mTORC1 regulates a lysosome-dependent adaptive shift in intracellular lipid species.

The mechanistic target of rapamycin complex 1 (mTORC1) senses and relays environmental signals from growth factors and nutrients to metabolic networks and adaptive cellular systems to control the synthesis and breakdown of macromolecules; however, beyond inducing de novo lipid synthesis, the role of mTORC1 in controlling cellular lipid content remains poorly understood. Here we show that inhibition of mTORC1 via small molecule inhibitors or nutrient deprivation leads to the accumulation of intracellular triglycerides in both cultured cells and a mouse tumor model. The elevated triglyceride pool following mTORC1 inhibition stems from the lysosome-dependent, but autophagy-independent, hydrolysis of phospholipid fatty acids. The liberated fatty acids are available for either triglyceride synthesis or ß-oxidation. Distinct from the established role of mTORC1 activation in promoting de novo lipid synthesis, our data indicate that mTORC1 inhibition triggers membrane phospholipid trafficking to the lysosome for catabolism and an adaptive shift in the use of constituent fatty acids for storage or energy production.

Multiomics assessment of dietary protein titration reveals altered hepatic glucose utilization.

Dietary protein restriction (PR) has rapid effects on metabolism including improved glucose and lipid homeostasis, via multiple mechanisms. Here, we investigate responses of fecal microbiome, hepatic transcriptome, and hepatic metabolome to six diets with protein from 18% to 0% of energy in mice. PR alters fecal microbial composition, but metabolic effects are not transferable via fecal transplantation. Hepatic transcriptome and metabolome are significantly altered in diets with lower than 10% energy from protein. Changes upon PR correlate with calorie restriction but with a larger magnitude and specific changes in amino acid (AA) metabolism. PR increases steady-state aspartate, serine, and glutamate and decreases glucose and gluconeogenic intermediates. 13C6 glucose and glycerol tracing reveal increased fractional enrichment in aspartate, serine, and glutamate. Changes remain intact in hepatic ATF4 knockout mice. Together, this demonstrates an ATF4-independent shift in gluconeogenic substrate utilization toward specific AAs, with compensation from glycerol to promote a protein-sparing response.

Age-dependent impact of two exercise training regimens on genomic and metabolic remodeling in skeletal muscle and liver of male mice.

Skeletal muscle adapts to different exercise training modalities with age; however, the impact of both variables at the systemic and tissue levels is not fully understood. Here, adult and old C57BL/6 male mice were assigned to one of three groups: sedentary, daily high-intensity intermittent training (HIIT), or moderate intensity continuous training (MICT) for 4 weeks, compatible with the older group's exercise capacity. Improvements in body composition, fasting blood glucose, and muscle strength were mostly observed in the MICT old group, while effects of HIIT training in adult and old animals was less clear. Skeletal muscle exhibited structural and functional adaptations to exercise training, as revealed by electron microscopy, OXPHOS assays, respirometry, and muscle protein biomarkers. Transcriptomics analysis of gastrocnemius muscle combined with liver and serum metabolomics unveiled an age-dependent metabolic remodeling in response to exercise training. These results support a tailored exercise prescription approach aimed at improving health and ameliorating age-associated loss of muscle strength and function in the elderly.

Short-Term Ketogenic Diet Induces a Molecular Response That Is Distinct From Dietary Protein Restriction.

There is increasing interest in utilizing short-term dietary interventions in the contexts of cancer, surgical stress and metabolic disease. These short-term diets may be more feasible than extended interventions and may be designed to complement existing therapies. In particular, the high-fat, low-carbohydrate ketogenic diet (KD), traditionally used to treat epilepsy, has gained popularity as a potential strategy for weight loss and improved metabolic health. In mice, long-term KD improves insulin sensitivity and may extend lifespan and healthspan. Dietary protein restriction (PR) causes increased energy expenditure, weight loss and improved glucose homeostasis. Since KD is inherently a low-protein diet (10% of calories from protein vs. >18% in control diet), here we evaluated the potential for mechanistic overlap between PR and KD via activation of a PR response. Mice were fed control, protein-free (PF), or one of four ketogenic diets with varying protein content for 8 days. PF and KD both decreased body weight, fat mass, and liver weights, and reduced fasting glucose and insulin levels, compared to mice fed the control diet. However, PF-fed animals had significantly improved insulin tolerance compared to KD. Furthermore, contrary to the PF-fed mice, mice fed ketogenic diets containing more than 5% of energy from protein did not increase hepatic Fgf21 or brown adipose Ucp1 expression. Interestingly, mice fed KD lacking protein demonstrated greater elevations in hepatic Fgf21 than mice fed a low-fat PF diet. To further elucidate potential mechanistic differences between PF and KD and the interplay between dietary protein and carbohydrate restriction, we conducted RNA-seq analysis on livers from mice fed each of the six diets and identified distinct gene sets which respond to dietary protein content, dietary fat content, and ketogenesis. We conclude that KD with 10% of energy from protein does not induce a protein restriction response, and that the overlapping metabolic benefits of KD and PF diets may occur via distinct underlying mechanisms.

SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis.

Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytes capable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolic drivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumption of numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and promotes peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of adipogenesis. In early adipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcrotonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adipose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes, suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. In summary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolic events that stimulate transcriptional reprogramming.

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

OBJECTIVE: The 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. METHODS: ATF4 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. RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver, in a hepatocyte-intrinsic manner by insulin in response to feeding. 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 nonessential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine. CONCLUSIONS: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in the liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

NQO1 protects obese mice through improvements in glucose and lipid metabolism.

Chronic nutrient excess leads to metabolic disorders and insulin resistance. Activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. Here, inducible activation of Nrf2 in mice and transgenesis of the Nrf2 target, NQO1, conferred protection from diet-induced metabolic defects through preservation of glucose homeostasis, insulin sensitivity, and lipid handling with improved physiological outcomes. NQO1-RNA interaction mediated the association with and inhibition of the translational machinery in skeletal muscle of NQO1 transgenic mice. NQO1-Tg mice on high-fat diet had lower adipose tissue macrophages and enhanced expression of lipogenic enzymes coincident with reduction in circulating and hepatic lipids. Metabolomics data revealed a systemic metabolic signature of improved glucose handling, cellular redox, and NAD+ metabolism while label-free quantitative mass spectrometry in skeletal muscle uncovered a distinct diet- and genotype-dependent acetylation pattern of SIRT3 targets across the core of intermediary metabolism. Thus, under nutritional excess, NQO1 transgenesis preserves healthful benefits.