The Molecular Circadian Clock Is a Target of Anti-cancer Translation Inhibitors.

Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.

Time-of-day-dependent variation of the human liver transcriptome and metabolome is disrupted in MASLD.

Background & Aims: Liver homeostasis is ensured in part by time-of-day-dependent processes, many of them being paced by the molecular circadian clock. Liver functions are compromised in metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), and clock disruption increases susceptibility to MASLD progression in rodent models. We therefore investigated whether the time-of-day-dependent transcriptome and metabolome are significantly altered in human steatotic and MASH livers. Methods: Liver biopsies, collected within an 8 h-window from a carefully phenotyped cohort of 290 patients and histologically diagnosed to be either normal, steatotic or MASH hepatic tissues, were analyzed by RNA sequencing and unbiased metabolomic approaches. Time-of-day-dependent gene expression patterns and metabolomes were identified and compared between histologically normal, steatotic and MASH livers. Results: Herein, we provide a first-of-its-kind report of a daytime-resolved human liver transcriptome-metabolome and associated alterations in MASLD. Transcriptomic analysis showed a robustness of core molecular clock components in steatotic and MASH livers. It also revealed stage-specific, time-of-day-dependent alterations of hundreds of transcripts involved in cell-to-cell communication, intracellular signaling and metabolism. Similarly, rhythmic amino acid and lipid metabolomes were affected in pathological livers. Both TNFα and PPARγ signaling were predicted as important contributors to altered rhythmicity. Conclusion: MASLD progression to MASH perturbs time-of-day-dependent processes in human livers, while the differential expression of core molecular clock components is maintained. Impact and implications: This work characterizes the rhythmic patterns of the transcriptome and metabolome in the human liver. Using a cohort of well-phenotyped patients (n = 290) for whom the time-of-day at biopsy collection was known, we show that time-of-day variations observed in histologically normal livers are gradually perturbed in liver steatosis and metabolic dysfunction-associated steatohepatitis. Importantly, these observations, albeit obtained across a restricted time window, provide further support for preclinical studies demonstrating alterations of rhythmic patterns in diseased livers. On a practical note, this study indicates the importance of considering time-of-day as a critical biological variable which may significantly affect data interpretation in animal and human studies of liver diseases.

Specific post-translational modifications of soluble tau protein distinguishes Alzheimer's disease and primary tauopathies.

Tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The tau isoforms observed in post mortem human brain aggregates is used to classify tauopathies. However, distinguishing tauopathies ante mortem remains challenging, potentially due to differences between insoluble tau in aggregates and soluble tau in body fluids. Here, we demonstrated that tau isoforms differ between tauopathies in insoluble aggregates, but not in soluble brain extracts. We therefore characterized post-translational modifications of both the aggregated and the soluble tau protein obtained from post mortem human brain tissue of patients with Alzheimer's disease, cortico-basal degeneration, Pick's disease, and frontotemporal lobe degeneration. We found specific soluble signatures for each tauopathy and its specific aggregated tau isoforms: including ubiquitination on Lysine 369 for cortico-basal degeneration and acetylation on Lysine 311 for Pick's disease. These findings provide potential targets for future development of fluid-based biomarker assays able to distinguish tauopathies in vivo.

Comparison of Different Ex-Vivo Preservation Strategies on Cardiac Metabolism in an Animal Model of Donation after Circulatory Death.

Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C-18 °C-25 °C-35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.

AMPK inhibits liver gluconeogenesis: fact or fiction?

Is there a role for AMPK in the control of hepatic gluconeogenesis and could targeting AMPK in liver be a viable strategy for treating type 2 diabetes? These are frequently asked questions this review tries to answer. After describing properties of AMPK and different small-molecule AMPK activators, we briefly review the various mechanisms for controlling hepatic glucose production, mainly via gluconeogenesis. The different experimental and genetic models that have been used to draw conclusions about the role of AMPK in the control of liver gluconeogenesis are critically discussed. The effects of several anti-diabetic drugs, particularly metformin, on hepatic gluconeogenesis are also considered. We conclude that the main effect of AMPK activation pertinent to the control of hepatic gluconeogenesis is to antagonize glucagon signalling in the short-term and, in the long-term, to improve insulin sensitivity by reducing hepatic lipid content.

Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

PPARs in liver physiology.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors and transcriptional modulators with crucial functions in hepatic and whole-body energy homeostasis. Besides their well-documented roles in lipid and glucose metabolism, emerging evidence also implicate PPARs in the control of other processes such as inflammatory responses. Recent technological advances, such as single-cell RNA sequencing, have allowed to unravel an unexpected complexity in the regulation of PPAR expression, activity and downstream signaling. Here we provide an overview of the latest advances in the study of PPARs in liver physiology, with a specific focus on formerly neglected aspects of PPAR regulation, such as tissular zonation, cellular heterogeneity, circadian rhythms, sexual dimorphism and species-specific features.

Effects of PKB/Akt inhibitors on insulin-stimulated lipogenesis and phosphorylation state of lipogenic enzymes in white adipose tissue.

We investigated acute effects of two allosteric protein kinase B (PKB) inhibitors, MK-2206 and Akti-1/2, on insulin-stimulated lipogenesis in rat epididymal adipocytes incubated with fructose as carbohydrate substrate. In parallel, the phosphorylation state of lipogenic enzymes in adipocytes and incubated epididymal fat pads was monitored by immunoblotting. Preincubation of rat epididymal adipocytes with PKB inhibitors dose-dependently inhibited the following: insulin-stimulated lipogenesis, increased PKB Ser473 phosphorylation, increased PKB activity and decreased acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. In contrast, the effect of insulin to decrease the phosphorylation of pyruvate dehydrogenase (PDH) at Ser293 and Ser300 was not abolished by PKB inhibition. Insulin treatment also induced ATP-citrate lyase (ACL) Ser454 phosphorylation, but this effect was less sensitive to PKB inhibitors than ACC dephosphorylation by insulin. In incubated rat epididymal fat pads, Akti-1/2 treatment reversed insulin-induced ACC dephosphorylation, while ACL phosphorylation by insulin was maintained. ACL and ACC purified from white adipose tissue were poor substrates for PKBα in vitro. However, effects of wortmannin and torin, along with Akti-1/2 and MK-2206, on recognized PKB target phosphorylation by insulin were similar to their effects on insulin-induced ACL phosphorylation, suggesting that PKB could be the physiological kinase for ACL phosphorylation by insulin. In incubated epididymal fat pads from wild-type versus ACC1/2 S79A/S212A knockin mice, effects of insulin to increase lipogenesis from radioactive fructose or from radioactive acetate were reduced but not abolished. Together, the results support a key role for PKB in mediating insulin-stimulated lipogenesis by decreasing ACC phosphorylation, but not by decreasing PDH phosphorylation.

TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise.

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

Genetic deletion of soluble 5'-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet.

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.

HBP1 phosphorylation by AKT regulates its transcriptional activity and glioblastoma cell proliferation.

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16INK4A and cyclin D1 was also affected. HBP1 had previously been shown to limit glioma cell growth. Accordingly, HBP1 silencing by small-hairpin RNA increased human glioblastoma cell proliferation. Conversely, HBP1 overexpression decreased cell growth and foci formation. This effect was amplified by mutations that prevented phosphorylation by AKT, and blunted by mutations that mimicked phosphorylation. In conclusion, our results suggest that HBP1 phosphorylation by AKT blocks its functions as transcriptional regulator and tumor suppressor.

Cellular uptake of proMMP-2:TIMP-2 complexes by the endocytic receptor megalin/LRP-2.

Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/ß in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2 in coupling glycolysis to cell proliferation and protein synthesis in this model.