Claude Atgié

Differential Regulation of GLUT1 and GLUT4 Glucose Transporters in Skeletal Muscle of a New Model of Type II Diabetes: The Obese SHR/N-cp Rat

The obese diabetic SHR/N-cp rat is a newly developed strain that inherits obesity as an autosomal recessive trait. These rats display early-onset hyperinsulinemia and hyperglycemia, which are hallmarks of type II diabetes. This study was undertaken to determine the expression and the subcellular distribution of the GLUT1 and GLUT4 glucose transporters in skeletal muscle of obese diabetic SHR rats. D-glucose-protectable cytochalasin-B binding to subcellular membrane fractions of hindlimb muscles was used to determine glucose transporter number. GLUT1 and GLUT4 glucose transporter isotypes were detected using antibodies to the COOH-terminal region of the GLUT1 and GLUT4 proteins. Glucose transporter number was significantly lower (–40%) in crude unfractionated membranes of obese diabetic SHR than of lean SHR muscles. When crude membranes were fractionated to separate plasma membranes and the intracellular membranes containing glucose transporters, the number of cytochalasin-B binding sites was found to be markedly lower (–50%) in intracellular membranes and slightly but not significantly reduced (–20%) in plasma membranes of muscle from obese diabetic SHR compared with lean SHR rats. Western blot analysis revealed that a lower GLUT4 protein abundance (–40%) accounts for the reduced glucose transporter number in intracellular membranes of obese diabetic SHR compared with lean SHR muscles. GLUT4 protein content was also reduced by 50% in plasma membranes from obese SHR muscles relative to lean rat muscles. In contrast, GLUT1 glucose transporters were more abundant (40d%) in plasma membranes of obese diabetic SHR rats compared with their lean counterparts. The latter finding may explain the lack of phenotypic difference in plasma membrane total glucose transporter number as measured by cytochalasin-B binding. GLUT4 and GLUT1 mRNA levels were both found to be significantly decreased in obese SHR rats compared with their lean littermates. These results demonstrate that 1) GLUT4 transporter expression is reduced in skeletal muscle of obese diabetic SHR rats, a model of type II diabetes; 2) the decreased GLUT4 abundance was localized to both the sarcolemmal and intracellular membrane fractions; 3) in contrast to the GLUT4 glucose transporter, GLUT1 protein content was greater in plasma membranes of skeletal muscle from obese diabetic SHR rats, suggesting that these transporter isoforms are inversely regulated in muscle of these diabetic animals; and 4) posttranscriptional mechanisms appear to be responsible for the differential expression of GLUT1 and GLUT4 proteins in muscle of obese diabetic SHR rats.

Vitamin A and lipid metabolism: relationship between hepatic stellate cells (HSCs) and adipocytes

Vitamin A or retinol plays a major role in the regulation of cellular homeostasis. Retinyl palmitate remains the main chemical form of vitamin A storage and is mainly located in hepatic stellate cells (HSCs) in lipid droplets resembling those found in adipose cells. White adipose tissue (WAT), is essentially involved in the regulation of lipid metabolism, through its role in lipid storage, and might also be considered as a vitamin A storage and metabolism site. WAT contains all the intracellular equipment for vitamin A metabolism and signaling pathways which allows retinol to be metabolized into retinoic acid, known to control genomic expression in WAT. The description of molecular mechanisms involved in the activation of HSCs and the differentiation of preadipocytes reveal similar cellular and molecular mechanisms. Indeed HSCs and adipocytes share a common expression of key transcription factors like PPAR-γ and RXR known to influence perilipin expression, which play fundamental roles in lipid droplet metabolism. Both cells are also sources of important endocrine signaling secretions influencing the expression of these transcription factors. The morphological and functional characteristics of HSCs and adipocytes, including the metabolism of vitamin A and other lipids and their related signaling pathways, are summarized and compared in this review. We highlight the complexity of the interrelationship between lipids and vitamin A metabolism and the role of the complex communication existing between HSCs and WAT in diseases such as non-alcoholic fatty liver disease which is the hepatic manifestation of the metabolic syndrome.

Effects of Chronic Treatment with Noradrenaline or a Specific β3-Adrenergic Agonist, CL 316 243, on Energy Expenditure and Epididymal Adipocyte Lipolytic Activity in Rat

1. The effects of 7 days exposure to a specific beta3-adrenergic agonist, CL 316 243 (1 mg/kg x 24 hr), or to the physiological hormone, noradrenaline (5 mg/kg x 24 hr), were tested on energy expenditure and on in vitro lipolysis in male Sprague-Dawley rats. 2. At the second day of treatment, the total energy expenditure and the resting metabolic rate were increased by 20 and 30%, respectively, in the CL-treated group. Under the same conditions, a dose five times higher of NA increased the resting metabolic rate by 11% without any significant change in the total daily energy expenditure. 3. The CL-treated group showed a lower weight gain, correlated with a significant reduction in retroperitoneal adipose tissue weight. Both treatments resulted in a marked desensitization (increased EC50 values) of the NA stimulated lipolysis of epididymal adipocytes. The effects of both treatments on maximal lipolysis were opposite. Indeed, chronic NA-treatment decreased the responsiveness of lipolysis while chronic treatment with CL increased the maximal stimulation of lipolysis to NA. Furthermore, dose-response curve for CL on lipolysis showed a marked functional desensitization of beta3-adrenergic response. 4. Our results demonstrate the high selectivity of beta3-adrenergic agonists to stimulate whole body energy expenditure and lipid mobilization in rodents. The present results point out for the first time an adrenergic desensitization of the lipolytic response after chronic administration of a beta3-agonist.

Effect of tyramine, a dietary amine, on glycerol and lactate release by isolated adipocytes from old rats.

Amine degradation by adipocyte amine oxidases leads to the production of metabolites that interact with lipid and glucose metabolisms and their hormonal regulations. To further investigate these interactions, we determined the effect of a dietary amine, tyramine (TYR), on glycerol and lactate releases, respectively taken as indices of lipolytic and glycolytic activities of isolated adipocytes. Old male Wistar rats were used to prepare adipocytes by collagenase dissociation of retroperitoneal fat pads. The two tested doses of tyramine (10 μM and 1 mM) had no effect on basal glycerol release. On the other hand, TYR, at the highest dose tested (1 mM), weakly but significantly increased basal lactate release, which was elevated in adipocytes from old rats. Norepinephrine (NE), highly stimulated adipocyte lipolysis with a submaximal effect at 1 μM which was slightly but significantly inhibited by TYR 1 mM. Insulin 1 nM (INS) also poorly inhibited the NE-stimulated lipolysis in adipocytes isolated from old rats. TYR was able to potentiate the poor antilipolytic efficiency of INS. Under similar conditions, a high dose of NE greatly reduced lactate production and TYR (1 mM) reversed this inhibition of lactate release. INS was also able to totally reverse the inhibitory effect of NE on lactate release, but there was no potentiation between insulin and tyramine effects. It can be concluded that high doses of TYR interact with norepinephrine and insulin, at least on the control of glycerol and lactate release, by counteracting catecholamine effects and by mimicking insulin actions.ResumenLa degradación de aminas por las aminooxidasas del tejido adiposo produce metabolitos capaces de influir sobre los metabolismos lipídico y glucídico y su regulación hormonal. Para investigar acerca de estos efectos, se ha estudiado el efecto de la tiramina (TIR), una amina presente en la dieta, sobre la producción de glicerol y de lactato, como índice respectivo de la lipolisis y de la glicolisis. A partir de tejido adiposo perirrenal de machos viejos de rata Wistar se aislaron adipocitos tras digestión con colagenasa. Se estudióin vitro el efecto de la tiramina a dos concentraciones, 10 μM y 1 mM, sobre la liberación de glicerol y de lactato. Ninguna de ellas modificó la liberación basal de glicerol, pero la mayor estimuló la liberación basal de lactato, ya bastante elevada. La norepinefrina (NE) estimuló fuertemente la lipolisis con un efecto submáximo a 1 μM, que fué inhibido parcial pero significativamente por TIR 1 mM. La producción basal de lactato se redujo por dosis elevadas de NE y esta inhibición se anuló por adición TIR 1 mM. Ademas, la tiramina fue capaz de reforzar la débil acción antilipolítica de la insulina en los adipocitos antilipolítica de la insulina en los adipocitos estimulados por norepinefrina de ratas viejas. Por otra parte, la insulina anuló el efecto inhibidor de la NE sobre la producción basal de lactato sin que se observara potenciación por la presencia de TIR. Estos resultados parecen indicar que la tiramina, a dosis elevadas, puede reemplazar parcialmente el efecto de la insulina e influir sobre el control de la liberación de glicerol y de lactato por la norepinefrina

Synergic effect of vitamin A and high-fat diet in adipose tissue development and nuclear receptor expression in young rats

In order to study the effects of dietary lipids and vitamin A on the development of adipose tissues, young rats were submitted for 8 d to a control or to two cafeteria diets with normal (Caf) or higher (Caf + ) vitamin A levels. Retinoid (retinoic acid receptor (RAR) a, RARg, retinoid X receptor(RXR) alpha) and fatty acid (PPARgamma) receptor mRNA was measured in the subcutaneous white adipose tissue (Swat) and in isolated mature adipocytes by RT-PCR. The stroma vascular fraction was cultured in vitro to test the capacities of the adipocyte precursors to proliferate and differentiate.The Caf diet enriched in vitamin A resulted in an increased adiposity, due to increased adipocyte hypertrophy. This was concomitant with a lower expression of RARa and RARg mRNA (234.6 and 238.6 %) and a higher expression of PPARgamma (+59 %) in the Swat and, to a less extent,in isolated adipocytes. Positive correlations were obtained between PPARgamma mRNA and Swat weights and between PPARgamma and RXRalpha mRNA. By contrast, RARgamma mRNA and Swat masses were negatively correlated. The adipocyte precursors from Caf + Swat proliferated more,in vitro, at the beginning of the culture. This difference progressively disappeared and was totally absent after 8 d of culture, but with a higher percentage of differentiated preadipocytes (+80.3 %) in the Caf + group. In conclusion, lipids and vitamin A act synergistically on the normal growth of the adipose tissue in young rats, concomitant with an imbalance in the pattern of the nuclear receptors. These changes influence the early normal development of the endogenous adipocyte precursors.

Characterization of a melatonin binding site in Siberian hamster brown adipose tissue

Melatonin has been shown, in various rodent species, to mediate photoperiodic effects on body weight and, consequently, fat mass. Pharmacological investigations indicated that the brown adipose tissue of Siberian hamsters possesses a melatonin binding site with a dissociation constant of 570+/-300 pM and a density of 3.2+/-1.8 fmol/mg protein. This binding site can also be detected on mature brown adipocyte membranes. The rank order of potency of a variety of drugs to displace 2-[125I]iodomelatonin from binding sites on Siberian hamster brown adipose tissue was as follows: 2-iodomelatonin > melatonin = prazosin > GR135531 (5-methoxycarbonylamino-N-acetyltryptamine) > N-acetylserotonin > 6-chloromelatonin > S20304 (N-(2-(1-naphthyl)ethyl)cyclobutanecarboxamide) >> methoxamine, phenylephrine, serotonin. Mel(1a) mRNA was not detected by RT-PCR (reverse transcription-polymerase chain reaction) in brown adipose tissue. Melatonin had no effect on either basal or stimulated lipolysis. Moreover, melatonin did not modify intracellular cAMP accumulation or inositol phosphate content. Together, these results suggest that the melatonin binding site characterized in brown adipose tissue is clearly different from the Mel(1) cloned subtype and has some features different from those of the Mel2 subtype.

Possible mechanisms of weight loss of Siberian hamsters (Phodopus sungorus sungorus) exposed to short photoperiod

Several weeks of short day photoperiod (SD) exposure promote a dramatic decrease of white adipose tissue (WAT) mass in Siberian hamsters(Phodopus sungorus sungorus). This slimming effect is accompanied by changes in the adipocyte responsiveness to adrenergic stimulation that are still under debate. We investigated whether possible changes in the antilipolytic responses, and/or lipogenic activities could be involved in such lipid deposition/mobilisation imbalance. Male Siberian hamsters were exposed for 11 weeks to SD or long day photoperiod and basal or stimulated lipolytic and lipogenic activities were measured on white adipocytes. As expected, the body mass of SD-animals was decreased. Besides a slight reduction in the basal lipolysis and in the maximal response to dibutyryl-cAMP, the responses to adrenergic and non-adrenergic lipolytic agents (forskolin, adenosine deaminase) were similar in both groups. Fat mass loss was likely not resulting from changes in the lipolytic responses of adipocytes to biogenic amines (e.g. octopamine), which were unaltered, or to a direct lipolytic stimulation by melatonin or histamine, which were inactive. Antilipolytic responses to insulin or tyramine were slightly decreased in SD-adipocytes. Basal or insulin-stimulated lipid accumulation in WAT, measured by glucose incorporation into lipids, did not change after SD-exposure. However, a significant decrease in the lipoprotein lipase activity was observed in the WAT of SDanimals. Despite the observed changes, the weight loss of SD-exposed Siberian hamsters was likely not resulting only from impaired antilipolytic orde novo lipogenic activities in white adipocytes, but either from other dramatic changes occurring during seasonal photoperiod-sensitive body weight regulation.

Effect of vitamin A content in cafeteria diet on the expression of nuclear receptors in rat subcutaneous adipose tissue

The aim of this study was to determine the effects of cafeteria diet containing control or elevated level of vitamin A on the expression of nuclear receptors in adipose tissue. Male Wistar rats were submitted to 3 experimental diets during 8 weeks, a standard diet and two hyper-energetic, hyperlipidic “cafeteria” diets containing normal (Caf) or higher (Caf+) vitamin A level. During the experiment, body weights and energy intakes were measured. At the end of the experimental period, subcutaneous adipose tissue (Swat) and all the fat mass were removed and weighted. Nuclear receptors mRNA levels of RARα, RARγ, RXRα, PPARγ were measured in the Swat by a real-time semi-quantitative RT-PCR method. We observed that energy intake of Caf+ and Caf groups was significantly higher than that of the control group. Despite a higher increase of the energy intake in the Caf group compared to the Caf+ group, no significant difference was observed in the body weight gain of the Caf+ compared to the Caf group. The Caf+ and Caf diets led to a significant increase of adipose tissue in cafeteria groups as observed in the Swat depot. The mRNA levels of PPARγ and RXRα were significantly increased in the Caf+ group as compared to control group, with a significant positive correlation between these two parameters. Expressions of RARα and RARγ were not modified in experimental groups compared to controls. In conclusion, 8-week exposure to cafeteria diets with normal and higher levels of vitamin A led to an increase of adiposity in rats, associated, only in the group fed with the higher vitamin A level cafeteria diet, with an increase of PPARγ and RXRα expressions in subcutaneous adipose tissue.ResumenEl objetivo del presente trabajo consiste en determinar las consecuencias de un alto contenido en vitamina A en dieta de cafetería sobre la expresión de receptores nucleares en el tejido adiposo. Así, ratas macho Wistar se dividieron en tres grupos: Durante 8 semanas, el grupo control se alimentó con pienso estándar, mientras que los grupos tratados recibieron una dieta rica en grasa (dieta de cafetería) enriquecida (Caf+) o no (Caf) con vitamina A. El peso corporal y la ingesta se determinaron durante todo el experimento. Al final del tratamiento, se pesó el tejido adiposo subcutáneo (Swat) y las otras reservas de grasa. Los niveles de ARNm de los receptores nucleares RARα, RARγ, RXRα, PPARγ se determinaron en el Swat con un método semi-cuantitativo de RT-PCR en tiempo real. Las ingestas energéticas de los grupos Caf+ y Caf fueron significativamente mayores que las del grupo control. A pesar del aumento en la ingesta del grupo Caf respecto del Caf+, no se observaron diferencias significativas en el aumento de peso corporal entre ambos grupos. Además, las dietas de los grupos Caf+ y Caf provocaron un claro aumento del tamaño de las reservas de grasa, incluido el peso del Swat. Los niveles de ARNm de PPARγ y RXRα se incrementaron significativamente en el grupo Caf+ respecto del control, con correlación positiva entre ambos. En cambio, no se modificó la expresión de RARα y RARγ. En suma, 8 semanas de alimentación con dieta de cafetería con niveles normales o elevados de vitamina A dan lugar a aumento de la adiposidad en la rata, asociado con aumento de la expresión de PPARγ y RXRα en el tejido adiposo subcutáneo solo en el grupo que recibió suplemento de vitamina A.

High lipolytic activity and dyslipidemia in a Spontaneous Hypertensive/NIH Corpulent (SHR/N-cp) rat: a genetic model of obesity and type 2 diabetes mellitus

In order to better understand the link between obesity and type 2 diabetes, lipolysis and its adrenergic regulation was investigated in various adipose depots of obese adult females SHR/N-cp rats. Serum insulin, glucose, free fatty acids (FFA), triglycerides (TG) and glycerol were measured. Adipocytes were isolated from subcutaneous (SC), parametrial (PM) and retroperitoneal (RP) fat pads. Total cell number and size, basal lipolysis or stimulated by norepinephrine (NE) and BRL 37344 were measured in each depot. Obese rats were hyperinsulinemic and hyperglycemic, suggesting high insulin resistance. They presented a marked dyslipidemia, attested by increased serum FFA and TG levels. High serum glycerol levels also suggest a strong lipolytic rate. Obese rats showed an excessive development of all fat pads although a more pronounced effect was observed in the SC one. The cellularity of this depot was increased 8 fold when compared to lean rats, but these fat cells were only 1.5 to 2-fold larger. SC adipocytes showed a marked increase in their basal lipolytic activity but a lack of change in responsiveness to NE or BRL 37344. The association between high basal lipolysis and increased cellularity yields to a marked adipose cell lipolytic rate, especially from the SC region. SHR/N-cp rats were characterized by a hyperplasic type of obesity with an excessive development of the SC depot. The dyslipidemia, attested by an altered serum lipid profile could be attributed to excessive lipolysis that contributes to increased FFA levels, and to early development of insulin resistance through a lipotoxicity effect.ResumenLa rata SHR/N-cp es un modelo genético que presenta obesidad y diabetes de tipo 2. A fin de comprender mejor el vínculo entre la obesidad y la diabetes, se investiga la lipólisis y su regulación adrenérgica en diversos depósitos adiposos de ratas hembras adultas obesas SHR/N-cp. Se midieron los niveles séricos de insulina, glucosa, ácidos grasos libres, triglicéridos y glicerol. Se utilizaron tres depósitos grasos, subeutáneo (SC), parametrial (PM) y retroperitoneal (RP), que se incubaron con colagenasa para obtener adipocitos aislados. Se determinó el número total de células y su tamanõ. Finalmente, se midió la lipólisis basal y la estimulada para noradrenalina y BRL 37344. El análisis de los parámetros biológicos confirmó que las ratas obesas eran hiperinsulinémicas e hiperglucémicas, lo que sugiere un alto grado de resistencia a la insulina. También presentaron una marcada dislipidemia, con elevados niveles de FFA y de TG. Los altos niveles de glicerol circulante sugieren una elevada tasa lipolítica en los depósitos. Las ratas obesas mostraron un excesivo desarrollo de todos los depósitos grasos, con un efecto más pronunciado en el SC. El número total de células de este depósito fue 8 veces mayor, pero el tamaño de los adipocitos fue sólo 1,5–2 veces superior. Los adipocitos SC mostraron un marcado aumento de su actividad lipolítica basal, pero ésta no cambió en respuesta a NE y BRL 37344. La asociación entre una elevada lipólisis basal y un aumento del número de células adiposas conllevó una mayor tasa lipolítica, especialmente SC. Las ratas SHR/N-cp se caracterizan por una obesidad de tipo hiperplásico, más pronunciada en el depósito subcutáneo. La marcada dislipidemia puede ser atribuida a una excesiva tasa lipolítica que contribuye a un incremento de los FFA circulantes, y, por tanto, a un desarrollo temprano de la resistencia a la insulina a través de un efecto lipotóxico.

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of β-adrenergic responses

Abstractβ3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a β3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of β-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, β3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to β1-, β2- and β3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the β3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and β3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts β3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all β-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of β3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical, use.ResumenLos agonistas β3-adrenérgicos son considerados potentes agentes anti-obesidad y antidiabéticos debido, fundamentalmente, a los efectos beneficiosos que producen en roedores obesos y diabeticos, descubiertos ya hace veinte años. El objetivo del presente estudio fue verificar si un tratamiento prolongado con agonists β3-adrenérgicos, conocidos estimulantes de la movilización lipídica, puede promover la desensibilización de las respuestas β-adrenérgicas. Para ello, se trataron ratas Wistar y cobayas con CL 316243 (CL, 1 mg/kg/d), administrado mediante el implante de minibombas osmóticas, durante una semana. En los adipocitos de ratas control, pero no en los de cobayas control, los agonistas β3-adrenérgicos produjeron efectos lipolíticos. El tratamiento con CL no modificó la ganancia de peso en ninguna de las dos especies, pero redujo los depósitos de grasa en ratas. En el tejido adiposo visceral y subcutáneo de las ratas tratadas con CL, la estimulación de la lipólisis por forskolina no se vió afectada, pero las respuestas a agonistas β1, β2, y β3 se redujeron. De manera análoga, el deterioro de la función insulínica, en lo que al transporte de glucosa y la lipogénesis se refiere, producido por los adrenérgicos β3 y que sólo se observa en los adipocitos de rata, disminuyó tras el tratamiento con CL. En los adipocitos de rata, la unión a [125I]ICYP y los niveles de ARNm del receptor adrenérgico β3 disminuyeron con la administración sostenida de CL. Estos resultados demuestran que el CL 316243 produce efectos lipolítico y antilipogénico únicamente en los adipocitos de rata. Estas acciones, muy probablemente relacionadas con la depleción de grasa observada en la rata, conducen a la desensibilización de todas las respuestas β-adrenérgicas. Esta desensibilización, junto con la ausencia de efecto adelgazante en cobayas, reduce la utilidad de los agonistas β3-adrenérgicos como agentes antiobesidad y podría explicar por qué no se han utilizado en la práctica clínica habitual como nuevos fármacos.