saline control; (2) hyperinsulinaemic hypoglycaemia (HH; i.v. bolus of 0.1 U/kg insulin); and (3) hyperinsulinaemic euglycaemia (HE; i.v. bolus of 0.1 U/kg insulin and 20% glucose). Inclusion criteria were that volunteers were healthy, aged >18 years, had a BMI between 19 and 26 kg/m2, and provided both written and oral informed consent. Exclusion criteria were the presence of a known chronic disease (including diabetes mellitus, epilepsy, ischaemic heart disease and cardiac arrhythmias) and regular use of prescription medication. The data was collected at the medical research facilities at Aarhus University Hospital, Denmark. The primary outcome was palmitic acid flux. Participants were blinded to intervention order, but caregivers were not. RESULTS: Adrenaline (epinephrine) and glucagon concentrations were higher during HH than during both HE and control treatments. NEFA levels and lipid oxidation rates (determined by indirect calorimetry) returned to control levels after 105 min. Palmitate flux was increased to control levels during HH (p = NS) and was more than twofold higher than during HE (overall mean difference between HH vs HE, 114 [95% CI 64, 165 µmol/min]; p < 0.001). In subcutaneous adipose tissue biopsies, we found elevated levels of hormone-sensitive lipase (HSL) and perilipin-1 phosphorylation 30 min after insulin injection during HH compared with both control and HE. There were no changes in the levels of adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58) or G0/G1 switch gene 2 (G0S2) proteins. Insulin-stimulated phosphorylation of Akt and mTOR were unaffected by hypoglycaemia. Expression of the G0S2 gene increased during HE and HH compared with control, without changes in ATGL (also known as PNPLA2) or CGI-58 (also known as ABHD5) mRNA levels. CONCLUSIONS/INTERPRETATION: These findings suggest that NEFAs become a major fuel source during insulin-induced hypoglycaemia and that lipolysis may be an important component of the counter-regulatory response. These effects appear to be mediated by rapid stimulation of protein kinase A (PKA) and HSL, compatible with activation of the ß-adrenergic catecholamine signalling pathway. TRIAL REGISTRATION: ClinicalTrials.gov NCT01919788 FUNDING: : The study was funded by Aarhus University, the Novo Nordisk Foundation and the KETO Study Group/Danish Agency for Science Technology and Innovation (grant no. 0603-00479, to NM).

Stabilization of Live Attenuated Influenza Vaccines by Freeze Drying, Spray Drying, and Foam Drying.

PURPOSE: The goal of this research is to develop stable formulations for live attenuated influenza vaccines (LAIV) by employing the drying methods freeze drying, spray drying, and foam drying. METHODS: Formulated live attenuated Type-A H1N1 and B-strain influenza vaccines with a variety of excipient combinations were dried using one of the three drying methods. Process and storage stability at 4, 25 and 37°C of the LAIV in these formulations was monitored using a TCID50 potency assay. Their immunogenicity was also evaluated in a ferret model. RESULTS: The thermal stability of H1N1 vaccine was significantly enhanced through application of unique formulation combinations and drying processes. Foam dried formulations were as much as an order of magnitude more stable than either spray dried or freeze dried formulations, while exhibiting low process loss and full retention of immunogenicity. Based on long-term stability data, foam dried formulations exhibited a shelf life at 4, 25 and 37°C of >2, 1.5 years and 4.5 months, respectively. Foam dried LAIV Type-B manufactured using the same formulation and process parameters as H1N1 were imparted with a similar level of stability. CONCLUSION: Foam drying processing methods with appropriate selection of formulation components can produce an order of magnitude improvement in LAIV stability over other drying methods.

Direct cytosolic delivery of polar cargo to cells by spontaneous membrane-translocating peptides.

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. D-amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.

Insulin Signaling Is Preserved in Skeletal Muscle During Early Diabetic Ketoacidosis.

BACKGROUND AND AIMS: During diabetic ketoacidosis (DKA), muscle tissue develops a profound insulin resistance that complicates reversal of this potentially lethal condition. We have investigated mediators of insulin action in human skeletal muscle during total insulin withdrawal in patients with type 1 diabetes, under the hypothesis that initial phases of DKA are associated with impaired postreceptor signaling. MATERIALS AND METHODS: Muscle biopsies were obtained during a randomized, controlled, crossover trial involving 9 patients with type 1 diabetes. The subjects were investigated during a high-dose insulin clamp preceded by either: (1) insulin-controlled euglycemia (control) or (2) total insulin withdrawal for 14 hours. Insulin action in skeletal muscle and whole-body substrate metabolism were investigated using western blot analysis and indirect calorimetry respectively. RESULTS: During insulin withdrawal, insulin-stimulated dephosphorylation of glycogen synthase decreased by ∼30% (P < .05) compared with the control situation. This was associated with a decrease in glucose oxidation by ∼30% (P < .05). Despite alterations in glucose metabolism, insulin transduction to glucose transport and protein synthesis (Akt, AS160, mammalian target of rapamycin, and eukaryotic translation initiation factor 4E binding protein) was intact, and glucose transporter (GLUT4) and mitochondrial proteins (succinate dehydrogenase complex, subunit A and prohibitin 1) protein expression were unaffected by the intervention. CONCLUSION: DKA impairs insulin-stimulated activation of glycogen synthase, whereas insulin signal transduction to glucose transport and protein synthesis remains intact. Reversal of insulin resistance during treatment of DKA should target postreceptor mediators of glucose uptake. CLINICAL TRIAL REGISTRATION NUMBER: NCT02077348.

Investigating effects of sodium beta-hydroxybutyrate on metabolism in placebo-controlled, bilaterally infused human leg with focus on skeletal muscle protein dynamics.

Systemic administration of beta-hydroxybutyrate (BHB) decreases whole-body protein oxidation and muscle protein breakdown in humans. We aimed to determine any direct effect of BHB on skeletal muscle protein turnover when administered locally in the femoral artery. Paired design with each subject being investigated on one single occasion with one leg being infused with BHB and the opposing leg acting as a control. We studied 10 healthy male volunteers once with bilateral femoral vein and artery catheters. One artery was perfused with saline (Placebo) and one with sodium-BHB. Labelled phenylalanine and palmitate were used to assess local leg fluxes. Femoral vein concentrations of BHB were significantly higher in the intervention leg (3.4 (3.2, 3.6) mM) compared with the placebo-controlled leg (1.9 (1.8, 2.1) mM) with a peak difference of 1.4 (1.1, 1.7) mM, p < 0.0005. Net loss of phenylalanine for BHB vs Placebo -6.7(-10.8, -2.7) nmol/min vs -8.7(-13.8, -3.7) nmol/min, p = 0.52. Palmitate flux and arterio-venous difference of glucose did not differ between legs. Under these experimental conditions, we failed to observe the direct effects of BHB on skeletal muscle protein turnover. This may relate to a combination of high concentrations of BHB (close to 2 mM) imposed systemically by spillover leading to high BHB concentrations in the saline-infused leg and a lack of major differences in concentration gradients between the two sides-implying that observations were made on the upper part of the dose-response curve for BHB and the relatively small number of subjects studied.

Gain enhancement of BiCMOS on-chip sub-THz antennas by mean of meta-cells.

A MM-loaded sub-THz on-chip antenna with a narrow beamwidth, 9 dB gain and a simulated peak efficiency of 76% at the center frequency of 300 GHz is presented. By surrounding the antenna with a single MM-cell ring defined solely on the top metal of the back-end of line, an efficient suppression of the surface waves is obtained. The on-chip antenna has been designed using IHPs 130 nm SiGe BiCMOS technology with a 7-layer metallization stack, combined with the local backside etching process aimed to creating an air cavity which is then terminated by a reflective plane. By comparing the measured MM-loaded antenna performances to its non-MM-loaded counterpart, an enhanced integrity of the main lobe due to the MM-cells shielding effect can be observed. An excellent agreement between the simulated and measured performances has been found, which makes the MM-loaded antennas a valid alternative for the upcoming next-generation sub-THz transceivers.

Ferrets as a model for tuberculosis transmission.

Even with the COVID-19 pandemic, tuberculosis remains a leading cause of human death due to a single infectious agent. Until successfully treated, infected individuals may continue to transmit Mycobacterium tuberculosis bacilli to contacts. As with other respiratory pathogens, such as SARS-CoV-2, modeling the process of person-to-person transmission will inform efforts to develop vaccines and therapies that specifically impede disease transmission. The ferret (Mustela furo), a relatively inexpensive, small animal has been successfully employed to model transmissibility, pathogenicity, and tropism of influenza and other respiratory disease agents. Ferrets can become naturally infected with Mycobacterium bovis and are closely related to badgers, well known in Great Britain and elsewhere as a natural transmission vehicle for bovine tuberculosis. Herein, we report results of a study demonstrating that within 7 weeks of intratracheal infection with a high dose (>5 x 103 CFU) of M. tuberculosis bacilli, ferrets develop clinical signs and pathological features similar to acute disease reported in larger animals, and ferrets infected with very-high doses (>5 x 104 CFU) develop severe signs within two to four weeks, with loss of body weight as high as 30%. Natural transmission of this pathogen was also examined. Acutely-infected ferrets transmitted M. tuberculosis bacilli to co-housed naïve sentinels; most of the sentinels tested positive for M. tuberculosis in nasal washes, while several developed variable disease symptomologies similar to those reported for humans exposed to an active tuberculosis patient in a closed setting. Transmission was more efficient when the transmitting animal had a well-established acute infection. The findings support further assessment of this model system for tuberculosis transmission including the testing of prevention measures and vaccine efficacy.

Prophylactic administration of bacterially derived immunomodulators improves the outcome of influenza virus infection in a murine model.

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.

A Human Randomized Controlled Trial Comparing Metabolic Responses to Single and Repeated Hypoglycemia in Type 1 Diabetes.

AIMS: Hypoglycemia hinders optimal glycemic management in type 1 diabetes (T1D). Long diabetes duration and hypoglycemia impair hormonal counter-regulatory responses to hypoglycemia. Our study was designed to test whether (1) the metabolic responses and insulin sensitivity are impaired, and (2) whether they are affected by short-lived antecedent hypoglycemia in participants with T1D. MATERIALS AND METHODS: In a randomized, crossover, 2x2 factorial design, 9 male participants with T1D and 9 comparable control participants underwent 30 minutes of hypoglycemia (p-glucose < 2.9 mmol/L) followed by a euglycemic clamp on 2 separate interventions: with and without 30 minutes of hypoglycemia the day before the study day. RESULTS: During both interventions insulin sensitivity was consistently lower, while counter-regulatory hormones were reduced, with 75% lower glucagon and 50% lower epinephrine during hypoglycemia in participants with T1D, who also displayed 40% lower lactate and 5- to 10-fold increased ketone body concentrations following hypoglycemia, whereas palmitate and glucose turnover, forearm glucose uptake, and substrate oxidation did not differ between the groups. In participants with T1D, adipose tissue phosphatase and tensin homolog (PTEN) content, hormone-sensitive lipase (HSL) phosphorylation, and muscle glucose transporter type 4 (GLUT4) content were decreased compared with controls. And antecedent hypoglycemic episodes lasting 30 minutes did not affect counter-regulation or insulin sensitivity. CONCLUSIONS: Participants with T1D displayed insulin resistance and impaired hormonal counter-regulation during hypoglycemia, whereas glucose and fatty acid fluxes were intact and ketogenic responses were amplified. We observed subtle alterations of intracellular signaling and no effect of short-lived antecedent hypoglycemia on subsequent counter-regulation. This plausibly reflects the presence of insulin resistance and implies that T1D is a condition with defective hormonal but preserved metabolic responsiveness to short-lived hypoglycemia.

Identification of Structurally Related Antibodies in Antibody Sequence Databases Using Rosetta-Derived Position-Specific Scoring.

The amount of antibody (Ab) variable gene sequence information is expanding rapidly, but our ability to predict the function of Abs from sequence alone is limited. Here, we describe a sequence-to-function prediction method that couples structural data for a single Ab/antigen (Ag) complex with repertoire data. We used a position-specific structure-scoring matrix (P3SM) incorporating structure-prediction scores from Rosetta to identify Ab variable loops that have predicted structural similarity to the influenza virus-specific human Ab CH65. The P3SM approach identified new members of this Ab class. Recombinant Ab expression, crystallography, and virus inhibition assays showed that the HCDR3 loops of the newly identified Abs possessed similar structure and antiviral activity as the comparator CH65. This approach enables discovery of new human Abs with desired structure and function using cDNA repertoires that are obtained readily with current amplicon sequencing techniques.

Pichinde virus induces microvascular endothelial cell permeability through the production of nitric oxide.

This report is the first to demonstrate infection of human endothelial cells by Pichinde virus (PIC). PIC infection induces an upregulation of the inducible nitric oxide synthase gene; as well as an increase in detectable nitric oxide (NO). PIC induces an increase in permeability in endothelial cell monolayers which can be abrogated at all measured timepoints with the addition of a nitric oxide synthase inhibitor, indicating a role for NO in the alteration of endothelial barrier function. Because NO has shown antiviral activity against some viruses, viral titer was measured after addition of the NO synthase inhibitor and found to have no effect in altering virus load in infected EC. The NO synthase inhibition also has no effect on levels of activated caspases induced by PIC infection. Taken together, these data indicate NO production induced by Pichinde virus infection has a pathogenic effect on endothelial cell monolayer permeability.

Effects of short-term prednisolone treatment on indices of lipolysis and lipase signaling in abdominal adipose tissue in healthy humans.

BACKGROUND: Glucocorticoid (GC) excess increases lipolysis, circulating free fatty acid concentrations and lipid oxidation rates in humans. In vitro and animal studies have shown that GCs increase adipocyte ATGL and HSL mRNA contents and HSL phosphorylations, but the effects of GC on in vivo lipase signaling in humans are uncertain. Our study was designed to test how GC administration affects ATGL and HSL related signals in human adipose tissue. MATERIAL AND METHODS: Nine healthy young men underwent 5 days administration of 37.5 mg prednisolone/d in a randomized, double-blinded, placebo-controlled crossover design. At the end of each 5 d period the subjects were studied after an overnight fast for 6.5 h including a basal period and a 2½â€¯h hyperinsulinemic euglycemic clamp. Adipose tissue biopsies were sampled from the abdominal subcutaneous adipose tissue at the end of the basal period and the clamp. RESULTS: GC treatment increased serum FFA concentrations and comparative gene identification-58 (CGI-58) mRNA - an ATGL activator - and decreased G0/G1 switch 2 gene (G0S2) mRNA - an ATGL inhibitor - in adipose tissue biopsies. In addition, pro-lipolytic ser563 HSL phosphorylations and protein kinase A (PKA) phosphorylation of PLIN1 (Perilipin-1) increased. The transcripts of ANGPTL4 (Angiopoietin-like 4) mRNA - a regulator of circulating triglycerides - were elevated by GC; as were CIDE (Cell-death Inducing DNA fragmentation factor-α-like Effector)-A and CIDE-C mRNA transcripts indicative of concurrent stimulation of lipolysis and lipogenesis. Finally GCs reduced insulin receptor phosphorylation, and Akt protein levels. CONCLUSIONS: High dose GC administration to humans leads to pro-lipolytic alterations of CGI-58, G0S2 and ANGPTL4 mRNA transcripts, increases PKA signaling to lipolysis and inhibits the insulin signal in adipose tissue. The increased CIDE-A and CIDE-C mRNA levels suggest concomitant stimulation of lipolysis and lipid storage.

Molecular and cellular adaptations to exercise training in skeletal muscle from cancer patients treated with chemotherapy.

BACKGROUND: A growing body of evidence suggests that exercise training has beneficial effects in cancer patients. The aim of the present study was to investigate the molecular basis underlying these beneficial effects in skeletal muscle from cancer patients. METHODS: We investigated expression of selected proteins involved in cellular processes known to orchestrate adaptation to exercise training by western blot. Skeletal muscle biopsies were sampled from ten cancer patients before and after 4-7 weeks of ongoing chemotherapy, and subsequently after 10 weeks of continued chemotherapy in combination with exercise training. Biopsies from ten healthy matched subjects served as reference. RESULTS: The expression of the insulin-regulated glucose transporter, GLUT4, increased during chemotherapy and continued to increase during exercise training. A similar trend was observed for ACC, a key enzyme in the biosynthesis and oxidation of fatty acids, but we did not observe any changes in other regulators of substrate metabolism (AMPK and PDH) or mitochondrial proteins (Cyt-C, COX-IV, SDHA, and VDAC). Markers of proteasomal proteolysis (MURF1 and ATROGIN-1) decreased during chemotherapy, but did not change further during chemotherapy combined with exercise training. A similar pattern was observed for autophagy-related proteins such as ATG5, p62, and pULK1 Ser757, but not ULK1 and LC3BII/LC3BI. Phosphorylation of FOXO3a at Ser318/321 did not change during chemotherapy, but decreased during exercise training. This could suggest that FOXO3a-mediated transcriptional regulation of MURF1 and ATROGIN-1 serves as a mechanism by which exercise training maintains proteolytic systems in skeletal muscle in cancer patients. Phosphorylation of proteins that regulate protein synthesis (mTOR at Ser2448 and 4EBP1 at Thr37/46) increased during chemotherapy and leveled off during exercise training. Finally, chemotherapy tended to increase the number of satellite cells in type 1 fibers, without any further change during chemotherapy and exercise training. Conversely, the number of satellite cells in type 2 fibers did not change during chemotherapy, but increased during chemotherapy combined with exercise training. CONCLUSIONS: Molecular signaling cascades involved in exercise training are disturbed during cancer and chemotherapy, and exercise training may prevent further disruption of these pathways. TRIAL REGISTRATION: The study was approved by the local Scientific Ethics Committee of the Central Denmark Region (Project ID: M-2014-15-14; date of approval: 01/27/2014) and the Danish Data Protection Agency (case number 2007-58-0010; date of approval: 01/28/2015). The trial was registered at http//www.clinicaltrials.gov (registration number: NCT02192216; date of registration 07/17-2014).

Alterations in intracellular potassium concentration by HIV-1 and SIV Nef.

BACKGROUND: HIV-1 mediated perturbation of the plasma membrane can produce an alteration in the transmembrane gradients of cations and other small molecules leading to cell death. Several HIV-1 proteins have been shown to perturb membrane permeability and ion transport. Xenopus laevis oocytes have few functional endogenous ion channels, and have proven useful as a system to examine direct effects of exogenously added proteins on ion transport. RESULTS: HIV-1 Nef induces alterations in the intracellular potassium concentration in CD4+ T-lymphoblastoid cells, but not intracellular pH. Two electrode voltage-clamp recording was used to determine that Nef did not form ion channel-like pores in Xenopus oocytes. CONCLUSION: These results suggest that HIV-1 Nef regulates intracellular ion concentrations indirectly, and may interact with membrane proteins such as ion channels to modify their electrical properties.

Insulin inhibits autophagy signaling independent of counterregulatory hormone levels but does not affect the effects of exercise.

Acute exercise increases autophagic signaling through Unc-51 like kinase-1 (ULK1) in human skeletal muscle during both anabolic and catabolic conditions. The aim of the present study was to investigate if changes in ULK1 Ser555 phosphorylation during exercise are reflected by changes in phosphorylation of a newly identified ULK1 substrate (ATG14 Ser29) and to elucidate the involvement of circulatory hormones in the regulation of autophagy in human skeletal muscle. We show that 1 h of cycling exercise increases ATG14 Ser29 phosphorylation during both hyperinsulinemic euglycemic and euinsulinemic euglycemic conditions. This could suggest that counterregulatory hormones stimulate autophagy in skeletal muscle, as circulating concentrations of these hormones are highly elevated during exercise. Furthermore, ATG14 Ser29 correlated positively with ULK1 phosphorylation, suggesting that ULK1 Ser555 (activating site) phosphorylation reflects ULK1 kinase activity. In a separate series of experiments, we show that insulin stimulates ULK1 phosphorylation at Ser757 (inhibitory site) in both hypoglycemic and euglycemic conditions, suggesting that counterregulatory hormones (such as epinephrine, norepinephrine, growth hormone, and glucagon) have limited effects on autophagy signaling in human skeletal muscle. In conclusion, 1 h of cycling exercise increases phosphorylation of ATG14 at Ser29 in a pattern that mirrors ULK1 phosphorylation at Ser555. Moreover, insulin effects on autophagy signaling in human skeletal muscle are independent of hypoglycemic and euglycemic conditions.NEW & NOTEWORTHY Autophagy signaling is regulated in a hierarchical order by exercise, insulin, and counterregulatory hormones. Exercise-induced autophagy signaling is stimulated by local factors in skeletal muscle rather than circulatory hormones. Unc-51 like kinase-1 (ULK1) phosphorylation at Ser555 reflects ULK1 kinase activity.

Characterization of Nanodiamond-based anti-HIV drug Delivery to the Brain.

Human Immunodeficiency Virus Type 1 (HIV-1) remains one of the leading causes of death worldwide. Present combination antiretroviral therapy has substantially improved HIV-1 related pathology. However, delivery of therapeutic agents to the HIV reservoir organ like Central nervous system (CNS) remains a major challenge primarily due to the ineffective transmigration of drugs through Blood Brain Barrier (BBB). The recent advent of nanomedicine-based drug delivery has stimulated the development of innovative systems for drug delivery. In this regard, particular focus has been given to nanodiamond due to its natural biocompatibility and non-toxic nature-making it a more efficient drug carrier than other carbon-based materials. Considering its potential and importance, we have characterized unmodified and surface-modified (-COOH and -NH2) nanodiamond for its capacity to load the anti-HIV-1 drug efavirenz and cytotoxicity, in vitro. Overall, our study has established that unmodified nanodiamond conjugated drug formulation has significantly higher drug loading capacity than surface-modified nanodiamond with minimum toxicity. Further, this nanodrug formulation was characterized by its drug dissolution profile, transmigration through the BBB, and its therapeutic efficacy. The present biological characterizations provide a foundation for further study of in-vivo pharmacokinetics and pharmacodynamics of nanodiamond-based anti-HIV drugs.