Consensus Delphi study on guidelines for the assessment of anterior cruciate ligament injuries in children.

BACKGROUND: Knee examination guidelines in minors are intended to aid decision-making in the management of knee instability. CLINICAL QUESTION: A Delphi study was conducted with a formal consensus process using a validated methodology with sufficient scientific evidence. A group consensus meeting was held to develop recommendations and practical guidelines for use in the assessment of instability injuries in children. KEY FINDINGS: there is a lack of evidence to analyse anterior cruciate ligament injuries in children and their subsequent surgical management if necessary. Diagnostic guidelines and clinical assessment of the patient based on a thorough examination of the knee are performed and a guide to anterior cruciate ligament exploration in children is developed. CLINICAL APPLICATION: In the absence of a strong evidence base, these established guidelines are intended to assist in that decision-making process to help the clinician decide on the most optimal treatment with the aim of benefiting the patient as much as possible. Following this expert consensus, surgical treatment is advised when the patient has a subjective sensation of instability accompanied by a pivot shift test ++, and may include an anterior drawer test + and a Lachman test +. If these conditions are not present, the conservative approach should be chosen, as the anatomical and functional development of children, together with a physiotherapy programme, may improve the evolution of the injury.

Early programming of reproductive health and fertility: novel neuroendocrine mechanisms and implications in reproductive medicine.

BACKGROUND: According to the Developmental Origins of Health and Disease (DOHaD) hypothesis, environmental changes taking place during early maturational periods may alter normal development and predispose to the occurrence of diverse pathologies later in life. Indeed, adverse conditions during these critical developmental windows of high plasticity have been reported to alter the offspring developmental trajectory, causing permanent functional and structural perturbations that in the long term may enhance disease susceptibility. However, while solid evidence has documented that fluctuations in environmental factors, ranging from nutrient availability to chemicals, in early developmental stages (including the peri-conceptional period) have discernible programming effects that increase vulnerability to develop metabolic perturbations, the impact and eventual mechanisms involved, of such developmental alterations on the reproductive phenotype of offspring have received less attention. OBJECTIVE AND RATIONALE: This review will summarize recent advances in basic and clinical research that support the concept of DOHaD in the context of the impact of nutritional and hormonal perturbations, occurring during the periconceptional, fetal and early postnatal stages, on different aspects of reproductive function in both sexes. Special emphasis will be given to the effects of early nutritional stress on the timing of puberty and adult gonadotropic function, and to address the underlying neuroendocrine pathways, with particular attention to involvement of the Kiss1 system in these reproductive perturbations. The implications of such phenomena in terms of reproductive medicine will also be considered. SEARCH METHODS: A comprehensive MEDLINE search, using PubMed as main interface, of research articles and reviews, published mainly between 2006 and 2021, has been carried out. Search was implemented using multiple terms, focusing on clinical and preclinical data from DOHaD studies, addressing periconceptional, gestational and perinatal programming of reproduction. Selected studies addressing early programming of metabolic function have also been considered, when relevant. OUTCOMES: A solid body of evidence, from clinical and preclinical studies, has documented the impact of nutritional and hormonal fluctuations during the periconceptional, prenatal and early postnatal periods on pubertal maturation, as well as adult gonadotropic function and fertility. Furthermore, exposure to environmental chemicals, such as bisphenol A, and maternal stress has been shown to negatively influence pubertal development and gonadotropic function in adulthood. The underlying neuroendocrine pathways and mechanisms involved have been also addressed, mainly by preclinical studies, which have identified an, as yet incomplete, array of molecular and neurohormonal effectors. These include, prominently, epigenetic regulatory mechanisms and the hypothalamic Kiss1 system, which likely contribute to the generation of reproductive alterations in conditions of early nutritional and/or metabolic stress. In addition to the Kiss1 system, other major hypothalamic regulators of GnRH neurosecretion, such as γ-aminobutyric acid and glutamate, may be targets of developmental programming. WIDER IMPLICATIONS: This review addresses an underdeveloped area of reproductive biology and medicine that may help to improve our understanding of human reproductive disorders and stresses the importance, and eventual pathogenic impact, of early determinants of puberty, adult reproductive function and fertility.

Neonatal exposure to androgens dynamically alters gut microbiota architecture.

Gonadal steroids strongly contribute to the metabolic programming that shapes the susceptibility to the manifestation of diseases later in life, and the effect is often sexually dimorphic. Microbiome signatures, together with metabolic traits and sex steroid levels, were analyzed at adulthood in neonatally androgenized female rats, and compared with those of control male and female rats. Exposure of female rats to high doses of androgens on early postnatal life resulted in persistent alterations of the sex steroid profile later on life, namely lower progesterone and higher estradiol and estrone levels, with no effect on endogenous androgens. Neonatally androgenized females were heavier (10% at early adulthood and 26% at adulthood) than controls and had impaired glucose homeostasis observed by higher AUC of glucose in GTT and ITT when subjected to obesogenic manipulations. Androgenized female displayed overt alterations in gut microbiota, indicated especially by higher Bacteroidetes and lower Firmicutes abundance at early adulthood, which disappeared when animals were concurrently overfed at adulthood. Notably, these changes in gut microbiota were related with the intestinal expression of several miRNAs, such as miR-27a-3p, miR-29a-5p, and miR-100-3p. Our results suggest that nutritional and hormonal disruption at early developmental periods not only alters the metabolic programming of the individual later in life but also perturbs the architecture of gut microbiota, which may interact with the host by a cross-talk mediated by intestinal miRNAs; phenomena that may contribute to amplify the metabolic derangement caused by obesity, as seen in neonatally androgenized female rats.

Intergenerational Influence of Paternal Obesity on Metabolic and Reproductive Health Parameters of the Offspring: Male-Preferential Impact and Involvement of Kiss1-Mediated Pathways.

Obesity and its comorbidities are reaching epidemic proportions worldwide. Maternal obesity is known to predispose the offspring to metabolic disorders, independently of genetic inheritance. This intergenerational transmission has also been suggested for paternal obesity, with a potential negative impact on the metabolic and, eventually, reproductive health of the offspring, likely via epigenetic changes in spermatozoa. However, the neuroendocrine component of such phenomenon and whether paternal obesity sensitizes the offspring to the disturbances induced by high-fat diet (HFD) remain poorly defined. We report in this work the metabolic and reproductive impact of HFD in the offspring from obese fathers, with attention to potential sex differences and alterations of hypothalamic Kiss1 system. Lean and obese male rats were mated with lean virgin female rats; male and female offspring were fed HFD from weaning onward and analyzed at adulthood. The increases in body weight and leptin levels, but not glucose intolerance, induced by HFD were significantly augmented in the male, but not female, offspring from obese fathers. Paternal obesity caused a decrease in luteinizing hormone (LH) levels and exacerbated the drop in circulating testosterone and gene expression of its key biosynthetic enzymes caused by HFD in the male offspring. LH responses to central kisspeptin-10 administration were also suppressed in HFD males from obese fathers. In contrast, paternal obesity did not significantly alter gonadotropin levels in the female offspring fed HFD, although these females displayed reduced LH responses to kisspeptin-10. Our findings suggest that HFD-induced metabolic and reproductive disturbances are exacerbated by paternal obesity preferentially in males, whereas kisspeptin effects are affected in both sexes.

Neonatal Androgen Exposure Causes Persistent Gut Microbiota Dysbiosis Related to Metabolic Disease in Adult Female Rats.

Alterations of gut microbiome have been proposed to play a role in metabolic disease, but the major determinants of microbiota composition remain ill defined. Nutritional and sex hormone challenges, especially during early development, have been shown to permanently alter adult female phenotype and contribute to metabolic disturbances. In this study, we implemented large-scale microbiome analyses to fecal samples from groups of female rats sequentially subjected to various obesogenic manipulations, including sex hormone perturbations by means of neonatal androgenization or adult ovariectomy (OVX), as a model of menopause, to establish whether these phenomena are related to changes in gut microbiota. Basic metabolic profiles concerning glucose/insulin homeostasis were also explored. The effects of the sex hormonal perturbations, either developmentally (androgenization) or in adulthood (OVX), clearly outshone the impact of nutritional interventions, especially concerning the gut microbiota profile. Notably, we observed a lower diversity in the androgenized group, with the highest Firmicutes to Bacteroidetes ratio, supporting the occurrence of durable alterations in gut microbiota composition, even in adulthood. Moreover, the elimination of adult ovarian secretions by OVX affected the richness of gut microbiota. Our data are the first to document the durable impact of sex steroid manipulations, and particularly early androgenization, on gut microbiota composition. Such dysbiosis is likely to contribute to the metabolic perturbations of conditions of obesity linked to gonadal dysfunction in the female.

Fibroblast activation protein (FAP) as a novel metabolic target.

OBJECTIVE: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice. METHODS: To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity. RESULTS: TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. CONCLUSIONS: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.

Age and sex dependent effects of early overnutrition on metabolic parameters and the role of neonatal androgens.

BACKGROUND: Males and females respond differently to diverse metabolic situations. Being raised in a small litter is reported to cause overnutrition that increases weight gain and predisposes an individual to metabolic disturbances in adulthood; however, existing data are inconsistent. Indeed, significant weight gain and/or metabolic disturbances, such as hyperinsulinemia and hyperleptinemia, are sometimes not encountered. We hypothesized that these inconsistencies could be due to the animal's sex and/or age at which metabolic parameters are measured. METHODS: To analyze the effects of neonatal overnutrition, male and female Wistar rats were raised in litters of 4 or 12 pups/dam and killed at postnatal days (PND) 10, 21, 30, 50, 85, or 150. In a second study to determine if neonatal sex steroid levels influence sex differences in metabolic parameters, female rats were treated with testosterone on PND1. Effects on weight, length, fat pads, adipokine production, and serum levels of glucose, metabolic hormones, and cytokines were analyzed in both studies. RESULTS: By PND10, both males and females raised in small litters had increased body weight, body length, adiposity, and serum glucose, insulin, leptin, and adiponectin levels. Females had a greater increase in inguinal fat, and males had higher expression of leptin messenger RNA (mRNA) and serum insulin, as well as increased testosterone levels. Most of the litter size effects diminished or disappeared after weaning and reappeared during adulthood in males, with sex differences in body size and adiposity being apparent postpubertally. Treatment of females with testosterone on PND1 tended to masculinize some metabolic parameters in adulthood such as increased body weight and serum leptin levels. CONCLUSIONS: Our results indicate that (1) both sex and age determine the response to neonatal overnutrition; (2) differences in neonatal sex steroid levels may participate in the development of sex differences in metabolic parameters in adulthood and possibly in the response to neonatal overnutrition; and (3) the comparison of circulating hormone and cytokine levels, even in normal control animals, should take into consideration the early neonatal nutritional environment.

Obesity-induced hypogonadism in the male: premature reproductive neuroendocrine senescence and contribution of Kiss1-mediated mechanisms.

Reproduction is sensitive to insufficient body energy reserves, especially in females. Metabolic regulation of the male reproductive axis is less obvious, and the impact of conditions of persistent energy excess has received moderate attention. Yet, the escalating prevalence of obesity and the clinical evidence of its deleterious effects on male fertility have raised considerable concerns. We report here phenotypic and mechanistic studies of the reproductive impact of postnatal nutritional manipulations (mainly overnutrition) coupled to a high-fat diet (HFD) after weaning. Metabolic and hormonal analyses in young (4 months old) and middle-aged (10 months old) animals revealed that HFD caused profound metabolic perturbations, including glucose intolerance, which were worsened by precedent postnatal overfeeding; these were detectable already in young males but aggravated in 10-month-old rats. Impairment of reproductive parameters took place progressively, and HFD alone was sufficient to explain most of these alterations, regardless of postnatal under- or overnutrition. In young males, testosterone (T) levels and steroidogenic enzyme expression were suppressed by HFD, without compensatory increases of LH levels, which were in fact partially inhibited in heavier males. In addition, obese males displayed suppressed hypothalamic Kiss1 expression despite low T, and HFD inhibited LH responses to kisspeptin. Overweight anticipated some of the neuroendocrine effects of aging, such as the suppression of hypothalamic Kiss1 expression and the decline in serum T and LH levels. Nonetheless, HFD per se caused a detectable worsening of key reproductive indices in middle-aged males, such as basal LH and FSH levels as well as LH responses to kisspeptin. Our study demonstrates that nutritional stress, especially HFD, has a profound deleterious impact on metabolic and gonadotropic function as well as on the Kiss1 system and precipitates neuroendocrine reproductive senescence in the male.

Phosphorylated S6K1 (Thr389) is a molecular adipose tissue marker of altered glucose tolerance.

Molecular tissue markers of altered glucose metabolism will be useful as potential targets for antidiabetic drugs. S6K1 is a downstream signal of insulin action. We aimed to evaluate (pThr389)S6K1 and total S6K1 levels in human and rat fat depots as candidate markers of altered glucose metabolism. (pThr389)S6K1 and total S6K1 levels were measured using enzyme linked immune sorbent assay (ELISA) in 49 adipose tissue samples from subjects with morbid obesity and in 18 peri-renal white adipose tissue samples from rats. The effects of high glucose and rosiglitazone have been explored in human preadipocytes. (pThr389)S6K1/(total)S6K1 in subcutaneous adipose tissue was significantly increased subjects with Type 2 diabetes (0.78 ± 0.26 vs. 0.55 ± 0.14, P=.02) and associated with fasting glucose (r=0.46, P=.04) and glycated hemoglobin (r=0.63, P=.02) in SAT. Similar associations with fasting glucose (r=0.43, P=.03) and IRS1 (r=-0.41, P=.04) gene expression were found in visceral adipose tissue. In addition, rat experiments confirmed the higher (pThr389)S6K1/totalS6K1 levels in adipose tissue in association with obesity-associated metabolic disturbances. (pThr389)S6K1/totalS6K1 was validated using western blot in rat adipose tissue. Both ELISA and western blot data significantly correlated (r=0.85, P=.005). In human preadipocytes, high glucose medium led to increased (pThr389)S6K1/total S6K1 levels in comparison with normal glucose medium, which was significantly decreased under rosiglitazone administration. In conclusion, in human and rat adipose tissue, phosphorylated S6K1 is a marker for increased glucose levels.

Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes.

Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity.

The anorexigenic neuropeptide, nesfatin-1, is indispensable for normal puberty onset in the female rat.

The hypothalamic peptide, nesfatin-1, derived from the precursor NEFA/nucleobindin 2 (NUCB2), was recently identified as anorexigenic signal, acting in a leptin-independent manner. Yet its participation in the regulation of other biological functions gated by body energy status remains unexplored. We show herein that NUCB2/nesfatin-1 is involved in the control of female puberty. NUCB2/nesfatin mRNA and protein were detected at the hypothalamus of pubertal female rats, with prominent signals at lateral hypothalamus (LHA), paraventricular (PVN), and supraoptic (SON) nuclei. Hypothalamic NUCB2 expression raised along pubertal transition, with detectable elevations of its mRNA levels at LHA, PVN, and SON, and threefold increase of its total protein content between late-infantile and peripubertal periods. Conditions of negative energy balance, such as 48 h fasting or sustained subnutrition, decreased hypothalamic NUCB2 mRNA and/or protein levels in pubertal females. At this age, central administration of nesfatin-1 induced modest but significant elevations of circulating gonadotropins, whose magnitude was notably augmented in conditions of food deprivation. Continuous intracerebroventricular infusion of antisense morpholino oligonucleotides (as-MONs) against NUCB2 along pubertal maturation, which markedly reduced hypothalamic NUCB2 protein content, delayed vaginal opening and decreased ovarian weights and serum luteinizing hormone (LH) levels. In contrast, in adult female rats, intracerebroventricular injection of nesfatin did not stimulate LH or follicle-stimulating hormone secretion; neither did central as-MON infusion alter preovulatory gonadotropin surges, despite suppression of hypothalamic NUCB2. In sum, our data are the first to disclose the indispensable role of NUCB2/nesfatin-1 in the central networks driving puberty onset, a function that may contribute to its functional coupling to energy homeostasis.

The Ppz protein phosphatases regulate Trk-independent potassium influx in yeast.

The Ppz protein phosphatases have been recently shown to negatively regulate the major potassium transport system in the yeast Saccharomyces cerevisiae, encoded by the TRK1 and TRK2 genes. We have found that, in the absence of the Trk system, Ppz mutants require abnormally high concentrations of potassium to proliferate. This can be explained by the observation that trk1 trk2 ppz1 or trk1 trk2 ppz1 ppz2 strains display a very poor rubidium uptake, with markedly increased Km values. These cells are very sensitive to the presence of several toxic cations in the medium, such as hygromicyn B or spermine, but not to lithium or sodium cations. At limiting potassium concentrations, addition of EGTA to the medium improves growth of these mutants. Therefore, our results indicate that, in addition to their role in regulating Trk potassium transporters, Ppz phosphatases (essentially Ppz1), positively affect the residual low affinity potassium transport mechanisms in yeast. These findings may provide a new way to elucidate the molecular nature of the low affinity potassium uptake system in yeast as well as a useful model to analyze the function of plant or mammalian potassium channels through heterologous expression in yeast.