A obesidade é um estado inflamatório crônico. As estatinas têm efeito antiinflamatório e podem afetar a homeostase glicêmica. Estudos, nesse sentido são contraditórios e pouco se sabe sobre os mecanismos moleculares envolvidos. Este estudo verificou em animais obesos por glutamato monossódico (MSG) que além de apresentaram resistência à insulina in vivo, o tecido adiposo branco (TAB) desses animais mostrou aumento de infiltração de macrófagos, fosforilação de IKK-a/b, expressão de mRNA de TNF-a and IL-6, e redução de mRNA e proteína de GLUT4. O tratamento com atorvastatina por 4 semanas restabeleceu a sensibilidade à insulina in vivo, reduziu a inflamação e restabeleceu a expressão de GLUT4 no TAB dos animais obesos. Adicionalmente, esse trabalho encontrou sítios de ligação de NF-kB no promotor do gene GLUT4, sugerindo ligação entre resistência à insulina e inflamação. Em conclusão, a obesidade induzida por MSG em camundongos acompanha-se de resistência à insulina in vivo e atividade inflamatória crônica no tecido adiposo, com prejuízo da expressão de GLUT4. A atorvastatina melhorou esses aspectos, sugerindo que essa estatina tenha efeitos antiinflamatórios que podem melhorar a resistência à insulina na obesidade. / Obesity is a chronic inflammatory state. Statins have anti-inflammatory effects and may affect glucose homeostasis; therefore, few are known about the molecular mechanisms. Considering that inflammation contributes to insulin resistance, the aim of the present study was to investigate if atorvastatin treatment has anti-inflammatory, and consequently insulin sensitization action in white adipose tissue (WAT) of obese mice. WAT of insulin-resistant obese mice showed increased macrophage infiltration, IKK-a and IKK-b phosphorylation, TNF-a and IL-6 mRNA expression and decreased GLUT4 mRNA and protein expression. Atorvastatin restored whole-body insulin sensitivity, decreased macrophage infiltration and normalized IKK-a/b phosphorylation, TNF-a, IL-6 and GLUT4 mRNA and GLUT4 protein to control levels. Moreover, NF-kB binding sites were found in GLUT4 gene promoter, pointing out an association between insulin resistance and inflammation. Together, atorvastatin anti-inflammatory effects on WAT may be important to its local and whole-body insulin sensitization effects.

A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-<font face=\"Symbol\">kB. / Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-<font face=\"Symbol\">kB.

A obesidade é um estado inflamatório crônico. As estatinas têm efeito antiinflamatório e podem afetar a homeostase glicêmica. Estudos, nesse sentido são contraditórios e pouco se sabe sobre os mecanismos moleculares envolvidos. Este estudo verificou em animais obesos por glutamato monossódico (MSG) que além de apresentaram resistência à insulina in vivo, o tecido adiposo branco (TAB) desses animais mostrou aumento de infiltração de macrófagos, fosforilação de IKK-a/b, expressão de mRNA de TNF-a and IL-6, e redução de mRNA e proteína de GLUT4. O tratamento com atorvastatina por 4 semanas restabeleceu a sensibilidade à insulina in vivo, reduziu a inflamação e restabeleceu a expressão de GLUT4 no TAB dos animais obesos. Adicionalmente, esse trabalho encontrou sítios de ligação de NF-kB no promotor do gene GLUT4, sugerindo ligação entre resistência à insulina e inflamação. Em conclusão, a obesidade induzida por MSG em camundongos acompanha-se de resistência à insulina in vivo e atividade inflamatória crônica no tecido adiposo, com prejuízo da expressão de GLUT4. A atorvastatina melhorou esses aspectos, sugerindo que essa estatina tenha efeitos antiinflamatórios que podem melhorar a resistência à insulina na obesidade. / Obesity is a chronic inflammatory state. Statins have anti-inflammatory effects and may affect glucose homeostasis; therefore, few are known about the molecular mechanisms. Considering that inflammation contributes to insulin resistance, the aim of the present study was to investigate if atorvastatin treatment has anti-inflammatory, and consequently insulin sensitization action in white adipose tissue (WAT) of obese mice. WAT of insulin-resistant obese mice showed increased macrophage infiltration, IKK-a and IKK-b phosphorylation, TNF-a and IL-6 mRNA expression and decreased GLUT4 mRNA and protein expression. Atorvastatin restored whole-body insulin sensitivity, decreased macrophage infiltration and normalized IKK-a/b phosphorylation, TNF-a, IL-6 and GLUT4 mRNA and GLUT4 protein to control levels. Moreover, NF-kB binding sites were found in GLUT4 gene promoter, pointing out an association between insulin resistance and inflammation. Together, atorvastatin anti-inflammatory effects on WAT may be important to its local and whole-body insulin sensitization effects.

Obesidade envolve redução da expressão do transportador de glicose GLUT4 e do gene codificador SLC2A4 em tecido adiposo, junto com aumento na produção de TNFA, que regula negativamente o SLC2A4. Objetivo: avaliar regulação de Slc2a4/GLUT4 em tecido adiposo perigonadal de camundongos controles (C-tratados com salina) e obesos (MSG-tratados com MSG), submetidos a dieta normolipídica (DN) ou hiperlipídica (DH) por 4, 8, 12 e 16 semanas. Em 4 semanas MSG-DN apresentaram aumento na na sensibilidade insulínica e no GLUT4. A partir de 8 semanas, esse quadro se inverteu, com redução de Slc2a4/GLUT4 e aumento de Tnfa, resistência à insulina, hiperinsulinemia e hiperglicemia. A DH acelerou/exacerbou essa evolução. Em síntese, a evolução da obesidade levou a um quadro de diabetes tipo 2, no qual redução do GLUT4 desempenha um importante papel. / Obesity involves reduction of the glucose transporter GLUT4 expression and the encoding gene SLC2A4 in adipose tissue along with increased production of TNFA that negatively regulates the SLC2A4. Objective: To evaluate regulation of SLC2A4/GLUT4 in perigonadal adipose tissue controls mice (C treated with saline) and obese (MSG treated with MSG) undergoing normolipidic diet (ND) or hyperlipidic diet (HD) for 4, 8, 12 and 16 weeks. In 4 weeks MSG-ND showed increased insulin sensitivity and GLUT4. From 8 weeks, this situation was reversed, with a reduction of SLC2A4/ GLUT4 and increased Tnfa, insulin resistance, hyperinsulinemia and hyperglycemia. The HD accelerated/exacerbated this trend. In summary, the development of obesity led to type 2 diabetes above in which reduction of GLUT4 plays an important role.

A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-<font face=\"Symbol\">kB. / Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-<font face=\"Symbol\">kB.

O ciclo estral das cadelas difere das demais espécies domésticas. Estudos demonstraram que o aumento da concentração plasmática de P4 durante a fase luteínica das cadelas pode levar a alterações metabólicas como a resistência insulínica, acarretando complicações como Diabetes mellitus. A glicose é uma molécula transportada, na maioria das células, por proteínas transportadoras. O processo de instalação da resistência insulínica é caracterizado por alterações teciduais da expressão de algumas proteínas transportadoras de glicose, como o GLUT4. Atualmente 13 isoformas de proteínas transportadoras já foram seqüenciadas (GLUT1 ao GLUT13). O GLUT4 está presente nos músculos e no tecido adiposo, principalmente. Para avaliar se a expressão do GLUT 4 está presente nas células luteínicas e se esta expressão relaciona-se à produção de P4 e E2, 28 cadelas foram divididas em 7 grupos de acordo com os dias após a ovulação -p.o. (de 10 à 70 dias, n = 4 por grupo). Os ovários foram dissecados e congelados em nitrogênio líquido, o RNA extraído e o cDNA confeccionado e submetido ao PCR em tempo real. O gene GAPDH foi utilizado como controle endógeno para padronização da expressão do gene alvo. Foi coletado sangue para dosagem da glicemia, insulinemia, progesterona e estradiol. Para avaliar a regulação positiva do GLUT4 avaliamos também a expressão do mRNA do HIF-1&alpha;, destas mesmas cadelas. A expressão do GLUT4 apresentou tendência a aumento de expressão aos 20 dias (p. o.), quando comparado aos 10, 30 e 40 dias, pico de expressão aos 50 dias (p.o.), e então apresentou tendência a queda aos 60 e 70 dias p.o. Já a expressão do HIF-1&alpha; manteve-se muito semelhante através dos dias, tendendo a queda aos 10 e aos 40 dias pós ovulação, quando comparado com os demais grupos. Os resultados de dosagem de P4 e E2 variaram dentro do esperado para o diestro e não apresentaram correlação com a expressão de GLUT 4; a glicemia e insulina, aqui expressas através do índice HOMA (insulina x glicose % 22,5), apresentou pico aos 40 dias. Sabe-se que quanto mais alto o índice HOMA, menos este animal é sensível à insulina, ou seja, mais resistente à ela. Observou-se que o índice HOMA apresentou-se mais alto aos 40 dias, associado aos menores valores de expressão do GLUT4. Por outro lado, obtivemos o pico de expressão de GLUT4 aos 50 dias, quando o índice HOMA apresentou valores baixos. Sugere-se que a queda da P4 associada à elevação do estradiol plasmático possa influenciar o índice HOMA. Pode-se concluir que a expressão do GLUT4 no corpo lúteo de cadelas segue o padrão observado para tecidos sensíveis à insulina, nos quais existe uma maior expressão durante a fase de maior sensibilidade à insulina e diminuição drástica em fase de pré ou já instalada resistência insulínica. / The canine estral cycle differs from other domestic species. Some studies demonstrated that the increase of the plasmatic concentration of progesterone during canine luteinic phase can lead to metabolic alterations, such as insulinic resistance and may cause complications such as Diabetes mellitus. Glucose is a molecule that is transported in most cells by transporting proteins. The process of installation of the insulinic resistance is characterized by tissue alterations of the expression of some glucose transporting proteins, as GLUT4. Currenly, 13 isoforms of transporting proteins were sequenced (GLUT1 to GLUT13). GLUT4 is present mainly in muscle and fat tissue. In order to assess if GLUT4 expression is present in luteal cells, and if this expression is related to P4 and E2 production, 28 bitches were divided into 7 groups, in accordance with the days after the ovulation -p.o. (from 10 to 70 days, n=4 for group). The ovaries were dissected and frozen in liquid nitrogen. The RNA was extracted and the cDNA was made and submitted to real time PCR. GAPDH gene was used as endogenous conntrol to standardization of target gene expression. Blood was collected to glycemia, insulinemia, P4, and E2&beta; dosage. To assess the positive regulation of GLUT4, we also assessed HIF-1&alpha; mRNA expression of the same bitches. GLUT4 expression showed a tendency to increase the expression on the twentieth day (p.o.), when compared to the 10th, 30th, and 40th days, expression top on the 50 th day (p.o.) , and then, it showed a tendency to foll on the 60th and 70th days p.o. HIF-1&alpha; expression was very similar over the days, tending to fall on the 10th and 40th days post ovulation, when compared to other groups. P4 and E2&beta; dosage results varied according to thr expectations in diestrus and have not shown correlation with GLUT4 expression; glycemia and insulin, here expressed by HOMA index (insulin x glucose % 22,5) showed crest (highest point) on the 40 th day. It is knows that the higher the HOMA index, the less sensitive this animal is to insulin, it is, more resistant to it. It was observed that HOMA index was higher on the 40th day, associated to small values of the GLUT4 expression. Otherwise, the got the top of GLUT4 expression on the 50th day, when HOMA index showed low values. It has been suggested that P4 fall associated to the plasmatic E2 increase may influence HOMA index. We may conclude that GLUT4 expression into the corpus luteum of bitches follows the standard observed in insulin-sensitive tissues, in which there is a higher expression over the phase of higher sensitiveness to insulin and remarkable decrease in pre or even installed insulinic resistance.

O estudo investigou a ação sensibilizadora à insulina da glimepirida (Gp), em ratos MSG, resistentes à insulina. Animais controle (C) e MSG, e tratados (CG e MSG/G) ou não com Gp, foram submetidos a ITT; análise do GLUT4 no tecido adiposo (TAB), EDL, sóleo e coração; ensaios de translocação e captação de glicose/incorporação ao glicogênio no sóleo; dosagem do glicogênio e p-GSK3 no fígado. No TAB, mRNA e proteína GLUT4 se elevaram nos MSG, o que foi revertido pela Gp, efeito importante para não agravar a obesidade. No sóleo, o mRNA aumentou nos MSG, mas a proteína GLUT4 foi igual a C, e se elevou com a Gp. Sob estímulo insulínico, GLUT4 em membranas plasmáticas aumentou em ratos C e MSG/G. Captação de glicose e incorporação ao glicogênio muscular no sóleo, conteúdo de glicogênio e de p-GSK3 no fígado foram menores nos MSG. A Gp aumentou a expressão/translocação de GLUT4 em músculo de ratos MSG, melhorando a captação de glicose e glicogeniogênese neste tecido. Estes efeitos, mais o aumento da glicogeniogênese hepática, conferem à Gp ação sensibilizadora à insulina. / The study investigated the insulin sensitizer action of the glimepiride (Gp) in insulin- resistant MSG rats. Control (C) and MSG animals, non-treated or treated with Gp (CG and MSG/G) were submitted to ITT; GLUT4 analysis in adipose tissue (WAT), EDL, soleus and heart; GLUT4 translocation assays and glucose uptake/glycogen incorporation in soleus; glycogen and p-GSK3 analysis in the liver. In WAT, GLUT4 mRNA and protein increase in MSG rats was abolished by Gp, an effect important to avoid the aggravation of the obesity. In MSG soleus, GLUT4 mRNA increased but protein was similar to C, elevating after Gp. Under insulin stimulation, GLUT4 in plasma membrane increased in C and MSG/G only. Glucose uptake and incorporation to muscular glycogen in soleus, and glycogen/p-GSK-3 content in the liver were all lower in MSG rats. Gp increased GLUT4 expression/translocation in MSG muscle, improving glucose uptake and glycogenesis in this tissue. These effects, added to the higher hepatic glycogenesis confer the Gp its insulin sensitizer action.

The incidence of cardiovascular diseases including cardiac hypertrophy and failure in pre-menopausal women is lower compared to age-matched men but the risk of heart disease increases substantially after the onset of menopause. It has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In animal studies including spontaneously hypertensive (SHR) rats, the development of cardiac hypertrophy is attenuated by 17&#946;-estradiol treatment. Cardiac energy metabolism is crucial for normal function of the heart. In cardiac hypertrophy and heart failure, the myocardium undergoes a metabolic shift from fatty acid as primary cardiac energy source to glucose, which re-introduces the fetal type of metabolism that representing the glucose as a major source of energy. Many studies have reported that the disruption of the balance between glucose and fatty acid metabolism plays an important role in cardiac pathologies including hypertrophy, heart failure, diabetes, dilative cardiomyopathy and myocardial infarction. Glucose enters cardiomyocytes via GLUT1 and GLUT4 glucose transporters and GLUT4 is the major glucose transporter which is insulin-dependent. Cardiac-selective GLUT4 deficiency leads to cardiac hypertrophy. This shows that the decrease in cardiac glucose uptake may play a direct role in the pathogenesis of cardiac hypertrophy. Estrogens modulate glucose homeostasis in the liver and the skeletal muscle. But it is not known whether estrogens affect also cardiac glucose uptake which could provide another mechanism to explain the prevention of cardiac hypertrophy by female sex hormones. In the present study, SHR Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17&#946;-estradiol. After 6 weeks of treatment, body weight, the serum levels of estrogen, insulin, intra-peritoneal glucose tolerance test (IP-GTT), myocardial glucose uptake by FDG-PET (2-(18F)-fluoro-deoxyglucose (18FDG) and Positron Emission Tomography), cardiac glucose transporter expression and localization and cardiac hexokinase activity were analyzed. As results of this study, PET analysis of female SHR revealed decreased cardiac glucose uptake in OVX animals compared to intact that was normalized by estrogen supplementation. Interestingly, there was no change in global glucose tolerance among the treatment groups. Serum insulin levels and cardiac hexokinase activity were elevated by E2 substitution. The protein content of cardiac glucose transporters GLUT-4 and GLUT-1, and their translocation as determined by fractionation studies and immuno-staining did not show any significant change by ovariectomy and estrogen replacement. Also levels of insulin receptor substrate-1 (IRS-1) and its tyrosine phosphorylation, which is required for activation and translocation of GLUT4, was un-affected in all groups of SHR. Cardiac gene expression analysis in SHR heart showed that ei4Ebp1 and Frap1 genes which are involved in the mTOR signaling pathway, were differentially expressed upon estrogen treatment. These genes are known to be activated in presence of glucose in the heart. As a conclusion of this study, reduced myocardial FDG uptake in ovariectomized spontaneously hypertensive rat is normalized by 17&#946;-estradiol treatment. Increased myocardial hexokinase appears as a potential mechanism to explain increased myocardial glucose uptake by 17&#946;-estradiol. Increased cardiac glucose uptake in response to 17&#946;-estradiol in ovariectomized SHR may provide a novel mechanism to explain the reduction of cardiac hypertrophy in E2 treated SHR. Therefore, 17&#946;-estradiol improves cardiac glucose utilization in ovariectomized SHR which may give rise to possible mechanism for its protective effects against cardiac hypertrophy. / Erkrankungen des kardiovaskulären Systems, wie beispielsweise Herzhypertrophie oder Herzinsuffizienz treten bei Frauen vor der Menopause im Vergleich zu gleichaltrigen Männern seltener auf. Das Risiko für eine solche kardiovaskuläre Erkrankung steigt jedoch drastisch mit dem Beginn der Menopause an. Aus diesem Grund wird angenommen, dass weibliche Geschlechtshormone kardioprotektive Wirkungen besitzen. Tierstudien an spontan hypertensiven Ratten (SHR) haben belegt, dass eine Herzhypertrophie durch die Behandlung der Tiere mit 17&#946;-Estradiol abgemildert werden kann. Entscheidend für die Funktion des Myokards ist sein Energiemetabolimus, der sich im Verlauf einer Hypertrophie oder Herzinsuffizienz vom primären Fettsäurestoffwechsel auf Glucosemetabolismus umschaltet. Diese Situation entspricht der des fetalen Herzens. Viele Studien haben belegt, dass eine Störung der Balance zwischen Glucose- und Fettsäurestoffwechsel oftmals ein erstes Anzeichen für einen pathologischen Zustand des Herzens, wie z.B. Hypertrophie, Herzinsuffizienz, Diabetes, dilative Kardiomyopathie und Myokardinfarkt ist. Im gesunden Herzen gelangt Glucose über die zwei Glucosetransporter GLUT1 und GLUT4 in die Zellen des Myokards, wobei der insulinabhängige Glut4-Transporter der Hauptglucosetransporter ist. Eine GLUT4-Defizienz führt daher ebenfalls zu einer Herzhypertrophie was wiederum zeigt, dass eine verminderte Glucoseaufnahme im direkten Zusammenhang mit pathologischen Zuständen des Herzens steht. Bisherige Studien haben gezeigt, dass Östrogen an der Glucosehomöostase in Leber und Skelettmuskeln beteiligt ist. Jedoch ist wenig darüber bekannt, ob Östrogen ebenfalls in die kardiale Glucosehomöostase eingreift und inwiefern die kardioprotektive Wirkung des Östrogens in diesem Zusammenhang steht.In der vorliegenden Arbeit wurden weibliche SH-Ratten ovariektomiert (OVX), nicht ovariektomiert (sham) oder ovariektomiert und zusätzlich subkutan mit 17&#946;-Estradiol behandelt. Nach einer Behandlungszeit von 6 Wochen wurden dann das Körpergewicht, die Serumspiegel von Östrogen, Insulin und IPGTT bestimmt, und die Glucoseaufnahme des Myokards mittels FDG-PET analysiert. Zusätzlich wurden Expression und zelluläre Lokalisation der kardialen Glucosetransporter sowie die kardiale Hexokinaseaktivität untersucht. Es konnte gezeigt werden, dass sich eine verminderte Glucoseaufnahme des Herzens bei ovariektomierten Tieren durch Östrogen-Supplementation normalisieren lässt. Eine Abweichung bezüglich der Glucosetoleranz der einzelnen Gruppen konnte nicht beobachtet werden. Jedoch konnte ein erhöhter Insulinspiegel des Serums und eine erhöhte kardiale Aktivität des Enzyms Hexokinase durch die Behandlung mit Östrogen bei den ovariektomierten Tieren beschrieben werden. Durch Fraktionierungen und immunhistologische Untersuchungen konnte kein signifikanter Unterschied in Bezug auf die Menge sowie die Translokation der Glucosetransporter GLUT1 und GLUT4 im Myokard zwischen den einzelnen Behandlungen der Tiere beschrieben werden. Ferner konnte zwischen den einzelnen Tiergruppen auch kein Unterschied zwischen dem Insulin Rezeptor Substrat-1 (IRS-1) und seiner Tyrosin-phosphorylierten Form festgestellt werden, die für die Aktivierung und Translokation des GLUT4 benötigt werden. Analysen der Genexpression in den Herzen der SH-Ratten konnten allerdings zeigen, dass die Gene ei4Ebp1 und Frap1, die im mTOR Signalweg involviert sind, bei den Östrogen-supplementierten Tieren ein abweichendes Expressionsmuster aufweisen. Über diese Gene ist bekannt, dass sie in der Gegenwart von Glucose im Herzen aktiviert werden und bei der Entstehung einer Herzhypertrophie mitwirken. Basierend auf den PET-Analysen und der Hexokinaseaktivität lässt sich als Resultat dieser Arbeit aussagen, dass Östrogen die kardiale Glucoseaufnahme in SH-Ratten fördert. Diese Ergebnisse könnten einen Hinweis auf einen noch unbekannten Mechanismus geben, um die protektive Wirkung des Östrogens im Hinblick auf die Herzhypertrophie zu erklären. Hinsichtlich der Tatsache, dass keine Veränderungen in der Translokation der GLUT4-Transporter in der Plasmamembran bei den einzelnen Behandlungen der Tiere zu verzeichnen sind, jedoch Veränderungen der Glucoseaufnahme durch die PET-Analysen dargestellt werden konnten, besteht jedoch noch Erklärungsbedarf. Es liegen diverse Studien vor, die diesen Unterschied damit erklären könnten, dass der GLUT4-Transporter in einer inaktiven Form in der Plasmamembran vorliegt bis die Glucoseaufnahme durch den GLUT4-Transporter mittels der Insulin Signaltransduktionskaskade reguliert wird.

Glucose transporter GLUT4 allows glucose uptake into muscle and adipose cells. Insulin promotes recruitment and plasma membrane insertion of GLUT4 vesicles that can recycle constitutively. Obesity and type 2 diabetes mellitus are associated with defects in insulin-induced GLUT4 recruitment. Knowledge of alternative modes and steps of GLUT4 traffic in L6-GLUT4myc muscle cells may reveal possible targets for therapeutic intervention in insulin-resistant patients. Hypertonicity and Platelet Derived Growth Factor also increase surface GLUT4 levels but it was unknown if they tap on the same intracellular GLUT4 depots as insulin. We explored whether GLUT4 vesicles recycle using different compartments and mechanisms for the surface gain elicited by each stimulus. We hypothesized that all vesicle fusion steps require NSF but depend on individual v-SNAREs. Specifically, we tested effects of ATPase-deficient NSF or VAMP7 siRNA transfections, and endosomal ablation on GLUT4 traffic. We show that VAMP7 was required for basal and hypertonic recycling, while VAMP2 is exclusively used in response to insulin. As insulin action bifurcates downstream of phosphatidylinositol 3-kinase, we also hypothesized that the Rac-to-actin and Akt-to-AS160 branches regulate distinct GLUT4 traffic steps. For this we determined GLUT4myc localization in rounded myoblasts relative to a surface marker. Interfering with Rac, actin dynamics or actin-binding α-actinin4 maintained GLUT4 in a perinuclear region even under insulin-stimulation. Interfering with AS160 allowed significant GLUT4 accumulation beneath the membrane, but not fusion. We propose that actin dynamics and α-actinin4 are required for cortical GLUT4 tethering mechanisms, and AS160 contributes to GLUT4 docking/fusion. We confirmed that VAMP2 facilitates GLUT4 fusion, as tetanus toxin-based cleavage did not inhibit peripheral GLUT4 recruitment. Finally, AS160 targets Rab8A and Rab14 in muscle respectively affected GLUT4 availability for membrane fusion, and basal GLUT4 retention. This work will lead to future testing of strategies to selectively enhance vesicle availability, tethering, or surface fusion, for bypassing insulin resistance.

Glucose transporter GLUT4 allows glucose uptake into muscle and adipose cells. Insulin promotes recruitment and plasma membrane insertion of GLUT4 vesicles that can recycle constitutively. Obesity and type 2 diabetes mellitus are associated with defects in insulin-induced GLUT4 recruitment. Knowledge of alternative modes and steps of GLUT4 traffic in L6-GLUT4myc muscle cells may reveal possible targets for therapeutic intervention in insulin-resistant patients. Hypertonicity and Platelet Derived Growth Factor also increase surface GLUT4 levels but it was unknown if they tap on the same intracellular GLUT4 depots as insulin. We explored whether GLUT4 vesicles recycle using different compartments and mechanisms for the surface gain elicited by each stimulus. We hypothesized that all vesicle fusion steps require NSF but depend on individual v-SNAREs. Specifically, we tested effects of ATPase-deficient NSF or VAMP7 siRNA transfections, and endosomal ablation on GLUT4 traffic. We show that VAMP7 was required for basal and hypertonic recycling, while VAMP2 is exclusively used in response to insulin. As insulin action bifurcates downstream of phosphatidylinositol 3-kinase, we also hypothesized that the Rac-to-actin and Akt-to-AS160 branches regulate distinct GLUT4 traffic steps. For this we determined GLUT4myc localization in rounded myoblasts relative to a surface marker. Interfering with Rac, actin dynamics or actin-binding α-actinin4 maintained GLUT4 in a perinuclear region even under insulin-stimulation. Interfering with AS160 allowed significant GLUT4 accumulation beneath the membrane, but not fusion. We propose that actin dynamics and α-actinin4 are required for cortical GLUT4 tethering mechanisms, and AS160 contributes to GLUT4 docking/fusion. We confirmed that VAMP2 facilitates GLUT4 fusion, as tetanus toxin-based cleavage did not inhibit peripheral GLUT4 recruitment. Finally, AS160 targets Rab8A and Rab14 in muscle respectively affected GLUT4 availability for membrane fusion, and basal GLUT4 retention. This work will lead to future testing of strategies to selectively enhance vesicle availability, tethering, or surface fusion, for bypassing insulin resistance.