Alexander A. Soukas

Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans

Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span.

C. elegans Major Fats Are Stored in Vesicles Distinct from Lysosome-Related Organelles

Genetic conservation allows ancient features of fat storage endocrine pathways to be explored in C. elegans. Multiple studies have used Nile red or BODIPY-labeled fatty acids to identify regulators of fat mass. When mixed with their food, E. coli bacteria, Nile red, and BODIPY-labeled fatty acids stain multiple spherical cellular structures in the C. elegans major fat storage organ, the intestine. However, here we demonstrate that, in the conditions previously reported, the lysosome-related organelles stained by Nile red and BODIPY-labeled fatty acids are not the C. elegans major fat storage compartment. We show that the major fat stores are contained in a distinct cellular compartment that is not stained by Nile red. Using biochemical assays, we validate oil red O staining as a method to assess major fat stores in C. elegans, allowing for efficient and accurate genetic and functional genomic screens for genes that control fat accumulation at the organismal level.

β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolic genes in skeletal muscle and contributes to the response of muscle to exercise. Muscle PGC-1α transgenic expression and exercise both increase the expression of thermogenic genes within white adipose. How the PGC-1α-mediated response to exercise in muscle conveys signals to other tissues remains incompletely defined. We employed a metabolomic approach to examine metabolites secreted from myocytes with forced expression of PGC-1α, and identified β-aminoisobutyric acid (BAIBA) as a small molecule myokine. BAIBA increases the expression of brown adipocyte-specific genes in white adipocytes and β-oxidation in hepatocytes both in vitro and in vivo through a PPARα-mediated mechanism, induces a brown adipose-like phenotype in human pluripotent stem cells, and improves glucose homeostasis in mice. In humans, plasma BAIBA concentrations are increased with exercise and inversely associated with metabolic risk factors. BAIBA may thus contribute to exercise-induced protection from metabolic diseases.

Site and mechanism of leptin action in a rodent form of congenital lipodystrophy

Lipodystrophy is characterized by the complete or partial absence of adipose tissue, insulin resistance, hepatic steatosis, and leptin deficiency. Here, we show that low-dose central leptin corrects the insulin resistance and fatty liver of lipodystrophic aP2-nSREBP-1c mice, while the same dose given peripherally does not. Central leptin also repressed stearoyl-CoA desaturase-1 (SCD-1) RNA and enzymatic activity, which were increased in livers of lipodystrophic mice. aP2-nSREBP-1c mice homozygous for an SCD-1 deletion had markedly reduced hepatic steatosis, increased saturated fatty acids, decreased acetyl-CoA carboxylase activity, and decreased malonyl-CoA levels in the liver. Despite the reduction in hepatic steatosis, these mice remained diabetic. A leptin dose-response curve showed that subcutaneous leptin improved hyperglycemia and hyperinsulinemia in aP2-nSREBP-1c mice at doses that did not substantially alter hepatic steatosis or hepatic SCD enzymatic activity. Leptin treatment at this dose improved insulin-stimulated insulin receptor and insulin receptor substrate 2 (IRS-2) phosphorylation, IRS-2-associated PI3K activity, and Akt activity in liver. Together, these data suggest that CNS-mediated repression of SCD-1 contributes to leptins antisteatotic actions. Intracerebroventricular leptin improves glucose homeostasis by improving insulin signal transduction in liver, but in this case the effect appears to be independent of SCD-1.

Identification of Akt-Independent Regulation of Hepatic Lipogenesis by Mammalian Target of Rapamycin (mTOR) Complex 2

Background: mTORC2 is an insulin-stimulated kinase that activates kinases such as Akt. Results: Hepatic mTORC2 deletion increased glucose output dependent on Akt-FoxO and blocked lipogenesis that was not restored by activation of Akt-FoxO signaling. Conclusion: mTORC2-dependent factors other than Akt are critical for hepatic lipogenesis. Significance: Understanding signals separating hepatic glucose output from lipogenesis is crucial for effective diabetes treatment. Mammalian target of rapamycin complex 2 (mTORC2) is a key activator of protein kinases that act downstream of insulin and growth factor signaling. Here we report that mice lacking the essential mTORC2 component rictor in liver (LrictorKO) are unable to respond normally to insulin. In response to insulin, LrictorKO mice failed to inhibit hepatic glucose output. LrictorKO mice also fail to develop hepatic steatosis on a high fat diet and manifest half-normal serum cholesterol levels. This is accompanied by lower levels of expression of SREBP-1c and SREBP-2 and genes of fatty acid and cholesterol biosynthesis. LrictorKO mice had defects in insulin-stimulated Akt Ser-473 and Thr-308 phosphorylation, leading to decreased phosphorylation of Akt substrates FoxO, GSK-3β, PRAS40, AS160, and Tsc2. LrictorKO mice also manifest defects in insulin-activated mTORC1 activity, evidenced by decreased S6 kinase and Lipin1 phosphorylation. Glucose intolerance and insulin resistance of LrictorKO mice could be fully rescued by hepatic expression of activated Akt2 or dominant negative FoxO1. However, in the absence of mTORC2, forced Akt2 activation was unable to drive hepatic lipogenesis. Thus, we have identified an Akt-independent relay from mTORC2 to hepatic lipogenesis that separates the effects of insulin on glucose and lipid metabolism.

Induction of Leptin Receptor Expression in the Liver by Leptin and Food Deprivation

Leptin resistance is a common feature of obesity and the metabolic syndrome. However, the regulated expression of the leptin receptor (Ob-R) has not been studied in detail. Expression profiling of liver mRNA in leptin-treated wild-type mice revealed a marked increase in leptin receptor mRNA levels, which had not previously been described. This was confirmed by isoform-specific real-time PCR, which showed a >25-fold increase in the mRNAs encoding the short forms (Ob-Ra, Ob-Rc) and a >10-fold increase in the mRNA encoding the long (Ob-Rb) form of the leptin receptor in liver. In parallel, we also observed induction of plasma-soluble leptin receptor (SLR) protein by leptin administration, pair feeding, and short term food restriction. However, induction of SLR by leptin is abolished in mice with selective deletion of Ob-R from liver using Cre-LoxP technology. These data suggest that the liver is a major source of Ob-R mRNA expression under conditions of negative energy balance. Membrane-bound Ob-R is then shed into the circulation as SLR. Our study thus reveals an unexpected role of the liver in modulating total circulating leptin levels and possibly its biological activity.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Cellular program controlling the recovery of adipose tissue mass: An in vivo imaging approach

The cellular program responsible for the restoration of adipose tissue mass after weight loss is largely uncharacterized. Leptin mRNA levels are highly correlated with adipose tissue mass, and leptin expression can thus be used as a surrogate for changes in the amount of adipose tissue. To further study the responses of adipocytes to changes in weight, we created a transgenic mouse expressing the luciferase reporter gene under the control of leptin regulatory sequences, which allows noninvasive imaging of the leptin expression of mice in vivo. We used these animals to show that weight loss induced by fasting or leptin treatment results in the retention of lipid-depleted adipocytes in adipose depots. To further study the cellular response to weight regain after leptin treatment, a leptin withdrawal protocol was used to induce a state of acute leptin deficiency in wild type mice. Acute leptin deficiency led to the transient deposition of large amounts of glycogen within pre-existing, lipid-depleted adipocytes. This was followed by rapid reaccumulation of lipid. Transcriptional profiling revealed that this cellular response was associated with induction of mRNAs for the entire pathway of enzymes necessary to convert glucose into acetyl-CoA and glycerol, key substrates for the synthesis of triglycerides.

Omega-3 and -6 fatty acids allocate somatic and germline lipids to ensure fitness during nutrient and oxidative stress in Caenorhabditis elegans

Significance Food availability in nature changes continually over an organism’s lifetime. As such, animals must diligently assess resource availability and appropriately allocate reserves that have been stored during times of feast for reproduction, to abate evolutionary pressures during times of famine. Our findings functionally link the availability of somatic (survival-promoting) and germline (reproduction-promoting) lipids to SKN-1 responses to oxidative and nutrient stress. We have defined this physiological response at the molecular, genetic, and organismal levels and identified a specific signaling system for regulating this process within intact animals. These findings will inform not only laboratory-based studies, but also ecological studies that have long sought to functionally integrate oxidative stress responses (like the SKN-1 pathway) into life-history traits. Animals in nature are continually challenged by periods of feast and famine as resources inevitably fluctuate, and must allocate somatic reserves for reproduction to abate evolutionary pressures. We identify an age-dependent lipid homeostasis pathway in Caenorhabditis elegans that regulates the mobilization of lipids from the soma to the germline, which supports fecundity but at the cost of survival in nutrient-poor and oxidative stress environments. This trade-off is responsive to the levels of dietary carbohydrates and organismal oleic acid and is coupled to activation of the cytoprotective transcription factor SKN-1 in both laboratory-derived and natural isolates of C. elegans. The homeostatic balance of lipid stores between the somatic and germ cells is mediated by arachidonic acid (omega-6) and eicosapentaenoic acid (omega-3) precursors of eicosanoid signaling molecules. Our results describe a mechanism for resource reallocation within intact animals that influences reproductive fitness at the cost of somatic resilience.

A non-canonical role for the C. elegans dosage compensation complex in growth and metabolic regulation downstream of TOR complex 2

The target of rapamycin complex 2 (TORC2) pathway is evolutionarily conserved and regulates cellular energetics, growth and metabolism. Loss of function of the essential TORC2 subunit Rictor (RICT-1) in Caenorhabditis elegans results in slow developmental rate, reduced brood size, small body size, increased fat mass and truncated lifespan. We performed a rict-1 suppressor RNAi screen of genes encoding proteins that possess the phosphorylation sequence of the AGC family kinase SGK, a key downstream effector of TORC2. Only RNAi to dpy-21 suppressed rict-1 slow developmental rate. DPY-21 functions canonically in the ten-protein dosage compensation complex (DCC) to downregulate the expression of X-linked genes only in hermaphroditic worms. However, we find that dpy-21 functions outside of its canonical role, as RNAi to dpy-21 suppresses TORC2 mutant developmental delay in rict-1 males and hermaphrodites. RNAi to dpy-21 normalized brood size and fat storage phenotypes in rict-1 mutants, but failed to restore normal body size and normal lifespan. Further dissection of the DCC via RNAi revealed that other complex members phenocopy the dpy-21 suppression of rict-1, as did RNAi to the DCC effectors set-1 and set-4, which methylate histone 4 on lysine 20 (H4K20). TORC2/rict-1 animals show dysregulation of H4K20 mono- and tri-methyl silencing epigenetic marks, evidence of altered DCC, SET-1 and SET-4 activity. DPY-21 protein physically interacts with the protein kinase SGK-1, suggesting that TORC2 directly regulates the DCC. Together, the data suggest non-canonical, negative regulation of growth and reproduction by DPY-21 via DCC, SET-1 and SET-4 downstream of TORC2 in C. elegans.