Pierre-Gilles Blanchard

Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue

OBJECTIVE The mammalian target of rapamycin (mTOR)/p70 S6 kinase 1 (S6K1) pathway is a critical signaling component in the development of obesity-linked insulin resistance and operates a nutrient-sensing negative feedback loop toward the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Whereas acute treatment of insulin target cells with the mTOR complex 1 (mTORC1) inhibitor rapamycin prevents nutrient-induced insulin resistance, the chronic effect of rapamycin on insulin sensitivity and glucose metabolism in vivo remains elusive. RESEARCH DESIGN AND METHODS To assess the metabolic effects of chronic inhibition of the mTORC1/S6K1 pathway, rats were treated with rapamycin (2 mg/kg/day) or vehicle for 15 days before metabolic phenotyping. RESULTS Chronic rapamycin treatment reduced adiposity and fat cell number, which was associated with a coordinated downregulation of genes involved in both lipid uptake and output. Rapamycin treatment also promoted insulin resistance, severe glucose intolerance, and increased gluconeogenesis. The latter was associated with elevated expression of hepatic gluconeogenic master genes, PEPCK and G6Pase, and increased expression of the transcriptional coactivator peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α) as well as enhanced nuclear recruitment of FoxO1, CRTC2, and CREB. These changes were observed despite normal activation of the insulin receptor substrate/PI 3-kinase/Akt axis in liver of rapamycin-treated rats, as expected from the blockade of the mTORC1/S6K1 negative feedback loop. CONCLUSIONS These findings unravel a novel mechanism by which mTORC1/S6K1 controls gluconeogenesis through modulation of several key transcriptional factors. The robust induction of the gluconeogenic program in liver of rapamycin-treated rats underlies the development of severe glucose intolerance even in the face of preserved hepatic insulin signaling to Akt and despite a modest reduction in adiposity.

Depot-specific effects of the PPARγ agonist rosiglitazone on adipose tissue glucose uptake and metabolism

We investigated mechanisms whereby peroxisome proliferator-activated receptor gamma (PPARgamma) agonism redistributes lipid from visceral (VF) toward subcutaneous fat (SF) by studying the impact of PPARgamma activation on VF and SF glucose uptake and metabolism, lipogenesis, and enzymes involved in triacylglycerol (TAG) synthesis. VF (retroperitoneal) and SF (inguinal) of rats treated or not for 7 days with rosiglitazone (15 mg/kg/day) were evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (or lipin-1), and diacylglycerol acyltransferase. Rosiglitazone increased SF glucose uptake, GLUT4 mRNA, and insulin-stimulated glucose oxidation, conversion to lactate, glycogen, and the glycerol and fatty acid components of TAG. In VF, only glucose incorporation into TAG-glycerol was stimulated by rosiglitazone and less so than in SF (1.5- vs. 3-fold). mRNA levels of proteins involved in glycolysis, Krebs cycle, glycogen synthesis, and lipogenesis were markedly upregulated by rosiglitazone in SF and again less so in VF. Rosiglitazone activated TAG-glycerol synthesis in vivo (2.8- vs. 1.9-fold) and lipin activity (4.6- vs. 1.5-fold) more strongly in SF than VF, whereas GPAT activity was increased similarly in both depots. The preferential increase in glucose uptake and intracellular metabolism in SF contributes to the PPARgamma-mediated redistribution of TAG from VF to SF, which in turn favors global insulin sensitization.

Major involvement of mTOR in the PPARγ-induced stimulation of adipose tissue lipid uptake and fat accretion

Evidence points to a role of the mammalian target of rapamycin (mTOR) signaling pathway as a regulator of adiposity, yet its involvement as a mediator of the positive actions of peroxisome proliferator-activated receptor (PPAR)γ agonism on lipemia, fat accretion, lipid uptake, and its major determinant lipoprotein lipase (LPL) remains to be elucidated. Herein we evaluated the plasma lipid profile, triacylglycerol (TAG) secretion rates, and adipose tissue LPL-dependent lipid uptake, LPL expression/activity, and expression profile of other lipid metabolism genes in rats treated with the PPARγ agonist rosiglitazone (15 mg/kg/day) in combination or not with the mTOR inhibitor rapamycin (2 mg/kg/day) for 15 days. Rosiglitazone stimulated adipose tissue mTOR complex 1 and AMPK and induced TAG-derived lipid uptake (136%), LPL mRNA/activity (2- to 6-fold), and fat accretion in subcutaneous (but not visceral) white adipose tissue (WAT; 50%) and in brown adipose tissue (BAT; 266%). Chronic mTOR inhibition attenuated the upregulation of lipid uptake, LPL expression/activity, and fat accretion induced by PPARγ activation in both subcutaneous WAT and BAT, which resulted in hyperlipidemia. In contrast, rapamycin did not affect most of the other WAT lipogenic genes upregulated by rosiglitazone. Together these findings demonstrate that mTOR is a major regulator of adipose tissue LPL-mediated lipid uptake and a critical mediator of the hypolipidemic and lipogenic actions of PPARγ activation.

Brown fat like gene expression in the epicardial fat depot correlates with circulating HDL-cholesterol and triglycerides in patients with coronary artery disease.

BACKGROUND Recent evidence indicates that epicardial adipose tissue (EAT) expresses uncoupling protein-1 (UCP1), a marker of brown adipocytes. However, the putative effects of the presence of brown adipocytes in EAT remain unknown. METHODS The mRNA expression of genes related to brown adipocyte-mediated thermogenesis was measured in the fat samples collected from the epicardial-, mediastinal- and subcutaneous-depots of patients undergoing coronary artery bypass grafting. Both univariate and multivariate analyses were then utilized to determine any association between gene expression and the anthropometrics and fasting blood chemistries of these patients. RESULTS EAT exhibited significantly higher expression of UCP1 and cytochrome c oxidase subunit-IV (COX-IV) compared to mediastinal- and subcutaneous-fat depots (P ≤ 0.05). EAT expression of UCP1 (r=0.50), COX-IV (r=0.37) and lipoprotein lipase (LPL) (r=0.58) positively associated with circulating levels of HDL-cholesterol (P ≤ 0.05). In addition, EAT expression of LPL, acyl coA dehydrogenase-short, -medium and -long chain genes associated negatively with circulating TG levels (P ≤ 0.05). CONCLUSIONS Abundance of UCP-1 in the EAT relative to other fat depots confirms the presence of brown adipocytes in human EAT. Furthermore, the correlations among the EAT expression of thermogenesis-related genes with the circulating HDL and TG levels indicate that presence of active brown adipocytes shares a functional association with the circulating plasma lipids in humans.

Control of Brown Adipose Tissue Glucose and Lipid Metabolism by PPARγ

Brown adipose tissue (BAT) non-shivering thermogenesis impacts energy homeostasis in rodents and humans. Mitochondrial uncoupling protein 1 in brown fat cells produces heat by dissipating the energy generated by fatty acid and glucose oxidation. In addition to thermogenesis and despite its small relative size, sympathetically activated BAT constitutes an important glucose, fatty acid, and triacylglycerol-clearing organ, and such function could potentially be used to alleviate dyslipidemias, hyperglycemia, and insulin resistance. To date, chronic sympathetic innervation and peroxisome proliferator-activated receptor (PPAR) γ activation are the only recognized inducers of BAT recruitment. Here, we review the major differences between these two BAT inducers in the regulation of lipolysis, fatty acid oxidation, lipid uptake and triacylglycerol synthesis, glucose uptake, and de novo lipogenesis. Whereas BAT recruitment through sympathetic drive translates into functional thermogenic activity, PPARγ-mediated recruitment is associated with a reduction in sympathetic activity leading to increased lipid storage in brown adipocytes. The promising therapeutic role of BAT in the treatment of hypertriglyceridemic and hyperglycemic conditions is also discussed.

The PPARγ agonist rosiglitazone enhances rat brown adipose tissue lipogenesis from glucose without altering glucose uptake

We investigated the mechanisms whereby peroxisome proliferator-activated receptor-gamma (PPARgamma) agonism affects glucose and lipid metabolism in brown adipose tissue (BAT) by studying the impact of PPARgamma activation on BAT glucose uptake and metabolism, lipogenesis, and mRNA levels plus activities of enzymes involved in triacylglycerol (TAG) synthesis. Interscapular BAT of rats treated or not with rosiglitazone (15 mg*kg(-1).day(-1), 7 days) was evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (PAP or lipin-1), and diacylglycerol acyltransferase (DGAT). Rosiglitazone increased BAT mass without affecting whole tissue glucose uptake. BAT glycogen content (-80%), its synthesis from glucose (-50%), and mRNA levels of UDP-glucose pyrophosphorylase (-40%), which generates UDP-linked glucose for glycogen synthesis, were all reduced by rosiglitazone. In contrast, BAT TAG-glycerol synthesis in vivo and glucose incorporation into TAG-glycerol in vitro were stimulated by the agonist along with the activities and mRNA levels of glycerol 3-phosphate-generating phosphoenolpyruvate carboxykinase and glycerokinase. Furthermore, rosiglitazone markedly increased the activities of GPAT and DGAT but not those of lipin-1-mediated PAP-1, enzymes involved in the sequential acylation of glycerol 3-phosphate and TAG synthesis. Because an adequate supply of fatty acids is essential for BAT nonshivering thermogenesis, the enhanced ability of BAT to synthesize TAG under PPARgamma activation may constitute an important mechanism by which lipid substrates are stored in preparation for an eventual thermogenic activation.

Basal adrenergic tone is required for maximal stimulation of rat brown adipose tissue UCP1 expression by chronic PPAR-γ activation

We investigated the involvement of basal sympathetic tone in brown adipose tissue (BAT) recruitment and gene expression profile induced by peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Innervated and surgically denervated BAT pads of rats treated or not with rosiglitazone (15 mg.kg(-1).day(-1), 7 days) were evaluated for weight, triacylglycerol (TAG) and DNA content, mitochondrial mass, and gene expression. Rosiglitazone induced BAT recruitment (increased mass, TAG and DNA content) and mRNA levels of lipolytic (adipose tissue triglyceride lipase and CGI58) and lipogenic (lipoprotein lipase, phosphoenolpyruvate carboxykinase, fatty acid binding protein 4, and diacylglycerol acyltransferase 1) proteins independently of tissue innervation status. Mitochondrial mass and mRNA levels of its regulators peroxisome proliferator-activated receptor coactivator-alpha and CCAAT/enhancer binding protein-beta were not affected by rosiglitazone, while being significantly reduced by denervation. By contrast, maximal stimulation of uncoupling protein 1 (UCP1) (thermogenesis), cell death-inducing DNA fragmentation factor-45-like effector A (inhibitor of UCP1 activity), monoacylglycerol lipase (lipolysis), small heterodimer partner (transcription), and glycerokinase (TAG synthesis) by rosiglitazone depended on the presence of intact BAT innervation. Cold exposure (5 degrees C, 24 h) significantly increased UCP1 mRNA levels in innervated BAT pads of untreated rats, without affecting the already high BAT UCP1 levels of rosiglitazone-treated animals. A similar pattern of response was found in denervated pads, but with markedly lower UCP1 expression than that in innervated BAT. In conclusion, whereas the mass (hyperplasia and hypertrophy), lipogenic, and lipolytic components of BAT recruitment induced by rosiglitazone occur independently of tissue sympathetic innervation, maximal UCP1 expression induced by PPAR-gamma in vivo depends on the presence of basal BAT adrenergic tone. The residual sympathetic tone found under rosiglitazone treatment is, therefore, involved in the modulation of a subset of major components of PPAR-gamma-mediated BAT recruitment.

Differential androgen and estrogen substrates specificity in the mouse and primates type 12 17 -hydroxysteroid dehydrogenase

Recently, we have shown that human and monkey type 12 17beta-hydroxysteroid dehydrogenases (17beta-HSD12) are estrogen-specific enzymes catalyzing the transformation of estrone (E(1)) into estradiol (E(2)). To further characterize this novel steroidogenic enzyme in an animal model, we have isolated a cDNA fragment encoding mouse 17beta-HSD12 and characterized its enzymatic activity. Using human embryonic kidney cells (HEK)-293 cells stably expressing mouse 17beta-HSD12, we found that in contrast with the human and monkey enzymes, which are specific for the transformation of E(1) to E(2), mouse 17beta-HSD12 also catalyzes the transformation of 4-androstenedione into testosterone (T), dehydroepiandroster-one (DHEA) into 5-androstene-3beta,17beta-diol (5-diol), as well as androsterone into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol). Previously, we have shown that the specificity of human and monkey 17beta-HSD12s for C18-steroid is due to the presence of a bulky phenylalanine (F) at position 234 creating steric hindrance, preventing the entrance of C19-steroids into the active site. To determine whether the smaller size of the corresponding leucine (L) in the mouse sequence is responsible for the entrance of androgenic substrates, we performed site-directed mutagenesis to substitute Leu 234 for Phe in the mouse enzyme. In agreement with our hypothesis, the mutated enzyme has a highly reduced ability to metabolize androgens. mRNA quantification in several mouse tissues using real-time PCR shows that mouse 17beta-HSD12 mRNA is highly expressed in the female clitoral gland, male preputial gland, as well as in retroperitoneal fat and adrenal of both sexes. The differential androgenic/estrogenic substrate specificity of type 12 17beta-HSD in the mouse and primates seems to agree with the observation that androgen and estrogen in the mouse are provided almost exclusively by gonads, while in primates an important part of these steroid hormones are produced locally from adrenal precursors.

Tissue-specific postprandial clearance is the major determinant of PPARγ-induced triglyceride lowering in the rat

Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonism potently reduces circulating triglycerides (TG) in rodents and more modestly so in humans. This study aimed to quantify in vivo the relative contribution of hepatic VLDL-TG secretion and tissue-specific TG clearance to such action. Rats were fed an obesogenic diet, treated with the PPARgamma full agonist COOH (30 mg.kg(-1).day(-1)) for 3 wk, and studied in both the fasted and refed (fat-free) states. Hepatic VLDL-TG secretion rate was not affected by chronic COOH in the fasted state and was only modestly decreased (-30%) in refed rats. In contrast, postprandial VLDL-TG clearance was increased 2.6-fold by COOH, which concomitantly stimulated adipose tissue TG-derived lipid uptake and one of its major determinants, lipoprotein lipase (LPL) activity, in a highly depot-specific manner. TG-derived lipid uptake and LPL were indeed strongly increased in subcutaneous inguinal white adipose tissue and in brown adipose tissue, independently of the nutritional state, whereas of the three visceral fat depots examined (epididymal, retroperitoneal, mesenteric) only the latter responded consistently to COOH. Robust correlations (0.5 < r < 0.9) were observed between TG-derived lipid uptake and LPL in adipose tissues. The agonist did not increase LPL in muscle, and its enhancing action on postprandial muscle lipid uptake appeared to be mediated by post-LPL processes involving increased expression of fatty acid binding/transport proteins (aP2, likely in infiltrated adipocytes, FAT/CD36, and FATP-1). The study establishes in a diet-induced obesity model the major contribution of lipid uptake by specific, metabolically safe adipose depots to the postprandial hypotriglyceridemic action of PPARgamma agonism, and suggests a key role for LPL therein.

Caenorhabditis elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17 -hydroxysteroid dehydrogenases

Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17beta-hydroxysteroid dehydrogenases (17beta-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17beta-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol. Previously, we have shown that amino acid F234 in human 17beta-HSD12 is responsible for the selectivity of the enzyme toward estrogens. To assess whether this amino acid position 234 in LET-767 could play a role in androgen-estrogen selectivity, we have changed the methionine M234 in LET-767 into F. The results show that the M234F change causes the loss of the ability to transform androstenedione into testosterone, while conserving the ability to transform estrone into estradiol, thus confirming the role of amino acid position 234 in substrate selectivity. To further analyze the structure-function relationship of this enzyme, we have changed the three amino acids corresponding to lethal mutations in let-767 gene. The data show that these mutations strongly affect the ability of LET-767 to convert estrone in to estradiol and abolish its ability to transform androstenedione into testosterone. The high conservation of the active site and amino acids responsible for enzymatic activity and substrate selectivity strongly suggests that LET-767 shares a common ancestor with human 17beta-HSD3 and 12.