OBJECTIVE
The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production.RESEARCH DESIGN AND METHODS
To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D157A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1312 [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo.RESULTS
Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production.CONCLUSIONS
These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.AMP-activated protein kinase (AMPK) is an evolutionarily conserved cellular energy sensor that regulates cellular metabolism (1). Consisting of a catalytic α subunit and two regulatory β and γ subunits, AMPK responds to an increase in intracellular AMP-to-ATP ratio and phosphorylates intracellular targets involved in cellular metabolism to promote ATP-generating processes and inhibit energy-consuming pathways. AMPK is expressed in a variety of tissues including the liver, skeletal muscles, adipose tissue, and the hypothalamus (1). AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) (1), which prevents the conversion of acetyl-CoA to malonyl-CoA. A decrease in malonyl-CoA relieves the inhibition of carnitine palmitoyltransferase-1 (2) and favors the transfer of long-chain fatty acyl-CoA (LCFA-CoA) into the mitochondria for β-oxidation. Conversely, direct inhibition of AMPK increases malonyl-CoA and LCFA-CoA levels (3).Studies have emerged implicating that AMPK in the hypothalamus integrates nutritional and hormonal signals to regulate food intake (4–8). In particular, direct inhibition of hypothalamic AMPK lowers food intake (8), whereas selective activation of hypothalamic AMPK negates the ability of leptin to activate hypothalamic ACC, increase hypothalamic malonyl-CoA levels, and lower food intake (9). In light of the fact that the hypothalamus integrates nutritional and hormonal signals to not only regulate energy (10–12) but also glucose (13–17) homeostasis, and that accumulation of hypothalamic malonyl-CoA and LCFA-CoA levels lowers food intake as well as hepatic glucose production (18–20), a possibility arises that direct inhibition of hypothalamic AMPK activity could alter hepatic glucose production (Fig. 1A). This working hypothesis was first tested in the current study.Open in a separate windowFIG. 1.Molecular knockdown of hypothalamic AMPK by the dominant-negative form of AMPK (DN AMPK) is sufficient to lower glucose production. A: Schematic representation of the working hypothesis: Inhibition of hypothalamic AMPK activity by DN AMPK or compound C leads to the lowering of hepatic glucose production. B: Experimental procedure and clamp protocol. A bilateral MBH catheter was implanted on day 0. Adenovirus tagged with GFP (Ad-GFP) or adenovirus-expressing DN AMPK (Ad-DN AMPK) was injected into the MBH of a group of rats immediately after MBH catheter implantation. Venous and arterial cannulations were done on day 5, and the pancreatic clamp protocol was performed on day 8. In the Ad-GFP and Ad-DN AMPK–injected rats, no MBH infusions were given during the clamp experiments. In rats with no adenovirus injection, 5% DMSO control or compound C was infused into the MBH during the clamps. C: Hypothalamic AMPK activity was significantly diminished in animals injected with Ad-DN AMPK, compared with control animals with injection of Ad-GFP (*P < 0.001). Hypothalamic injection of Ad-DN AMPK led to an increase in glucose infusion rate (D) (*P < 0.01) and a decrease in glucose production (E) (*P < 0.001) compared with the GFP control. F: Suppression of glucose production during the clamp period (180–210 min) expressed as percentage reduction from basal steady state (60–90 min) (*P < 0.01 vs. GFP control). G: Glucose uptake was not significantly different from that of GFP control. Values are shown as means ± SEM. (A high-quality color representation of this figure is available in the online issue.)Second, hypothalamus glucose metabolism to lactate, and the subsequent conversion of lactate to pyruvate and acetyl-CoA, have been reported to lower hepatic glucose production (21). However, the downstream biochemical pathways that mediate the ability of hypothalamic glucose/lactate sensing to lower glucose production remain unclear, although it was hypothesized that the formation of malonyl-CoA via the enhanced flux of acetyl-CoA could be a necessary step (3,15). Given the well-established regulatory role of AMPK on the formation of malonyl-CoA from acetyl-CoA and that hypothalamic malonyl-CoA regulates glucose production (18), we next tested the possibility that direct activation of hypothalamic AMPK negates the ability of central nervous system glucose/lactate sensing to regulate glucose production.In summary, we tested the hypothesis that molecular and pharmacological changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to regulate glucose production in vivo. 相似文献Methods: Sixty-four rabbits were instrumented for measurement of left ventricular pressure, cardiac output, and myocardial infarct size (IS). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits underwent a treatment period consisting of either no intervention for 35 min (control group, n = 12) or 15 min of 1 minimum alveolar concentration desflurane inhalation followed by a 10-min washout period (desflurane group, n = 12). Four additional groups received the tyrosine kinase inhibitor genistein (5 mg/kg) or lavendustin A (1.3 mg/kg) at the beginning of the treatment period with (desflurane-genistein group, n = 11; desflurane-lavendustin A group, n = 12) or without desflurane inhalation (genistein group, n = 9; lavendustin A group, n = 8).
Results: Hemodynamic values were similar in all groups during baseline (left ventricular pressure, 87 +/- 14 mmHg [mean +/- SD]; cardiac output, 198 +/- 47 ml/min), during coronary artery occlusion (left ventricular pressure, 78 +/- 12 mmHg; cardiac output, 173 +/- 39 ml/min), and after 2 h of reperfusion (left ventricular pressure, 59 +/- 17; cardiac output, 154 +/- 43 ml/min). IS in the control group was 55 +/- 10% of the area at risk. The tyrosine inhibitors had no effect on IS (genistein group, 56 +/- 13%; lavendustin A group, 49 +/- 13%; each P = 1.0 vs. control group). Desflurane preconditioning reduced IS to 40 +/- 15% (P = 0.04 vs. control group). Tyrosine kinase inhibitor administration had no effect on IS reduction (desflurane-genistein group, 44 +/- 13%; desflurane-lavendustin A group, 44 +/- 16%; each P = 1.0 vs. desflurane group). 相似文献
Methods: To determine whether any of these kinase cascades were activated by opioids, mu, delta, or kappa opioid receptors were transiently introduced into COS-7 cells together with MAPKs tagged to allow recognition by specific antibodies, and then exposed to opioids. Mitogen-activated protein kinase activation was determined by an in vitro MAPK activation assay. In addition, C6 glioma cells with either mu, delta, or kappa receptors stably introduced were exposed to opioids and MAPK activation determined by in vitro activation assay or antibody detection of activated forms.
Results: Transient experiments in COS cells revealed potent stimulation of ERK by mu and delta receptor activation, weak stimulation of stress-activated protein kinase by all receptor types, and no activation of p38. In stably transfected C6 glioma cells, only ERK activation was observed. Extracellular signal-related kinase induction was rapid, peaking 5 min after stimulation, and its activation was receptor-type specific. Mu and delta receptor stimulation activated ERK, but kappa stimulation did not. 相似文献
Methods. Langendorff-perfused rabbit hearts received: (1) control; (2) dimethyl sulfoxide (vehicle); (3) acetylcholine (0.55 mmol/L; PKC agonist); (4) 1,2-s,n-dioctanoylglycerol (DOG; 22 mmol/L; PKC agonist); (5) chelerythrine (0.8 mmol/L; PKC antagonist); or (6) DOG–chelerythrine followed by a 2-hour ischemic period, using modified St. Thomas cardioplegia and a 45-minute reperfusion period. The period of ischemia was chosen so as to allow for improvement by appropriate agonists. To observe metabolic changes, tissue nucleotides and nucleosides were measured. Membrane and cytosolic fractions of PKC were determined by an anti-PKC antibody directed against the PKC δ isozyme. Lactate levels and myocardial pH were measured.
Results. The PKC agonists DOG and acetylcholine showed the greatest recovery of developed pressure (68% ± 2%, 60% ± 9%, respectively). Although pH, lactate, and nucleotide levels were similar between groups at all times, myocyte PKC translocation demonstrated 25% of PKC δ isoforms on cell membrane sites during baseline, which shifted to 67% ± 17% with unprotected ischemia. DOG mimicked this shift with 58% ± 12% of PKC δ isoforms on membranes, which was also blocked by chelerythrine to 35% ± 7%.
Conclusions. These data demonstrate that PKC translocation results in improved postischemic function, not by alteration of energetics or metabolism, and deserves further investigation. 相似文献