The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10⁻⁹ M and as early as 4 h. The phosphatidylinositol 3′ (PI3′)-kinase inhibitors wortmannin (at concentrations of 10⁻⁸ M) and LY294002 (10⁻⁶ M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70^S6K) inhibitor rapamycin (10⁻⁸ M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3′-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10⁻⁵ M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.

Obesity is frequently associated with metabolic disease. Here, we show that obesity changes the miRNA profile of plasma exosomes in mice, including increases in miR-122, miR-192, miR-27a-3p, and miR-27b-3p. Importantly, treatment of lean mice with exosomes isolated from obese mice induces glucose intolerance and insulin resistance. Moreover, administration of control exosomes transfected with obesity-associated miRNA mimics strongly induces glucose intolerance in lean mice and results in central obesity and hepatic steatosis. Expression of the candidate target gene Ppara is decreased in white adipose tissue but not in the liver of mimic-treated (MIMIC) mice, and this is accompanied by increased circulating free fatty acids and hypertriglyceridemia. Treatment with a specific siRNA targeting Ppara transfected into exosomes recapitulates the phenotype induced by obesity-associated miRNAs. Importantly, simultaneously reducing free fatty acid plasma levels in MIMIC mice with either the lipolysis inhibitor acipimox or the PPARα agonist fenofibrate partially protects against these metabolic alterations. Overall, our data highlight the central role of obesity-associated exosomal miRNAs in the etiopathogeny of glucose intolerance and dyslipidemia. Significance The presence of extracellular miRNAs in body fluids has been exploited as a brand-new source of biomarkers for different diseases. A fraction of those extracellular miRNAs, contained in extracellular vesicles and exosomes, are additionally being revealed as novel mediators of intercellular communication. Here, we show that systemic injection of exosomes transfected with synthetic miRNAs simulating those enriched in the plasma of obese mice robustly induces glucose intolerance, adipose inflammation, and hepatic steatosis in lean mice. These results support a role for exosomal miRNAs in the modulation of glucose and lipid metabolism in mice and may help us uncover thus far unexplored pathological mechanisms and provide us with novel therapeutic targets.

Mutations in the human genes encoding hepatocyte nuclear factors (HNF) 1α, 1β, 4α, and IPF1(PDX1/IDX1/STF1) result in pancreatic β cell dysfunction and diabetes mellitus. In hepatocytes, hnf4α controls the transcription of hnf1α , suggesting that this same interaction may operate in β cells and thus account for the common diabetic phenotype. We show that, in pancreatic islet and exocrine cells, hnf4α expression unexpectedly depends on hnf1α . This effect is tissue-specific and mediated through direct occupation by hnf1α of an alternate promoter located 45.6 kb from the previously characterized hnf4α promoter. Hnf1α also exerts direct control of pancreatic-specific expression of hnf4 γ and hnf3γ . Hnf1α dependence of hnf4α , hnf4γ , hnf3γ , and two previously characterized distal targets ( glut2 and pklr ) is established only after differentiated cells arise during pancreatic embryonic development. These studies define an unexpected hierarchical regulatory relationship between two genes involved in human monogenic diabetes in the cells, which are relevant to its pathophysiology. Furthermore, they indicate that hnf1α is an essential component of a transcription factor circuit whose role may be to maintain differentiated functions of pancreatic cells.

Implementation of regular physical activity helps in the maintenance of a healthy metabolic profile both in humans and mice through molecular mechanisms not yet completely defined. Here, we show that high-intensity interval training (HIIT) modifies the microRNA (miRNA) profile of circulating exosomes in mice, including significant increases in miR-133a and miR-133b. Importantly, treatment of sedentary mice with exosomes isolated from the plasma of trained mice improves glucose tolerance, insulin sensitivity, and decreases plasma levels of triglycerides. Moreover, exosomes isolated from the muscle of trained mice display similar changes in miRNA content, and their administration to sedentary mice reproduces the improvement of glucose tolerance. Exosomal miRNAs up-regulated by HIIT target insulin-regulated transcription factor forkhead box O1 (FoxO1) and, accordingly, expression of FoxO1 is decreased in the liver of trained and exosome-treated mice. Treatment with exosomes transfected with a miR-133b mimic or with a specific siRNA targeting FoxO1 recapitulates the metabolic effects observed in trained mice. Overall, our data suggest that circulating exosomes released by the muscle carry a specific miRNA signature that is modified by exercise and induce expression changes in the liver that impact whole-body metabolic profile. Significance The beneficial metabolic effects of exercise are mediated at least in part by the release of soluble factors by the muscles. Exosomes, small vesicles that facilitate the exchange of biological components among cells and tissues, may constitute one of these factors. Here, we show that exercise triggers the release of exosomes by the trained muscle, carrying a specific miRNA signature that induces gene expression changes in the liver, finally contributing to increased insulin sensitivity. Molecular characterization of exercise-induced exosomal miRNAs and their effects may drive the design of novel therapeutic strategies to alleviate insulin resistance and other aging-related ailments in an increasingly older society.

Diabetes is frequently diagnosed late, when the development of complications is almost inevitable, decreasing the quality of life of patients. However, early detection of affected individuals would allow the implementation of timely and effective therapies. Here we set to describe the profile of circulating microRNAs (miRNAs) in prediabetic patients with the intention of identifying novel diagnostic and therapeutic tools. We used real-time RT-PCR to measure the abundance of 176 miRNAs in serum of a cohort of 92 control and prediabetic individuals with either impaired fasting glucose or impaired glucose tolerance, as well as newly diagnosed diabetic patients. We validated the results in a second cohort of control and prediabetic subjects undergoing a therapeutic exercise intervention, as well as in a mouse model of glucose intolerance. We identified two miRNAs, miR-192 and miR-193b, whose abundance is significantly increased in the prediabetic state but not in diabetic patients. Strikingly, these miRNAs are also increased in plasma of glucose-intolerant mice. Moreover, circulating levels of miR-192 and miR-193b return to baseline in both prediabetic humans and glucose-intolerant mice undergoing a therapeutic intervention consisting in chronic exercise, which succeeded in normalizing metabolic parameters. Our data show that the pattern of circulating miRNAs is modified by defects in glucose metabolism in a similar manner in mice and humans. This circulating miRNA signature for prediabetes could be used as a new diagnostic tool, as well as to monitor response to intervention.

Epigenetic mechanisms, in particular the enzymatic modification of histones, are a crucial element of cell differentiation, a regulated process that allows a precursor cell basically to turn into a different cell type while maintaining the same genetic equipment. We have previously described that the promoters of adipogenic genes display significant levels of dimethylation at the Lys(4) of histone H3 (H3K4) in preadipocytes, where these genes are still silenced, thus maintaining the chromatin of the precursor cell in a receptive state. Here, we show that the expression of several histone demethylases and methyltransferases increases during adipogenesis, suggesting an important role for these proteins in this process. Knockdown of the H3K4/K9 demethylase LSD1 results in markedly decreased differentiation of 3T3-L1 preadipocytes. This outcome is associated with decreased H3K4 dimethylation and increased H3K9 dimethylation at the promoter of transcription factor cebpa, whose expression must be induced >200-fold upon stimulation of differentiation. Thus, our data suggest that LSD1 acts to maintain a permissive state of the chromatin in this promoter by opposing the action of a H3K9 methyltransferase. Knockdown of H3K9 methyltransferase SETDB1 produced the opposite results, by decreasing H3K9 dimethylation and increasing H3K4 dimethylation levels at the cebpa promoter and favoring differentiation. These findings indicate that the histone methylation status of adipogenic genes as well as the expression and function of the proteins involved in its maintenance play a crucial role in adipogenesis.