Effect of stress on the chronotropic and inotropic responses to ß-adrenergic agonists in isolated atria of KOß2 mice.

Altered sensitivity to the chronotropic and inotropic effects of catecholamines and reduction in ß1/ß2-adrenoceptor (ß1/ß2-AR) ratio were reported in failing and in senescent human heart, as well as in isolated atria and ventricle of rats submitted to stress. This was due to downregulation of ß1-AR with or without up-regulation of ß2-AR. AIMS: To investigate the stress-induced behavior of ß1-AR in the heart of mice expressing a non-functional ß2-AR subtype. The guiding hypothesis is that the absence of ß2-AR signaling will not affect the behavior of ß1-AR during stress and that those are independent processes. MATERIALS AND METHODS: The chronotropic and inotropic responses to ß-AR agonists in isolated atria of stressed mice expressing a non-functional ß2-AR were analyzed. The mRNA and protein expressions of ß1- and ß2-AR were also determined. KEY FINDINGS: No deaths were observed in mice under stress protocol. Atria of stressed mice displayed reduced sensitivity to isoprenaline compared to the controls, an effect that was abolished by the ß2- and ß1-AR antagonists 50 nM ICI118,551 and 300 nM CGP20712A, respectively. Sensitivity and maximum response to the ß-agonists dobutamine and salbutamol were not altered by stress or ICI118,551. The responses to dobutamine and salbutamol were prevented by CGP20712A. The expression of ß1-AR was reduced at protein levels. SIGNIFICANCE: Collectively, our data provide evidence that the cardiac ß2-AR is not essential for survival in a stressful situation and that the stress-induced reduction of ß1-AR expression was independent of the ß2-AR presence.

Synergistic electrocatalytic activity of In2O3@FMWCNTs nanocomposite for electrochemical quantification of dobutamine in clinical patient blood and in injection dosage form.

Dobutamine (DBT) is a sympathomimetic amine drug that was designed as an inotropic agent for use in congestive heart failure. Hence, there was an impetus to develop a rapid and accurate method for monitoring the concentration of DBT within clinical samples. To address this critical need, a novel In2O3 and functionalized multi-walled carbon nanotubes nanocomposite (In2O3@FMWCNTs) was successfully prepared and applied in an electrochemical sensor to detect DBT. The resulting sensor displayed electrocatalytic toward the oxidation of DBT, which attributed to the synergistic effect of In2O3 and FMWCNTs. Electrochemical impedance spectroscopy (EIS) studies revealed that the smaller charge transfer resistance value (Rct) was observed at In2O3@FMWCNTs modified glassy carbon spherical (GCS) paste electrode (PE) as compared to that of In2O3NPs/GCSPE, FMWCNTs/GCSPE and GCSPE, which authenticates its good conductivity. Furthermore, the calculated value of standard rate constant (ks) for the modified electrode demonstrates the fast electron transfer between DBT and the electrode surface. The fabricated electrochemical sensor indicated high selectivity and sensitivity for DBT determination over the oxidation of uric acid and ascorbic acid. The limit of detection of DBT at In2O3@FMWCNTs/GCSPE was found to be 1.42 × 10-10 M. The proposed sensor is effectively used for the detection of DBT in biological fluids, clinical patient blood and in injection dosage form.

Embryonic developmental oxygen preconditions cardiovascular functional response to acute hypoxic exposure and maximal ß-adrenergic stimulation of anesthetized juvenile American alligators (Alligator mississippiensis).

The effects of the embryonic environment on juvenile phenotypes are widely recognized. We investigated the effect of embryonic hypoxia on the cardiovascular phenotype of 4-year-old American alligators (Alligator mississippiensis). We hypothesized that embryonic 10% O2 preconditions cardiac function, decreasing the reduction in cardiac contractility associated with acute 5% O2 exposure in juvenile alligators. Our findings indicate that dobutamine injections caused a 90% increase in systolic pressure in juveniles that were incubated in 21% and 10% O2, with the 10% O2 group responding with a greater rate of ventricular relaxation and greater left ventricle output compared with the 21% O2 group. Further, our findings indicate that juvenile alligators that experienced embryonic hypoxia have a faster rate of ventricular relaxation, greater left ventricle stroke volume and greater cardiac power following ß-adrenergic stimulation, compared with juvenile alligators that did not experience embryonic hypoxia. When juveniles were exposed to 5% O2 for 20 min, normoxic-incubated juveniles had a 50% decline in left ventricle maximal rate of pressure development and maximal pressure; however, these parameters were unaffected and decreased less in the hypoxic-incubated juveniles. These data indicate that embryonic hypoxia in crocodilians alters the cardiovascular phenotype, changing the juvenile response to acute hypoxia and ß-adrenergic stimulation.

Effect of gender on the prognostic value of dobutamine stress myocardial contrast echocardiography.

BACKGROUND: Dobutamine stress contrast echo (DSCE) has a well-established prognostic value in the context of coronary artery disease (CAD). However, data regarding its prognostic capability separately in men and women are scarce. The aim of the current study was to assess gender-related differences in the prognostic performance of DSCE. METHODS: DSCE was performed in 2645 consecutive patients, who were classified into two groups depending on gender. Follow-up lasted 57.1±10.1 months. End points included all-cause mortality, cardiac death, late revascularization, and hospitalizations. Survival analysis was performed comparing men and women. RESULTS: Of the 2645 patients (59.3±8.7 years), 69.1% were men. DSCE was positive in 23.4% of male patients, while in females, the respective percentage was 14.3%. There was statistically significant difference between the two groups with regard to end point occurrence (11.6% vs. 6.1%, p<0.05). Multivariate analysis revealed that the DSCE response was the strongest predictor of adverse outcomes (Exp(B)=51.9, p<0.05) in both groups. The predictive model including DSCE results along with clinical data performed well without significant differences between males and females (C-index 0.93 vs. 0.87 respectively, p=NS). CONCLUSION: DSCE has a strong prognostic value for patients with known or suspected CAD, regardless of patient gender. This makes DSCE an attractive screening option for women in whom CAD assessment can be challenging.

Ventricular function and cardiac reserve in contemporary Fontan patients.

BACKGROUND/OBJECTIVE: Total cavopulmonary connection (TCPC) has been the preferred treatment for patients with univentricular hearts. Current TCPC-techniques are the intra-atrial lateral tunnel (ILT) and the extracardiac conduit (ECC). We aimed to determine ventricular function during rest and stress, and to compare results for both techniques and for left (LV) versus right ventricular (RV) dominance. METHODS: 99 patients, aged 12.5 ± 4.0 years underwent echocardiography and magnetic resonance imaging (MRI), and 69 patients underwent stress MRI. RESULTS: Echocardiography showed impaired systolic and diastolic function. MRI parameters were comparable between ILT and ECC at rest. During dobutamine there was a decrease in end-diastolic volume (EDVi) (91 ± 21 vs. 80 ± 20 ml/m(2) p<0.001). Ejection fraction (EF) and cardiac index (CI) during dobutamine were lower for ILT patients (59 ± 11 (ILT) vs. 64 ± 7% (ECC), p=0.027 and 4.2 ± 1.0 (ILT) vs. 4.9 ± 1.0L/min/m(2) (ECC), p=0.006), whereas other parameters were comparable. TEI-index was higher in ILT-patients (0.72 ± 0.27 (ILT) vs. 0.56 ± 0.22 (ECC), p=0.002). Diastolic function was frequently impaired in patients with a dominant RV (67% (RV) vs. 39% (LV), p=0.011). Patients with dominant LV's had smaller end-systolic volume (ESVi) (40 ± 13 (LV) vs. 47 ± 16 (RV) ml/m(2), p=0.030) and higher EF (55 ± 8 (LV) vs. 49 ± 9 ml/m(2) (RV), p=0.001) and contractility (2.3 ± 0.8 (LV) vs. 1.9 ± 0.7 mmHg/ml/m(2) (RV), p=0.050) during rest and higher EF during dobutamine (63 ± 8 (LV) vs. 58 ± 10 ml/m(2) (RV), p=0.043). CONCLUSION: Ventricular function is relatively well preserved in modern-day Fontan patients. With dobutamine stress there is a decrease in EDVi. ECC patients have higher CI and EF during stress. Patients with a dominant RV have lower systolic, including impaired contractility, and diastolic function.

Hydrogen profiles of dobutamine hydrochloride and fentanyl citrate solutions according to intravenous administration systems, temperature, and luminosity conditions.

Factors such as temperature, light exposure, drug concentration, ionic strength, time of infusion, and duration of drug association can influence the effectiveness of pharmacological solutions, which can compromise the solutions' quality, resulting in unstable solutions and drug incompatibility. The aim of this study was to determine the pH of solutions of dobutamine hydrochloride, fentanyl citrate, and their combination in 5% dextrose in water (D5W) under various light exposures and temperature conditions over time. The analysis was performed by measuring the pH of the substances in both pharmacological (commercial) preparations and in D5W under dark fluorescent light in the presence or absence of sunlight exposures, intravenous apparatus packaging (clear and amber burettes), and temperature (22°C and 37°C). Samples were collected immediately after preparation and after 0.5, 1, 2, 3, 4, and 24 hours of exposure to the various conditions; data were analyzed using mean standard deviations. Of the 260 pH values obtained, 50 (19.2%) were from commercial preparations and 210 (80.8%) from solutions exposed to various experimental conditions. Significant pH differences were found among the vials of the commercial preparation drugs. The largest pH value difference (0.88) was observed for fentanyl citrate, in which a pH increase of 0.88 (4.23 ± 0.62) was observed. The combination of drugs in D5W resulted in more acidic values than those of fentanyl citrate and of D5W and fentanyl citrate in D5W, but they were closer to what was observed for the solution of dobutamine hydrochloride in D5W. This solution was more acidic than fentanyl citrate diluted in D5W. The lower acidity of fentanyl citrate had a minor influence on the final pH of the combined drug solution in D5W. Under most conditions, the drug solutions kept at 22°C had pH values that were more acidic and less variable. Temperature was a major factor controlling the chemical behavior of the solutions analyzed. Analysis of chemical behavior in response to light exposure indicated that the solutions were more stable over time when kept in the dark.

Lipophilicity of amine neurotransmitter precursors, metabolites and related drugs estimated on various TLC plates.

The retention behavior for a series of amine neurotransmitters, their precursors, metabolites and structurally related drugs has been investigated in reversed-phase thin-layer chromatography using RP-18, RP-8, RP-2, CN and Diol stationary phases and mixtures of phosphate buffer and methanol as mobile phase. According to the computed lipophilicity of the neutral form of the investigated compounds, the most lipophilic compound is dobutamine (log P(N) = 3.78), while the less lipophilic is norepinephrine (log P(N) = -0.14). The experimental results also show dobutamine as the most lipophilic compound in the case of RP-18 and CN stationary phases (RM0(RP-18) = 1.58 and RM(CN) = 1.21), while RP-8 indicates norepinephrine as the less lipophilic one (RM0(RP8) = -0.70). Both the theoretical computation and the experimental data revealed that only one ionic form of the compounds prevails in the used chromatographic conditions. In addition, the evaluation of the experimental results showed that a similar chromatographic behavior could be assumed in the case of RP-18, RP-8 and CN stationary phases. Moreover, the mRM (mean of the RM values) and PC1/RM (scores of the first principal component) experimental lipophilicity indices showed a high correlation with the computed lipophilicity indices.

In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1-13C] and [2-13C]pyruvate.

Hyperpolarized (13)C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl-coenzyme A (acetyl-CoA). [1-(13)C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in (13)C-bicarbonate production after dichloroacetate (DCA) administration. With [1-(13)C]pyruvate, the (13)C label is released as (13 CO2 /(13)C-bicarbonate, and, hence, does not allow us to follow the fate of acetyl-CoA. Pyruvate labeled in the C2 position has been used to track the (13)C label into the TCA (tricarboxylic acid) cycle and measure [5-(13)C]glutamate as well as study changes in [1-(13)C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl-CoA in response to metabolic interventions of DCA-induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the (13)C labeling of [5-(13)C]glutamate, and a considerable increase in [1-(13)C]acetylcarnitine and [1,3-(13)C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2-(13)C]lactate, [2-(13)C]alanine and [5-(13)C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC-mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate.

Myocardial 123I-MIBG scintigraphy predicts an impairment in myocardial functional reserve during dobutamine stress in patients with idiopathic dilated cardiomyopathy.

PURPOSE: We investigated whether myocardial (123)I-metaiodobenzylguanidine ((123)I-MIBG) scintigraphy predicts impairment of myocardial functional reserve in response to dobutamine stress in patients with idiopathic dilated cardiomyopathy (DCM). METHODS: Forty DCM patients (LVEF 39 ± 12 %) underwent myocardial (123)I-MIBG scintigraphy, echocardiography, and cardiac catheterization. Myocardial (123)I-MIBG uptake was quantified as the delayed heart to mediastinum (H/M) ratio and washout rate (WR). Local denervation was evaluated on polar map images. LV dP/dt (max) and T(1/2) were determined from left ventricular pressure curves at baseline and during dobutamine infusion (15 µg/kg /min). Patients were classified into two groups as follows: group A comprised 21 patients showing a delayed H/M ratio of <1.9 (median value); group B comprised 19 patients showing a delayed H/M ratio of ≥ 1.9. RESULTS: The percentage change in heart rate (%HR), LV dP/dt (max) (%LV dP/dt (max)), and T (1/2) (%T (1/2)) from baseline to dobutamine stress were significantly more reduced in group A than in group B (39.3 ± 20.2 %, 55.2 ± 24.1 %, p < 0.01; 102.3 ± 46.3 %, 152.0 ± 72.3 %, p < 0.05; 38.7 ± 15.3 %, 46.9 ± 15.4 %, p < 0.05, respectively). No significant differences between the two groups were observed in the echocardiographic parameters or baseline cardiac catheterization parameters. Significant correlations were found between delayed H/M ratio and %HR (r = 0.35, p < 0.05), %LV dP/dt (max) (r = 0.45, p < 0.05) and %T (1/2) (r = 0.34, p < 0.05). Significant inverse correlations were also found between WR and %HR (r = -0.37, p < 0.05), %LV dP/dt (max) (r = -0.60, p < 0.0001), and %T (1/2) (r = -0.34, p < 0.05). SPECT images revealed enhanced denervation from the inferoposterior to anterior wall in accordance with the advancement of global denervation. CONCLUSION: Reduced (123)I-MIBG uptake and increased washout were related to impairment in adrenergic myocardial functional reserve in idiopathic DCM.

The structural basis for agonist and partial agonist action on a ß(1)-adrenergic receptor.

ß-adrenergic receptors (ßARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit ßARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) ß(1)-adrenergic receptor (ß(1)AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser(5.42) and Ser(5.46)), but partial agonists only interact with Ser(5.42) (superscripts refer to Ballesteros-Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

How many and which amino acids are responsible for the large activity differences between the highly homologous UDP-glucuronosyltransferases (UGT) 1A9 and UGT1A10?

The amino acid sequences of the human UDP-glucuronosyltransferases (UGTs) 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. For example, the regioselectivity in propranolol glucuronidation, the regioselectivity in dobutamine glucuronidation, and the glucuronidation rate of alpha- and beta-estradiol differ greatly between UGT1A9 and UGT1A10. To identify the residue responsible for the activity differences, we divided the N-terminal half of the two UGTs into five comparable segments by inserting four unique restriction sites at identical positions in both genes and constructing chimeras in which segments of UGT1A9 were individually replaced by the corresponding segments from UGT1A10. Activity analyses of the resulting mutants, 910A [1A10((1-83))/1A9((84-285))], 910B [1A9((1-83))/1A10((84-147))/1A9((148-285))], 910C [1A9((1-147))/1A10((148-181))/1A9((182-285))], 910D [1A9((1-181))/1A10((182-235))/1A9((236-285))], and 910E [1A9((1-235))/1A10((236-285))] indicated that more than one residue is responsible for the differences between UGT1A9 and UGT1A10. We next prepared four double chimeras, in which two of the above UGT1A9 segments were replaced simultaneously by the corresponding UGT1A10 segments. However, none of the double chimeras glucuronidated either estradiol, propranolol, or dobutamine at rates that resembled those of UGT1A10. On the other hand, studying the kinetics of 1-naphthol glucuronidation yielded more focused results, indicating that residues within segment B (84-147) contribute directly to the K(m) value for this substrate. Further mutagenesis and activity assays suggested that Phe117 of UGT1A9 participates in 1-naphthol binding. In addition, it appears that residues within segment C of the N-terminal domain, mainly at positions 152 and 169, contribute to the higher glucuronidation rates of UGT1A10.

Functional rescue of beta-adrenoceptor dimerization and trafficking by pharmacological chaperones.

Site-directed mutagenesis guided by evolutionary trace analysis revealed that substitution of V179 and W183 within a cluster of evolutionarily important residues on the surface of the fourth transmembrane domain of the beta(1)-adrenergic receptor (beta(1)AR) significantly reduced the propensity of the receptor to self-assemble into homodimers as assessed by bioluminescence resonance energy transfer in living cells. These results suggest that mutation of V179 and W183 result in conformational changes that reduce homodimerization either directly by interfering with the dimerization interface or indirectly by causing local misfolding that result in reduced self-assembly. However, the mutations did not cause a general misfolding of the beta(1)AR as they did not prevent heterodimerization with the beta(2)AR. The homodimerization-compromised mutants were significantly retained in the endoplasmic reticulum (ER) and could not be properly matured and trafficked to the cell surface. Lipophilic beta-adrenergic ligands acted as pharmacological chaperones by restoring both dimerization and plasma membrane trafficking of the ER-retained dimerization-compromised beta(1)AR mutants. These results clearly indicate that homodimerization occurs early in the biosynthetic process in the ER and that pharmacological chaperones can promote both dimerization and cell surface targeting, most likely by stabilizing receptor conformations compatible with the two processes.

Measurement of myocardial fatty acid esterification using [1-11C]palmitate and PET: comparison with direct measurements of myocardial triglyceride synthesis.

BACKGROUND: The purpose of the present study was to assess the accuracy of rates of myocardial fatty acid esterification (MFAE) obtained using positron emission tomography (PET). METHODS AND RESULTS: Sixteen dogs were studied after an overnight fast (FAST), during a euglycemic hyperinsulinemic clamp (CLAMP), or during infusion of intralipid (IL) or IL plus dobutamine (IL/DOB). MFAE was quantified using [1-(11)C]palmitate and PET and compared to the rate of triglyceride (TG) synthesis measured using [1-(13)C]palmitate and tissue sampling. Plasma free fatty acid (FFA) concentration varied approximately 20-fold across groups, with this variation in FFA availability accompanied by a approximately 20-fold range in TG synthesis. MFAE varied approximately 12-fold across groups, and was significantly correlated with TG synthesis (R = 0.80, P < .001). MFAE, however, was 3- to 4-fold higher than TG synthesis in FAST, CLAMP, and IL, but only approximately 50% higher when cardiac work was increased in IL/DOB, suggesting that MFAE reflects, in part, the incorporation of label into amino acids via TCA cycle exchange reactions. CONCLUSIONS: Changes in MFAE parallel changes in TG synthesis, at least in the basal state. Although the data need to be interpreted cautiously, such measurements should be useful for quantifying acute changes in FFA storage by the heart in various pathophysiological states.

Rest and low-dose dobutamine Tc-99m-mibi gated-SPECT for early prediction of left ventricular remodeling after a first reperfused myocardial infarction.

BACKGROUND: Left ventricular (LV) remodeling after myocardial infarction (MI) occurs frequently despite successful percutaneaous coronary intervention (PCI) but cannot be predicted by simple clinical parameters. METHODS AND RESULTS: This prospective study tested the value of rest and low-dose dobutamine (LDD) Tc-99m-mibi gated-SPECT for early prediction of LV remodeling in patients treated by PCI in the acute phase of a first MI. Infarct size, infarct severity, regional wall motion abnormality (RWMA), and wall thickening score (WTs) were assessed at rest and on LDD by SPECT 6 +/- 2 days after MI in 40 patients. LV remodeling was defined as 20% increase at 6 months in LV end-diastolic volume assessed by MRI. Infarct severity at rest showed the best predictive values for left remodeling (PPV: 86%, NPV: 88%, accuracy: 88%; AUC: 0.750). Functional parameters at neither rest nor LDD study further improved predictive values of the SPECT imaging. CONCLUSIONS: Infarct severity assessed by Tc-99m-sestamibi gated-SPECT performed in the subacute phase of a first STEMI predicts LV remodeling with high accuracy without incremental value nor of functional parameters nor of LDD. Therefore, our results suggest that LDD should not be used in this setting.

Comparison of dobutamine stress echocardiography, dobutamine SPECT, and adenosine SPECT myocardial perfusion imaging in patients with end-stage renal disease.

OBJECTIVES: We sought to assess and compare the diagnostic accuracy and prognostic value of dobutamine stress echocardiography (DSE), dobutamine SPECT, and adenosine SPECT myocardial perfusion imaging (MPI) in patients with end-stage renal disease (ESRD). BACKGROUND: The optimal stress imaging modality for patients with ESRD has not yet been determined. METHODS: Forty-nine patients with ESRD underwent DSE, dobutamine SPECT MPI, and adenosine SPECT MPI. The primary endpoint of the trial was concordance between stress tests with respect to the presence or absence of ischemia. RESULTS: Agreement on the presence or absence of ischemia between adenosine SPECT MPI and DSE was 69% (kappa = .25, P = NS). Agreement on the presence or absence of ischemia between adenosine and dobutamine SPECT MPI was 77% (kappa = .37, P = <.009). Summed stress scores for adenosine and dobutamine SPECT MPI studies were highly correlated (r = .9, P = <.0001). DSE and SPECT MPI results provided incremental prognostic information when added to clinical variables. CONCLUSIONS: There is moderate concordance between DSE and adenosine SPECT MPI in ESRD patients referred for stress testing. Interobserver agreement was higher for SPECT MPI compared to DSE. Based on these observations, the optimal approach for diagnosing severe coronary artery disease and assessing risk in patients with ESRD has yet to be determined, but appears to warrant further investigation.

PET detection of the impact of dobutamine on myocardial glucose metabolism in women with type 1 diabetes mellitus.

BACKGROUND: Our objective was to determine, in the hearts of women with type 1 diabetes mellitus (T1DM), whether the fate of extracted glucose is altered and, if so, what the impact of dobutamine is on myocardial substrate metabolism. In experimental models of T1DM, myocardial glycolysis and glucose oxidation are reduced with the impairment becoming more pronounced with dobutamine. Whether similar changes occur in humans with T1DM is unclear. METHODS AND RESULTS: Myocardial perfusion, oxygen consumption, and glucose and fatty acid metabolism were measured with positron emission tomography in 19 women, 7 normal volunteers (NVs) and 12 with T1DM. The NVs and 6 T1DM (DM1) patients were studied under baseline metabolic conditions and 6 T1DM patients were studied during hyperinsulinemic-euglycemic clamp (DM1-C), both at rest and during dobutamine. At rest, myocardial glucose uptake, glycolysis, glycogen storage, and oxidation were reduced by similar levels in DM1 patients compared with NVs (P < .05). During dobutamine, although myocardial glucose uptake was not different from DM1 patients at rest, fractional glycolysis was lower compared with NVs or DM1-C patients and reflected a lower glucose oxidation rate (P < .001). Measurements of myocardial glucose metabolism at rest and during dobutamine were comparable between NVs and DM1-C patients. During dobutamine, myocardial fatty acid uptake and oxidation increased in all 3 groups. CONCLUSIONS: In women with T1DM, (1) myocardial glucose metabolism is impaired downstream from initial uptake, (2) these abnormalities become more pronounced with dobutamine and are paralleled by an increase in myocardial fatty acid metabolism, and (3) insulin restores glucose metabolism to levels observed in normal control subjects.

Protein kinase C-dependent antilipolysis by insulin in rat adipocytes.

Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the beta1-adrenergic receptor (beta1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and alpha1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the beta1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and alpha1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCalpha- and beta-specific inhibitor Gö 6976 and PKCbeta-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCalpha, betaI, and delta, and wortmannin decreases PKCbetaI, betaII, and delta in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.

Antibodies against the beta1-adrenergic receptor induce progressive development of cardiomyopathy.

Different immune disturbances have been found among patients with dilated cardiomyopathy (DCM), including antibodies directed against different cardiac antigens, such as the second extracellular loop of the beta(1)-adrenergic receptor. The aim of our study was to investigate antibodies directed against the second extracellular loop of the beta(1)-adrenergic receptor effect on cardiac functions at an early and late stage during DCM development. This was made in a mouse model, in which DCM was induced by immunization with the second extracellular loop of the beta(1)-adrenergic receptor. Mice were immunized for 14 or 25 weeks respectively with the second extracellular loop of the beta(1)-adrenergic receptor. At 14 weeks, there was no decreased heart function reviled by echocardiography at rest, but when dobutamine stress echocardiography was used, a lower cardiac reserve was shown in the mice with antibodies against the second extracellular loop of the beta(1)-adrenergic receptor. By 25 weeks, decreased heart function, dilatation of the left ventricle and thinner left ventricular posterior wall were observed. Further biochemical analyses at 25 weeks showed increased mRNA expressions for beta(1)-adrenergic receptor kinase, monocyte chemoattractant protein-1 and the brain natriuretic peptide as well as increased concentrations of complement factor 3 in sera in the immunized animals. Our data suggest a cardiotoxic effect of antibodies directed against the second extracellular loop of the beta(1)-adrenergic receptor and a capacity to induce DCM with progressive remodeling, decreased cardiac function, altered beta(1)AR signaling and upregulation of proinflammatory components.