Obesity affects about 34% of adults in the United Sates, and 25% of these individuals will become insulin resistance. Studies have provided evidence to suggest that subcutaneous adipose tissue is protective against insulin resistance and type 2 diabetes. Anti-diabetic drugs, thiazolidinediones (TZDs), improve insulin sensitivity by redistributing fat storage into the subcutaneous fat depots and away from visceral fat depots. Unlike subcutaneous fat, visceral fat is highly lipolytic, releasing elevated concentrations of free fatty acids (FFAs) into circulation. Elevated free fatty acids are a common phenotype of insulin resistance, causing lipotoxicity in tissues and impairing insulin signaling in insulin-responsive cells. For example, hepatic lipotoxicity inhibits insulin's ability to suppress gluconeogenesis, in addition to acetyl-CoA entering into the gluconeogenic pathway. Thus, it is important to prevent the intrusion of lipids into tissues and preserve insulin action. However, not all obese individuals become insulin resistant.

It has been reported that a significant portion of obese individuals are metabolically healthy. The purpose of this dissertation was to investigate potential mechanisms that protect some obese individuals from becoming insulin resistant. Using ²H₂O in BMI-matched obese insulin sensitive (IS) and insulin resistant (IR) individuals, we quantified subcutaneous adipose lipid kinetics, cellular proliferation and the synthesis of proteins involved in metabolic pathways, all of which may play an imperative role in the etiology of insulin resistance.

We hypothesized that dysfunctional subcutaneous adipose triglyceride storage is an initiating factor in the development of systemic insulin resistance in human obesity. We tested this hypothesis using ²H₂O to quantify, in vivo, triglyceride (TG) synthesis, de novo lipogenesis (DNL), and adipocyte proliferation in subcutaneous adipose cells of insulin-sensitive (IS) and insulin-resistant (IR) subjects. Adipose cell size distribution was quantified using Beckman Multisizer III. Plasma free fatty acid (FFA) concentrations were measured under fasting and postprandial insulin concentrations during a steady state plasma glucose (SSPG) test as a measurement of insulin suppression of lipolysis. Total TG synthesis and DNL were significantly lower in adipose cells of IR compared to IS subjects, and correlated inversely with the proportion of small adipocytes. FFA concentrations were significantly greater in IR versus IS during fasting and postprandial insulin concentrations. Adipocyte proliferation did not differ significantly between groups. These results strongly support the hypothesis that decreased capacity for lipid storage in subcutaneous adipose tissue is a defining feature of obesity-associated insulin resistance and may contribute to insulin resistance in peripheral tissues due to ectopic fat deposition.

Following our findings in a predominately Caucasian cohort, we sought to determine if adipose dysfunction is predictive of insulin resistance amongst different ethnicities. In the United States, African-Americans (AA) have 51% higher rates of obesity than Caucasians (C), and the highest rates of type 2 diabetes and cardiovascular disease Obesity is associated with insulin resistance, yet the physiological mechanisms of this association and fat regional distribution as a potential contributor is not yet completely understood, particularly in minority populations most affected by this disease. Increased visceral adipose tissue (VAT) mass a significant risk factor for insulin-resistance, but recent studies have discovered that this phenotype may not be a global risk factor. VAT tends to be consistently lower in AA compared to C, yet individuals in this group are more insulin resistant for a given degree of body fat. Thus, we aimed to further investigate differences in adipose lipid synthesis between African-Americans and Caucasians. We administered ²H₂O to five African-Americans and seven Caucasians for the quantification of TG synthesis, DNL and adipocyte proliferation

Using ²H₂O, we aimed to apply dynamic proteomics to quantify protein turnover rates of proteins from isolated subcutaneous mature adipocytes. Dynamic proteomics is the large-scale measurement of protein expression and turnover in a single specimen, biological tissue or bodily fluid. Dynamic proteomics applied to humans may allow us to discover the adipoycte proteome of insulin resistance. We investigated the adipocyte proteome of 3 IS and 3 IR, equally obese subjects at steady state after 4 weeks of 2H2O consumption. Peptides were submitted through LC/MS-MS analysis to separate and identify tryspinized peptides by size and charge, followed by Spectrum Mill analysis to organize peptides based on their corresponding protein. A total of 620 proteins were identified amongst the six subjects, but after filtering for criteria (1

Proteins are the ultimate focal point for organismal composition and the basis for many biological functions that occur within. It is well known that proteins in a biological system are not static; there is flux through synthesis and degradation pathways, resulting in a dynamic steady-state that underlies both stable and adaptive levels of proteins. For many biological functions to occur, the rate of either protein synthesis or degradation deviates from normal to allow for changes to occur .occur. Accordingly, when protein pool size adjusts to new levels a kinetic signature in the form of a fraction of newly synthesized proteins may be present at each stage. In the Hellerstein Lab, we utilize an innovative methodology and approach to measure this signature directly in- vivo with stable isotope labeling.The approach used in this study to measure protein dynamics in- vivo involves administering deuterium (2H) from deuterated “heavy” water (2H2O) to research subjects or experimental animals. During the labeling period, newly synthesized proteins will incorporate the available 2H into amino acids in newly synthesized protein molecules, resulting in “heavy” proteins. These are functionally identical to their non- 2H-incorporated “light” counterparts, and only differ in subtle alterations in the pattern and amount of deuterium atoms in the constituent amino acids of which a peptide chain is composed. Protein mass isotopomers are then quantified through analysis by high performance high-performance liquidliquid chromatography (HPLC) coupled single (MS) and tandem (MS/MS) mass spectrometry and protein flux rates are calculated by use of Mass Isotopomer Distribution Analysis (MIDA). With this approach, we investigated here non-invasive biomarkers of Duchenne muscular dystrophy (DMD) as well as the unique stages of muscle tissue regeneration and effects on tissues distant from acute injury. DMD is the most severe form of muscular dystrophy, with debilitating symptoms that cause patients to succumb to their complications early in life. Currently, there are no existing a few approved treatments for DMD, but these therapies mainly treat the symptoms of the disease and have shown mild improvements in a clinical population so far. and approved therapies have shown little promise in a clinical population. DMD is one of the most studied genetic diseases, as there is a pressing demand for proper treatment or therapy for this indication. Here, we have discovered and validated a clinical biomarker of whole muscle protein synthesis, which is a key marker of skeletal muscle health in DMD, from non-invasive methods. This marker was first discovered in a clinical population, but then reverse translated to an animal model of DMD to validate that it is a faithful marker of whole muscle protein synthesis rates. Our data clearly demonstrate that certain muscle- specific proteins can escape into circulation from the dystrophic muscle and be captured from urinary excretion to understand changes in whole musclewhole-muscle protein flux rates. These findings may provide crucial information to clinicians and researchers alike about a key metric that can determine disease progression or response to treatment/therapy. Both skeletal and smooth muscle tissue have the capacity to regenerate after injury or damage to help maintain optimal functional properties. This regenerative capacity is an important process, and its dysregulation is implicated in many diseases, such as muscular dystrophy and pathological atrophy. Muscle regeneration is a widely studied topic, but no one has utilized the method of in- vivo stable isotope labeling to categorize the global, ontology-groupedgrouped, and individual flux rates of proteins at each sequential unique stage of regeneration. In the work presented here, we examine how protein flux rates change over time as skeletal muscle tissue regenerates from an acute injury. Also, we explore the relationship between protein flux measurements and abundance-based gene expressions to understand their correlation. There is evidence to support the notion that other muscle tissue that is not directly injured will respond to systemic circulating factors from acute tissue injury and enter an altered functional state. Therefore, we also explore the effect that acute local injury has on distant muscle tissue that is undamaged. Our data show clear changes in global, ontology-grouped, and individual protein flux changes at each unique stage of muscle regeneration, and that there is no correlation between measured protein flux changes in regenerating muscle and gene expression of the same proteins. Interestingly, changes are further demonstrated in global, ontology-grouped, and individual protein fluxes in muscle that are distant to local acute injury, which don’t do not reflect the same tissue gene expression rates. This data provides further evidence that other muscle tissue that is not directly injured will respond to systemic circulating factors from acute tissue injury. Therefore, setting up future experiments to deepen our understanding of the systemic communication network that our body utilizes during injury or regenerative events.

The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases, including non-alcoholic fatty liver disease. Here, we first characterize the UPRER induced metabolic changes in mouse liver through in vivo metabolic labeling and mass spectrometric analysis of proteome and lipid fluxes. We induced ER stress in vivo via tunicamycin treatment and measured rates of proteome-wide protein synthesis, de novo lipogenesis and cholesterol synthesis serially over a three-day period, thereby generating a metabolic “signature” of the UPRER over time. Synthesis of most proteins was suppressed under ER stress conditions, including proteins involved in lipogenesis, consistent with reduced de novo lipogenesis at 48 and 72 hours. The reduction in de novo lipogenesis was explicable by reduced food intake, shown in pair-feeding studies. Despite the lower de novo lipogenesis rates, electron microscopy revealed striking morphological changes to ER and H&E staining showed lipid droplet enriched livers under ER stress. Pre-labeling of adipose tissue prior to ER stress induction revealed mobilization of lipids from adipose to the liver. Interestingly, the source of these lipids in liver was uptake of free fatty acids, not whole triglycerides or phospholipids from lipoproteins, as demonstrated by replacement of the triglyceride-glycerol moiety in liver concurrently with increased incorporation of labeled palmitate from adipose tissue. We also induced ER stress by a high-fat diet and observed similar metabolic flux signatures, suggesting that this mechanism may play a role in the progression of fatty liver disease. Using these same stable isotope labeling approaches, several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared to young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was only seen in the triglyceride fraction. These data indicate that aged mice have an exaggerated response to ER stress, which may indicate less effective mechanisms of protein clearance after inducing the UPRER. These flux-based approaches provide a powerful tool to identify novel regulators of ER stress and potential targets for pharmacological intervention. We applied the same stable isotope labeling techniques to generate region-specific metabolic flux signatures of mouse brains. Assessing how metabolic flux signatures of the brain are perturbed through different interventions, including anti-aging therapeutics and preventative lifestyle interventions, could provide a powerful tool to assess cell-specific and region-specific changes with age, and provide a kinetic metric to assess intervention effectiveness.

My broad goals are to use new stable isotopic metabolic labeling techniques with tandem mass spectrometric analysis to measure in both preclinical models and human subjects, the synthesis and breakdown rates of low abundance proteins that play key roles in common metabolic disorders such as heart disease, diabetes, non-alcoholic fatty liver disease and counterregulatory hormones on muscle protein metabolism. First, I developed a mass spectrometry method to measure the turnover rates of the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). We revealed that lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. Secondly, I developed a non-invasive technique using 2H2O labeling and mass spectrometry for measuring the synthesis of pancreatic beta cell insulin in circulation as a tool to monitor the progression of diabetes in human beings. The significance of this work is that a) we have revealed how long pancreatic beta cell insulin lives for, and b) the effect of prediabetes on insulin biogenesis. Our third objective was to develop a method to determine the kinetics of PNPLA3, Patatin-like phospholipase domain-containing protein 3. This gene single nucleotide polymorphisms confers susceptibility for non-alcoholic fatty liver disease (NAFLD) by accumulating PNPLA3 proteins on lipid droplets. To understand the kinetic basis of this genetic polymorphisms associated with NAFLD, we would like to determine how PNPLA3 turnover rates are altered in the liver in NAFLD patients with different PNPLA3 genetic backgrounds. Our fourth objective investigated how the treatment with acetyl-CoA carboxylase inhibitors (ACCi) increases plasma triglyceride (TG) concentrations in patients with non-alcoholic steatohepatitis (NASH), with variable results reported for concentrations of plasma apolipoprotein B (ApoB). We determined the effects of treatment with the ACCi, firsocostat in NASH on production and clearance rates of plasma LDL ApoB-containing particles. For the last part of this dissertation, I used stable isotopic labeling with 2H2O combined with mass spectrometry to measure the turnover rates of proteins across the proteome in response to glucocorticoid treatment and/or in a Pik3r1 (a glucocorticoid response gene) knockout mouse model. We revealed a suppressive effect on lowering protein turnover rates in muscle of Pik3r1 knockout mice when given dexamethasone. This dynamic proteomic study revealed how Pik3r1 regulates skeletal muscle protein metabolism. Collectively, these studies are anticipated to shed insight into the metabolic physiology of common metabolic diseases such as atherosclerotic cardiovascular disease (ASCVD), type 2 diabetes mellitus (T2D), non-alcoholic fatty liver disease, and muscle protein metabolism.

Glucocorticoids are widely prescribed to treat autoimmune and inflammatory diseases. While they are extremely potent, their utility in clinical practice is limited by a variety of adverse effects, including osteoporosis, skin thinning and increased susceptibility to bruising, muscle wasting, fat redistribution, cognitive impairments and mood disorders, and insulin resistance and diabetes. Development of compounds that retain the potent immunomodulating and anti-inflammatory properties of classic glucocorticoids while exhibiting reduced adverse actions is therefore a priority. Much attention has been focused on developing selective glucocorticoid receptor modulators (SGRMs) that would dissociate adverse effects from therapeutic immunomodulatory and anti-inflammatory effects.

Here we present several approaches to the field of SGRM development: characterization of a novel putative SGRM (compound L5); characterization of the effects of a glucocorticoid receptor mutant mouse (the GRdim/dim mouse); and comparison of acute vs. chronic effects of glucocorticoids using a model of chronic glucocorticoid excess (the CRH-transgenic mouse). The phenotypic actions of glucocorticoids were measured in these mice by a heavy water labeling strategy combined with mass spectrometry that allows measurement of fluxes through multiple target metabolic pathways concurrently. Pathways monitored included collagen synthesis in bone and skin, protein synthesis in skeletal muscle, triglyceride dynamics in adipose tissue and liver, and proliferation of cells in spleen, pancreatic islets, and hippocampal neuronal precursors. To quantify accumulation or distribution of bone mass, lean tissue mass, and fat mass, we used DEXA and microCT. To quantify changes in glucose homeostasis, we used the deuterated glucose disposal test, a modified oral glucose tolerance test that also reveals glucose uptake and metabolism by peripheral tissues in addition to the standard measurements of glucose and insulin concentrations.

We demonstrate for the first time that L5, a compound belonging to a novel class of potential SGRMs, exhibits clearly selective actions on disease-relevant pathways compared to prednisolone. Prednisolone reduced bone collagen synthesis, skin collagen synthesis, muscle protein synthesis, and splenic lymphocyte counts, proliferation, and cell death, whereas L5 had none of these actions. In contrast, L5 was a more rapid and potent inhibitor of hippocampal neurogenesis than was prednisolone; and L5 and prednisolone induced insulin resistance equally.

We also demonstrate that, contrary to expectations, some metabolic effects of glucocorticoid treatment are exaggerated in adipose tissue of GRdim/dim mice compared to wildtype mice. The GRdim/dim mouse has a mutation in the dimerization domain of GR, and has been shown to have attenuated transactivation with intact repression. The predominant, current hypothesis is that GRdim/dim mice should experience fewer or less severe effects of glucocorticoids, because the GRdim/dim mutation is a loss of function mutation, from a molecular standpoint. Thus, it would be surprising, a priori, if GRdim/dim mice were to show any exaggerated responses to glucocorticoids. Our labeling studies demonstrate that adipose tissue triglyceride synthesis and de novo lipogenesis exhibit greater stimulation by glucocorticoid treatment in GRdim/dim mice than in wildtype controls. More concordant with the predominant hypothesis were our findings that glucocorticoid-dependent inhibition of bone and skin collagen synthesis and decreases in insulin sensitivity were diminished in GRdim/dim mice compared to wildtype mice. Wildtype and GRdim/dim mice were equally sensitive to glucocorticoid-dependent decreases in muscle protein synthesis. Thus the consequences of mutation in the dimerization domain of the glucocorticoid receptor vary on a pathway-by-pathway basis, and are not restricted to reduced responses to glucocorticoids.

Finally, we demonstrated that increased futile cycling between triglycerides and free fatty acids occurs in both abdominal and subcutaneous fat depots in a model of chronic glucocorticoid exposure, CRH-transgenic (CRH-Tg+) mice, and in these mice triglyceride accumulation is favored over net lipolysis. In these studies, we characterized multiple pathways in mice exposed to both chronically and acutely-elevated levels of glucocorticoids (CRH-Tg+ mice and wildtype mice administered glucocorticoids, respectively) to explore how glucocorticoids act on these pathways over time. In bone and skin, fractional collagen synthesis rates were dramatically inhibited by acute glucocorticoid exposure, but in CRH-Tg+ mice, which had reduced bone mass and thinner skin compared to controls, fractional collagen synthesis rates were only modestly lower in bone and are similar in skin compared to controls. In muscle, fractional synthesis of total muscle protein was not dramatically inhibited by either acute or chronic exposure to glucocorticoids, although CRH-Tg+ mice had measurably less muscle mass relative to controls. In adipose tissue there were no remarkable changes in triglyceride dynamics in young mice given glucocorticoids acutely, but in CRH-Tg+ mice we observed more triglyceride synthesis in subcutaneous and abdominal fat depots, more de novo lipogenesis in the abdominal depot only, more triglyceride accumulation in both fat depots and no difference in glyceroneogenesis in either depot compared to controls. Fat accumulation had occurred much more slowly than measured triglyceride synthesis in both depots in CRH-Tg+ mice, thus while gradual accumulation, rather than reduction, of fat is favored, both triglyceride synthesis and lipolysis occurs simultaneously at a high rate. We conclude that futile cyling between triglycerides and free fatty acids occurs at a high rate, in response to chronic, but not acute, glucocorticoid exposure, and that chronic, endogenous glucocorticoid exposure has different metabolic consequences than acute, exogenous administration.

We draw several conclusions relevant to novel glucocorticoid agents. First, ligands for the glucocorticoid receptor can exhibit phenotypic selectivity. Second, although L5 may not be a therapeutically attractive candidate, it is a SGRM, and further testing on other compounds of the same class is warranted. Third, dose response ranges vary depending on the ligand, the pathway measured, and the tissue in which it is measured, so a variety of pathways need to be measured in different tissues to accurately establish therapeutic index. Fourth, while the expectation is that SGRMs will activate only a subset of glucocorticoid receptor-dependent pathways, it is important to assess the tissue-specific effects of all potential SGRMs on global transcription and GC-dependent metabolic pathways in many different tissues to accurately establish therapeutic indices of compounds. Fifth, targeting the glucocorticoid receptor dimerization domain pharmacologically is not likely to result in simple diminution of glucocorticoid actions due to attenuated transactivation with intact repression, but may result in stimulation of certain processes. Sixth, there are several advantages of this heavy water labeling method combined with mass spectrometry, in particular that small amounts of drug, subjects, and tissues are required relative to the large amount of information gained. Finally, this method can be adapted to characterize ligands for the glucocorticoid receptor and other nuclear hormone receptors, can be applied to disease models and transgenic mice, and can be integrated into existing clinical studies.

Calorie restriction (CR), reducing caloric intake without malnutrition, increases lifespan and delays the onset of age-related diseases. Characterizing the underlying mechanisms that mediate the effects of calorie restriction on aging and lifespan will provide insight into the fundamental biology of aging, as well as guide research into the development of therapeutics for age-related diseases. It seems likely that some combination of physiologic, metabolic and molecular adaptations to CR lead to cellular responses that in-turn increase the longevity of the organism. Thus the goal of this thesis work was to combine a kinetic biomarker strategy with classic physiologic and molecular techniques to determine the role of physiologic adaptations, fat metabolism and molecular signaling on biomarkers of CR-induced longevity in mice. The data presented here demonstrate that CR leads to significant reductions in cell proliferation rates in keratinocytes, liver cells, mammary epithelial cells and splenic T-cells. These reductions in cell proliferation rates cannot be accounted for by reductions in food intake, energy expenditure, fat mass or body weight. In addition, the CR-induced reduction in cell proliferation is not dependent on Sirt1 expression, nor can it be mimicked by resveratrol treatment. However, reductions in cell proliferation rates were associated with a CR-induced increase in whole body fatty acid oxidation and have a strong negative correlation with circulating IGF-1 levels. Taken together these results suggest that increased reliance on fatty acid oxidation and reductions in IGF-1 signaling may be metabolic pathways that mediate the effects of CR on aging and longevity. These results also point to molecular mediators that can translate changes in substrate utilization to regulation of growth factor signaling as potential regulatory nodes necessary for the CR-induced effects on cell proliferation and longevity.

Cellular energy homeostasis contributes to normal cell growth, functioning as a biological checkpoint of life. There have been numerous genes and metabolic pathways known to regulate the energy status of the cell, and how they contribute to cellular maintenance is of great interest; Caloric Restriction (CR) takes advantage of these metabolic pathways, retarding the aging process by slowing down the metabolic rate. However, despite the many studies on cellular energy homeostasis, much work still needs to be done.

Many of the applications of CR in various organisms have established life-extending benefits by regulating age-related diseases through convergent mechanisms. In fact, a number of signaling pathways, as well as master regulator proteins, act through these mechanisms to interact and regulate protein expressions. CR, as well as other lifespan extending interventions such as rapamycin treatment and inhibition of insulin growth factors, are subject to translational regulation. Increased caloric intake and obesity related comorbidities, in turn, can also work through these pathways to induce translation and cause disease, indicating the role of protein synthesis in health and disease is of great importance. Stable isotope-labeling proteomics are a powerful strategy that enable the assessment of proteome-wide dynamic fluxes in energy regulating biological interventions such as CR and mimetics. Using mass spectrometric (MS) strategies has afforded scientists a never before seen understanding of how proteome dynamics stand at the center of phenotype, physiologic adaptation, and disease pathogenesis. Through this approach, researches have been able to measure protein synthesis and turnover rates, both for targeted proteins an unbiased screening.

In this dissertation, I present a thorough review of our current understanding of metabolic pathways in the context of lifespan extension interventions like CR. I also discuss the underlying principles for measuring protein dynamics, focusing on metabolic labeling with 2H2O (heavy water) combined with tandem MS analysis of mass isotopomer abundances. Next, I demonstrate its application in four separate studies. First, I use 2H2O labeling in CR mice in a time-course to identify whether the protein turnover rate slowdown occurs immediately, gradually, or within a discreate time period. Next, I explore separately whether two potential CR mimetics, exercise and metformin administration, are able to slow down the fractional synthesis rate of proteins in a manner comparable to CR. Finally, I discuss a potential CR modulator in Nitric Oxide, a short-lived bioactive molecule known to be induced under CR conditions and reduced during aging and disease. Overall, the work reported in this dissertation demonstrates how global proteome dynamics require NO for proper regulation, CR mimetics influence the proteome in similar mode, aerobic exercise has very different effects and CR effects on proteome fluxes are activated within a narrow and discrete time period.

Proteins are the ultimate laborers in biological systems. The behavior of a cell is governed by the dynamic interactions between thousands of proteins; a tissue is governed by the interactions of thousands of cells; and an organism is governed by the interaction of multiple tissues. The maintenance of protein homeostasis (proteostasis) is therefore a critical component of proper cellular function and the response to constantly changing environmental signals.

The dynamic proteomic approach used in these rodent studies involves the administration of 2H2O in the drinking water to achieve a target body water enrichment of 5%. During the labeling period, in vivo protein synthesis occurs, resulting in the creation of "heavy" proteins identical to their "light" counterparts in function, differing only in the incorporation of deuterium atoms at specific positions in the constituent amino acids from which they are comprised. These peptides are then resolved by high-affinity liquid chromatography mass spectrometry (LC-MS), resulting in the identification of hundreds to thousands of proteins from a single tissue sample. Using this experimental approach, we investigated the effect of calorie restriction (CR), insulin resistance, and diabetes on dynamic protein synthesis.

To date, CR has taken center stage as the most effective intervention in lifespan extension. Despite this, the biological mechanisms underlying increased health and longevity have yet to be fully described. While a growing body of evidence suggests that CR promotes significant gain-of-function attributes in mitochondria, a verdict on whether CR promotes mitochondrial biogenesis has not been established. In order to elucidate the controversy in the field regarding the response of mitochondria to reduced energy intake, we designed an experiment to study the effect of long-term CR on hepatic protein turnover. Our data provide conclusive evidence that mitochondrial protein turnover, concentration, and overall flux are reduced in response to CR, and may play central a role in mediating the health and longevity benefits of reduced energy intake.

Insulin resistance and islet cell failure are the two fundamental processes underlying type 2 diabetes. Alterations in mitochondrial protein turnover have been implicated in the pathology of type 2 diabetes, however the specific effect of insulin resistance and diabetes on the intracellular dynamic islet proteome have yet to be described. We investigated the effects of insulin resistance and diabetes on the synthesis of proteins from isolated rat islets for the first time, using both 2H2O (heavy water) labeling and SILAM quantitative proteomics. Using this approach, we measured fractional and absolute synthesis rates of cytoskeletal, glycolytic, mitochondrial, ER, and ribosomal proteins, the principal pathways responsible for glucose stimulated insulin secretion (GSIS). We found that insulin resistance increased the fractional synthesis rates (FSR) of 97% of all measured islet proteins, and the subsequent transition to diabetes resulted in the selective impairment of ribosomal protein synthesis. Our findings suggest that the rapid rate of islet cell proliferation due to insulin resistance is accompanied by increased fractional and absolute synthesis of critical GSIS proteins, and that the failure of islets results mainly in impaired ribosomal pathway flux, independent of alterations in mitochondrial metabolism. Our data suggest that the rapid rate of islet cell proliferation due to insulin resistance is accompanied by increased fractional and absolute synthesis of critical GSIS components, and that the failure of islet cells in diabetes results mainly in impaired ribosomal pathway flux, independent of alterations in mitochondrial protein metabolism.

Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor alpha (PPAR alpha) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR alpha activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling.

PPAR alpha is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR alpha ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR alpha similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR alpha activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR alpha. Consistent with a role for vascular lipases in the activation of PPAR alpha, administration of a lipase inhibitor (p-407) prevented PPAR alpha dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR alpha ligands. Our results support a role for the local generation of PPAR alpha ligands by lipase activity.

The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins.

Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists may be of therapeutic value.

Overall, these findings shed light on the endogenous mechanisms of PPAR alpha activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease.