Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders

Metabolic Activation of Intrahepatic CD8+ T Cells and NKT Cells Causes Nonalcoholic Steatohepatitis and Liver Cancer via Cross-Talk with Hepatocytes

SummaryHepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8+ T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography–mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance

Molekulare Grundlagen der Nierenkanzerogenese

Einleitung:Krebserkrankungen wie etwa Nierenkrebs werden häufig durch Fremdstoffe verursacht. Zur Bestimmung des kanzerogenen Potentials dieser Fremdstoffe sind 2-jährige Kanzerogenesestudien in Nagetieren vom Gesetzgeber vorgeschrieben. Derartige Studien sind jedoch zeitaufwendig und teuer, verursachen einen hohen Tierverbrauch und setzen einen hohen Bedarf an Testsubstanzen voraus. Hinzu kommt, dass die Ergebnisse dieser Langzeit-Studien oft durch unspezifische Hochdosiseffekte, eine altersabhängige Pathologie oder durch nagetierspezifische Effekte verfälscht werden. Umgekehrt vernachlässigen sie den Einfluss zusätzlicher Krebsrisikofaktoren beim Menschen, etwa eine fetthaltige Diät und Fettleibigkeit. Im Rahmen dieser Arbeit sollte daher untersucht werden, ob Nieren-kanzerogene Substanzen bereits in Kurzeit-In-Vivo-Studien durch Microarray-Transkriptomanalysen identifiziert werden können. Des weiteren sollten molekulare Mechanismen bei der Entwicklung von Fremdstoff-induzierten Nierentumoren charakterisiert werden. Schließlich sollten die Einflüsse einer fettreichen Diät sowie erhöhter Fettleibigkeit auf die Nierenkanzerogenese in der Ratte bestimmt werden. Experimenteller Aufbau: I) TSC2(+/+) Wildtyp- sowie TSC2(+/-) Eker Ratten wurden täglich mit den genotoxischen Nierenkanzerogenen Aristolochia Säure (AA), Methylazoxymethanol Azetat (MAMAc), oder dem nicht-genotoxischen Nierenkanzerogen Ochratoxin A (OTA) gavagiert. Nach 1, 3, 7 und 14 Tagen wurden von Nierenkortex-Homogenaten aller behandelten und Kontroll-Tiere Genexpressionsprofile mittels Microarrays erstellt. Paraffinschnitte wurden zur Untersuchung von Histopathologie und Zellproliferation verwendet. II) Eker Ratten wurden mit AA, MAMAc und OTA über 3 und 6 Monate gavagiert und histopathologische Veränderungen und Zellproliferationsraten bestimmt. Parallel hierzu wurde ein Protokoll entwickelt, welches erstmals reproduzierbare und verlässliche Genexpressionsanalysen von mikrodissektierten Nierentubuli ermöglichte. Nach diesem Protokoll wurden Transkriptionsanalysen aus Präneoplasien sowie aus gesunden Nierentubuli AA-, OTA- und Vehikel-behandelter Eker Ratten erstellt. Die zentralen Ergebnisse dieser Genexpressionsanalysen wurden mittels immunhistochemischen Färbungen verifiziert. III) Adverse Effekte auf Nierenpathologie und zelluläre Signalwege wurden in zwei Subpopulationen von Wistar-Ratten mit hoher Sensitivitaet bzw. Resistenz gegenüber nahrungsinduzierter Adipositas nach 11-monatiger Exposition mit fetthaltiger Diät, sowie in Wistar-Ratten nach Exposition mit fettarmer Diät untersucht. Ergebnisse/Diskussion: Histopatholgische Untersuchungen sowie Genexpressionsanalysen aus Nierenkortexhomogenaten zeigten, dass früheste substanzspezifische Effekte Nieren-kanzerogener Substanzen bereits nach 1 bis 14-tägiger Exposition mit AA, MAMAc bzw. OTA erkennbar sind. Besagte Genexpressionsprofile erlaubten die Unterscheidung von genotoxischen und nicht-genotoxischen Substanzen und erwiesen sich als prediktiv für die substanzspezifische Inzidenz und Zahl von präneoplastischen Läsionen in Eker Ratten nach 3- bzw. 6-monatiger Exposition. Genexpressionsprofile aus mikrodissektierten präneoplastischen Läsionen von Eker Ratten nach 6-monatiger Exposition mit AA, OTA oder dem Vehikel zeigten im Vergleich zu mikrodissektierten gesunden Tubuli von Vehikel-behandelten Ratten eine stark erhöhte Deregulation gleicher Gene. Des weiteren konnten nur geringfügige Genexpressionveränderungen in gesunden Tubuli von AA- und OTA-behandelten Ratten gemessen werden. Diese Daten weisen darauf hin, dass die Anzahl der präneoplastischen Läsionen, die während einer begrenzten kritischen Phase der Substanzbehandlung entstehen, ausschlaggebend für die Inzidenz und Anzahl von Tumoren am Ende der 2-jährigen Kanzerogenese Studie sein könnte. Die klonale Expansion dieser Läsionen ist demgegenüber eher ein substanzunabhängiger Prozess, der möglicherweise durch eine Deregulation des mTOR Signalwegs verstärkt wird. Im letzten Teil der Arbeit konnte gezeigt werden, dass eine stark fetthaltige Diät eine deutlich schädliche Wirkung auf Rattennieren ausübt. Der Grad der Adipositas nahm im Vergleich zu den adversen Effekten der über die Nahrung aufgenommenen Lipide nur eine untergeordnete Rolle ein. Immunohistochemische Färbungen der Nieren wiesen zudem auf eine Rolle des mTOR Signalweges bei der Lipid-induzierten Pathologie der Niere. Fazit: Microarrays erlauben die Charakterisierung von frühesten molekularen Vorgängen bei der Entstehung von Fremdstoff-induzierten Nierentumoren in Kurzzeit-In-Vivo-Studien. Dabei erfüllen sie wichtigen Vorraussetzungen für eine verlässliche Identifizierung von Nieren-kanzerogenenen Substanzen, nämlich eine hohe Sensitivität, Spezifität und Prediktivität. Langfristig könnte somit unter Verwendung neuer und sensitiver Detektionstechniken 2-jährige Kanzerogenesestudien deutlich verkürzt werden, was auch eine Reduzierung des Tierverbrauchs ermöglichen würde

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treated for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H&E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230_2.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats

L-BMAA induced ER stress and enhanced caspase 12 cleavage in human neuroblastoma SH-SY5Y cells at low nonexcitotoxic concentrations

The cyanobacterial L-BMAA (β-N-methylamino-L-alanine) is described as a low potency excitotoxin, possibly a factor in the increased incidence of amyotrophic lateral sclerosis (ALS) and Parkinsonism dementia complex (PDC) on Guam. The latter association is intensively disputed, as L-BMAA concentrations required for toxic effects exceed those assumed to occur via food. The question thus was raised whether L-BMAA leads to neurodegeneration at non-excitotoxic conditions. Using human SH-SY5Y neuroblastoma cells, L-BMAA-transport, incorporation into proteins and subsequent impairment of cellular protein homeostasis were investigated. Binding of L-BMAA to intracellular proteins, but no clear protein incorporation was detected in response to (14)C-L-BMAA exposures. Nevertheless, low L-BMAA concentrations (≥0.1 mM, 48 hours) increased protein ubiquitination, 20S proteasomal and caspase 12 activity, expression of the ER-stress marker CHOP, and enhanced phosphorylation of elf2α in SH-SY5Y. In contrast, high L-BMAA concentrations (≥1 mM, 48 hours) increased ROS and protein oxidization, which were partially ameliorated by co-incubation vitamin E. L-BMAA mediated cytotoxicity was observable 48 hours following ≥2 mM L-BMAA treatment. Consequently, the data presented here suggest that low L-BMAA concentrations result in a dysregulation of the cellular protein homeostasis with ensuing ER stress that is independent from high concentration effects such as excitotoxicity and oxidative stress. Thus, the latter could be a contributing factor in the onset and slow progression of ALS/PDC on Guam

Inkretinbasierte Ko- und Triagonisten: innovative Polypharmakologie zur Behandlung von Adipositas und Diabetes

Background The worldwide rise in overweight and obesity is paralleled by an increasing prevalence of type-2 diabetes. Apart from bariatric surgery, treatment options to decrease body weight are often underwhelming. Innovative pharmacological options are required to cope with the global "diabesity" pandemic. Objectives Particular novel pharmacological approaches are discussed, with a special focus on polyagonist-based pharmacotherapies. Materials and methods Articles on co- and tri-agonists for the treatment of obesity and diabetes are presented and discussed. Results Unimolecular peptides have been developed for the treatment of obesity and type-2 diabetes. These peptides activate the receptors of multiple hormones and bundle their positive effects in one single molecule. In preclinical studies, polyagonists targeting the receptors for glucagon-like peptide-1 (GLP-1), glucagon, or glucose-dependent insulinotropic peptide (GIP) were promising to reduce body weight and blood glucose. GLP-1-mediated delivery of the nuclear hormones estrogen or dexamethasone also yielded beneficial effects in preclinical studies of obesity. Conclusions Polyagonists represent an innovative strategy for the development of novel pharmacotherapies to treat obesity and diabetes

Microcystin congener– and concentration-dependent induction of Murine neuron apoptosis and Neurite degeneration

Cyanobacterial microcystins (MCs) represent a toxin group with > 100 variants, requiring active uptake into cells via organic anion–transporting polypeptides, in order to irreversibly inhibit serine/threonine-specific protein phosphatases. MCs are a human health hazard with repeated occurrences of severe poisonings. In the well-known human MC intoxication in Caruaru, Brazil (1996), patients developed signs of acute neurotoxicity, e.g., deafness, tinnitus, and intermittent blindness, as well as subsequent hepatotoxicity. The latter data, in conjunction with some animal studies, suggest that MCs are potent neurotoxins. However, there is little data to date demonstrating MC neuron-specific toxicity. MC exposure–induced cytotoxicity, caspase activity, chromatin condensation, and microtubule-associated Tau protein hyperphosphorylation (epitopes serine199/202 and serine396) were determined. Neurite degeneration was analyzed with confocal microscopy and neurite length determined using image analysis. MC-induced apoptosis was significantly increased by MC-LF and MC-LW, however, only at high concentrations (≥ 3MyM), whereas significant neurite degeneration was already observed at 0.5MyM MC-LF. Moreover, sustained hyperphosphorylation of Tau was observed with all MC congeners. The concentration- and congener-dependent mechanisms observed suggest that low concentrations of MC-LF and MC-LW can induce subtle neurodegenerative effects, reminiscent of Alzheimer’s disease type human tauopathies, and thus should be taken more seriously with regard to potential human health effects than the apical cytotoxicity (apoptosis or necrosis) demonstrated at high MC concentrations