Extra-cellular matrix rigidity may dictate the fate of injury outcome.

After injury, while regeneration can be observed in hydra, planaria and some vertebrates, regeneration is rare in mammals and particularly in humans. In this paper, we investigate the mechanisms by which biological tissues recover after injury. We explore this question on adipose tissue, using the mathematical framework recently developed in Peurichard et al., J. Theoret. Biol. 429 (2017), pp. 61-81. Our assumption is that simple mechanical cues between the Extra-Cellular Matrix (ECM) and differentiated cells can explain adipose tissue morphogenesis and that regeneration requires after injury the same mechanisms. We validate this hypothesis by means of a two-dimensional Individual Based Model (IBM) of interacting adipocytes and ECM fiber elements. The model successfully generates regeneration or scar formation as functions of few key parameters, and seems to indicate that the fate of injury outcome could be mainly due to ECM rigidity.

Simple mechanical cues could explain adipose tissue morphology.

The mechanisms by which organs acquire their functional structure and realize its maintenance (or homeostasis) over time are still largely unknown. In this paper, we investigate this question on adipose tissue. Adipose tissue can represent 20 to 50% of the body weight. Its investigation is key to overcome a large array of metabolic disorders that heavily strike populations worldwide. Adipose tissue consists of lobular clusters of adipocytes surrounded by an organized collagen fiber network. By supplying substrates needed for adipogenesis, vasculature was believed to induce the regroupment of adipocytes near capillary extremities. This paper shows that the emergence of these structures could be explained by simple mechanical interactions between the adipocytes and the collagen fibers. Our assumption is that the fiber network resists the pressure induced by the growing adipocytes and forces them to regroup into clusters. Reciprocally, cell clusters force the fibers to merge into a well-organized network. We validate this hypothesis by means of a two-dimensional Individual Based Model (IBM) of interacting adipocytes and extra-cellular-matrix fiber elements. The model produces structures that compare quantitatively well to the experimental observations. Our model seems to indicate that cell clusters could spontaneously emerge as a result of simple mechanical interactions between cells and fibers and surprisingly, vasculature is not directly needed for these structures to emerge.

A gerophysiology perspective on healthy ageing.

Improvements in public health and health care have resulted in significant increases in lifespan globally, but also in a significant increase in chronic disease prevalence. This has led to a focus on healthy ageing bringing a shift from a pathology-centered to an intrinsic capacity and function-centered view. In parallel, the emerging field of geroscience has promoted the exploration of the biomolecular drivers of ageing towards a transverse vision by proposing an integrated set of molecular hallmarks. In this review, we propose to take a step further in this direction, highlighting a gerophysiological perspective that considers the notion of homeostasis/allostasis relating to robustness/fragility respectively. While robustness is associated with homeostasis achieved by an optimal structure/function relationship in all organs, successive repair processes occurring after daily injuries and infections result in accumulation of scar healing leading to progressive tissue degeneration, allostasis and frailty. Considering biological ageing as the accumulation of scarring at the level of the whole organism emphasizes three transverse and shared elements in the body - mesenchymal stroma cells/immunity/metabolism (SIM). This SIM tryptich drives tissue and organ fate to regulate the age-related evolution of body functions. It provides the basis of a gerophysiology perspective, possibly representing a better way to decipher healthy ageing, not only by defining a composite biomarker(s) but also by developing new preventive/curative strategies.

Immuno-metabolism and adipose tissue: The key role of hematopoietic stem cells.

The field of immunometabolism has come a long way in the past decade, leading to the emergence of a new role for white adipose tissue (WAT) that is now recognized to stand at the junction of immune and metabolic regulations. Interestingly, a crucial role of the abundant and heterogeneous immune population present in WAT has been proposed in the induction and development of metabolic diseases. Although a large body of data focused on mature immune cells, only few scattered studies are dedicated to leukocyte production, and the activity of hematopoietic stem cells (HSC) in these pathological states. Considering that blood cell production and the differentiation of HSCs and their progeny is orchestrated, in part, by complex interacting signals emanating from their microenvironment, it thus seems worth to better understand the relationships between metabolism and HSC. This review discusses the alterations of hematopoietic process described in metabolic diseases and focused on the emerging data concerning HSC present in WAT.

Target cells of gamma3-melanocyte-stimulating hormone detected through intracellular Ca2+ responses in immature rat pituitary constitute a fraction of all main pituitary cell types, but mostly express multiple hormone phenotypes at the messenger ribonucleic acid level. Refractoriness to melanocortin-3 receptor blockade in the lacto-somatotroph lineage.

Gamma3-MSH has recently been shown to be a biologically active peptide in the rat anterior pituitary. It induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a relatively small population of immature pituitary cells. The present study was intended to identify the target cells of this peptide and to discern the signal-transducing melanocortin (MC) receptor. In dispersed pituitary cells from 14-day-old rats, increasing doses of gamma3-MSH (0.1, 1, and 10 nM) evoked a sustained oscillating [Ca2+]i rise in an increasing number of cells (up to 14.5%). Within the responsive cells, 53% showed GH immunoreactivity (-ir), 12% showed PRL-ir, 2% showed TSHbeta-ir, 5% showed LHbeta-ir, and 10% showed ACTH-ir, whereas 18% did not express any hormone-ir to a detectable level. As assessed by single cell RT-PCR for the presence of pituitary hormone messenger RNA (mRNA), 26% of the gamma3-MSH-responsive cells contained only GH mRNA, 5% contained only PRL mRNA, and 4% contained only TSHbeta mRNA. Twenty-two percent contained mRNA of GH, PRL, and TSHbeta in various dual or triple combinations. About 24% of the gamma3-MSH-responsive cells expressed POMC mRNA, mostly together with other mRNAs, i.e. with GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSHbeta. Eighteen percent of the responsive cells expressed LHbeta, all of them together with mRNA of GH, PRL, and TSHbeta in various combinations. The absence of hormone mRNA was found in less than 1% of the responsive cells. In cells chosen at random (representative of the total pituitary cell population), the proportion of cells expressing two or multiple hormone mRNAs was twice as low as that in the gamma3-MSH-responsive population, whereas the proportion of cells expressing a single hormone mRNA was twice as high (about two thirds of all cells). Moreover, unlike in the gamma3-MSH-responsive cell population, randomly chosen cells were found that coexpressed POMC mRNA with LHbeta mRNA. The effect of gamma3-MSH on [Ca2+]i was blocked by the MC-3 receptor antagonist SHU9119 (used up to a 1000-fold excess) in 46% or less of the responsive cells. SHU9119 failed to block the [Ca2+]i response to gamma3-MSH in PRL-, GH-, and TSHbeta-ir cells, but it did block the response in most ACTH-ir cells and in cells expressing no hormone to a detectable level. Single cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected in only 16% of gamma3-MSH-responsive cells. The present data suggest that the target cells of gamma3-MSH in terms of [Ca2+]i responses in the immature rat pituitary constitute subpopulations of all main pituitary cell types, including nonhormonal (or low expression hormonal) cells. However, in contrast to the total pituitary cell population, most of these cells display multilineage gene activation at the mRNA level, i.e. express mRNA of GH, PRL, TSHbeta, POMC, and LHbeta in dual, triple, or quadruple combinations. Although gamma3-MSH may act through the MC-3 receptor in a portion of these cells, most of these cells (mainly in the lacto-somatotroph lineage) may transduce the signal through another receptor or through an MC-3 receptor with unconventional binding characteristics.

Intracellular calcium concentration and hormone secretion are controlled differently by TRH in rat neonatal lactotrophs and somatotrophs.

We studied the effects of TRH on the cytosolic free calcium concentration ([Ca2+]i) of female rat pituitary prolactin-secreting (lactotroph) and GH-secreting (somatotroph) cells in the early postnatal period, i.e. at postnatal days 5 and 10. [Ca2+]i of single identified lactotrophs and somatotrophs was recorded by dual-emission microspectrofluorimetry using the intracellular fluorescent calcium probe indo 1. An application of TRH (100 nM, 10 s) induced a marked [Ca2+]i increase in 65% of neonatal lactotrophs and 34% of neonatal somatotrophs while the remaining cells were unaffected. Most of the responsive cells, both lactotrophs and somatotrophs, exhibited a similar biphasic Ca2+ response, made up of an initial rapid large increase in [Ca2+]i followed by sustained [Ca2+]i fluctuations. In both cell types, removal of Ca2+ from the extracellular medium or addition of the Ca2+ channel blocker, cadmium chloride (500 microM), inhibited the second phase whereas the first phase persisted. Furthermore, in both cell types, protein kinase C (PKC) depletion by incubation in phorbol myristate acetate (1 microM) for 24 h abolished the second phase but did not inhibit the first phase. Conversely, when cells were pretreated with the Ca(2+)-ATPase inhibitor, thapsigargin (100 nM), all TRH-induced [Ca2+]i changes in both cell types disappeared. TRH therefore induces a biphasic increase in [Ca2+]i involving intra- and extracellular Ca2+ in neonatal lactotrophs and somatotrophs as it does in adult lactotrophs. The first phase is presumably due to mobilization of Ca2+ from intracellular stores whereas the second phase presumably results from a PKC-sensitive influx of Ca2+. TRH action on membrane potential was then investigated using the patch-clamp technique in the whole-cell mode. TRH-induced changes in membrane potential consisted of an initial hyperpolarization followed by depolarization and action potential firing. We also investigated TRH action on prolactin and GH secretion by neonatal pituitary cells using RIA. Surprisingly, static assays of prolactin and GH revealed only stimulation of prolactin release by TRH but no effect on GH secretion, although, as expected, GH-releasing factor was a potent agonist of GH secretion. Our results suggest that TRH regulates neonatal lactotrophs and somatotrophs differently, in that the [Ca2+]i changes do not correlate with stimulation of exocytosis in the latter cell type.

Stimulation of Ca2+ entry in lactotrophs and somatotrophs from immature rat pituitary by N-terminal fragments of proopiomelanocortin.

We have previously shown that 10-12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC1-76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC1-26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca2+]i) in response to POMC1-76 and POMC1-26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca2+) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC1-76 or POMC1-26 induced a maintained oscillating [Ca2+]i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca2+]i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC1-76 or 5 nM POMC1-26, and 11-13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca2+]i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca2+]i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca2+ response to POMC1-76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC1-26. The rise of [Ca2+]i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca2+ stores by thapsigargin. Removal of Ca2+ from the extracellular medium or addition of cadmium completely abolished both the POMC1-76- and POMC1-26-induced [Ca2+]i increase. Nifedipine inhibited the Ca2+ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca2+ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC1-76- or POMC1-26-induced [Ca2+]i increase. The present data show that, in immature pituitary cells, POMC1-76 induces an increase in [Ca2+]i through extracellular Ca2+ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC1-76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC1-76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.

Acute intracarotid glucose injection towards the brain induces specific c-fos activation in hypothalamic nuclei: involvement of astrocytes in cerebral glucose-sensing in rats.

The detection of changes in glucose level constitutes the first step of the control of glucose homeostasis. Glucose sensors are therefore expected to be present in different parts of the body and particularly in the central nervous system. Some studies have already attempted to determine glucose-sensitive cerebral structures either after a glucoprivic stimulus or after prolonged hyperglycaemia. By analogy to beta cells, it was postulated that the glucose sensors in the brain could involve GLUT2, glucokinase and/or ATP-sensitive K(+) channels. Surprisingly, GLUT2 was mainly found in astrocytes. Thus, the aims of the present investigation were to determine, in awake rats: (i) the hypothalamic areas that respond to acute hyperglycaemic condition induced by an intracarotid injection of glucose and (ii) the involvement of astrocytes in glucose-sensing by the use of a glial drug, methionine sulfoximine. Rats were given intracarotid injections of glucose solution to trigger a transient insulin secretion without change in peripheral glycaemia, thus involving only central nervous regulation. Hypothalamic activation was determined by immunodetection of the immediate early gene c-fos protein. Acute glucose injection induces significant activation of arcuate and paraventricular nuclei. This stimulation mainly affects neurones in both nuclei, but also astrocytes in the former as illustrated by double immunohistochemistry (Fos and neuronal nuclei or glial fibrillary acidic protein). After specific impairment of astrocyte metabolism by methionine sulfoximine, cerebral activation disappears in the arcuate nucleus, correlated with the lack of cerebral glucose-induced insulin secretion. Therefore, arcuate and paraventricular hypothalamic nuclei are able to detect acute cerebral hyperglycaemia, leading to a peripheral stimulation of insulin secretion. Arcuate nucleus and more especially astrocytes in this nucleus play a pivotal role in glucose-sensing.

Detection of melanocortin-3 receptor mRNA in immature rat pituitary: functional relation to gamma3-MSH-induced changes in intracellular Ca2+ concentration?

We have previously shown that gamma3-MSH stimulates DNA replication in lactotrophs, somatotrophs and thyrotrophs of early postnatal rat pituitary in culture. Since the melanocortin-3 (MC-3) receptor is the only known receptor displaying high affinity for gamma3-MSH, the present study explored whether mRNA of the latter receptor is present in the pituitary and whether the receptor is functional. RT-PCR of RNA extracts from 14-day-old rat pituitary revealed the presence of MC-3 receptor mRNA in both the anterior and the neurointermediate lobe. The identity of the amplified products was confirmed by sequence analysis. Dispersed cells of 14-day-old female rats (24 h in culture) were exposed to gamma3-MSH, and changes in intracellular free calcium levels ([Ca2+]i) were assessed by means of fluo-3 video imaging. Gamma3-MSH evoked a rapid and maintained oscillating [Ca2+]i increase in 5%, 10% and 15% of the cells at a dose of 0.1, 1 and 10 nM, respectively. The MC-3/MC-4 receptor antagonist Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (SHU 9119) blocked the effect of gamma3-MSH in about 50% of the responsive cells. The present data suggest that the MC-3 receptor is expressed in the rat pituitary but that this receptor mediates only half of the effects of its putative ligand, gamma3-MSH, on [Ca2+]i. Part of the effect of gamma3-MSH may be mediated by a MC-3 receptor in a functional state different from the one studied previously in transfected cell lines or by a hitherto unidentified MC receptor.

Cerebral insulin increases brain response to glucose.

The hypothalamus participates in the regulation of carbohydrate metabolism involving a feedback loop between the brain and the periphery in which glucose-sensitive hypothalamic areas appear to be involved. We have previously shown that a glucose injection (9 mg/kg) in the carotid artery toward the brain, in an amount that did not modify glycaemia, caused a rapid and transient increase in plasma insulin concentrations. To determine whether central insulin could influence this response, we investigated the change in central glucose-induced insulin secretion in intracerebroventricular (i.c.v) insulin-injected rats and in hyperinsulinaemic obese Zucker rats. Central glucose-induced insulin secretion was increased by 50% in i.c.v. insulin-injected rats compared to control rats. When a similar test was performed at a lower dose of glucose (3 mg/kg), a significant insulin secretion was observed only in rats submitted to a prior central insulin injection. These data indicate an increase in the brain response to glucose after insulin treatment. Using an identical lower glucose dose, we also demonstrated an enhanced brain glucose sensitivity in hyperinsulinaemic and insulin-resistant obese Zucker rats. Together, these results indicate that acute i.c.v. insulin or pathological hyperinsulinaemic state (i.e. obese Zucker rat) modulates the nervous control of insulin secretion by increasing the brain response to glucose.

The autonomic nervous system, adipose tissue plasticity, and energy balance.

In most mammals, two types of adipose tissue, white and brown, are present. Both are able to store energy in the form of triacylglycerols and to hydrolyze them into free fatty acids and glycerol. Whereas white adipose tissue can provide lipids as substrates for other tissues according to the needs of the organism, brown adipose tissue will use fatty acids for heat production. Over the long term, white fat mass reflects the net balance between energy expenditure and energy intake. Even though these two parameters are highly variable during the life of an individual, most adult subjects remain relatively constant in body weight throughout their lives. This observation suggests that appetite, energy expenditure, and basal metabolic rate are linked. An important characteristic of the adipose tissue is its enormous plasticity for volume and cell-number variations and an apparent change in phenotype between the brown and white adipose tissues. The present review focuses on the cellular mechanisms participating in the plasticity of adipose tissues and their regulation by the autonomic nervous system. There is compelling evidence with regard to the importance of the nervous system in the regulation of adipose tissue mass, either brown or white, by acting on the metabolic pathways and on the plasticity (proliferation, differentiation, transdifferentiation, apoptosis) of these tissues. A better comprehension of the different mechanisms involved in the feedback loop linking the brain and these two types of adipose tissue will lead to a better understanding of the pathophysiology of various disorders including obesity, cachexia, anorexia, and type II diabetes mellitus.

Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons.

Adiponectin is involved in the control of energy homeostasis in peripheral tissues through Adipor1 and Adipor2 receptors. An increasing amount of evidence suggests that this adipocyte-secreted hormone may also act at the hypothalamic level to control energy homeostasis. In the present study, we observed the gene and protein expressions of Adipor1 and Adipor2 in rat hypothalamus using different approaches. By immunohistochemistry, Adipor1 expression was ubiquitous in the rat brain. By contrast, Adipor2 expression was more limited to specific brain areas such as hypothalamus, cortex, and hippocampus. In arcuate and paraventricular hypothalamic nuclei, Adipor1, and Adipor2 were expressed by neurons and astrocytes. Furthermore, using transgenic green fluorescent protein mice, we showed that Adipor1 and Adipor2 were present in pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) neurons in the arcuate nucleus. Finally, adiponectin treatment by intracerebroventricular injection induced AMP-activated protein kinase (AMPK) phosphorylation in the rat hypothalamus. This was confirmed by in vitro studies using hypothalamic membrane fractions. In conclusion, Adipor1 and Adipor2 are both expressed by neurons (including POMC and NPY neurons) and astrocytes in the rat hypothalamic nuclei. Adiponectin is able to increase AMPK phosphorylation in the rat hypothalamus. These data reinforced a potential role of adiponectin and its hypothalamic receptors in the control of energy homeostasis.

Short applications of gamma-aminobutyric acid increase intracellular calcium concentrations in single identified rat lactotrophs.

We have investigated the direct effect of GABA receptor agonists on the cytosolic free calcium concentration ([Ca2+]i) and the membrane potential of rat lactotrophs in primary culture. [Ca2+]i was recorded in single identified lactotrophs by dual emission microspectrofluorimetry using indo-1 as intracellular fluorescent calcium probe. Whole cell and perforated patch-clamp were performed. A short application of GABA (10(-5) M, 10 s) induced a marked transient [Ca2+]i increase in 66% of lactotrophs, which could be readily mimicked by muscimol (10(-5) M). By contrast, neither L-homocarnosine (10(-3) M) nor baclofen (10(-5) M), a GABAB agonist, had any effect on [Ca2+]i. The GABA-induced [Ca2+]i increase was antagonized by picrotoxin (10(-5) M), bicuculline methiodide (10(-5) M) and strychnine (10(-4) M), demonstrating GABAA receptor specificity. Furthermore, clonazepan (1.5 x 10(-4) M) could potentiate the GABA effect on [Ca2+]i. The [Ca2+]i increase disappeared in the absence of Ca2+ in the extracellular medium or in the presence of Ca2+ channel blockers (cadmium, PN 200-110). GABA and muscimol depolarized the membrane potential with a concomitant fall in cell input resistance, thus suggesting, as in other cell types, the opening of receptor-operated chloride channels. When Ca2+ entry was prevented by the use of cadmium (500 x 10(-6) M), GABA still elicited membrane depolarization but did not raise [Ca2+]i. Our results suggest that a short application of GABA leads to Ca2+ entry through voltage-gated Ca2+ channels in single lactotrophs. This Ca2+ influx is due to depolarization of the prolactin cell.

Long-term GABAA receptor activation increases [Ca2+]i in single lactotrophs.

Prolactin (PRL) release by pituitary lactotrophs is inhibited by gamma-aminobutyric acid (GABA). We have investigated the effect of long-lasting activation of GABAA receptors on membrane potential and cytosolic free calcium concentration ([Ca2+]i) in single identified lactotrophs. Membrane potential was recorded using the perforated patch-clamp technique and [Ca2+]i using indo 1 as a fluorescent Ca2+ probe. When cells were bathed in muscimol (10 microM) for 30 min, [Ca2+]i unexpectedly increased in 53% of the lactotrophs. This was due to a Ca2+ influx, since it was inhibited by Ca(2+)-free extracellular medium or by Ca2+ channel blockers such as the dihydropyridine PN 200-110. In cells incubated in muscimol, wash of muscimol from the cell membrane potential and reduced [Ca2+]i to the levels found in control cells. This effect was mimicked by picrotoxin, a GABA-operated Cl- channel blocker, thus supporting the involvement of a muscimol-induced Cl- flux. Conversely, under similar experimental conditions, static assays of PRL release revealed an inhibition of release by muscimol, unaffected by the dihydropyridine PN 200-110. Our observations suggest that GABAA, receptors may not regulate the process of exocytosis within the Ca(2+)- regulated steps.