Ketamine reduces temporal expectation in the rhesus monkey.

RATIONALE: Ketamine, a well-known general dissociative anesthetic agent that is a non-competitive antagonist of the N-methyl-D-aspartate receptor, perturbs the perception of elapsed time and the expectation of upcoming events. OBJECTIVE: The objective of this study was to determine the influence of ketamine on temporal expectation in the rhesus monkey. METHODS: Two rhesus monkeys were trained to make a saccade between a central warning stimulus and an eccentric visual target that served as imperative stimulus. The delay between the warning and the imperative stimulus could take one of four different values randomly with the same probability (variable foreperiod paradigm). During experimental sessions, a subanesthetic low dose of ketamine (0.25-0.35 mg/kg) was injected i.m. and the influence of the drug on movement latency was measured. RESULTS: We found that in the control conditions, saccadic latencies strongly decreased with elapsed time before the appearance of the visual target showing that temporal expectation built up during the delay period between the warning and the imperative stimulus. However, after ketamine injection, temporal expectation was significantly reduced in both subjects. In addition, ketamine also increased average movement latency but this effect could be dissociated from the reduction of temporal expectation. CONCLUSION: In conclusion, a subanesthetic dose of ketamine could have two independent effects: increasing reaction time and decreasing temporal expectation. This alteration of temporal expectation could explain cognitive deficits observed during ketamine use.

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.

Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.

Dysfunctional mTORC1 signaling is associated with a number of human pathologies owing to its central role in controlling cell growth, proliferation, and metabolism. Regulation of mTORC1 is achieved by the integration of multiple inputs, including those of mitogens, nutrients, and energy. It is thought that agents that increase the cellular AMP/ATP ratio, such as the antidiabetic biguanides metformin and phenformin, inhibit mTORC1 through AMPK activation of TSC1/2-dependent or -independent mechanisms. Unexpectedly, we found that biguanides inhibit mTORC1 signaling, not only in the absence of TSC1/2 but also in the absence of AMPK. Consistent with these observations, in two distinct preclinical models of cancer and diabetes, metformin acts to suppress mTORC1 signaling in an AMPK-independent manner. We found that the ability of biguanides to inhibit mTORC1 activation and signaling is, instead, dependent on the Rag GTPases.

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-gamma coactivator-1alpha (PGC-1alpha) 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.

Functional significance of skeletal muscle adiponectin production, changes in animal models of obesity and diabetes, and regulation by rosiglitazone treatment.

Endocrine effects of adipose-derived adiponectin on skeletal muscle have been shown to account, at least in part, for the anti-diabetic effects of this adipokine. Recently, the concept of myokines has gained credence, and the potential for skeletal muscle to produce adiponectin has been suggested. Here we demonstrated an increased level of adiponectin mRNA and protein expression as well as protein secretion in response to rosiglitazone treatment in L6 muscle cells. This correlated with the ability of rosiglitazone to enhance insulin sensitivity for stimulation of protein kinase B (Akt) phosphorylation and glucose transport; rosiglitazone also corrected high-glucose-induced insulin resistance in L6 cells. Overexpression of adiponectin confirmed the functional significance of local production of adiponectin in muscle cells via elevated glucose uptake and increased insulin sensitivity. In obese diabetic db/db mice, there was a change in the adiponectin expression profile in soleus and extensor digitorum longus (EDL) muscle with less high molecular weight (HMW) and more medium (MMW)/low (LMW) molecular weight species detected. Induction of obesity and insulin resistance in rats by feeding a high-fat high-sucrose diet also led to decreased muscle HMW adiponectin content that could be corrected by rosiglitazone treatment. In summary, we show the ability of skeletal muscle cells to produce adiponectin, which can mediate autocrine metabolic effects, thus establishing adiponectin as a bona fide myokine. We also demonstrate that skeletal muscle adiponectin production is altered in animal models of obesity and diabetes and that these changes can be corrected by rosiglitazone.

Rosiglitazone-induced heart remodelling is associated with enhanced turnover of myofibrillar protein and mTOR activation.

We investigated cardiac hypertrophy elicited by rosiglitazone treatment at the level of protein synthesis/degradation, mTOR, MAPK and AMPK signalling pathways, cardiac function and aspects of carbohydrate/lipid metabolism. Hearts of rats treated or not with rosiglitazone (15 mg/kg day) for 21 days were evaluated for gene expression, protein synthesis, proteasome and calpain activities, signalling pathways, and function by echocardiography. Rosiglitazone induced eccentric heart hypertrophy associated with increased expression of ANP, BNP, collagen I and III and fibronectin, reduced heart rate and increased stroke volume. Rosiglitazone robustly increased heart glycogen content ( approximately 400%), an effect associated with increases in glycogenin and UDPG-PPL mRNA levels and glucose uptake, and a reduction in glycogen phosphorylase expression and activity. Cardiac triglyceride content, lipoprotein lipase activity and mRNA levels of enzymes involved in fatty acid oxidation were also reduced by the agonist. Rosiglitazone-induced cardiac hypertrophy was associated with an increase in myofibrillar protein content and turnover (increased synthesis and an enhancement of calpain-mediated myofibrillar degradation). In contrast, 26S beta5 chymotryptic proteasome activity and mRNA levels of 20S beta2 and beta5 and 19S RPN 2 proteasome subunits along with the ubiquitin ligases atrogin and CHIP were all reduced by rosiglitazone. These morphological and biochemical changes were associated with marked activation of the key growth-promoting mTOR signalling pathway, whose pharmacological inhibition with rapamycin completely blocked cardiac hypertrophy induced by rosiglitazone. The study demonstrates that both arms of protein balance are involved in rosiglitazone-induced cardiac hypertrophy, and establishes the mTOR pathway as a novel important mediator therein.

Identification of IRS-1 Ser-1101 as a target of S6K1 in nutrient- and obesity-induced insulin resistance.

S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. This effect can be triggered directly by nutrients such as amino acids or by insulin through a homeostatic negative-feedback loop. However, the role of S6K1 in mediating IRS-1 phosphorylation in a physiological setting of nutrient overload is unresolved. Here we show that S6K1 directly phosphorylates IRS-1 Ser-1101 in vitro in the C-terminal domain of the protein and that mutation of this site largely blocks the ability of amino acids to suppress IRS-1 tyrosine and Akt phosphorylation. Consistent with this finding, phosphorylation of IRS-1 Ser-1101 is increased in the liver of obese db/db and wild-type, but not S6K1(-/-), mice maintained on a high-fat diet and is blocked by siRNA knockdown of S6K1 protein. Finally, infusion of amino acids in humans leads to the concomitant activation of S6K1, phosphorylation of IRS-1 Ser-1101, a reduction in IRS-1 function, and insulin resistance in skeletal muscle. These findings indicate that nutrient- and hormonal-dependent activation of S6K1 causes insulin resistance in mice and humans, in part, by mediating IRS-1 Ser-1101 phosphorylation.

Structure of the bovine VASAP-60/PRKCSH gene, functional analysis of the promoter, and gene expression analysis.

Vacuolar system-associated protein-60 (VASAP-60) constitutes the bovine ortholog of the human "protein kinase C substrate 80K-H" (PRKCSH or 80K-H). We characterized the bovine VASAP-60/PRKCSH gene structure and promoter, identified cis-acting elements controlling VASAP-60 expression, searched for mRNA splice variants, and analyzed mRNA expression in ovarian follicles. Expression of VASAP-60 mRNA showed a 2.4-fold increase (P<0.0001) in granulosa cells of dominant follicles compared to small follicles (2-4 mm) or ovulatory follicles, and no mRNA splice variant was identified. The bovine VASAP-60 gene encompasses 12.5 kb and is composed of 18 exons and 17 introns. Primer extension analysis revealed a single transcription initiation site, and the promoter lacks a TATA box. Promoter activity assays were performed with a series of deletion constructs in different bovine cell lines (endometrial epithelial glandular, kidney epithelial and aortic endothelial) to identify cis-acting elements. The -53/+16 bp fragment (+1 = transcription start site) conferred minimal promoter activity whereas activator and repressor elements were located in the -200/-53 bp and -653/-200 bp fragments, respectively. Analysis of cis-acting elements in the -200/-53 bp activation domain revealed by gel shift assays and chromatin immunoprecipitation assay that transcription factor YY1 binds to VASAP-60 promoter. This study is the first to report that VASAP-60 is up-regulated in granulosa cells of dominant follicles, to document the primary structure of the bovine VASAP-60 gene and promoter, and to demonstrate that YY1 binds to the VASAP-60 proximal promoter and may act as a positive transcriptional regulator.

Immunolocalization of vacuolar system-associated protein-60 (VASAP-60).

We have characterized the localization of the protein termed VASAP-60 in different bovine tissues and cell lines, and have investigated if VASAP-60 interacts with other proteins. Monospecific polyclonal antibodies were raised against distinct fragments of VASAP-60: NH(2) (V(22) to Q(234)), central (A(246) to S(418)), and COOH (L(416) to L(533)). These three antibodies recognized an 88-kDa protein in immunoblotting analysis. The calculated Mr of VASAP-60 derived from its cDNA (60.1 kDa) was significantly lower than its Mr estimated by SDS-PAGE, and this was mainly attributed to the glutamic acid- and aspartic acid-rich composition of its central region (A(246) to S(418)). A 58-kDa proteolytically processed form of VASAP-60 was also identified. Immunocytochemistry demonstrated that VASAP-60 is found predominantly in the perinuclear region, colocalized with calnexin in the endoplasmic reticulum (ER), and partially colocalized with the endocytic marker DAMP. Immunohistochemical localization of VASAP-60 also demonstrated its presence within specialized vesicular structures not related to the ER. Immunoprecipitation using extracts prepared from S(35)Met/Cys metabolically labeled cells demonstrates that VASAP-60 interacts with 116-, 48.5-, and 26.5-kDa proteins. Therefore, VASAP-60 was found to be more widely distributed in the vacuolar system than anticipated, suggesting that VASAP-60 may function in intracellular transport events, rather than being an exclusive component of the quality control mechanism of newly synthesized proteins as thought previously.

Serine protease inhibitor-E2 (SERPINE2) is differentially expressed in granulosa cells of dominant follicle in cattle.

The objective was to analyze gene expression in bovine granulosa cells of the dominant follicle by mRNA differential display. Total RNA was extracted from granulosa cells of