Sperm not only delivers the paternal genome to the oocyte but also regulatory small RNA (sRNA). However, the role of sRNA in fertilisation and human embryo development remains poorly understood. Here, couples undergoing IVF are recruited, and sperm sRNA analysed to investigate their role in IVF treatment. Differential expression of mitochondrial sRNA and Y-RNA are observed in relation to sperm concentration. For fertilisation rate, sRNA from a single locus are significantly changed. Expression of microRNA (miRNA) and ribosomal sRNA correlates positively and negatively, respectively, to high-quality embryos. Notably, the top miRNA have an area under ROC of >0.8. Predicted targets of these miRNA are relevant for development, suggesting a role for sperm-borne miRNA in embryo development. In conclusion, sperm-borne sRNA are biomarkers for sperm concentration and embryo quality in IVF. These findings may contribute to clinical strategies improving embryo quality, lowering costs and reducing the need for additional treatment cycles.

Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity.Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms.Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event.Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility.

In the recent years there has been a tremendous increase in our understanding of chromatin, transcription and the importance of metabolites in their regulation. This review highlights what is currently sparse information that suggest existence of a refined system integrating metabolic and chromatin control. We indicate possible regulatory modes, such as feed forward amplification, that may help effect and stabilize long-lasting phenotypic decisions within and even across generations using adipogenesis as the primary context.

The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.

We have examined the concentration of beta-carotene in the fat of isolated abdominal subcutaneous adipocytes obtained from lean (BMIless than23 kg/m(2)), non-obese with higher BMI (23 less than= BMIless than28 kg/m(2)), obese (BMI greater than= 28 kg/m(2)), and from a group of obese subjects with type 2 diabetes. The concentration of b-carotene was 50% lower in the adipocytes from the obese and obese/diabetic groups compared with the lean and non-obese groups. Interestingly, the total amount of beta-carotene in the adipocyte stores of each subject was constant among all groups. Triacylglycerol constituted 92 +/- 1% (by weight) of the adipocyte lipids in the lean group and this was increased to 99 +/- 2% in the obese group with diabetes (pless than0.05). The concentration of cholesteryl esters was in all cases less than0.1 g per 100 g of total lipids, demonstrating that mature human adipocytes have negligible stores of cholesteryl ester. Our findings demonstrate that adipocyte concentrations of beta-carotene are reduced in obese subjects. The lower concentrations in adipocytes from subjects with type 2 diabetes apparently reflect subjects obesity. Our finding that whole-body stores of beta-carotene in adipocytes are constant raises new questions regarding what function it serves, as well as the mechanisms for maintaining constant levels in the face of varied adipose tissue mass among individuals over a period of time.

In both insects and vertebrates, each olfactory sensory neuron (OSN) expresses one odorant receptor (OR) from a large genomic repertoire. How a receptor is specified is a tantalizing question addressing fundamental aspects of cell differentiation. Here, we demonstrate that the corepressor Atrophin (Atro) segregates OR gene expression between OSN classes in Drosophila. We show that the knockdown of Atro result in either loss or gain of a broad set of ORs. Each OR phenotypic group correlated with one of two opposing Notch fates, Notch responding, Nba (N(on)), and nonresponding, Nab (N(off)) OSNs. Our data show that Atro segregates ORs expressed in the Nba OSN classes and helps establish the Nab fate during OSN development. Consistent with a role in recruiting histone deacetylates, immunohistochemistry revealed that Atro regulates global histone 3 acetylation (H3ac) in OSNs and requires Hdac3 to segregate OR gene expression. We further found that Nba OSN classes exhibit variable but higher H3ac levels than the Nab OSNs. Together, these data suggest that Atro determines the level of H3ac, which ensures correct OR gene expression within the Nba OSNs. We propose a mechanism by which a single corepressor can specify a large number of neuron classes.-Alkhori, L., Öst, A., Alenius, M. The corepressor Atrophin specifies odorant receptor expression in Drosophila.

The protein kinase mammalian target of rapamycin (mTOR) mediates insulin control ofprotein synthesis, autophagy, mitochondrial function, and, through feedback signaling tophosphorylation of IRS1 at serine residues, mTOR directly controls insulin signaling. Weshow that in adipocytes from patients with type 2 diabetes (T2D) insulin activation of mTORis attenuated and that the resultant phenotype is compatible with, and can be mimicked by,loss of mTOR activation. In T2D adipocytes mitochondrial function is impaired andautophagy strongly upregulated, with concomitant increased autophagic destruction ofmitochondria and lipofuscin particles, and a dependence on autophagy for ATP production.Conversely, mitochondrial dysfunction attenuates insulin activation of mTOR, enhancesautophagy and attenuates feedback to IRS1. Our findings put mTOR in the driver´s seat of aninsulin resistance that in adipocytes can be fuelled by mitochondrial dysfunction,inflammation, ER-stress, or hypoxia.

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta(3)-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KID of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained P-32-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta(3)-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KID. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.

Energy storage in the adipose tissue, to an extent leading to obesity, is associated with local as well assystemic insulin resistance. When insulin-producing beta-cells in the pancreas gradually fail tocompensate, plasma levels of glucose rise and overt type 2 diabetes is diagnosed. Adipocytes are largecells, mostly consisting of one big central lipid droplet, with the surrounding plasma membrane full ofsmall invaginations called caveolae. As caveolae contain the insulin receptor and several other insulinsignallingproteins, we have investigated several aspects of caveolae. We have also mapped mechanismsand defects in the insulin-signalling network in adipocytes from type 2 diabetic patients.

In paper I, we show that a subtype of caveolae has the capability to synthesize triglycerides from fattyacids and glycerol-3-phosphate. The triglyceride-synthesizing caveolae subtype also contains perilipin,suggesting the existence of a mechanism to protect newly made triglycerides from hydrolysis.

In paper II, we demonstrate that adipocytes from patients with type 2 diabetes have an attenuated insulinstimulatedphosphorylation of IRS-1 at Ser-307 (human sequence), which correlates with reduced insulinstimulatedphosphorylation of IRS-1 at tyrosine residues. Insulin-stimulated phosphorylation of IRS-1 atSer-307 is dependent on the nutrient sensor TORC1. This finding indicates that adipocytes from type 2diabetic patients have reduced TORC1 activity.

In paper III, we focus on the mechanisms for RBP4-induced insulin resistance. We also continue ourmapping of insulin-resistance in adipocytes from type 2 diabetes. These cells exhibit, in addition toimpaired insulin-stimulated glucose uptake and the defects presented in paper I, impaired insulinstimulatedphosphorylation of ERK. We do, however, not see any defects in PKB signalling. Neither dowe se any enhanced insulin-stimulated phosphorylation of IRS-1 at Ser-312 (human sequence), a site thatin mice is hyper-stimulated in response to high-fat feeding. Incubation with RBP4 recapitulates all defectswe so far have seen in type 2 diabetes except reduced insulin-stimulated glucose uptake. These results aremirrored by blockade of endogenously produced RBP4 in the incubations with adipocytes from type 2diabetic patients. In other words, RBP4-blocking antibodies restore all insulin-signalling defects we havefound in adipocytes from type 2 diabetic patients, except insulin-stimulated glucose uptake.

In paper IV we show by several approaches that TORC1 activation is down-regulated in adipocytes fromtype 2 diabetic patients. The main finding is that there is enhanced autophagy in those adipocytes.Interestingly, autophagy may be a mechanism to enhance the breakdown of stored triglycerides in theadipocyte.

In conclusion, our data suggest that caveolae, in addition to being micro-domains for insulin-signallingare metabolic platforms. We describe defects in insulin-signalling in adipocytes from type 2 diabeticpatients where the main finding is enhanced autophagy in these obese patients. The perceived starvationin adipose tissue might via secretion of adipokines, such as RBP4, have implications for local as well assystemic insulin-resistance.

Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m²) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.