The HPA axis response to critical illness: New study results with diagnostic and therapeutic implications.

For decades, elevated plasma cortisol concentrations in critically ill patients were exclusively ascribed to a stimulated hypothalamus-pituitary-adrenal axis with increased circulating adrenocorticotropic hormone (ACTH) inferred to several-fold increase adrenal cortisol synthesis. However, 'ACTH-cortisol dissociation' has been reported during critical illness, referring to low circulating ACTH coinciding with elevated circulating cortisol. It was recently shown that metabolism of cortisol is significantly reduced in critically ill patients explained by a suppression of the activity and expression of cortisol metabolizing enzymes in kidney and liver. This reduced cortisol breakdown determines hypercortisolemia, much more than increased cortisol production, in the critically ill. Although the low plasma ACTH concentrations, evoked by the elevated plasma cortisol via feedback inhibition, are part of this adaptation, they may negatively affect adrenocortical structure and function in the prolonged phase of critical illness. These new insights have implications for diagnosis and treatment of adrenal insufficiency in critically ill patients.

Anterior pituitary morphology and hormone production during sustained critical illness in a rabbit model.

Prolonged critical illness is hallmarked by striking alterations in the somatrope, thyrotrope, and lactotrope axes, the severity of which is associated with the risk of morbidity and mortality. The exact role of the pituitary gland in these alterations is unknown. We studied the impact of sustained critical illness on pituitary morphology and hormone production in a standardized rabbit model of prolonged (7 days) burn injury-induced critical illness. In healthy and prolonged critically ill rabbits, we determined pituitary weight, size, morphology and orientation of the somatrope, lactotrope and thyrotrope cells and the pituitary expression of GH, PRL, and TSH at gene and protein level. The weight of the pituitary gland was unaltered by 7 days of critical illness. Also, spatial orientation and morphology of the GH, PRL, and TSH producing cells remained normal. In prolonged critically ill rabbits GH mRNA levels were higher and PRL mRNA levels were lower than in healthy controls, whereas TSH mRNA was not affected. The sizes of GH, PRL, or TSH producing cells and the pituitary content of GH, PRL, and TSH proteins were unaltered. In conclusion, in this rabbit model of prolonged critical illness, the morphology of the pituitary gland and the pituitary GH, PRL, and TSH content was normal. The alterations in pituitary hormone mRNA levels with sustained critical illness are compatible with altered hypothalamic and peripheral regulation of pituitary hormone release as previously suggested indirectly by responses to exogenous releasing factors.

Endocrine and metabolic disturbances in critical illness: relation to mechanisms of organ dysfunction and adverse outcome.

Critically ill patients face a high risk of death, which is mostly due to non-resolving multiple organ failure. The plethora of endocrine and metabolic disturbances that hallmark critical illness may play a key role. The major part of our research performed during the period 2004-2009 focused on the disturbed glucose metabolism that commonly develops during critical illness. The onset of this research interest was the landmark randomized clinical study on strict blood glucose control (80-110 mg/ dl) with intensive insulin therapy performed by Prof. Van den Berghe and our clinical team members. This study, published in 2001 in the New England Journal of Medicine, showed reduced morbidity and improved survival with intensive insulin therapy versus toleration of hyperglycemia up to 215 mg/dl. This review summarizes our findings in both patients and animal models on mechanisms contributing to the clinical benefits of strict blood glucose control. Intensive insulin therapy appeared to lower blood glucose levels by ameliorating insulin sensitivity and stimulation of glucose uptake in skeletal muscle, whereas hepatic insulin resistance was not affected. The therapy also improved the lipid profile and the immune response and attenuated inflammation. Maintenance of strict normoglycemia appeared essentially most important, rather than elevating insulin levels. Avoiding hyperglycemia protected the endothelium and the mitochondria. In our animal model, nutritional interventions counteracted the hypercatabolic state of critical illness and insulin improved myocardial contractility, but only when normoglycemia was maintained. Interestingly, we identified the adipose tissue as a functional storage depot for toxic metabolites during critical illness.

Melanocortin peptides stimulate prolactin gene expression and prolactin accumulation in rat pituitary aggregate cell cultures.

Treatment for 40 h of reaggregate pituitary cell cultures from 14-day-old female rats with nanomolar concentrations of gamma3-melanocyte-stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). During the 40 h incubation, gamma3-MSH stimulated prolactin accumulation in the culture medium. alpha-MSH, a potent agonist of the rat melanocortin-3 receptor (MC3R) and Ala(8)-gamma2-MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as gamma3-MSH. The effect of gamma3-MSH on prolactin mRNA expression was abolished when aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol. By contrast, oestradiol abolished the stimulatory effect of Ala(8)-gamma2-MSH on prolactin mRNA expression. In GH3 cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3-kb prolactin promoter fragment, a dose as low as 1 nMgamma3-MSH, added for 24 h, significantly increased eGFP fluorescence. Agouti-related protein (AgRP(83-132)), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by gamma3-MSH or Ala(8)-gamma2-MSH. On its own, AgRP(83-132) significantly increased prolactin mRNA expression level and prolactin accumulation. Both gamma2-MSH and Ala(8)-gamma2-MSH increased [S(35)]GTPgammaS binding in membrane preparations of 14-day-old rat pituitaries and of GH3 cells. Whereas MC3R and MC5R mRNA were detectable by RT-PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 cells using various oligonucleotide primer sets. The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved. AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.

Stimulation of intracellular free calcium in GH3 cells by gamma3-melanocyte-stimulating hormone. Involvement of a novel melanocortin receptor?

The melanocortin (MC) gamma3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca(2+)](i)) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that gamma3MSH and some analogs increase [Ca(2+)](i) in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pM gamma3MSH induced an oscillating [Ca(2+)](i) increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nM. gamma2MSH, alphaMSH, and NDP-alphaMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca(2+)](i) rise in response to gamma3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of gamma3MSH showed low potency in increasing [Ca(2+)](i) in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the gamma2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca(2+)](i) in GH3 cells, but was about 50 times less potent than gamma2- or gamma3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of gamma3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for gamma3MSH.

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.