BACKGROUND: Elevation of glucagon levels and increase in alpha-cell mass are associated with states of hyperglycemia in diabetes. However, little is known about the mechanisms that control glucagon secretion and alpha-cell mass expansion in normal or diabetogenic conditions. Glucagon is secreted during the fasting state, when glucose levels are low, to stimulate glycogenolysis and gluconeogenesis in the liver to increase the blood glucose level. Amino acids, also, stimulate-glucagon secretion and alpha-cell mass. Amino acids increase glucagon secretion via activation of mTORC1 in alpha-cells. A critical step for mTOR activation is the localization of mTORC1 to the lysosome where it meets Rheb for activation. Amino acids are unique in their ability to localize mTORC1 to the lysosomal membrane for activation through their interaction with a variety of amino acid sensors, such as Sestrin2, which modulates mTORC1 activity via its interaction with GATOR2. Integral to mTORC1’s localization is the Ragulator complex, more specifically, p18, which provides the essential scaffolding necessary for lysosomal docking. Amino acids sensors work upstream of mTORC1 and sense amino acid concentrations with different affinities and are specific to certain amino acids and relay this information to mTORC1. OBJECTIVE: To investigate the role that p18, a component of the Ragulator complex, and GATOR2, a component of an amino acid sensor complex, play in amino-acid dependent mTORC1 lysosomal localization and its effect on alpha-cell function and glucagon secretion. METHODS: Generation of Knockout mice for p18 and GATOR2 in alpha-cells were produced by crossing Glu-Cre mice with P18(flox/flox) and GATOR2(flox/flox). Blood glucose, glucagon, and insulin levels were evaluated during fed, fasting, and insulin-induced hypoglycemic conditions to evaluate glucagon secretion. Isolated islets were also exposed to media containing different glucose or nutrient concentrations to evaluate the effect on glucagon secretion. RESULTS: Our data shows that knockdown experiments in alpha-cells for p18 and GATOR2 have demonstrated the role of these proteins in amino acid dependent localization of mTORC1 to the lysosomal membrane. More specifically, our data demonstrate that animals with a knockdown for p18 or GATOR2 demonstrated decreased glucagon secretion during hypoglycemic conditions. Mice with a knockdown for p18 also demonstrate decreased glucagon secretion in the presence of glucagon secretion stimulators such as arginine and also demonstrated decreased insulin secretion. CONCLUSIONS: Loss of essential components of the amino acid signaling and lysosomal localization in the mTORC1 pathway results in impaired function of alpha-cells and glucagon secretion. Loss of p18 in alpha-cells potentially results in an inability of mTORC1 to dock and bind to the lysosomal membrane, whereas loss of GATOR2 potentially results in chronic inhibition of mTORC1 via GATOR1. Loss of each of these components results in the lost or impaired ability for mTORC1 to migrate and bind to the lysosomal membrane. / 2022-06-02T00:00:00Z

Spleen tyrosine kinase (SYK) regulates mitogenic signaling, inflammatory responses and cell fate in a number of diverse cell types. KRAS is a proto-oncogene that controls cell growth and proliferation through several mitogenic pathways. In pancreatic cancer, KRAS is frequently mutated, resulting in constitutive activation in 90% of pancreatic cancer cell lines. We previously showed that SYK is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. We demonstrated that SYK kinase inhibition with PRT062607 (SYKi) causes decreased cell proliferation of PDAC cell lines. Furthermore, combined SYKi and MEK inhibitor (MEKi) treatment promotes additive effects on suppression of PDAC cell proliferation and clonogenic growth. Mechanistically, SYK activates the mTORC1 kinase complex as shown by reduced phosphorylation of ribosomal S6 protein and its upstream kinase p70 S6 kinase (p70S6K) following SYKi treatment in PDAC cell lines. SYK-mediated mTORC1 activation occurs independently of MEK/ERK and PI3K/AKT effector signaling pathways. The mTORC1 complex suppresses lysosome biogenesis and macroautophagy (autophagy). Consequently, mTORC1 suppression via SYK inhibition or shRNA-mediated depletion causes accumulation of autolysosomes. These effects are mediated by the enhanced nuclear localization of MITF, a key transcriptional regulator of genes involved in lysosome biogenesis and autophagy pathway activation. In summary, SYK positively regulates mTORC1 activation in a subset of PDAC cell lines to suppress hyperactivation of autophagy. These findings open new avenues for further exploration of SYK as a critical regulator of the autophagy pathway in KRAS/mTORC1-dependent PDAC, and how this may be exploited for therapeutic benefit.

Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.

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High fat feeding increases hepatic fat accumulation and is associated with hepatic insulin resistance. AMP Activated Protein Kinase (AMPK) is thought to inhibit lipid synthesis by the acute inhibition of glycerol-3-phosphate acyltransferase (GPAT) activity and transcriptional regulation via SREBP-1c. The purpose of this study was to determine if chronic activation of AMPK prevented an increase in GPAT1 activity in rats fed a high fat diet. Rats were fed a control (C), or a high fat (HF) diet (60% fat) for 6 weeks and injected with saline or a daily AICAR dose of 0.5 mg/g body weight. Chronic AMPK activation by AICAR injections resulted in a significant reduction in hepatic triglyceride accumulation in both the C and HF fed animals (C, 5.5±0.7; C+AICAR, 2.7 ±0.3; HF, 21.8±3.3; and HF+AICAR, 8.0±1.8 mg/g liver). HF feeding caused an increase in total GPAT and GPAT1 activity which was not affected by chronic AMPK activation (GPAT1 activity vs. C, C+AICAR, 92±19%; HF, 186±43%; HF+AICAR, 234±62%). Markers of oxidative capacity, including citrate synthase activity and cytochrome c abundance, were not affected by chronic AICAR treatment. Interestingly, HF feeding caused a significant increase in LCAD (up 66% from C), a marker of fatty acid oxidation capacity. These results suggest that chronic AMPK activation limits hepatic triglyceride accumulation independent of a reduction in total GPAT1 activity.

The 52 kDa intermediate filament protein desmin plays an important role in force transmission in skeletal muscle by connecting myofibrils at Z-lines and to the sarcolemma. Desmin content in muscle adapts to contractile activity and may be involved in cellular signaling mechanisms responsible for muscle growth. Purpose: To compare signaling responses of the mTOR pathway in wild type (WT) vs desmin knock out (KO) mice. Methods: WT (n=12) and KO (n=12) mice were exposed to high frequency electric stimulation of the left hindlimb to elicit an acute response of the mTOR pathway. Non-stimulated right hindlimbs were used as a within animal control. Right and left TA and EDL muscles were dissected 30 min post-stimulation and examined for changes in mTOR, 4E-BP1 and p70S6K. Results: Relative to WT control samples, total mTOR and total 4E-BP1 content was higher in KO control samples. Electrical stimulation resulted in an increase p70S6K phosphorylation in WT and KO animals however there was no difference between the groups. 4E-BP1 phosphorylation was increased in WT but not KO following electrical stimulation. There was no change in mTOR phosphorylation in response to stimulation in WT or KO. Conclusion: The absence of desmin in skeletal muscle does not impair the phosphorylation of p70S6K demonstrating that a tensile load on the muscle will likely result in an increase in protein synthesis. Elevated levels of total mTOR and 4E-BP1 may imply an adaptation to increase sensitivity to growth stimuli in the muscle.

Skeletal muscle has the ability to increase in size (hypertrophy) after resistance is placed upon it. This hypertrophy is marked by significant upregulation of the mammalian target of rapamycin (mTOR) and its downstream targets. The upstream kinases, protein kinase B (also known as Akt) and AMP-activated protein kinase (AMPK), are two of the many regulators of the mTOR pathway. Recent studies suggest that the widely consumed neuroactive compound caffeine could potentially inhibit mTOR by acting through Akt and/or AMPK. The purpose of this thesis was to: 1) determine if caffeine can inhibit the mTOR pathway and ultimately attenuate skeletal muscle hypertrophy and 2) determine if this inhibition is through LKB1, an upstream regulator of AMPK. First, 3 month old male rats underwent unilateral tenotomy of the gastrocnemius, resulting in overloading (OVLD) of the synergistic plantaris muscle. The contralateral limb was sham-operated (SHAM) on. Rats were given ad libitum access to tap water or tap water + caffeine (1 g/L). The OVLD procedure resulted in significant hypertrophy of the plantaris which was attenuated after 1 wk of caffeine treatment. However, after two wks this effect was not observed. mTOR targets were examined in both the SHAM and OVLD plantaris muscle which showed significant upregulation with OVLD but no impact with caffeine treatment. Akt and AMPK was also assessed in the plantaris muscle which showed diminished Akt phosphorylation in 1 wk treated rats while the phosphorylation of AMPK remained relatively unaffected. Notably, caffeine caused decreased atrophy of the tenotomized gastrocnemius after 1 wk along with decreased body weight gains, food consumption, and retroperitoneal fat pad weight in both 1 and 2 wk treated rats. Second, to elucidate how caffeine could be impacting the mTOR pathway and how LKB1/AMPK might be involved, skeletal muscle specific LKB1 knockout (skmLKB1-KO) mice were subjected to high-frequency electrical stimulation (HFES) of the sciatic nerve resulting in contraction of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles against the larger gastrocnemius. All mice were given an intraperitoneal injection of saline or saline + caffeine (20 mg/kg BW at 1 g/L). HFES resulted in marked upregulation of mTOR targets in the TA/EDL of mice 0, 3, and 8 h post HFES. mTOR targets remained relatively unchanged with caffeine treatment. We also observed that these markers were consistently upregulated in our skmLKB1-KO mice with or without HFES. Our findings indicate that caffeine, at physiological concentrations, does not impact anabolic signaling. Furthermore, diminished LKB1 levels resulted in increased levels and activation of markers of protein synthesis.

Although poorly understood, normal cells and cancerous cells of the same type exhibit different patterns of nutrient consumption, processing and utility of metabolic substrates. Differences in substrate uptake, preference, and alternately emphasized metabolic pathways offer opportunities for selective targeting of cancer versus stroma. This may be accomplished by using a sequential approach of nutrient deprivation and pharmaceutical perturbation of metabolic pathways to inhibit cellular proliferation. The purpose of this study was to investigate the effects of restricting glucose and glutamine concentrations, in vitro, to levels that resemble a potential human fasting state. The mammalian target of rapamycin (mTOR), a mediator of nutrient sensation, was then inhibited with rapamycin in the nutrient-restricted conditions. Because active Akt/mTOR is implicated in cancer cell pro-survival, the hypothesis is that pharmaceutical inhibition of active Akt/mTOR signaling in combination with the stress of restricted nutrient supply will be more effective than nutrient deprivation alone at disrupting metabolic processes to impair cancer cell proliferation and/or pro-survival mechanisms. Untreated and treated conditions were tested to determine if an additive or synergistic effect would result from a sequential insult of nutrient deprivation followed by inhibited mTORC1 signaling. The cell line used for this study was cultivated from a murine pancreatic intraepithelial neoplasia (PANIN) derived from a transgenic mouse with pancreatic tissue-specific expression of constitutively active Akt. The transgene of Akt, isoform 1, contains a myristoyl tag that facilitates co-localization of Akt to the plasma membrane, thereby promoting the activation of this signaling protein.; This aberrantly activated Akt represents a prosurvival condition observed in most cancers, and impacts metabolic balance with increased downstream signaling to metabolic sensors and regulators, including mTORC1. Several methods were used to evaluate changes in metabolic and physiological response to nutrient deprivation and mTORC1 inhibition. These included tetrazolium reduction/absorbance readings to qualitatively evaluate differences in cell proliferation, and Western immunoblots for observing changes in protein expression and phosphorylation. ATP luminescence assays were applied to quantify intracellular ATP content, and citrate synthase spectrophotometry used to quantify specific activity/indicate changes in the TCA/OXPHOS production of ATP. Results from the above methods suggest that, individually, nutrient deprivation and rapamycin treatment share some similar effects on metabolically-related protein phosphorylation and in reducing cellular proliferation. Collectively, nutrient deprivation plus rapamycin treatment, however, resulted in unanticipated metabolic alterations under conditions used for this study, the complexities of which would need to be delineated in future studies.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway appears to be the primary regulator of protein synthesis and growth. While resistance exercise paradigms are often divided into hypertrophy (HYP) and strength (STR) protocols, it is unknown whether these protocols differentially stimulate mTORC1 signaling. The purpose of this study was to examine mTORC1 signaling in conjunction with circulating hormone concentrations following a typical lower-body HYP and STR resistance exercise protocol. Ten resistance-trained men (24.7±3.4y; 90.1±11.3kg; 176.0±4.9cm) performed each resistance exercise protocol in a random, counterbalanced order. Blood samples were obtained at baseline (BL), immediately (IP), 30 minutes (30P), 1 hour (1H), 2 hours (2H), and 5 hours (5H) post-exercise. Fine needle muscle biopsies were completed at BL, 1H, and 5H. Electromyography of the vastus lateralis was also recorded during each protocol. HYP and STR produced a similar magnitude of muscle activation across sets. Myoglobin and lactate dehydrogenase concentrations were significantly greater following STR compared to HYP (p=0.01-0.02), whereas the lactate response was significantly higher following HYP compared to STR (p=0.003). The GH, cortisol, and insulin responses were significantly greater following HYP compared to STR (p=0.0001-0.04). No significant differences between protocols were observed for the IGF-1 or testosterone response. Intramuscular anabolic signaling analysis revealed a significantly greater (p=0.03) phosphorylation of IGF-1 receptor at 1H following HYP compared to STR. Phosphorylation status of all other signaling proteins including mTOR (mammalian target of rapamycin), p70S6k (ribosomal S6 kinase 1), and RPS6 (ribosomal protein S6) were not significantly different between trials. Despite significant differences in markers of muscle damage and the endocrine response following STR and HYP, both protocols appeared to elicit similar mTORC1 activation in resistance-trained men.