Experimental dog model for assessment of fasting and postprandial fatty acid metabolism: pitfalls and feasibility.

The dog is a widely-used model for conducting metabolic studies. This is mainly due to its large size and its physiology which is relatively similar to that of humans. Here, we attempted to optimize a postprandial metabolic study protocol used in dogs. Following acclimatization, female mongrel dogs underwent 9 h profiling for time-course baseline plasma data on triglyceride, adrenocorticotropic hormone (ACTH) and cortisol levels. One week later, carotid and jugular catheters were surgically inserted for sampling and infusions. Initial post-operative care, based on the literature (Protocol 1), consisted of analgesia (buprenorphine every 8-12 h and 2-3 doses/day of acepromazine), restriction by Pavlov harness within cages, and a two- to three-day recovery period. Throughout the experiment, dogs received a lipid tracer diluted in 5% bovine serum albumin (BSA). Compared with baseline, animals vomited (n = 6/6) and exhibited high ACTH + cortisol levels (stress biomarkers), resulting in blunted triglyceride peak levels. To avoid these undesirable effects, post-operative care was modified (Protocol 2) as follows: animals (n = 19) were given a single dose of buprenorphine and no acepromazine, were unrestrained and free to move within cages, the recovery period was extended to seven days, and the lipid tracer was diluted in 0.002% versus 5% BSA. Using this modified protocol, postprandial plasma-triglyceride and ACTH/cortisol patterns were similar to baseline values. Controlling for stressors, as well as for factors which may alter proper digestion, is critical for all postprandial metabolic studies. Our results show that an optimized postprandial metabolic protocol used in dogs reduces experimental variability, while improving animal care and comfort.

Lasting alterations of the sodium current by short-term hyperlipidemia as a mechanism for initiation of cardiac remodeling.

Clinical and animal studies indicate that increased fatty acid delivery to lean tissues induces cardiac electrical remodeling and alterations of cellular calcium homeostasis. Since this may represent a mechanism initiating cardiac dysfunction during establishment of insulin resistance and diabetes or anaerobic cardiac metabolism (ischemia), we sought to determine if short-term exposure to high plasma concentration of fatty acid in vivo was sufficient to alter the cardiac sodium current (INa) in dog ventricular myocytes. Our results show that delivery of triglycerides and nonesterified fatty acids by infusion of Intralipid + heparin (IH) for 8 h increased the amplitude of INa by 43% and shifted its activation threshold by -5 mV, closer to the resting membrane potential. Steady-state inactivation (availability) of the channels was reduced by IH with no changes in recovery from inactivation. As a consequence, INa "window" current, a strong determinant of intracellular Na+ and Ca2+ concentrations, was significantly increased. The results indicate that increased circulating fatty acids alter INa gating in manners consistent with an increased cardiac excitability and augmentation of intracellular calcium. Moreover, these changes could still be measured after the dogs were left to recover for 12 h after IH perfusion, suggesting lasting changes in INa. Our results indicate that fatty acids rapidly induce cardiac remodeling and suggest that this process may be involved in the development of cardiac dysfunctions associated to insulin resistance and diabetes.

Central (mainly) actions of GPCRs in energy homeostasis/balance: view from the Chair.

To maintain a constant body weight, energy intake must equal energy expenditure; otherwise, there is a risk of overweight and obesity. The hypothalamus is one of the primary brain regions where multiple nutrient-related signals from peripheral and central sources converge and become integrated to regulate both short- and long-term nutritional states. The aim of the afternoon session of the 15th Annual International Symposium of the Laval University Obesity Research Chair held in Quebec City on 9 November 2012 was to present the most recent insights into the complex molecular mechanisms regulating food intake. The aims were to emphasize on the interaction between central and peripheral actions of some of the key players acting not only at the hypothalamic level but also at the periphery. Presentations were focused on melanocortin-3 receptor (MC3R) and melanin-concentrating hormone (MCH) as anorexigenic and orexigenic components of the hypothalamus, on endocannabinoid receptors, initially as a central neuromodulatory signal, and on glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) as peripheral signals. What becomes clear from these four presentations is that the regulation of food intake and energy homeostasis involves several overlapping pathways, and that we have only touched the tip of the iceberg. From the examples presented in this symposium, it could be expected that in the near future, in addition to a low-fat diet and exercise, a combination of appropriate peptides and small molecules is likely to become available to improve/facilitate the objectives of long-term maintenance of energy balance and body weight.

Fyn is involved in angiotensin II type 2 receptor-induced neurite outgrowth, but not in p42/p44mapk in NG108-15 cells.

In NG108-15 cells, activation of p42/p44(mapk) is essential for induction of neurite outgrowth by angiotensin II (Ang II) type 2 receptor (AT(2)). The aim was to verify whether Fyn, a member of the Src family kinases (SFK), is involved in neurite outgrowth induced by AT(2) activation. Preincubation of cells with PP1, a general inhibitor of the SKF, decreased activation of Rap1 and p42/p44(mapk) and abolished TrkA activation by Ang II or by the AT(2) agonist, CGP42112A. NG108-15 cells were transfected with a Fyn-WT and a Fyn-DN expressing vector. Fyn-WT was sufficient to induce neurite outgrowth, although transfection with Fyn-DN abolished neurite elongation. However, the Fyn-DN form failed to affect activation of TrkA, Rap1 or p42/p44(mapk) by Ang II. Thus, although SKF activity is required to achieve AT(2)-induced activation of TrkA, Rap1 and p42/p44(mapk), Fyn is essential for AT(2) receptor-induced neurite outgrowth, but not in AT(2) signaling leading to p42/p44(mapk) activation.

A connection between extracellular matrix and hormonal signals during the development of the human fetal adrenal gland.

The human adrenal cortex, involved in adaptive responses to stress, body homeostasis and secondary sexual characters, emerges from a tightly regulated development of a zone-specific secretion pattern during fetal life. Its development during fetal life is critical for the well being of pregnancy, the initiation of delivery, and even for an adequate adaptation to extra-uterine life. As early as from the sixth week of pregnancy, the fetal adrenal gland is characterized by a highly proliferative zone at the periphery, a concentric migration accompanied by cell differentiation (cortisol secretion) and apoptosis in the central androgen-secreting fetal zone. After birth, a strong reorganization occurs in the adrenal gland so that it better fulfills the newborn's needs, with aldosterone production in the external zona glomerulosa, cortisol secretion in the zona fasciculata and androgens in the central zona reticularis. In addition to the major hormonal stimuli provided by angiotensin II and adrenocorticotropin, we have tested for some years the hypotheses that such plasticity may be under the control of the extracellular matrix. A growing number of data have been harvested during the last years, in particular about extracellular matrix expression and its putative role in the development of the human adrenal cortex. Laminin, collagen and fibronectin have been shown to play important roles not only in the plasticity of the adrenal cortex, but also in cell responsiveness to hormones, thus clarifying some of the unexplained observations that used to feed controversies.

The angiotensin type 2 receptor of angiotensin II and neuronal differentiation: from observations to mechanisms.

The angiotensin II (Ang II) type 2 receptor (AT(2)) is a member of the seven-transmembrane domain, G-protein coupled receptor family. This receptor is ubiquitously distributed in the fetus but, in most tIssues, its expression dramatically falls in the first few hours after birth. Based on this observation, the hypothesis that this receptor could be involved in fetal development was raised and, over the past ten Years, many studies have tried to identify a role for the AT(2) receptor using many different tIssues and cell lines. To date, one of the major roles associated with the Ang II AT(2) receptor concerns its ability to induce neuronal differentiation. Indeed, in cells of neuronal origin, activation of the AT(2) receptor was shown to induce neurite outgrowth and elongation, modulate neuronal excitability, promote cellular migration and, in particular conditions, induce neuronal cell death. Regarding its signaling mechanisms, the AT(2) receptor still represents one of the most controversial G-protein coupled receptors since it does not stimulate the production of any of the classical second messengers. This review summarizes knowledge of the functions and the signaling mechanisms involved in the actions of the AT(2) receptor in neurons and cells of neuronal origin. Based on its altered expression in neurological disorders, a role for the AT(2) receptor in control of neuronal plasticity is proposed.

Fibronectin, laminin, and collagen IV interact with ACTH and angiotensin II to dictate specific cell behavior and secretion in human fetal adrenal cells in culture.

Whereas collagen IV is expressed throughout the fetal adrenal gland during the second trimester of human development, fibronectin, and laminin demonstrate a rather mirror-image distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. In the present study, extracellular matrices were able to modulate the profile of steroid secretion in primary cultures: collagen IV favored cortisol secretion following adrenocorticotropin (ACTH) or angiotensin II (Ang II) stimulation while specific stimulation of the AT2 receptor of Ang II elicited dehydroepiandrostenedione (DHEA) production. These effects were correlated by changes in mRNA levels of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior: collagen IV and laminin enhanced cell proliferation whereas fibronectin incited cell death. These results indicate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.

Expression and regulation of adenylyl cyclase isoforms in the human adrenal gland.

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the betagamma-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.

Ca(2+)-dependent sensitization of adenylyl cyclase activity.

It has been shown that intracellular Ca(2+) concentrations have multiple modulatory influences on hormone-stimulated adenylyl cyclase activity. Here, we report that increasing intracellular Ca(2+) concentration by treating cells with the Ca(2+) ionophore A23187 leads to a sensitization of the beta-adrenoceptor-stimulated adenylyl cyclase activity in Ltk(-) cells expressing the human beta(2)-adrenoceptor. The ionophore treatment led to a 20+/-5% increase of the maximal isoproterenol-stimulated cyclase activity that could be prevented by chelation of the extracellular Ca(2+) using EGTA. A similar Ca(2+)-mediated sensitization (20+/-6%) was observed when the adenylyl cyclase was directly stimulated by the diterpene forskolin indicating that the catalytic activity of the enzyme itself is modulated by the change in Ca(2+) concentration. Sensitization of both the isoproterenol- and forskolin-stimulated adenylyl cyclase activities were completely blocked by treatment with KN-62[1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine], an inhibitor of the Ca(2+)-calmodulin-dependent protein kinase (CamKinase). Taken together, our data reveal the existence of a CamKinase-dependent sensitization process acting at the level of the adenylyl cyclase catalytic moiety.

Identification of extracellular matrix components and their integrin receptors in the human fetal adrenal gland.

The development of the human fetal adrenal gland is characterized by a gradient of mitotic activity, cell migration, and cell apoptosis, all of which dictate its particular function. Such plasticity may possibly be under the control of the extracellular environment. The goal of this study was to identify components of the extracellular matrix in second-trimester fetal adrenal glands. Whereas collagen IV was expressed evenly throughout the gland, both fibronectin and laminin demonstrated a mirror-imaged distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. The integrin subunit alpha1 was found mainly in the definitive zone and the alpha2-subunit mainly in the transitional zone, whereas integrin alpha3 (which binds both fibronectin and laminin) was detected only in the fetal zone. The beta2-subunit was observed solely in chromaffin cells. Such specific gradients of integrin and MEC component expression suggest that the extracellular environment does play a definite role during adrenal gland development. Indeed, compared with that in untreated plastic dishes, ACTH stimulation of dehydroepiandrosterone sulfate and cortisol was enhanced by collagen IV. In addition, fibronectin enhanced dehydroepiandrosterone sulfate but decreased cortisol secretion, compared with collagen IV substrates. These results provide fundamental insight into the contribution of the microenvironment in cellular processes leading to fetal adrenal gland development.

Localization of G protein alpha-subunits in the human fetal adrenal gland.

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.

A Ras-dependent chloride current activated by adrenocorticotropin in rat adrenal zona glomerulosa cells.

In the present study, we report that ACTH induces a transient chloride current. The lack of correlation between ACTH-induced cAMP production and amplitude of the Cl- current, as well as the absence of stimulation by forskolin or 8Br-cAMP indicated that the ACTH-induced current was not cAMP-dependent. We explored the possibility that one or several elements of the Ras/Raf MAPK cascade were involved. Indeed, we found that ACTH at 10(-10) M induced activation of Ras. Inhibition of the current by QEHA peptide, a Gbetagamma sequestrant, demonstrated that Gbetagamma subunits transduced the message. Blockage of the Ras activation using an inhibitor of farnesyl transferase (BZA-5B) or the monoclonal antibody H-Ras(259) abrogated the current. Moreover, the addition of Ras-GTPyS in the pipette medium gave rise to the Cl- current. Treatment of the cells with BZA decreased the aldosterone secretion induced by 10(-10) M ACTH but not that induced by 10(-8) M ACTH, confirming the involvement of Ras in steroid secretion. We conclude that ACTH triggers a Cl- current through the activation of the Ras protein by Gbetagamma subunits. This current, activated at physiological ACTH concentrations (1 to 100 pM) where cAMP production is very low, could play a significant role in aldosterone production.

Occurrence and effect of PACAP in the human fetal adrenal gland.

In the study reported in this paper, we characterized PACAP in the human fetal adrenal gland and we investigated the effect of PACAP on steroid secretion from cultured fetal adrenal cells. The adrenal gland from 20-week-old fetuses contained substantial concentrations of PACAP-immunoreactive material (88.6 ng/g wet tissue). HPLC analysis of adrenal extracts revealed the presence of both PACAP27 and PACAP38, the latter being the predominant form. Incubation of cultured fetal adrenal cells with PACAP38 (10(-7) M) significantly increased cortisol and DHEAS secretion. Administration of the beta-adrenoreceptor agonist isoproterenol mimicked the stimulatory effect of PACAP on both steroid secretion whereas preincubation of fetal cells with the beta-adrenoreceptor antagonist propranolol suppressed the steroidogenic effect of PACAP. These data, together with the observation that PACAP receptors are exclusively located on chromaffin cells, suggest that, in the fetal human adrenal gland, the effect of PACAP on steroid secretion is mediated via the local release of catecholamines.

Involvement of the angiotensin II type 2 receptor in apoptosis during human fetal adrenal gland development.

The aim of this study was to establish a link between the highly expressed angiotensin II (Ang II) type 2 receptor (AT2) in human fetal adrenal cells and the proposed apoptotic activity in the center of the gland. There was an important increase in apoptotic DNA fragmentation with age in adrenal glands of fetuses from 15-20 weeks gestation. Adrenal cells showing the characteristic apoptotic internucleosomal DNA fragmentation were localized in the central portion of the fetal zone. In cells cultured for 24 h, Ang II, via the AT2 receptor, induced DNA fragmentation and cleavage of the DNA repair enzyme, poly-(ADP-ribose) polymerase. Furthermore, characteristic membrane blebbing was observed specifically on cells of the fetal zone. Immunofluorescence studies demonstrated that stimulation with Ang II or CGP 42112 (an agonist of the AT2 receptor) strongly modified the actin network, now localized exclusively along the plasma membrane, with a predominance of labeling at the base of the bleb formation. This rearrangement of actin distribution was different in cells from the definitive zone, corroborating the observation that these cells express many more Ang II type 1 receptors (AT1) than AT2 receptors. Taken together, our data indicate that the AT2 receptor is involved in the apoptotic process observed in the human fetal adrenal gland and could participate in the morphological changes occurring after birth, leading to involution of the fetal zone.

Activation of the AT(2) receptor of angiotensin II induces neurite outgrowth and cell migration in microexplant cultures of the cerebellum.

Microexplant cultures from three-day-old rats were used to investigate whether angiotensin II (Ang II), through its AT(1) and AT(2) receptors, could be involved in the morphological differentiation of cerebellar cells. Specific activation of the AT(2) receptor during 4-day treatment induced two major morphological changes. The first was characterized by increased elongation of neurites. The second change was cell migration from the edge of the microexplant toward the periphery. Western blot analyses and indirect immunofluorescence studies revealed an increase in the expression of neuron-specific betaIII-tubulin, as well as an increase in expression of the microtubule-associated proteins tau and MAP2. These effects were demonstrated by co-incubation of Ang II with 1 microM DUP 753 (AT(1) receptor antagonist) or with 10 nM CGP 42112 (AT(2) receptor agonist) but abolished when Ang II was co-incubated with 1 microM PD 123319 (AT(2) receptor antagonist), indicating that differentiation occurs through AT(2) receptor activation and that the AT(1) receptor inhibits the AT(2) effect. Taken together, these results demonstrate that Ang II is involved in cerebellum development for both neurite outgrowth and cell migration, two important processes in the organization of the various layers of the cerebellum.

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic adenosine monophosphate in rat and human glomerulosa cells.

The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells.

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex.

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca2+ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone. Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.