Flexible care in breast cancer.

Treatment of patients with cancer in hospitals or clinics is resource-intensive and imposes a burden on patients. 'Flexible care' is a term that can be used to describe treatment administered outside the oncology ward, oncological outpatient clinic or office-based oncologist setting. Programmes that reduce travel burden by bringing cancer treatment to the patient's home, workplace or closer to the patient's home, in the form of satellite clinics or mobile cancer units, expand treatment capacity and are well received. Clinical trial data show that, compared with intravenous administration, subcutaneous (s.c.) administration of trastuzumab is preferred by patients with breast cancer (BC), saves healthcare professionals' (HCPs) time, reduces drug preparation and administration time and reduces direct and indirect costs. As such, s.c. trastuzumab is well suited to flexible care. The results of a Belgian study (BELIS) show that home administration of s.c. trastuzumab is feasible and preferred by patients with BC. Numerous programmes and pilot studies in Europe show that s.c. trastuzumab can be administered effectively in the patient's home, in primary care settings or local hospitals. Such programmes require planning, training, careful patient selection and technology to link patients, caregivers and specialists in oncology clinics. Once these elements are in place, flexible care offers patients with BC a choice of how treatment may be delivered and lead to improved quality of life, while reducing pressure on HCPs and hospitals. The concept of flexible care is particularly relevant amid the COVID-19 pandemic where guidelines have been developed encouraging remote care.

SU-E-I-38: Optimizing CT Slice Width Measurements.

PURPOSE: For almost four decades bead and wire ramps have been used in the evaluation of slice width as part of QA testing for CT scanners. While each of these approaches have been recognized and accepted as reliable, in this paper we investigate the differences, advantages and limitations of these tools. Moreover, we study the effect of varying the field of view (FOV) and focal spot size. METHODS: The Catphan® 700 phantom includes two pairs of bead ramps (coarse and fine) and a pair of wire ramps in the same module providing an ideal setting for comparing bead ramps and wire ramps. The phantom was scanned using three devices from two different manufacturers. The data set consisted of 428 slices of 0.5,1,2,4,8 and 10 mm thickness. For the study of FOV and focal spot, 512 slices from the Catphan® 600 were acquired. All images were analyzed using Image Owl Catphan® QA software. RESULTS: For 0.5mm slices, bead ramps gave more accurate and precise (lower variance) estimation of the thickness than wire ramps. For 2-4 mm slices, the two approaches performed on equal terms while for the thickest slices (8 and 10mm), the wires gave more precise results. For thin slices, a small FOV (100mm) gave better results and lower spread than a large FOV (240mm). Finally, a small focal spot gave significantly better results than a large one using wire ramps for 0.5 and 1mm slices. CONCLUSIONS: For measuring thin slices, the use of bead ramps, with adequately small FOV and a small focal spot should be advised. For measuring thick slices, wire ramps will give less variability although bead ramps give equally accurate results on average. Funding provided by The Phantom Laboratory, Incorporated and Image Owl, Incorporated.

SU-E-I-37: A Renewed Look at CT Numbers as Encountered in the Diagnostic and Therapeutic Range.

PURPOSE: With the development of RT treatment planning systems that use the relationship between CT number and Electron Density (ED), rather than the Linear Attenuation Coefficient (LAC) of a given material, a number of authors have pointed out the divergence between the linearity of CT numbers vs. LAC in the diagnostic range compared to the relationship between the CT number and ED. This paper will review the differences and similarities, and describe a new set of phantom test objects and automated software that can be used to automatically assess both scales. In particular the relative importance of atomic number (Z) and the relative impact of Coherent vs. Incoherent effects at high Z levels will be evaluated. METHODS: A newly developed phantom (Catphan® 700) has an expanded set of sensitometric samples of known density, electron density, and chemical composition (Z effective). CT scans of the phantom were obtained at various energies (kVp) and the measured CT numbers were compared to the known physical characteristics mentioned above. Regressions between measured CT numbers, linear attenuation coefficients (with and without coherent scattering effects), and electron density for the materials were performed. Effects of different materials and the inclusion of coherent scattering on linearity scale and effective energy were established. RESULTS: The linearity scale and effective energy are shown to be dependent on the selection of materials scanned and the inclusion/exclusion of coherent scattering effects in the linear attenuation coefficients. Electron density deviates significantly from a linear relationship with CT number. CONCLUSIONS: Caveats accompanying high Z materials are reinforced regarding application to the RT relationship between CT number and electron density. Interesting results were obtained for the influence of coherent vs. incoherent scattering, which appears to be important as the number of slices and scanning volume increases in CT. Funding provided by The Phantom Laboratory, Incorporated and Image Owl, Incorporated.

WE-G-217BCD-11: A New Phantom to Study Combined Effects of In-Plane (x,y) and Z Axis Resolution for 3-D Imaging.

PURPOSE: The aim of this work is to develop phantoms that can be used to sample the radial and 3D properties of a CT image, including in-plane (x,y) and z-axis information. The Phantom is amenable to mathematical analysis of the x, y, and z axis resolution properties separately and combined. METHODS: A periodic pattern of a pair of opposed (30°) angled ramps is configured to produce a waveform profile across the CT image. A perfect CT image (with no loss of resolution) of the test object would produce a consistent geometric pattern of the intersection of a line with the pair of angled ramps. However, due to the finite resolution (x, y and z), the CT waveform profile will not yield the perfect profile; rather it will be influenced by slice thickness, and in-plane resolution (PSF, MTF), as well as noise limitations, and other sources of non-uniformity such as beam hardening etc. Various characteristics of the waveform profile including, amplitude, frequency, and slope (rate of climb) of the peaks, can be studied using mathematical analysis such as the Fourier transform. It will be shown how these performance characteristics are encoded in the wave pattern. RESULTS: The waveform profiles are visually examined and mathematically analyzed, to demonstrate the effect of Slice Thickness (z axis) and changes of In-Plane (x,y) Resolution and non-uniformity across the image field; moreover, the harmonic analysis of the waveform is used to predict, either the in-plane resolution (MTF), or the z-axis MTF when one of the two is already known. CONCLUSIONS: The Wave pattern phantom offers a way to consider 3-D imaging characteristics of a CT scanner by scanning a single repetitive test object that encodes both in-plane resolution and z-axis resolution and also offers a way to study non-uniformity effects throughout the CT plane (volume). DJG is a consultant to The Phantom Laboratory and Image OWL, Salem, NY. Funding of other authors is supplied by Image OWL Salem, NY.

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

Report and recommendations of the workshop of the European Centre for the Validation of Alternative Methods for Drug-Induced Cardiotoxicity.

Cardiotoxicity is among the leading reasons for drug attrition and is therefore a core subject in non-clinical and clinical safety testing of new drugs. European Centre for the Validation of Alternative Methods held in March 2008 a workshop on "Alternative Methods for Drug-Induced Cardiotoxicity" in order to promote acceptance of alternative methods reducing, refining or replacing the use of laboratory animals in this field. This review reports the outcome of the workshop. The participants identified the major clinical manifestations, which are sensitive to conventional drugs, to be arrhythmias, contractility toxicity, ischaemia toxicity, secondary cardiotoxicity and valve toxicity. They gave an overview of the current use of alternative tests in cardiac safety assessments. Moreover, they elaborated on new cardiotoxicological endpoints for which alternative tests can have an impact and provided recommendations on how to cover them.

A cell-based sensor system for toxicity testing using multiwavelength fluorescence spectroscopy.

A novel cell-based fluorometric sensor system for toxicity monitoring is described, which uses functional spontaneously contracting cardiomyocytes (HL-1 cell line) as the biological recognition element. Based on these highly specialized cells, it has the potential of providing a sensitive and relevant analytical in vitro toxicity testing method. The system was configured by propagating the surface-attaching HL-1 cardiomyocytes in the wells of a 96-well microtiter plate and connecting the plate via an optical fiber to a fluorescence spectrometer capable of excitation-emission matrix scanning. The fluorescence data were analyzed using a conventional spectral analysis software program. The performance of the system for detection of general cytotoxicity to the cells was evaluated using three well-known drugs: verapamil, quinidine, and acetaminophen. The dose-response curves were assessed and the EC(50) values were determined (0.10+/-0.007, 0.23+/-0.025, and 12.32+/-2.40 mM, respectively). Comparison with in vitro and in vivo reference data for the drugs showed good correlations, suggesting that this cell-based sensor system could be a useful tool in pharmacological in vitro drug testing.

A novel approach to data processing of the QT interval response in the conscious telemetered beagle dog.

INTRODUCTION: Drug-induced QT interval prolongation may lead to ventricular arrhythmias. The aim of the study was to optimize QT interval data processing to quantify drug-induced QT interval prolongation in the telemetry instrumented conscious dog model. METHODS: The test substances cisapride, dofetilide, haloperidol, and terfenadine and corresponding vehicles were given to male and female beagle dogs during two consecutive 90-min intravenous infusions. Cardiovascular parameters were recorded for 24 h and exposure to the drugs was measured. The delayed response in the QT interval after an abrupt change in heart rate was investigated. Eight mathematical models to describe the QT interval-heart rate relationship were compared and different sets of covariates were used to quantify the drug-induced effect on the QT interval. RESULTS: After an abrupt decrease in heart rate, a 75% adaptation of the QT interval was reached after 54+/-9 s. A linear model was preferred to correct the drug-induced effect on the QT interval for heart rate, vehicle effect, serial correlation, plasma concentration and time of day. All test substances significantly prolonged the QT interval. DISCUSSION: To optimize the processing of QT interval data, the delay in QT interval response after an abrupt change in heart rate should be considered. The QT interval-heart rate relationship and vehicle response were individual-specific and corrections were therefore made individually. When estimating the drug-induced effect on the QT interval it is considered advantageous to use plasma concentration as a covariate, as well as adjusting for vehicle effect and serial correlation in measurements. The conscious dog model detected significant increases in the QT interval for all test substances investigated.

A polymorphism in the adiponectin gene influences adiponectin expression levels in visceral fat in obese subjects.

OBJECTIVE: Reduced serum adiponectin levels have been found in obesity and type 2 diabetes and variations in the adiponectin gene (APM1) have been associated with type 2 diabetes and features of the metabolic syndrome in different populations. STUDY DESIGN: Here, we investigated the expression of APM1 in adipose tissue and studied the relationship between variation in APM1 expression, the APM1 G276T polymorphism, the common PPARG Pro12Ala polymorphism and clinical features of 36 morbidly obese (body mass index (BMI) 41.5 +/- 4.9 kg/m2) nondiabetic subjects. RESULTS: APM1 mRNA expression in visceral fat was correlated with serum adiponectin levels (r = 0.54, P = 0.012). In visceral, but not in subcutaneous, adipose tissue APM1 mRNA level was 38% higher among carriers of the APM1 G276T T allele (G/T and T/T) than among carriers of the G/G genotype (0.91 +/- 0.06 for G/T and T/T carriers vs 0.66 +/- 0.09 for G/G carriers, P = 0.013). Carriers of the T allele also had significantly higher body fat percent compared to G/G carriers (65 +/- 6 vs 56 +/- 10%, P = 0.011). CONCLUSION: Our results indicate that genetic variation in APM1 influences the expression of the gene in visceral adipose tissue and suggest a potential role for such variation in regulation of body fat accumulation in obese subjects.

Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption.

To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression.

Norepinephrine but not hypoxia stimulates HIF-1alpha gene expression in brown adipocytes.

The cellular response to hypoxic stress is mainly mediated via activation of the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). In the present study, the sympathetically controlled brown adipose tissue was used to investigate the effect of norepinephrine on HIF-1alpha gene expression. Norepinephrine increased HIF-1alpha mRNA levels in cultured brown adipocytes, whereas the hypoxia-mimic cobalt was without effect. Cold exposure of mice increased HIF-1alpha gene expression in brown adipose tissue. In UCP1-ablated mice, which are incapable of inducing thermogenic oxygen consumption in brown adipose tissue, cold exposure generated a significantly higher elevation of HIF-1alpha mRNA levels than in wild-type. These results demonstrate that cold-induced HIF-1alpha gene expression is independent of thermogenic oxygen consumption leading to hypoxia, but is consistent with a norepinephrine regulation of HIF-1alpha gene expression. Thus, by elevating HIF-1alpha gene expression, norepinephrine may mediate an increased potential to respond to hypoxia in brown adipose tissue and possibly in other tissues.

Women and men have similar amounts of liver and intra-abdominal fat, despite more subcutaneous fat in women: implications for sex differences in markers of cardiovascular risk.

AIMS/HYPOTHESIS: Fat accumulation in the liver has been shown to be closely correlated with hepatic insulin resistance and features of insulin resistance, also independently of body weight. It remains to be established how fat in the liver correlates with that in other depots, and whether any association differs between men and women. METHODS: Liver fat (assessed using proton spectroscopy), intra-abdominal and subcutaneous fat (measured using magnetic resonance imaging) and markers of insulin resistance, including serum adiponectin, were determined in 132 non-diabetic subjects: 66 men (age 41+/-1 years) and 66 women (age 42+/-1 years). RESULTS: Although the women had almost twice as much subcutaneous fat as the men (5045+/-207 vs 2610+/-144 cm3, p<0.0001), amounts of intra-abdominal fat (1305+/-80 vs 1552+/-111 cm3, NS) and liver fat (6.7+/-0.8 vs 8.9+/-1.2%, NS) were similar. In this study, no sex differences were observed with respect to serum insulin, adiponectin, triglyceride and HDL cholesterol concentrations. Of all measures of body composition, liver fat was best correlated with serum insulin (r=0.58, p<0.001), with no difference observed between men and women. Serum adiponectin was inversely correlated with liver fat content (r=-0.21, p<0.05). Multiple linear regression analysis revealed that intra-abdominal fat was significantly associated with liver fat, independently of serum adiponectin and subcutaneous fat. Liver fat, but not intra-abdominal fat, significantly explained the variation in serum insulin concentrations. CONCLUSIONS/INTERPRETATION: Intra-abdominal fat is independently associated with liver fat, whereas subcutaneous fat is not. Liver fat, but not intra-abdominal fat, is independently associated with serum insulin. Men and women with similar amounts of intra-abdominal and liver fat do not exhibit sex differences in markers of insulin resistance (serum insulin, triglycerides, HDL cholesterol and adiponectin).

Parallel manifestation of insulin resistance and beta cell decompensation is compatible with a common defect in Type 2 diabetes.

AIMS/HYPOTHESIS: The aim of the study was to evaluate the relationship between insulin sensitivity, beta cell function and glucose tolerance, and its dependence on variants in the newly identified Type 2 diabetes susceptibility gene, calpain-10 ( CAPN10). METHODS: We studied 203 men of the same age but with varying degrees of glucose tolerance. These men participated in (i) an oral glucose tolerance test, (ii) a euglycaemic clamp combined with indirect calorimetry and infusion of [3-(3)H]-glucose and (iii) a stepwise assessment of acute insulin response to arginine (AIR) at three different glucose concentrations (fasting, 14 and 28 mmol/l). RESULTS: There was a linear increase in NEFA levels ( p<0.0005) and WHR ( p<0.0005) and decrease in glucose uptake due to a reduction in glucose storage over the entire range of glucose tolerance ( r=-0.404; p<0.005). No increase in endogenous glucose production (EGP) was seen until patients had manifest diabetes. However, when EGP was expressed relative to fasting insulin concentrations, there was a linear deterioration of basal hepatic insulin sensitivity ( r=-0.514; p<0.005). The AIR followed a bell-shaped curve with an initial rise and subsequent decrease. However, AIR adjusted for insulin sensitivity (disposition index) showed a linear decrease with increasing glucose concentrations ( r=-0.563; p<0.001) starting already in subjects with normal glucose tolerance. There was an inverse correlation between increase in WHR and NEFA and peripheral as well as hepatic insulin sensitivity. Subjects with the genotype combination of CAPN10 consisting of SNP44 TT and SNP43 GG genotypes had significantly lower insulin-stimulated glucose uptake than carriers of the other genotype combinations (5.3+/-0.4 vs 7.2+/-0.4 mg.ffm kg(-1).min(-1).mU.l(-1); p<0.005). CONCLUSIONS/INTERPRETATION: We conclude that the pre-diabetic state is characterised by a similar linear deterioration of peripheral and hepatic insulin sensitivity as beta cell function and that variants in the CAPN10 gene modify this relationship. These findings are compatible with a common defect in muscle, liver and beta cells in the pathogenesis of Type 2 diabetes.

Characterization of the human skeletal muscle glycogen synthase gene (GYS1) promoter.

BACKGROUND: Impaired activation of the human skeletal muscle glycogen synthase by insulin is typical for type 2 diabetic patients. Regulation of glycogen synthase occurs mainly by phosphorylation/dephoshorylation but little is known whether there also is transcriptional regulation. Therefore we studied transcriptional regulation of the human skeletal muscle glycogen synthase gene (GYS1) and evaluated the effects of insulin and forskolin on the promoter activity. METHODS: Seven promoter fragments were expressed in C2C12 myoblasts and myotubes and in HEK293 cells, and the luciferase assay was used to determine transcriptional activity. RESULTS: The highest luciferase activity, 350-fold of the promoterless vector, was obtained with nucleotides -692 to +59 in myotubes (P < 0.001), while the nucleotides -250 to +59 provided the highest, 45-fold, activity in the HEK293 cells (P < 0.001). Longer promoter constructs (nucleotides -971, -1707 and -2158 to +59, respectively) had low promoter activity in both cell types. Forskolin treatment for 24 h resulted in approximately 30% decreased promoter activity in myotubes (P < 0.05). Insulin treatment for 0.5-3 h did not increase GYS1 promoter activity; instead the activity was slightly but significantly decreased after 24 h in myotubes (P < 0.005). CONCLUSIONS: From our results we conclude that basal GYS1 promoter activity is obtained from the first 250 nucleotides of the promoter, while the nucleotides -692 to -544 seem to be responsible for muscle-specific expression, and nucleotides -971 to -692 for negative regulation. In myotubes, the GYS1 promoter was sensitive to negative regulation by forskolin, whereas insulin did not increase GYS1 transcription.

Vaccinations may induce diabetes-related autoantibodies in one-year-old children.

Vaccinations have been discussed as one among many environmental candidates contributing to the immune process that later may lead to type 1 diabetes. ABIS (All Babies in Southeast Sweden) is a prospective cohort study following a nonselected birth cohort of general population. In a randomly selected sample collection from 4400 children, GADA and IA-2A have been determined at the age of 1 year. The information on vaccinations was collected from questionnaires answered by the parents and was related to beta cell autoantibodies. When studying the induction of autoantibodies using the autoantibody level of 90th percentile as cutoff level, hemophilus influenza B (HIB) vaccination appeared to be a risk factor for IA-2A [OR 5.9 (CI 1.4-24.4; p = 0.01)] and for GADA [OR 3.4 (CI 1.1-10.8; p = 0.04)] in logistic regression analyses. Furthermore, the titers of IA-2A were significantly higher (p < 0.01 in Mann-Whitney test) in those children who had got HIB vaccination. When 99th percentile was used as cutoff to identify the children at risk of type 1 diabetes, BCG vaccination was associated with increased prevalence of IA-2A (p < 0.01). We conclude that HIB vaccination may have an unspecific stimulatory polyclonal effect increasing the production of GADA and IA-2A. This might be of importance under circumstances when the beta cell-related immune response is activated by other mechanisms.

A novel pathway for adrenergic stimulation of cAMP-response-element-binding protein (CREB) phosphorylation: mediation via alpha1-adrenoceptors and protein kinase C activation.

Because of the central role of adrenergic mechanisms in the expression of crucial genes during brown adipocyte differentiation, we examined the activation (phosphorylation) of CREB (cAMP-response-element-binding protein) in mouse brown adipocytes in primary culture. We found that noradrenaline ('norepinephrine') stimulated CREB phosphorylation rapidly (maximum effect in < or =5 min with slow decay) and efficiently (EC(50), 6 nM). The increase in CREB phosphorylation coincided with increased expression of an artificial cAMP-response-element-containing reporter construct. CREB phosphorylation was partly inhibitable, both by the beta-adrenergic antagonist propranolol and by the alpha(1)-adrenergic antagonist prazosin. Adenylate cyclase hyperactivation (by forskolin) could stimulate CREB phosphorylation to the same extent as noradrenaline. The alpha(1)-adrenergic agonist cirazoline also increased CREB phosphorylation. An increase in intracellular [Ca(2+)] had, however, no effect, but protein kinase C activation by PMA was a potent stimulator. The cirazoline-stimulated (alpha(1)-adrenergic) CREB phosphorylation was inhibited by a desensitizing pretreatment with PMA, demonstrating that the alpha(1)-stimulation was mediated via protein kinase C activation; neither Src nor extracellular-signal-regulated kinases 1 and 2 activation was involved in the signalling process. We conclude that CREB phosphorylation in brown adipocytes is mediated not only through the classical beta-adrenergic/cAMP pathway but also through a novel alpha(1)-adrenergic/protein kinase C/CREB pathway, which has not been described previously in any tissue.

Norepinephrine specifically stimulates ribonucleotide reductase subunit R2 gene expression in proliferating brown adipocytes: mediation via a cAMP/PKA pathway involving Src and Erk1/2 kinases.

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.

Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of beta(3)-adrenergic signalling rather than beta(3)-adrenoceptor antagonism by H89.

Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC(50) of approx. 40 microM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 microM). However, H89 has been reported to be an adrenergic antagonist on beta(1)/beta(2)-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via beta(3)-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on beta(3)-ARs. It was found that EC(50) values for beta(3)-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC(50) of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective beta(3)-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on beta(3)-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via beta(3)-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis.

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.

Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta -adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2.

To identify the signaling pathway that mediates the adrenergic stimulation of the expression of the gene for vascular endothelial growth factor (VEGF) during physiologically induced angiogenesis, we examined mouse brown adipocytes in primary culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced VEGF expression 3-fold, in a dose- and time-dependent manner (EC(50) approximately 90 nm). Also, the hypoxia-mimicking agent cobalt, as well as serum and phorbol ester, induced VEGF expression, but the effect of NE was additive to each of these factors, implying that a separate signaling mechanism for the NE-mediated induction was activated. The NE effect was abolished by propranolol and mimicked by isoprenaline or BRL-37344 and was thus mediated via beta-adrenoreceptors. The NE-induced VEGF expression was fully cAMP mediated, an effect which was inhibited by H-89 and thus was dependent on protein kinase A activity. Involvement of other adrenergic signaling pathways (alpha(1)-adrenoreceptors, Ca(2+), protein kinase C, alpha(2)-adrenoreceptors, and pertussis toxin-sensitive G(i)-proteins) was excluded. The specific inhibitor of Src tyrosine kinases, PP2, markedly reduced the stimulation by NE, which demonstrates that a cAMP-dependent Src-mediated pathway is positively connected to VEGF expression. However, inhibition of Erk1/2 MAP kinases by PD98059 was without effect. NE did not prolong VEGF mRNA half-life and its effect was thus transcriptional, and was independent of protein synthesis. These results demonstrate that adrenergic stimulation, through beta-adrenoreceptor/cAMP/protein kinase A signaling, recruits a pathway that branches off from the NE-activated Src-Erk1/2 cascade to enhance transcription of the VEGF gene.