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The medicinal uses of Calotropis procera are diverse, yet some of them are based on effects that still lack scientific support. Control of diabetes is one of them. Recently, latex proteins from C. procera latex (LP) have been shown to promote in vivo glycemic control by the inhibition of hepatic glucose production via AMP-activated protein kinase (AMPK). Glycemic control has been attributed to an isolated fraction of LP (CpPII), which is composed of cysteine peptidases (95%) and osmotin (5%) isoforms. Those proteins are extensively characterized in terms of chemistry, biochemistry and structural aspects. Furthermore, we evaluated some aspects of the mitochondrial function and cellular mechanisms involved in CpPII activity. The effect of CpPII on glycemic control was evaluated in fasting mice by glycemic curve and glucose and pyruvate tolerance tests. HepG2 cells was treated with CpPII, and cell viability, oxygen consumption, PPAR activity, production of lactate and reactive oxygen species, mitochondrial density and protein and gene expression were analyzed. CpPII reduced fasting glycemia, improved glucose tolerance and inhibited hepatic glucose production in control animals. Additionally, CpPII increased the consumption of ATP-linked oxygen and mitochondrial uncoupling, reduced lactate concentration, increased protein expression of mitochondrial complexes I, III and V, and activity of peroxisome-proliferator-responsive elements (PPRE), reduced the presence of reactive oxygen species (ROS) and increased mitochondrial density in HepG2 cells by activation of AMPK/PPAR. Our findings strongly support the medicinal use of the plant and suggest that CpPII is a potential therapy for prevention and/or treatment of type-2 diabetes. A common epitope sequence shared among the proteases and osmotin is possibly the responsible for the beneficial effects of CpPII.

Human islet amyloid polypeptide (hIAPP or amylin) is a hormone co-secreted with insulin by pancreatic β-cells, and is the main component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes and may be involved in β-cell dysfunction and death, observed in this disease. Thus, counteracting islet amyloid toxicity represents a therapeutic approach to preserve β-cell mass and function. In this sense, thiazolidinediones (TZDs), as rosiglitazone, have shown protective effects against other harmful insults to β-cells. For this reason, we investigated whether rosiglitazone could protect β-cells from hIAPP-induced cell death and the underlying mechanisms mediating such effect. Here, we show that rosiglitazone improved the viability of hIAPP-exposed INS-1E cells. This benefit is not dependent on the insulin-degrading enzyme (IDE) since rosiglitazone did not modulate IDE protein content and activity. However, rosiglitazone inhibited hIAPP fibrillation and decreased hIAPP-induced expression of C/EBP homologous protein (CHOP) (CTL 100.0 ± 8.4; hIAPP 182.7 ± 19.1; hIAPP + RGZ 102.8 ± 9.5), activating transcription factor-4 (ATF4) (CTL 100.0 ± 3.1; hIAPP 234.9 ± 19.3; hIAPP + RGZ 129.6 ± 3.0) and phospho-eukaryotic initiation factor 2-alpha (p-eIF2α) (CTL 100.0 ± 31.1; hIAPP 234.1 ± 36.2; hIAPP + RGZ 150.4 ± 18.0). These findings suggest that TZDs treatment may be a promising approach to preserve β-cell mass and function by inhibiting islet amyloid formation and decreasing endoplasmic reticulum stress hIAPP-induced.

Background and aims Malnutrition in the early stages of life may lead to changes in the glycemic metabolism during adulthood, such as pancreatic beta cells dysfunction and failure. Therefore, this study aimed to evaluate the effects of an in vitro amino acid restriction model on the function and viability of pancreatic beta cells. Methods Insulin-producing cells (INS-1E) were maintained in control or amino acid restricted culture medium containing 1 × or 0.25 × of amino acids, respectively, for 48 h. Results Amino acid restricted group showed lower insulin secretion and insulin gene expression, reduced mitochondrial oxygen consumption rate and reactive oxygen species production. Besides, amino acid restricted group also showed higher levels of endoplasmic reticulum stress and apoptosis markers and enhanced Akt phosphorylation. However, even with higher levels of apoptosis markers, amino acid restricted group did not show higher levels of cell death unless the PI3K/Akt pathway was inhibited. Conclusion Amino acid restricted beta cell viability seems to be dependent on the PI3K/Akt pathway. Supplementary Information The online version contains supplementary material available at 10.1007/s00394-021-02568-2.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a- let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a- let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

Objective MicroRNAs (miRNA) are known to regulate the expression of genes involved in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation, and cell death. Methods Using “in silico” analyses, we identified 219 unique miRNAs that potentially bind to the 3′UTR region of a critical mitochondrial regulator, the peroxisome proliferator-activated receptor gamma coactivator (PGC) 1 alpha (Pgc1α). Of the 219 candidate miRNAs, miR-696 had one of the highest interactions at the 3′UTR of Pgc1α, suggesting that miR-696 may be involved in the regulation of Pgc1α. Results Consistent with this hypothesis, we found that miR-696 was highly expressed in the skeletal muscle of STZ-induced diabetic mice and chronic high-fat-fed mice. C2C12 muscle cells exposed to palmitic acid also exhibited a higher expression of miR-696. This increased expression corresponded with a reduced expression of oxidative metabolism genes and reduced mitochondrial respiration. Importantly, reducing miR-696 reversed decreases in mitochondrial activity in response to palmitic acid. Using C2C12 cells treated with the AMP-activated protein kinase (AMPK) activator AICAR and skeletal muscle from AMPKα2 dominant-negative (DN) mice, we found that the signaling mechanism regulating miR-696 did not involve AMPK. In contrast, overexpression of SNF1-AMPK-related kinase (SNARK) in C2C12 cells increased miR-696 transcription while knockdown of SNARK significantly decreased miR-696. Moreover, muscle-specific transgenic mice overexpressing SNARK exhibited a lower expression of Pgc1α, elevated levels of miR-696, and reduced amounts of spontaneous activity. Conclusions Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle cells, which in turn inhibits Pgc1α, reducing mitochondrial function. SNARK plays a role in this process as a metabolic stress signaling molecule inducing the expression of miR-696., Highlights • MicroRNA miR-696 is expressed under metabolic stress. • miR-696 downregulates Pgc1α in skeletal muscle. • Downregulation of miR-696 increases mitochondrial function. • AMPK-related kinase SNARK, but not AMPK, regulates miR-696 expression in skeletal muscle. • SNARK plays a novel role in mediating mitochondrial function.

The endoplasmic reticulum (ER) stress is one of the mechanisms related to decreased insulin secretion and beta cell death, contributing to the progress of type 2 diabetes mellitus (T2D). Thus, investigating agents that can influence this process would help prevent the development of T2D. Recently, the growth-hormone-releasing hormone (GHRH) action has been demonstrated in INS-1E cells, in which it increases cell proliferation and insulin secretion. As the effects of GHRH and its agonists have not been fully elucidated in the beta cell, we proposed to investigate them by evaluating the role of the GHRH agonist, MR-409, in cells under ER stress. Our results show that the agonist was unable to ameliorate or prevent ER stress. However, cells exposed to the agonist showed less oxidative stress and greater survival even under ER stress. The mechanisms by which GHRH agonist, MR-409, leads to these outcomes require further investigation.

Pioglitazone belongs to the class of drugs thiazolidinediones (TZDs) and is an oral hypoglycemic drug, used in the treatment of type 2 diabetes, which improves insulin sensitivity in target tissues. Adipose tissue is the main target of pioglitazone, a PPARg and PPARa agonist; however, studies also point to skeletal muscle as a target. Non-PPAR targets of TZDs have been described, thus we aimed to study the direct effects of pioglitazone on skeletal muscle and the possible role of microRNAs as targets of this drug. Pioglitazone treatment of obese mice increased insulin-mediated glucose transport as a result of increased fatty acid oxidation and mitochondrial activity. PPARg blockage by treatment with GW9662 nullified pioglitazone's effect on systemic and muscle insulin sensitivity and citrate synthase activity of obese mice. After eight weeks of high-fat diet, miR-221–3p expression in soleus muscle was similar among the groups and miR-23b-3p and miR-222–3p were up-regulated in obese mice compared to the control group, and treatment with pioglitazone was able to reverse this condition. In vitro studies in C2C12 cells suggest that inhibition of miR-222–3p protects C2C12 cells from insulin resistance and increased non-mitochondrial respiration induced by palmitate. Together, these data demonstrate a role of pioglitazone in the downregulation of microRNAs that is not dependent on PPARg. Moreover, miR-222 may be a novel PPARg-independent mechanism through which pioglitazone improves insulin sensitivity in skeletal muscle.

Key points We report that the peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-α (PGC-1α)/PPARβ axis is a crucial mediator of uncoupling protein 3 (UCP3) expression in skeletal muscle cells via the transactivativation of a distal PPAR response element at the Ucp3 gene promoter. This mechanism is activated during the myogenic process and by high concentrations of fatty acids independent of PGC-1α protein levels. Ucp3 is essential for PGC-1α-induced oxidative capacity and the adaptive mitochondrial response to fatty acid exposure. These findings provide further evidence for the broad spectrum of the coactivator action in mitochondrial homeostasis, positioning the PGC-1ɑ/PPARβ axis as an essential component of the molecular regulation of Ucp3 gene in skeletal muscle cells. Abstract Uncoupling protein 3 (UCP3) has an essential role in fatty acid metabolism and mitochondrial redox regulation in skeletal muscle. However, the molecular mechanisms involved in the expression of Ucp3 are poorly known. In the present study, we show that the peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-α (PGC-1α)/PPARβ axis is a crucial mediator of Ucp3 expression in skeletal muscle cells. In silico analysis of the UCP3 promoter and quantitative chromatin immunoprecipitation experiments revealed that the induction of the UCP3 transcript is mediated by the transactivation of a distal PPAR response element at the Ucp3 gene promoter by the coactivator PGC-1α. This mechanism is activated during myogenesis and during metabolic stress induced by fatty acids independent of PGC-1α protein levels. We also provide evidence that Ucp3 is essential for PGC-1α-induced oxidative capacity. Taken together, our results highlight PGC-1ɑ/PPARβ as an essential component of the molecular regulation of Ucp3 gene in skeletal muscle cells.

The ability to respond to fluctuations of reactive oxygen species (ROS) within the cell is a central aspect of mammalian physiology. This dynamic process depends on the coordinated action of transcriptional factors to promote the expression of genes encoding for antioxidant enzymes. Here, we demonstrate that the transcriptional coregulators, PGC-1α and NCoR1, are essential mediators of mitochondrial redox homeostasis in skeletal muscle cells. Our findings reveal an antagonistic role of these coregulators in modulating mitochondrial antioxidant induction through Sod2 transcriptional control. Importantly, the activation of this mechanism by either PGC-1α overexpression or NCoR1 knockdown attenuates mitochondrial ROS levels and prevents cell death caused by lipid overload in skeletal muscle cells. The opposing actions of coactivators and corepressors, therefore, exert a commanding role over cellular antioxidant capacity.