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.

Motivation: Feature-based counting is commonly used in RNA-sequencing (RNA-seq) analyses. Here, sequences must align to target features (like genes or non-coding RNAs) and related sequences with different compositions are counted into the same feature. Consequently, sequence integrity is lost, making results less traceable against raw data.Small RNA (sRNA) often maps to multiple features and shows an incredible diversity in form and function. Therefore, applying feature-based strategies may increase the risk of misinterpretation. We present a strategy for sRNA-seq analysis that preserves the integrity of the raw sequence making the data lineage fully traceable. We have consolidated this strategy into Seqpac: An R package that makes a complete sRNA analysis available on multiple platforms. Using published biological data, we show that Seqpac reveals hidden bias and adds new insights to studies that were previously analyzed using feature-based counting.We have identified limitations in the concurrent analysis of RNA-seq data. We call it the traceability dilemma in alignment-based sequencing strategies. By building a flexible framework that preserves the integrity of the read sequence throughout the analysis, we demonstrate better interpretability in sRNA-seq experiments, which are particularly vulnerable to this problem. Applying similar strategies to other transcriptomic workflows may aid in resolving the replication crisis experienced by many fields that depend on transcriptome analyses.

Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, we show that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV w(m4h) reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Small RNA (sRNA) sequencing has been critical for our understanding of many cellular processes, including gene regulation. Nonetheless, the varying biochemical properties of sRNA, such as 5 nucleotide modifications, make many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy that captures a more complete picture of sRNA. By tagging 5 P sRNA during library preparation, 5XP-seq combines an open approach that includes all types of 5MODIFIER LETTER PRIME-terminal modifications (5 X), with a selective approach for 5-phosphorylated sRNA (5 P). We show that 5XP-seq not only enriches phosphorylated miRNA and piRNA but successfully discriminates these sRNA from all other sRNA species. We further demonstrate the importance of this strategy by successful inter-species validation of sRNAs that would have otherwise failed, including human to insect translation of several tRNA (tRFs) and rRNA (rRFs) fragments. By combining 5 insensitive library strategies with 5 sensitive tagging, we have successfully tackled an intrinsic bias in modern sRNA sequencing that will help us reveal the true complexity and the evolutionary significance of the sRNA world.

The world has recently experienced a decline in male reproductive (e.g. sperm counts and motility) and metabolic (e.g. obesity and diabetes) health. Accumulated evidence from animal models also shows that the metabolic health of the father may influence the metabolic health in his offspring. Vectors for such paternal intergenerational metabolic responses (IGMRs) involve small noncoding RNAs (sncRNAs) that often increase in spermatozoa during the last days of maturation in the epididymis. We and others have shown that the metabolic state - depending on factors such as diet, obesity and physical exercise - may affect sperm quality and sperm sncRNA. Together, this suggests that there are overlapping aetiologies between the male metabolic syndrome, male factor infertility and intergenerational responses. In this review, we present a theoretical framework for an overlap of these aetiologies by exploring the advances in our understanding of the roles of sncRNA in spermatogenesis and offspring development. A special focus will lie on novel findings about tRNA-derived small RNA (tsRNA), rRNA-derived small RNA (rsRNA) and small mitochondrial RNA (mitoRNA), and their emerging roles in intergenerational metabolic and reproductive health.

The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.

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.

The plasma membrane of cells functions as a barrier to the environment. Caveolae are minute invaginations of the membrane that selectively carry out the exchange of information and materials with the environment, by functioning as organizers of signal transduction and through endocytosis. Recent findings of uptake of different metabolites and of lipid metabolism occurring in caveolae, point to a new general function of caveolae. As gateways for the uptake of nutrients across the plasma membrane, and as platforms for the metabolic conversion of nutrients, especially in adipocytes, caveolae are now emerging as active centers for many aspects of intermediary metabolism, with implications for our understanding of obesity, diabetes and other metabolic disorders.

Aims/hypothesis: Several studies have suggested that large fat cells are less responsive to insulin than small fat cells. However, in these studies, large fat cells from obese individuals were compared with smaller fat cells from leaner participants, in effect making it impossible to draw conclusions about whether there is a causal relationship between fat cell size and insulin sensitivity. We hypothesised that small fat cells might be more insulin-responsive than large adipocytes when obtained from the same individual.

Materials and methods: We developed a method of sorting isolated primary human fat cells by using nylon filters of two different pore sizes. The cells were stained to visualise DNA, which allowed discrimination from artefacts such as lipid droplets. The sorted cells were left to recover overnight, since we had previously demonstrated that this is necessary for correct assessment of insulin response.

Results: We found similar amounts of the insulin receptor (IR), IRS-1 and GLUT4 when we compared small and large adipocytes from the same volunteer by immunoblotting experiments using the same total cell volume from both cell populations. Activation of IR, IRS-1 and Akt1 (also known as protein kinase B) by insulin was similar in the two cell populations. However, immunofluorescence confocal microscopy of plasma membrane sheets did not reveal any increase in the amount of GLUT4 in the plasma membrane following insulin stimulation in the large fat cells, whereas we saw a twofold increase in the amount of GLUT4 in the small fat cells.

Conclusions/interpretation: Our results support a causal relationship between the accumulation of large fat cells in obese individuals and reduced insulin responsiveness.

Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.

ZnO nanorods with 80 nm diameter and 700 nm length and grown on the tip of a borosilicate glass capillary (0.7 μm in diameter) were used to create a highly sensitive pH sensor for monitoring in vivo biological process within single cells. The ZnO nanorods, functionalized by proton H3 O+ and hydroxyl O H- groups, exhibit a pH -dependent electrochemical potential difference versus a AgAgCl microelectrode. The potential difference was linear over a large dynamic range (4-11), which could be understood in terms of the change in surface charge during protonation and deprotonation. These nanoelectrode devices have the ability to enable analytical measurements in single living cells and have the capability to sense individual chemical species in specific locations within a cell. © 2007 American Institute of Physics.

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC50 for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t1/2 of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.

Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO₂ at 37 °C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA-IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using β-cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation. Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.