Shikonin Increases Glucose Uptake in Skeletal Muscle Cells and Improves Plasma Glucose Levels in Diabetic Goto-Kakizaki Rats

Glucose is the most common substrate for energy metabolism. Despite the varying demands for glucose, the body needs to regulate its internal environment and maintain a constant and stable condition. Glucose homeostasis requires harmonized interaction between several tissues, achieving equilibrium between glucose output and uptake. In this thesis we aimed to investigate factors modulating glucose homeostasis in a rat model of type 2 diabetes, the Goto-Kakizaki (GK) rat. In addition, we investigated sex differences in hepatic carbohydrate and lipid metabolism in healthy rats. In Paper I, three-week but not three-day treatment with a Southeast Asian herb, Gynostemma pentaphyllum (GP), significantly reduced plasma glucose (PG) levels in GK rats. An intra-peritoneal glucose tolerance test (IPGTT) was significantly improved in GP-treated compared to placebo-treated group. In the GP treated rats, the glucose response in an intra-peritoneal pyruvate tolerance test was significantly lower, indicating decreased gluconeogenesis, and hepatic glucose output (HGO) was reduced. GP-treatment significantly reduced hepatic glycogen content, but not glycogen synthase activity. The study provides evidence that the GP extract exerted anti-diabetic effect in GK rats, reducing PG levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis. In Paper II, shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increased glucose uptake in L6 myotubes, but did not phosphorylate Akt. Furthermore we found no evidence for the involvement of AMP activated protein kinase (AMPK) in shikonin induced glucose uptake. Shikonin increased the intracellular levels of calcium in these cells and stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myotubes. In GK rats treated with shikonin once daily for 4 days, PG levels were significantly decreased. In an insulin sensitivity test, the absolute PG levels were significantly lower in the shikonin-treated rats. These findings suggest that shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. In Paper III, GK and control Wistar rats were injected daily for up to 4 weeks with either a non-hematopoietic erythropoietin analog ARA290 or with placebo. PG levels in GK but not Wistar rats were significantly lower in ARA290-treated compared to placebo. After 2 and 4 weeks, the IPGTT was significantly improved in ARA290 treated GK rats. In insulin and pyruvate tolerance tests, glucose responses were similar in ARA290 and placebo groups. In isolated GK rat islets, glucose-stimulated insulin release was two-fold higher and islet intracellular calcium concentrations in response to several secretagogues were significantly higher in ARA290-treated than in placebo-treated GK rats. These findings indicate that treatment with ARA290 significantly improved glucose tolerance in diabetic GK rats, most likely due to improvement of insulin release. In Paper IV, sex differences in hepatic carbohydrate and lipid metabolism were characterized in healthy rats. No sex-differences were observed regarding hepatic triglyceride content, fatty acid oxidation rates or insulin sensitivity. Male rats had higher ratios of insulin to glucagon levels, increased hepatic glycogen content, a lower degree of AMPK phosphorylation, a higher rate of glucose production and higher expression levels of gluconeogenic genes, as compared to female rats. A sex-dependent response to mild starvation was observed with males being more sensitive. In conclusion, sex-differences reflect a higher capacity of the healthy male rat liver to respond to increased energy demands. Key words: glucose homeostasis, type 2 diabetes, GK rats, L6 myotubes, hepatic glucose output, insulin sensitivity, sex differences

β-Adrenergic Inhibition of Contractility in L6 Skeletal Muscle Cells

The β-adrenoceptors (β-ARs) control many cellular processes. Here, we show that β-ARs inhibit calcium depletion-induced cell contractility and subsequent cell detachment of L6 skeletal muscle cells. The mechanism underlying the cell detachment inhibition was studied by using a quantitative cell detachment assay. We demonstrate that cell detachment induced by depletion of extracellular calcium is due to myosin- and ROCK-dependent contractility. The β-AR inhibition of L6 skeletal muscle cell detachment was shown to be mediated by the β2-AR and increased cAMP but was surprisingly not dependent on the classical downstream effectors PKA or Epac, nor was it dependent on PKG, PI3K or PKC. However, inhibition of potassium channels blocks the β2-AR mediated effects. Furthermore, activation of potassium channels fully mimicked the results of β2-AR activation. In conclusion, we present a novel finding that β2-AR signaling inhibits contractility and thus cell detachment in L6 skeletal muscle cells by a cAMP and potassium channel dependent mechanism

The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis

ObjectiveSimultaneous activation of β2- and β3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of β1-ARs – and thus inducing cardiovascular complications – are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel β2-and β3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models.MethodsIn the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective β-AR agonist isoprenaline across various rodent β-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis.ResultsExposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis.ConclusionsOur results demonstrate that ATR-127 is a highly effective, novel β2- and β3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies

Effects of Alzheimer s and Parkinson s disease gene mutation on cell signaling

Alzheimer s disease (AD) and Parkinson s disease (PD) are the major neurodegenerative disorders affecting the elderly. The discovery of genes causing familial forms of these disorders has contributed enormously towards our understanding of underlying pathogenic mechanisms. This thesis explores mechanisms by which Phospholipase C (PLC) mediated signaling is regulated by presenilin (PS) and parkin, these being genes in which mutations cause familial AD and PD respectively. In Paper I, mouse embryonic fibroblasts (MEFs) lacking PS1, PS2 or both, were used to evaluate the dependence of PS on PLC and Protein Kinase C (PKC). Results revealed a dependence of both PS1 and PS2 on m-3M3FBS stimulated PLC activities. Also, PLC stimulated PKCalpha and PKCgamma activities were dependent on the presence of both PSs. Protein levels of PKCalpha/gamma were decreased in PS double knockout MEFs, while PKCdelta levels were elevated in the same cells. These results were also verified by transfecting back PS and by using another cell line lacking PS. Furthermore PKCalpha levels were shown to be dependent on amyloid precursor protein (APP) and APP intracellular domain. It is concluded that PS modulate PLC and PKC activity and differently regulates PKC protein levels by both APP dependent and independent mechanisms. Paper II explored the dependence of PS on Extracellular regulated kinase 1/2 (ERK1/2) activities. ERK1/2 has previously been implicated in the pathogenesis of AD by different mechanisms. Using MEF cells lacking PSs (same as in paper I) it was found that ERK1/2 activities were increased in PS1 or PS2 knockout MEFs. PKCalpha inhibition could reverse these elevated ERK1/2 activities. Results also revealed a lower PLC or PKC stimulation in PS double knockout cells. The total levels of ERK were only downregulated after phorbol-12,13-dibutyrate (PdBu) treatment in PS double knockout. These findings show that PS regulates ERK1/2 activity via a PKCalpha dependent manner. Lack of both PSs disrupts PLC/PKC signaling and this is also reflected in the lower downstream activation of ERK1/2. In Paper III, PLCgamma1 was found to interact with parkin, in cells transfected with parkin WT, mutants G328E and R42P. This interaction was also detected in cortical, striatal and nigral human brain homogenates. PLCgamma1 protein levels were found to be higher in parkin knockout mice and lower in parkin WT cells. Also, parkin mutants cells showed a disrupted ubiquitination. In summary, PLCgamma1 was identified as a novel substrate for parkin by using interaction and ubiquitination studies of PLCgamma1 and parkin. Paper IV describes functional studies that revealed an increased PLC phosphorylation and activity in parkin mutant cells. Also, intracellular calcium levels were elevated in these cells, which could be reversed by the PLC inhibitor Neomycin and ryanodine receptor inhibitor dantrolene, suggesting a deregulated PLC activity. Parkin WT was shown to be protective against 6-OH-dopamine toxicity. The toxicity seen in parkin mutants after 6-OH-dopamine, could be reversed by dantrolene treatment, suggesting that ryanodine receptor calcium deregulation contributes to toxicity. In conclusion, these studies provide evidence that PS and parkin regulate PLC mediated signaling

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days, and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide aditional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.Funding agencies|Gustav V and Queen Victoria Foundation||County Council of Ostergotland||Stiftelsen Olle Engkvist Byggmastare||Stifielsen for Gamla Tjanarinnor||Gunoch Bertil Stohnes Stiftelse||Lions forskningsfond||Svenska Lundbeckstiftelsen||Karolinska Institute Fund for Geriatric Research||Alice och Knut Wallenberg Stiftelse||Swedish Alzheimer Foundation||Swedish Brain Power|

β3-Adrenergically induced glucose uptake in brown adipose tissue is independent of UCP1 presence or activity: Mediation through the mTOR pathway

Objective: Today, the presence and activity of brown adipose tissue (BAT) in adult humans is generally equated with the induced accumulation of [2-18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in adipose tissues, as investigated by positron emission tomography (PET) scanning. In reality, PET-FDG is currently the only method available for in vivo quantification of BAT activity in adult humans. The underlying assumption is that the glucose uptake reflects the thermogenic activity of the tissue. Methods: To examine this basic assumption, we here followed [18F]FDG uptake by PET and by tissue [3H]-2-deoxy-d-glucose uptake in wildtype and UCP1(−/−) mice, i.e. in mice that do or do not possess the unique thermogenic and calorie-consuming ability of BAT. Results: Unexpectedly, we found that β3-adrenergically induced (by CL-316,243) glucose uptake was UCP1-independent. Thus, whereas PET-FDG scans adequately reflect glucose uptake, this acute glucose uptake is not secondary to thermogenesis but is governed by an independent cellular signalling, here demonstrated to be mediated via the previously described KU-0063794-sensitive mTOR pathway. Conclusions: Thus, PET-FDG scans do not exclusively reveal active BAT deposits but rather any tissue possessing an adrenergically-mediated glucose uptake pathway. In contrast, we found that the marked glucose uptake-ameliorating effect of prolonged β3-adrenergic treatment was UCP1 dependent. Thus, therapeutically, UCP1 activity is required for any anti-diabetic effect of BAT activation. Keywords: Brown adipose tissue, Uncoupling protein 1, Glucose uptake, Adrenergic signaling, Positron emission tomograph