Marc Bedoucha

Diabetic KKAy mice exhibit increased hepatic PPARγ1 gene expression and develop hepatic steatosis upon chronic treatment with antidiabetic thiazolidinediones

BACKGROUND/AIMS Peroxisome proliferator-activated receptor-gamma, which is involved in the regulation of lipid homeostasis, is upregulated in the liver of obese and diabetic mice, but the biological consequences of this induction are largely unknown. This study was aimed at further characterizing this upregulation and exploring the downstream biological effects of specific activators on hepatic lipid metabolism. METHODS Hepatic expression of peroxisome proliferator-activated receptor-gamma1 and gamma2 mRNA and protein was analyzed by real-time polymerase chain reaction and Western immunoblotting in KKAy mice and ob/ob mice. KKAy mice were treated with thiazolidinediones, and hepatic triglyceride content and lipid distribution were analyzed biochemically and by histopathology. RESULTS KKAy mice exhibited a marked increase in hepatic peroxisome proliferator-activated receptor-gamma1 mRNA and protein levels, whereas the gamma2 isoform was upregulated in ob/ob mice. Treatment of KKAy mice with troglitazone or rosiglitazone resulted in severe microvesicular periacinar steatosis, whereas lean control mice did not develop any pathological liver changes. Hepatic triglyceride levels, however, were not altered by the treatment. CONCLUSIONS In mice with obesity-associated upregulated hepatic peroxisome proliferator-activated receptor-gamma expression, thiazolidinediones may produce hepatic steatosis. Under pathophysiological conditions, such as non-insulin-dependent diabetes, the liver may thus become sensitized towards peroxisome proliferator-activated receptor-gamma-activating drugs.

Toxicological consequences of altered peroxisome proliferator-activated receptor γ (PPARγ) expression in the liver: insights from models of obesity and type 2 diabetes

The pivotal role of peroxisome proliferator-activated receptor gamma (PPARgamma) in the liver, although important for the regulation of genes involved in glucose and lipid metabolism, has generally not been fully appreciated. This may be due to the fact that PPARgamma, in contrast to PPARalpha or PPARdelta, is not abundantly expressed in liver under normal conditions. However, recent findings have revealed that in several murine models of obesity and type 2 diabetes mellitus (T2DM), PPARgamma mRNA and receptor protein are highly up-regulated in the liver, and that the receptor causes increased transcriptional activity as demonstrated by the activation of PPARgamma-responsive genes in the liver. Prolonged treatment of obese and diabetic mice, but not of lean control mice, with the selective PPARgamma ligands and activators, thiazolidinediones (TZDs), including troglitazone, rosiglitazone, or pioglitazone, has resulted in the development of severe hepatic centrilobular steatosis. In contrast to these effects in hepatocytes, TZD-mediated effects on Kupffer cells (down-regulation of proinflammatory cytokines) seem to be PPARgamma-independent. In view of the findings that sustained hepatic steatosis can lead to steatohepatitis and/or fibrosis and that troglitazone (but not the other TZDs) has been associated with rare but serious hepatotoxicity in patients, further insight into PPARgamma-mediated versus non-PPARgamma-mediated effects of TZDs is desirable. It is concluded that liver-specific effects associated with TZD antidiabetics may become relevant under conditions of selective PPARgamma up-regulation in the liver. Therefore, receptor expression in human liver tissue of obese and T2DM patients should deserve increased consideration in the future.

Down-regulation by troglitazone of hepatic tumor necrosis factor-α and interleukin-6 mRNA expression in a murine model of non-insulin-dependent diabetes∗

Troglitazone, a novel thiazolidinedione drug used to treat non-insulin-dependent diabetes mellitus, is a selective ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Recent results indicate that PPARgamma activation by thiazolidinediones regulates adipose tissue- and monocyte/peritoneal macrophage-derived cytokine expression in vitro. We evaluated whether troglitazone may also negatively regulate cytokine expression in the liver, which harbors the majority of the bodys resident macrophages but which only weakly expresses PPARgamma. Lean C57BL6 mice and genetically obese KKA(y) mice were chronically treated with troglitazone (100 mg/kg/day for 2 weeks). At the end of treatment, hepatic expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA was quantitatively determined by kinetic polymerase chain reaction both under basal conditions and after stimulation with lipopolysaccharide (LPS). Both untreated lean and obese mice exhibited low levels of baseline TNF-alpha and IL-6 mRNA expression and responded with a dramatic increase in hepatic cytokine transcripts and TNF-alpha protein expression following a challenge with LPS. Similar to the effects on white adipose tissue, troglitazone not only down-regulated the baseline levels of hepatic TNF-alpha and IL-6, but also greatly attenuated the inducing effects of LPS. The extent of this inhibitory effect of troglitazone was higher in obese KKA(y) mice than in lean mice and was also reflected by markedly down-regulated hepatic TNF-alpha protein expression. These data demonstrate that chronic administration of troglitazone is associated with a greatly attenuated responsiveness towards inducers of hepatic TNF-alpha and IL-6 production. The possible biological consequences of these effects, however, have not yet been assessed.

Incretin-like effects of small molecule trace amine-associated receptor 1 agonists

Objective Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. Methods We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. Results TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. Conclusions We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.

Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

BackgroundThe major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation.ResultsThe designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins.ConclusionThe current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.