Françoise Kühne

Uncoupling Protein-3 Expression in Rodent Skeletal Muscle Is Modulated by Food Intake but Not by Changes in Environmental Temperature

A new member of the uncoupling protein (UCP) family called UCP3 has recently been cloned and shown to be highly expressed in skeletal muscle of rodents and humans. In the present study, UCP3 was overexpressed in C2C12myoblasts where it acts as an uncoupling protein. Changes in UCP3 mRNA expression were examined in rodent muscles under conditions known to modulate thermogenesis in brown adipose tissue. In skeletal muscle, UCP3 expression did not change in response to 48 h of cold exposure (6 °C), whereas it was decreased by 81% or increased 5.6-fold by 1 week of 50% food restriction or fasting, respectively. It was also decreased by 36% in soleus muscle of obese (fa/fa) as compared with lean Zucker rats. The unexpected rise of UCP3 mRNA level induced by fasting did not change in vitro muscle basal heat production rate but decreased by 31% the capacity to produce heat in response to the uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone. This decrease may reflect underlying uncoupling by UCP3. Up-regulation of UCP3 mRNA after a 24-h fast was still observed in mice exposed at thermoneutrality. These results show that the increase in UCP3 expression induced by fasting is associated with the maintenance of thermogenesis measured in muscle in vitro and is not modulated by environmental temperature. The notion that UCP3 expression is modulated by food intake is of importance to better understand the pathophysiology of obesity in humans.

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Sertoli cell Dicer is essential for spermatogenesis in mice

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

A phytoestrogen-rich diet increases energy expenditure and decreases adiposity in mice

Background Obesity is an increasingly prevalent health problem, and natural effective therapeutic approaches are required to prevent its occurrence. Phytoestrogens are plant-derived compounds with estrogenic activities; they can bind to both estrogen receptors α and β and mimic the action of estrogens on target organs. Objectives The purpose of this study was to examine the influence of soy-derived phytoestrogens on energy balance and metabolism. Methods Male outbred mice (CD-1) were allowed ad libitum access to either a high soy-containing diet or a soy-free diet from conception to adulthood. We measured circulating serum isoflavone levels using reverse-phase solid-phase extraction for subsequent liquid chromatography electrospray tandem mass spectrometry analysis. Adult animals were analyzed for body composition by dual-energy X-ray absorptiometry, locomotor activity by running-wheel experiments, respiratory exchange rate by indirect calorimetry, and food intake using metabolic cages. Quantitative reverse transcriptase-polymerase chain reaction was performed to determine the expression of hypothalamic neuropeptide genes. Results We found that adult mice fed a soy-rich diet had reduced body weight, adiposity, and resistance to cold. This lean phenotype was associated with an increase in lipid oxidation due to a preferential use of lipids as fuel source and an increase in locomotor activity. The modulation of energy balance was associated with a central effect of phytoestrogens on the expression of hypothalamic neuropeptides, including agouti-related protein. Conclusion The data suggest that dietary soy could have beneficial effects on obesity, but they also emphasize the importance of monitoring the phytoestrogen content of diets as a parameter of variability in animal experiments.

Loss of Dicer in Sertoli Cells Has a Major Impact on the Testicular Proteome of Mice

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3′UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.

Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.

Single-cell transcriptomics of the mouse gonadal soma reveals the establishment of sexual dimorphism in distinct cell lineages

Sex determination is a unique process that allows the study of multipotent progenitors and their acquisition of sex-specific fates during differentiation of the gonad into a testis or an ovary. Using time-series single-cell RNA sequencing (scRNA-seq) on ovarian Nr5a1-GFP+ somatic cells during sex determination, we identified a single population of early progenitors giving rise to both pre-granulosa cells and potential steroidogenic precursor cells. By comparing time-series scRNA-seq of XX and XY somatic cells, we demonstrate that the supporting cells emerge from the early progenitors with a non-sex-specific transcriptomic program, before pre-granulosa and Sertoli cells acquire their sex-specific identity. In XX and XY steroidogenic precursors similar transcriptomic profiles underlie the acquisition of cell fate, but with a delay in XX cells. Our data provide a novel framework, at single-cell resolution, for further interrogation of the molecular and cellular basis of mammalian sex determination.

Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insülin receptor (INSR) and type‐I insülin‐like growth factor receptor (IGF1R), have not, to oür knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double‐mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell‐lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell‐autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.—Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stevant, I., Griffeth, R. J., Pitetti, J.‐L., Andric, S. A., Hü, M.‐C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adült Leydig cells. FASEB J. 32, 3321–3335 (2018). www.fasebj.org