Biology subjects

Manning, B. D.

Publications and source records attributed to Manning, B. D..

7 recordsLinked to original sources

bioRxiv2025

Dietary protein governs the role of insulin signaling in the postprandial regulation of hepatic mTORC1

The nutrient-sensing mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway controls cellular and organismal growth and metabolism, while aberrant activation is linked to human disease, including metabolic disease. Cellular studies have established several regulatory mechanisms influencing mTORC1 activation, but the physiological signals that control mTORC1 at the organismal and tissue levels are less well defined. mTORC1 is dynamically regulated by fasting and feeding in metabolic tissues, with both nutrients and insulin proposed to activate mTORC1 in response to feeding. Here, a liver-specific mouse model that disconnects mTORC1 activation from AKT-mediated TSC2 phosphorylation is employed. This genetic mouse model demonstrates that AKT-mediated TSC2 phosphorylation is the predominant mechanism of hepatic mTORC1 induction by insulin but is dispensable for activation by feeding. Furthermore, dietary protein is critical and dictates the insulin-responsiveness of hepatic mTORC1 signaling. Contrary to dogma, hepatic mTORC1 signaling was not elevated in response to diet-induced obesity associated with the phenotypes of type-2 diabetes, including hyperinsulinemia, systemic insulin resistance, and hyperglycemia, and blocking hepatic AKT-TSC-mTORC1 signaling did not prevent these metabolic impairments. Evidence is also provided supporting a role for glucagon in hepatic mTORC1 suppression during fasting. This study reveals a hierarchy of physiological signals regulating hepatic mTORC1.

physiology↗

bioRxiv2025

Growth Factor-Independent mTORC1 Signaling Promotes Primary Cilia Length via Suppression of Autophagy

ABSTRACT/SUMMARYThe mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), as a sensor of growth signals that subsequently controls cell growth, has been predominantly studied in actively proliferating cells. Primary cilia are sensory organelles present on most quiescent cells, where they play essential roles in receiving environmental and developmental signals. Given that ciliated cells are non-proliferative, we investigated whether mTORC1 signaling influences the growth of primary cilia. Here, we show that mTORC1 promotes primary cilia elongation, without effects on ciliogenesis or cell growth, by suppressing autophagy. Inhibition of mTORC1 signaling through pharmacological, nutritional, or genetic interventions gave rise to shortened primary cilia, while activation of the pathway resulted in elongation. Furthermore, pharmacological or genetic inhibition of autophagy, a key downstream process blocked by mTORC1, also elongated primary cilia and rendered them resistant to mTORC1 inhibition. Notably, these mTORC1-mediated effects on primary cilia extend to mouse neurons ex vivo and in vivo. These findings highlight a previously unrecognized role for mTORC1 signaling in the control of primary cilia length that may contribute to diseases where ciliary function is altered, referred to as ciliopathies.

cell biology↗

bioRxiv2024

AKT-mediated phosphorylation of TSC2 controls stimulus and tissue-specific mTORC1 signaling and organ growth

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

cell biology↗

bioRxiv2021

Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin

ObjectiveThe mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism. MethodsATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice. ResultsWe demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver in response to feeding and in a hepatocyte-intrinsic manner by insulin. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of non-essential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. ConclusionThe results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

cell biology↗

bioRxiv2020

The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the adaptive stress-responsive transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, how ATF4 functions in response to these distinct modes of regulation and its broader role as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon activation of mTORC1 or the ISR. The mTORC1-ATF4 pathway stimulated the expression of only a subset of the ATF4 target genes stimulated by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

cell biology↗

bioRxiv2019

IMPDH inhibitors for anti-tumor therapy in tuberous sclerosis complex

Structured AbstractO_ST_ABSPurposeC_ST_ABSmTORC1 is a master regulator of anabolic cell growth and proliferation that is activated in the majority of human tumors. We recently demonstrated that elevated mTORC1 activity in cells and tumors can confer dependence on IMPDH, the rate-limiting enzyme in de novo guanylate nucleotide synthesis, to support increased ribosome biogenesis and cell viability. Pharmacological agents that inhibit IMPDH, such as mizoribine and mycophenolic acid (CellCept), are in wide clinical use as immunosuppressants. However, whether these agents can be repurposed for anti-tumor therapy requires further investigation in preclinical models, including direct comparisons to identify the best candidate(s) for advancement. Experimental DesignDistinct IMPDH inhibitors were tested on cell and mouse tumor models of tuberous sclerosis complex (TSC), a genetic tumor syndrome featuring widespread lesions with uncontrolled mTORC1 activity. Growth and viability were assessed in cells and tumors lacking the TSC2 tumor suppressor, together with drug pharmacokinetics and pharmacodynamics, target inhibition, and effects on tumor, tissue, and plasma metabolic biomarkers. ResultsMizoribine, used throughout Asia, exhibited greater selectivity in specifically targeting TSC2-deficient cells with active mTORC1 compared to the FDA-approved IMPDH inhibitors mycophenolic acid or ribavirin, or approved inhibitors of other nucleotide synthesis enzymes. In distinct tumor models, mizoribine demonstrated robust anti-tumor efficacy that is superior to mycophenolic acid, despite similar immunosuppressive effects. ConclusionsThese results provide pre-clinical rationale for repurposing mizoribine as an anti-tumor agent in tumors with active mTORC1, such as in TSC. Our findings also suggest that IMPDH inhibitors should be revisited in cancer models where MMF has shown modest efficacy. Statement of translational relevanceIMPDH inhibitors have been used clinically for decades as safe and effective immunosuppressants. Recent studies in pre-clinical tumor models establish IMPDH as a viable target for anti-tumor therapy, but the relative efficacies of approved IMPDH inhibitors in tumors have not been directly compared. Our data demonstrate a clear advantage of the IMPDH inhibitor mizoribine, used clinically throughout Asia, over the FDA-approved IMPDH inhibitor mycophenolate mofetil (or CellCept, a prodrug of mycophenolic acid) in mouse models of tuberous sclerosis complex (TSC) exhibiting mTORC1-driven tumor growth. While these IMPDH inhibitors elicit similar immunosuppressive effects, mizoribine has far superior anti-tumor activity in these models, indicating the potential for repurposing this drug for TSC and perhaps cancer treatment. We also identify the purine synthesis intermediate AICAR as an in vivo metabolic biomarker specific for effective inhibition of IMPDH with mizoribine, which can be readily detected in blood plasma shortly after mizoribine administration.

cancer biology↗

bioRxiv2017

Variable dose analysis: A novel RNAi-based method for detection of synthetic lethal interactions

Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells but such screens generally suffer from low reproducibility. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called variable dose analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC) and generated a SS/L network for TSC. Using this network, we identified four FDA-approved drugs that selectively affect viability of TSC deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.

cancer biology↗