Mechanisms and predictors of menses resumption once normal weight is reached in anorexia nervosa.

BACKGROUND: In cases of Anorexia Nervosa (AN), achieving weight gain recovery beyond the lower limits set by the World Health Organization and normalizing classical nutritional markers appears to be essential for most patients. However, this is not always adequate to restore menstrual cycles. This discrepancy can cause concern for both patients and healthcare providers, and can impact the medical management of these individuals. Thus, the purpose of this study was to assess the ability of anthropometric and hormonal factors to predict the resumption of menstrual cycles in individuals with anorexia nervosa upon reaching a normal body weight. METHOD: Patients with AN who had achieved a normal Body Mass Index but had not yet resumed their menstrual cycles (referred to as ANRec) were evaluated on two occasions: first at visit 1 and then again 6 months later, provided their body weight remained stable over this period (visit 2). Among the 46 ANRec patients who reached visit 2, they were categorized into two groups: 20 with persistent amenorrhea (PA-ANRec) and 26 who had regained their menstrual cycles (RM-ANRec). Anthropometric measurements, several hormone levels, Luteinizing Hormone (LH) pulsatility over a 4-h period, and LH response to gonadotropin-releasing hormone injection (LH/GnRH) were then compared between the two groups at visit 1. RESULTS: Patients in the RM-ANRec group exhibited higher levels of follicular stimulating hormone, estradiol, inhibin B, LH/GnRH, and lower levels of ghrelin compared to those in the PA-ANRec group. Analysis of Receiver Operating Characteristic curves indicated that having ≥ 2 LH pulses over a 4-h period, LH/GnRH levels ≥ 33 IU/l, and inhibin B levels > 63 pg/ml predicted the resumption of menstrual cycles with a high degree of specificity (87%, 100%, and 100%, respectively) and sensitivity (82%, 80%, and 79%, respectively). CONCLUSIONS: These three hormonal tests, of which two are straightforward to perform, demonstrated a high predictive accuracy for the resumption of menstrual cycles. They could offer valuable support for the management of individuals with AN upon achieving normalized weight. Negative results from these tests could assist clinicians and patients in maintaining their efforts to attain individualized metabolic targets. TRIAL REGISTRATION: IORG0004981.

Once a minimally normal weight has been reached during eating disorder recovery for female patients with anorexia nervosa (AN), the persistence of amenorrhea can be a cause for concern both patient and practitioner. In our study, we have discovered that positive results in biological blood tests, which can be conveniently conducted in an ambulatory setting, offer valuable predictive insights. Specifically, parameters such as LH pulse numbers exceeding 2, LH response to GnRH injection surpassing 33 UI/L, or Inhibin B levels in the blood exceeding 63 pg/mL, can accurately predict the resumption of menstrual cycles in the upcoming months, provided that the patient does not experience weight loss or engage in intense exercise. Conversely, negative results from these tests at this critical juncture in the recovery process can serve as valuable tools to encourage and motivate both the healthcare provider and the patient. By maintaining their efforts and continuing to increase their weight, patients can work towards a more comprehensive restoration of their menstrual cycles.

Lower leptin level at discharge in acute anorexia nervosa is associated with early weight-loss.

OBJECTIVE: Predictive values of acute phase metabolic abnormalities of anorexia nervosa (AN) have seldom been studied. As early postrestoration weight loss is associated with poor outcome, discharge biologic parameters were assessed to detect an association with 2-month follow-up weight loss as a proxy to poor outcome. METHOD: Fasting plasma levels of leptin, acyl-ghrelin, obestatin, PYY, oxytocin and BDNF were measured in 26 inpatients, at inclusion, at discharge and 2 months later. A body mass index less than 18 2-month postdischarge was considered a poor outcome. RESULTS: Nineteen patients (73%) had a fair outcome and seven (27%) had a poor one with a mean loss of 0.69 versus 4.54 kg, respectively. Only discharge leptin levels were significantly higher in fair versus poor outcome patients (14.1 vs. 7.0 ng/ml, p = 0.006). The logistic regression model using discharge leptin, acyl-ghrelin, obestatin, oxytocin, PYY and BDNF levels as predictors of outcome disclosed a nearly significant effect of leptin (p < 0.10). Receiver operating characteristic analysis showed 11.9 ng/ml was the best value of threshold. Neither clinical variables differed according to outcome. CONCLUSION: Leptin level may be a biomarker of early weight relapse after acute inpatient treatment of AN. Its clinical usefulness in monitoring care in AN should further be determined.

Unexpected Association of Desacyl-Ghrelin with Physical Activity and Chronic Food Restriction: A Translational Study on Anorexia Nervosa.

Anorexia nervosa (AN) is a severe metabopsychiatric disorder characterised by caloric intake restriction and often excessive physical exercise. Our aim is to assess in female AN patients and in a rodent model, the co-evolution of physical activity and potential dysregulation of acyl-(AG) and desacyl-(DAG) ghrelin plasma concentrations during denutrition and weight recovery. AN inpatients were evaluated at inclusion (T0, n = 29), half-(T1) and total (T2) weight recovery, and one month after discharge (T3, n = 13). C57/Bl6 mice with access to a running wheel, were fed ad libitum or submitted to short-(15 days) or long-(50 days) term quantitative food restriction, followed by refeeding (20 days). In AN patients, AG and DAG rapidly decreased during weight recovery (T0 to T2), AG increased significantly one-month post discharge (T3), but only DAG plasma concentrations at T3 correlated negatively with BMI and positively with physical activity. In mice, AG and DAG both increased during short- and long-term food restriction. After 20 days of ad libitum feeding, DAG was associated to persistence of exercise alteration. The positive association of DAG with physical activity during caloric restriction and after weight recovery questions its role in the adaptation mechanisms to energy deprivation that need to be considered in recovery process in AN.

Resistance to lean mass gain in constitutional thinness in free-living conditions is not overpassed by overfeeding.

BACKGROUND: Constitutional thinness (CT), a non-malnourished underweight state with no eating disorders, is characterized by weight gain resistance to high fat diet. Data issued from muscle biopsies suggested blunted anabolic mechanisms in free-living state. Weight and metabolic responses to protein caloric supplementation has not been yet explored in CT. METHODS: A 2 week overfeeding (additional 600 kcal, 30 g protein, 72 g carbohydrate, and 21 g fat) was performed to compare two groups of CTs (12 women and 11 men) to normal-weight controls (12 women and 10 men). Bodyweight, food intake, energy expenditure, body composition, nitrogen balance, appetite hormones profiles, and urine metabolome were monitored before and after overfeeding. RESULTS: Before overfeeding, positive energy gap was found in both CT genders (309 ± 370 kcal in CT-F and 332 ± 709 kcal in CT-M) associated with higher relative protein intake per kilo (1.74 ± 0.32 g/kg/day in CT-F vs. 1.16 ± 0.23 in C-F, P < 0.0001; 1.56 ± 0.36 in CT-M vs. 1.22 ± 0.32 in C-M, P = 0.03), lower nitrogen (7.26 ± 2.36 g/day in CT-F vs. 11.41 ± 3.64 in C-F, P = 0.003; 9.70 ± 3.85 in CT-M vs. 14.14 ± 4.19 in C-M, P = 0.02), but higher essential amino acids urinary excretion (CT/C fold change of 1.13 for leucine and 1.14 for arginine) in free-living conditions. After overfeeding, CTs presented an accentuated positive energy gap, still higher than in controls (675 ± 540 in CTs vs. 379 ± 427 in C, P = 0.04). Increase in lean mass was induced in both controls genders but not in CTs (a trend was noticed in CT women), despite a similar nitrogen balance after overfeeding (5.06 ± 4.33 g/day in CTs vs. 4.28 ± 3.15 in controls, P = 0.49). Higher anorectic gut hormones' tone, glucagon-like peptide 1 and peptide tyrosine tyrosine, during test meal and higher snacking frequency were noticed before and after overfeeding in CTs. CONCLUSIONS: The blunted muscle energy mechanism, previously described in CTs in free-living state, is associated with basal saturated protein turn over suggested by the concordance of positive nitrogen balance and an increased urine excretion of several essential amino acids. This saturation cannot be overpassed by increasing this spontaneous high-protein intake suggesting a resistance to lean mass gain in CT phenotype.

Postprandial Hyperglycemia Stimulates Neuroglial Plasticity in Hypothalamic POMC Neurons after a Balanced Meal.

Mechanistic studies in rodents evidenced synaptic remodeling in neuronal circuits that control food intake. However, the physiological relevance of this process is not well defined. Here, we show that the firing activity of anorexigenic POMC neurons located in the hypothalamus is increased after a standard meal. Postprandial hyperactivity of POMC neurons relies on synaptic plasticity that engages pre-synaptic mechanisms, which does not involve structural remodeling of synapses but retraction of glial coverage. These functional and morphological neuroglial changes are triggered by postprandial hyperglycemia. Chemogenetically induced glial retraction on POMC neurons is sufficient to increase POMC activity and modify meal patterns. These findings indicate that synaptic plasticity within the melanocortin system happens at the timescale of meals and likely contributes to short-term control of food intake. Interestingly, these effects are lost with a high-fat meal, suggesting that neuroglial plasticity of POMC neurons is involved in the satietogenic properties of foods.

Ghrelin acylation by ghrelin- O-acyltransferase can occur in healthy part of oncological liver in humans.

Activation of ghrelin is controlled by the enzyme ghrelin- O-acyl transferase (GOAT). In humans, localization of this acylation is poorly understood. The aim of this study is to explore GOAT localization and activation in the human liver by evaluating both bioactive and non-bioactive ghrelin in the bloodstream entering and leaving the liver and to simultaneously evaluate GOAT mRNA expression in the liver. A healthy part of oncologic hepatic tissue collected from nine patients undergoing hepatectomy was used to evaluate GOAT mRNA expression by quantitative real-time polymerase chain reaction (RT-qPCR). Simultaneously, blood from the portal vein, the suprahepatic vein, the subclavicular vein, and the radial artery was also sampled to assay total and acylated ghrelin. Acylated ghrelin level was significantly increased in the suprahepatic vein compared with the portal vein level (385 ± 42 ng/ml vs. 268 ± 24 ng/ml, P = 0.04). Suprahepatic-to-portal vein ratio for acylated ghrelin (acylation ratio) is 1.4 ± 0.1. Mean expression of GOAT mRNA in the liver, expressed as 2-∆Ct·µg total RNA-1·1 µl of liver tissue-1 was at 0.042 ± 0.021 arbitrary units. GOAT mRNA expression in the liver was correlated with acylated-to-total ghrelin ratio in the suprahepatic vein ( P = 0.016, R = 0.75) and with the acylation liver ratio ( P = 0.05, R = 0.61). Blood concentration of acylated ghrelin was found significantly increased after its passage through the liver, suggesting that acylation can occur in the liver. RT-qPCR data confirmed the presence of GOAT in the liver, with a positive correlation between GOAT expression and acylated ghrelin liver ratio. This study strongly suggests that the liver is a site of ghrelin acylation in humans. NEW & NOTEWORTHY Although the activation of ghrelin by the enzyme ghrelin- O-acyl transferase (GOAT) is yet well demonstrated, its localization, especially in humans, remains poorly understood. We explored GOAT localization and activation in the human liver by simultaneously evaluating both bioactive and non-bioactive ghrelin in the bloodstream entering and leaving the liver and also GOAT mRNA expression in the liver. We therefore showed for the first time, to our knowledge, that GOAT localized in the liver is active and takes part in ghrelin activation.

Somatostatin-IRES-Cre Mice: Between Knockout and Wild-Type?

The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3'UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression (serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

Combination of Selective Immunoassays and Mass Spectrometry to Characterize Preproghrelin-Derived Peptides in Mouse Tissues.

Preproghrelin is a prohormone producing several preproghrelin-derived peptides with structural and functional heterogeneity: acyl ghrelin (AG), desacyl ghrelin (DAG), and obestatin. The absence of selective and reliable assays to measure these peptides simultaneously in biological samples has been a limitation to assess their real proportions in tissues and plasma in physiological and pathological conditions. We aimed at reliably measure the ratio between the different preproghrelin-derived peptides in murine tissues using selective immunoassays combined with a highly sensitive mass spectrometry method. AG-, DAG-, and obestatin-immunopositive fractions from the gastrointestinal tract of adult wild-type and ghrelin-deficient mice were processed for analysis by mass spectrometry (MS) with a Triple Quadrupole mass spectrometer. We found that DAG was predominant in mouse plasma, however it only represented 50% of total ghrelin (AG+DAG) production in the stomach and duodenum. Obestatin plasma levels accounted for about 30% of all circulating preproghrelin-derived peptides, however, it represented <1% of total preproghrelin-derived peptides production (AG+DAG+Obestatin) in the stomach. Assays were validated in ghrelin-deficient mice since neither ghrelin nor obestatin immunoreactivities were detected in their stomach, duodenum nor plasma. MS analyses confirmed that obestatin-immunoreactivity in stomach corresponded to the C-terminal amidated form of the peptide but not to des(1-10)-obestatin, nor to obestatin-Gly. In conclusion, specificity of ghrelin and obestatin immunoreactivities in gastrointestinal tissues using selective immunoassays was validated by MS. Obestatin was less abundant than AG or DAG in these tissues. Whether this is due to inefficient processing rate of preproghrelin into mature obestatin in gastrointestinal mouse tissues remains elusive.

Ghrelin-Derived Peptides: A Link between Appetite/Reward, GH Axis, and Psychiatric Disorders?

Psychiatric disorders are often associated with metabolic and hormonal alterations, including obesity, diabetes, metabolic syndrome as well as modifications in several biological rhythms including appetite, stress, sleep-wake cycles, and secretion of their corresponding endocrine regulators. Among the gastrointestinal hormones that regulate appetite and adapt the metabolism in response to nutritional, hedonic, and emotional dysfunctions, at the interface between endocrine, metabolic, and psychiatric disorders, ghrelin plays a unique role as the only one increasing appetite. The secretion of ghrelin is altered in several psychiatric disorders (anorexia, schizophrenia) as well as in metabolic disorders (obesity) and in animal models in response to emotional triggers (psychological stress …) but the relationship between these modifications and the physiopathology of psychiatric disorders remains unclear. Recently, a large literature showed that this key metabolic/endocrine regulator is involved in stress and reward-oriented behaviors and regulates anxiety and mood. In addition, preproghrelin is a complex prohormone but the roles of the other ghrelin-derived peptides, thought to act as functional ghrelin antagonists, are largely unknown. Altered ghrelin secretion and/or signaling in psychiatric diseases are thought to participate in altered appetite, hedonic response and reward. Whether this can contribute to the mechanism responsible for the development of the disease or can help to minimize some symptoms associated with these psychiatric disorders is discussed in the present review. We will thus describe (1) the biological actions of ghrelin and ghrelin-derived peptides on food and drugs reward, anxiety and depression, and the physiological consequences of ghrelin invalidation on these parameters, (2) how ghrelin and ghrelin-derived peptides are regulated in animal models of psychiatric diseases and in human psychiatric disorders in relation with the GH axis.

Physiological roles of preproghrelin-derived peptides in GH secretion and feeding.

Among the factors playing a crucial role in the regulation of energy metabolism, gastro-intestinal peptides are essential signals to maintain energy homeostasis as they relay to the central nervous system the informations about the nutritional status of the body. Among these factors, preproghrelin is a unique prohormone as it encodes ghrelin, a powerful GH secretagogue and the only orexigenic signal from the gastrointestinal tract and obestatin, a proposed functional ghrelin antagonist. These preproghrelin-derived peptides may contribute to balance energy intake, metabolism and body composition by regulating the activity of the GH/IGF-1 axis and appetite. Whereas the contribution of ghrelin has been well characterized, the role of the more recently identified obestatin, in this regulatory process is still controversial. In this chapter, we describe the contribution of these different preproghrelin-derived peptides and their receptors in the regulation of GH secretion and feeding. Data obtained from pharmacological approaches, mutant models and evaluation of the hormones in animal and human models are discussed.

Ghrelin and obestatin circadian levels differentiate bingeing-purging from restrictive anorexia nervosa.

CONTEXT: Anorexia nervosa (AN) patients present with restrictive food behavior (AN-R). Some of them develop episodes of bulimia (AN-BP) without any clear pathophysiological explanation to date. Their clinical differentiation is important but not easily performed. Orexigenic/anorexigenic peptides measurements could provide some clues for that matter. OBJECTIVE: The objective of the study was to determine whether the circadian profile of total and acylated ghrelin, obestatin, and peptide YY (PYY) levels is different in AN-R subjects when compared with AN-BP patients. DESIGN AND SETTINGS: This was a cross-sectional study in an endocrinological unit. PATIENTS AND CONTROL SUBJECTS: Four groups of age-matched young women: 22 AN-R, 10 AN-BP, 16 normal-weight bulimia nervosa (BN), and nine controls. MAIN OUTCOME MEASURES: Twelve-point circadian profiles of plasma total and acylated ghrelin, obestatin, and PYY were measured. RESULTS: Total and acylated ghrelin and obestatin circadian levels were increased in AN-R when compared with controls but decreased in both AN-BP and BN groups (P < 0.001). PYY was decreased in all groups with eating disorders. Acylated to total ghrelin ratio was decreased in AN-BP and BN (P < 0.001), whereas obestatin to acylated ghrelin and PYY to acylated ghrelin ratios were increased in both groups with bingeing-purging behavior (P < 0.0001). CONCLUSIONS: Patients with AN-associated bingeing-purging behavior present a very different profile of appetite regulatory peptides when compared with the pure restrictive type. The assessment of ghrelin (and eventually obestatin) could be of particular interest for differential diagnosis. Very low ghrelin levels and increased anorexigenic to orexigenic peptide ratios suggest either a lack of adaptation to a starvation state or a higher facility to cope with undernutrition.

Role of the ghrelin/obestatin balance in the regulation of neuroendocrine circuits controlling body composition and energy homeostasis.

Ghrelin and obestatin are two peptides isolated from the gastrointestinal tract and encoded by the same preproghrelin gene. They convey to the central nervous system informations concerning the nutritional status and/or the energy stores. Ghrelin, mostly acting through the GH secretagogue receptor GHS-R, is a potent GH secretagogue, an orexigenic peptide and a long-term regulator of energy homeostasis. Obestatin was initially described for its anorexigenic effects and its binding to the G protein-coupled receptor 39 (GPR39). However, the role of obestatin is still controversial and the nature of the obestatin receptor remains an open question. This review is focussed on the possible implication of the ghrelin/obestatin system in psychiatric diseases with particular emphasis on eating disorders.

Ghrelin/obestatin ratio in two populations with low bodyweight: constitutional thinness and anorexia nervosa.

Constitutional thinness (CT) and anorexia nervosa (AN) are two categories of severely underweight subjects. Some appetite-regulating hormones display opposite levels in AN and CT. While levels of ghrelin, an orexigenic hormone, fit with the normal food intake in CT, the lack of efficacy of increased ghrelin levels in AN is not clear. Obestatin is a recently described peptide derived from the preproghrelin gene, reported to inhibit appetite in contrast to ghrelin. The aim of this study was to determine whether the circadian profile of obestatin, total and acylated ghrelin levels is different in CT subjects when compared with AN patients. Six-points circadian profiles of plasma obestatin, acylated ghrelin, total ghrelin and other hormonal and nutritional parameters were evaluated in four groups of young women: 10 CT, 15 restricting-type AN, 7 restored from AN and 9 control subjects. Obestatin circadian levels were significantly higher in AN (p<0.0001) while no difference was found between CT and control subjects. Acylated and total ghrelin were found increased in AN. Acylated ghrelin/obestatin and total ghrelin/obestatin were found decreased in AN compared to CT or C subjects (p<0.05). The percentage of acylated ghrelin was found decreased in CT group (p<0.05). The decreased ghrelin/obestatin ratio found in AN might participate in the restraint in nutriment intake of these patients. In contrast, in CT a lower percentage of acylated over total ghrelin might be considered in the aetiology of this condition.

Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature.

The growth hormone (GH) secretagogue receptor (GHSR) was cloned as the target of a family of synthetic molecules endowed with GH release properties. As shown recently through in vitro means, this receptor displays a constitutive activity whose clinical relevance is unknown. Although pharmacological studies have demonstrated that its endogenous ligand--ghrelin--stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR-dependent pathways remains an open question that gives rise to much controversy. We report the identification of a GHSR missense mutation that segregates with short stature within 2 unrelated families. This mutation, which results in decreased cell-surface expression of the receptor, selectively impairs the constitutive activity of the GHSR, while preserving its ability to respond to ghrelin. This first description, to our knowledge, of a functionally significant GHSR mutation, which unveils the critical importance of the GHSR-associated constitutive activity, discloses an unusual pathogenic mechanism of growth failure in humans.

[Ghrelin: a striking example of neuroendocrine peptide pleiotropy]. / La ghréline, un exemple saisissant de pléïotropie des peptides neuroendocriniens.

Ghrelin, a peptide predominantly produced by the stomach, has been discovered as a natural ligand of the growth hormone secretagogue receptor (GHS-R) type 1a. Shortly there after, it attracted enormous interest since it appeared as the first peripheral orexigenic factor. Besides, ghrelin exerts other neuroendocrine metabolic and non-endocrine actions (e.g. cardiovascular activities) that may rely on the widespread distribution of ghrelin and its receptor (GHS-R). The existence of several GHS-R subtypes and evidences that neuroendocrine and metabolic but not all other ghrelin actions are dependent on acylation on serine 3 add further complexity to the system whose major physiological role remains to be definitely elucidated. Ghrelin knockout(-/-) mice are neither anorectic nor dwarf though GHS-R-/- are slightly underweight and do not respond to ghrelin with increased GH secretion or appetite. Thus, the continuation of the fascinating ghrelin story as well as its potential pathophysiological implications in endocrinology and internal medicine remain open avenues for future investigations.

The role of the small bowel in the regulation of circulating ghrelin levels and food intake in the obese Zucker rat.

Circulating levels of ghrelin, a stomach peptide that promotes food intake, rise before and fall after meal. We aimed to investigate whether there is an independent contribution of the small bowel to the regulation of ghrelin and appetite. A duodenal-jejunal bypass (DJB) with preservation of normal gastric volume and exposure to nutrients was performed in 12-wk-old obese Zucker ZDF fa/fa rat. Food intake, weight gain, 48-h fasting, and 24-h refeeding levels of total and acylated ghrelin were measured. The DJB was challenged against gastric banding (GB), diet, and a sham operation in matched animals. Normal controls were age-matched Wistar rats, which underwent either DJB or a sham operation. The Zucker obese animals showed a paradoxical increase of acylated ghrelin levels after refeeding (+30% with respect to fasting levels; P = 0.001), an abnormality that was completely reversed only by the DJB (-30%; P = 0.01) but not after GB, diet, or sham operation. In obese rats, the DJB resulted in significantly less food intake and weight gain compared with both GB (P < 0.05) and sham operation (P < 0.01). In sharp contrast, the DJB did not alter food intake and weight gain in normal rats. The DJB does not physically restrict the flow of food but restores meal-induced suppression of acylated ghrelin and significantly reduces food intake in Zucker obese rats. These findings suggest an independent intestinal contribution to the regulation of the dynamic ghrelin response to eating and the possibility that defective signaling from the proximal bowel could be involved in the pathogenesis of obesity/hyperphagia.

Altered morphology of rod bipolar cell axonal terminals in the retinas of mice carrying genetic deletion of somatostatin subtype receptor 1 or 2.

Somatostatin (SRIF), similar to other neuropeptides, is likely to influence the morpho-functional characteristics of neurons. We studied possible morphological alterations of mouse retinal neurons following genetic deletion of SRIF subtype receptor 1 [sst1 knockout (KO)] or 2 (sst2 KO). In sst1 KO retinas, axonal terminals of rod bipolar cells (RBCs), identified with protein kinase C immunoreactivity, were 25% larger than in controls. In contrast, in sst2 KO retinas, RBC axonal terminals were significantly smaller (-14%). No major ultrastructural differences were observed between control and KO RBCs. In sst2 KO retinas, SRIF levels decreased by about 35%, while both sst1 receptor mRNA and protein increased by about 170% and 100%, respectively. This compares to previous results reporting an increase of both retinal SRIF and sst2 receptors following sst1 receptor deletion. Together, these findings suggest that, on the one hand, sst1 receptor deletion induces over-expression of sst2 receptors, and vice versa; on the other hand, that an imbalance in sst1 and sst2 receptor expression and/or changes in the levels of retinal SRIF induced by sst1 or sst2 receptor deletion are responsible for the morphological changes in RBC axonal terminals. Similar alterations of RBC terminals were observed in KO retinas at 2 weeks of age (eye opening). In addition, reverse transcription-polymerase chain reaction analysis of the expression of sst2 and sst1 receptors in developing sst1 and sst2 KO retinas, respectively, demonstrated that these receptors are up-regulated at or near eye opening. These findings suggest that the integrity of the somatostatinergic system during development is necessary for proper RBC maturation.

Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina.

In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst(1-5)). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain.