Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes

Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic–mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase–Akt–mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.Adipose tissue plays an essential role in maintaining whole-body energy homeostasis by storing or releasing nutrients. This balance is controlled by opposing signaling pathways where anabolic processes are activated by insulin (INS) and catabolic actions are activated by catecholamines. An important unanswered question in adipose biology is how catecholamine-induced β-adrenergic signaling opposes insulin-stimulated glucose uptake (1–6). Surprisingly, the underlying mechanism for this well-established physiological response in adipocytes is still unknown.When nutrients are plentiful, insulin is released by the pancreas and stimulates the absorption of glucose and fatty acids in adipose tissue, where they are packaged and stored as triacylglycerol (TAG) in cellular lipid droplets. Insulin signaling in adipocytes is mediated by the phosphoinositide 3-kinase (PI3K)–Akt–mTOR pathway. mTOR is a highly conserved serine/threonine protein kinase that functions in either of two distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is defined primarily by the association of mTOR with raptor, whereas mTORC2 includes mTOR with rictor (7). Importantly, mTORC2 phosphorylation of Akt at S473 is required for Akt activity on AS160, which is necessary for glucose uptake in response to insulin (8–11). Of note, for both mTORC1 and mTORC2, the integrity of these protein complexes is essential for kinase substrate specificity and proper signaling (12, 13).During periods of fasting or stress, catecholamines are released by the sympathetic nervous system to activate lipolysis. Stimulation of the β-adrenergic receptor on adipocytes activates adenylyl cyclase (AC), leading to elevated cAMP and protein kinase A (PKA) activity. PKA initiates lipolysis by direct phosphorylation of hormone-sensitive lipase (HSL) and perilipin (14–16) and indirect activation of adipose triglyceride lipase (ATGL) (17–19). Lipolysis involves hydrolysis of TAG stored in the lipid droplet to produce diacylglycerol (DAG), monoacylglycerol (MAG), fatty acids, and glycerol. These lipolytic products are important energy substrates that can act as precursors for other lipids and impact cellular signaling. However, their potential role as signaling molecules has been underappreciated (20).In this study, we provide insight into the mechanisms that link β-adrenergic stimulation to the inhibition of insulin-stimulated glucose uptake. Namely, we show that activation of lipolysis is crucial. Moreover, we find that products of lipolysis themselves cause mTOR inhibition by complex dissociation, which inhibits glucose uptake in adipocytes. This mechanism of mTOR regulation (i.e., by complex dissociation) has major implications in the regulation of cellular metabolism and likely contributes to stress-induced hyperglycemia and obesity-induced insulin resistance.     

Timing-Invariant CT Angiography Derived from CT Perfusion Imaging in Acute Stroke: A Diagnostic Performance Study

BACKGROUND AND PURPOSE:Timing-invariant (or delay-insensitive) CT angiography derived from CT perfusion data may obviate a separate cranial CTA in acute stroke, thus enhancing patient safety by reducing total examination time, radiation dose, and volume of contrast material. We assessed the diagnostic accuracy of timing-invariant CTA for detecting intracranial artery occlusion in acute ischemic stroke, to examine whether standard CTA can be omitted.MATERIALS AND METHODS:Patients with suspected ischemic stroke were prospectively enrolled and underwent CTA and CTP imaging at admission. Timing-invariant CTA was derived from the CTP data. Five neuroradiologic observers assessed all images for the presence and location of intracranial artery occlusion in a blinded and randomized manner. Sensitivity and specificity of timing-invariant CTA and standard CTA were calculated by using an independent expert panel as the reference standard. Interrater agreement was determined by using κ statistics.RESULTS:We included 108 patients with 47 vessel occlusions. Overall, standard CTA and timing-invariant CTA provided similar high diagnostic accuracy for occlusion detection with a sensitivity of 96% (95% CI, 90%–100%) and a specificity of 100% (99%–100%) for standard CTA and a sensitivity of 98% (95% CI, 94%–100%) and a specificity of 100% (95% CI, 100%–100%) for timing-invariant CTA. For proximal large-vessel occlusions, defined as occlusions of the ICA, basilar artery, and M1, the sensitivity and specificity were 100% (95% CI, 100%–100%) for both techniques. Interrater agreement was good for both techniques (mean κ value, 0.75 and 0.76).CONCLUSIONS:Timing-invariant CTA derived from CTP data provides diagnostic accuracy similar to that of standard CTA for the detection of artery occlusions in acute stroke.

Stroke imaging research currently focuses on prediction of patient outcome and identifying patients who are suitable for neurointerventional treatment.^1,2 For these purposes, advanced stroke imaging protocols typically add CT perfusion imaging or diffusion-weighted MR imaging to the traditional work-up, consisting of noncontrast CT and CT angiography.^2,3 Noncontrast CT is used to differentiate hemorrhagic stroke from ischemic stroke and to assess early signs of ischemia. CTA is used to localize arterial occlusions and to identify proximal large-vessel occlusions that may be suitable for endovascular treatment. CT perfusion imaging and DWI are used to assess the extent and severity of hypoperfusion and particularly increase the sensitivity of imaging in the early stages of ischemic stroke.⁴ The practical advantages of CT perfusion imaging are that it is widely available and does not delay treatment decisions because it is fast and most patients already undergo CT scanning.³Currently, CTA can be derived from CT perfusion data. Such an approach allows the enhancement of patient safety by reducing the total scanning time, radiation dose, and amount of contrast material needed.⁵ In CT perfusion imaging, multiple scans after intravenous injection of contrast material are obtained with time, generating a 4D dataset, which is used to derive cerebral perfusion maps such as the cerebral blood flow, cerebral blood volume, and arrival times. When imaging is performed on a CT scanner with large spatial coverage, however, this 4D data can also be used to provide CT angiographic information, referred to as 4D-CTA or dynamic CTA. Previous studies have assessed whether 4D-CTA can be used for detection of vascular occlusion in a stroke setting but found that image quality was moderate and diagnostic performance for stroke assessment was limited because large-vessel occlusions may be missed.^5–8 Recently, a different approach to obtain CTA from CT perfusion source data was presented that combines the whole 4D-CTA dataset into 1 high-quality 3D-CTA dataset by displaying maximum contrast enhancement with time.⁵ This technique is referred to as “timing-invariant CTA” because it is insensitive to delayed contrast arrival and was shown to provide similar-to-superior image quality compared with standard CTA.⁵The aim of our study was to test the diagnostic performance of timing-invariant CTA for stroke evaluation, to assess whether standard CTA can be omitted when CT perfusion imaging has been performed.     

MiR‐34a deficiency accelerates medulloblastoma formation in vivo

Previous studies have evaluated the role of miRNAs in cancer initiation and progression. MiR‐34a was found to be downregulated in several tumors, including medulloblastomas. Here we employed targeted transgenesis to analyze the function of miR‐34a in vivo. We generated mice with a constitutive deletion of the miR‐34a gene. These mice were devoid of mir‐34a expression in all analyzed tissues, but were viable and fertile. A comprehensive standardized phenotypic analysis including more than 300 single parameters revealed no apparent phenotype. Analysis of miR‐34a expression in human medulloblastomas and medulloblastoma cell lines revealed significantly lower levels than in normal human cerebellum. Re‐expression of miR‐34a in human medulloblastoma cells reduced cell viability and proliferation, induced apoptosis and downregulated the miR‐34a target genes, MYCN and SIRT1. Activation of the Shh pathway by targeting SmoA1 transgene overexpression causes medulloblastoma in mice, which is dependent on the presence and upregulation of Mycn. Analysis of miR‐34a in medulloblastomas derived from ND2:SmoA1(tg) mice revealed significant suppression of miR‐34a compared to normal cerebellum. Tumor incidence was significantly increased and tumor formation was significantly accelerated in mice transgenic for SmoA1 and lacking miR‐34a. Interestingly, Mycn and Sirt1 were strongly expressed in medulloblastomas derived from these mice. We here demonstrate that miR‐34a is dispensable for normal development, but that its loss accelerates medulloblastomagenesis. Strategies aiming to re‐express miR‐34a in tumors could, therefore, represent an efficient therapeutic option.     

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

High‐resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi‐core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9‐ and 1.3‐ppm signals) and metabolite quantification using HR MAS ¹H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16–0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one‐way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.