C1q/TNF-related protein 3 (CTRP3) is a secreted hormone that modulates hepatic glucose and lipid metabolism. Its circulating levels are reduced in human and rodent models of obesity, a metabolic state accompanied by chronic low-grade inflammation. Recent studies have demonstrated an anti-inflammatory role for recombinant CTRP3 in attenuating LPS-induced systemic inflammation, and its deficiency markedly exacerbates inflammation in a mouse model of rheumatoid arthritis. We used genetic mouse models to explore the immunomodulatory function of CTRP3 in response to acute (LPS challenge) and chronic (high-fat diet) inflammatory stimuli. In a sublethal dose of LPS challenge, neither CTRP3 deficiency nor its overexpression in transgenic mice had an impact on IL-1β, IL-6, TNF-α, or MIP-2 induction at the serum protein or mRNA levels, contrary to previous findings based on recombinant CTRP3 administration. In a metabolic context, we measured 71 serum cytokine levels in wild-type and CTRP3 transgenic mice fed a high-fat diet or a matched control low-fat diet. On a low-fat diet, CTRP3 transgenic mice had elevated circulating levels of multiple chemokines (CCL11, CXCL9, CXCL10, CCL17, CX3CL1, CCL22 and sCD30). However, when obesity was induced with a high-fat diet, CTRP3 transgenic mice had lower circulating levels of IL-5, TNF-α, sVEGF2, and sVEGFR3, and a higher level of soluble gp130. Contingent upon the metabolic state, CTRP3 overexpression altered chemokine levels in lean mice, and attenuated systemic inflammation in the setting of obesity and insulin resistance. These results highlight a context-dependent immunomodulatory role for CTRP3.

Diabetes is the seventh leading cause of death in the United States, and nearly 34% of U.S. adults are prediabetic. CTRP3 is an adipose secreted protein that has shown to play a key role in glucose metabolism and insulin sensitivity, however, the research on CTRP3 total levels and its relationship to type 2 diabetes is controversial. The oligomeric state (protein structure) of CTRP3 in relation to metabolic dysfunction has not been studied. This study will be the first analysis of the circulating forms of CTRP3 in human blood. Hypothesis: The relative circulating amounts of the three oligomeric states of CTRP3 will differ in patients with type 2 diabetes. Methods: Human serum samples are analyzed using western blotting under native, reduced non-denaturing, and denaturing conditions. Results: In reducing non-denaturing conditions, three oligomeric states of CTRP3 were visualized in human serum: the high molecular weight (HMW) oligomer, the low molecular weight (LMW) oligomer, and the trimer. Conclusion: Reduced, non-denaturing conditions appear to yield the most effective separation of the three oligomeric states of CTRP3, and further studies aim to observe a difference in oligomeric state with a diabetic phenotype. Investigating the relationship of CTRP3’s oligomeric state with diabetic phenotype could present novel understanding of this protein’s possible protective effects against certain metabolic disorders.

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions.

INTRODUCTION: The liver is the primary organ responsible for the removal of toxic substances from the body by means of a variety of metabolic pathways. One class of proteins responsible for much of the body’s xenobiotic drug and alcohol metabolism is the Cytochrome P450 family of proteins. One protein, Cytochrome P450 Class E Subclass 2 (Cyp2E1), has an integral role in alcohol metabolism by the liver. Cyp2E1 becomes fully activated after an organism has consumed excessive amounts of alcohol excessive alcohol and works with aldehyde dehydrogenase (ALDH) to metabolize ethanol to acetaldehyde. Another metabolic protein, C1q TNF Related Protein 3 (CTRP3), has been shown to effectively prevent alcoholic fatty liver disease (AFLD), specifically with long-term alcohol-induced lipid accumulation. METHODS: In this experiment, 12-week old male mice were fed a Lieber-Decarli alcohol diet (5% ETOH by volume) for 6-weeks. The food intake and body weight of the mice was recorded each day. The mice in the experiment included both wild type and transgenic CTRP3 overexpressing mice. At the end of the 6-week period the mice were euthanized, and the liver was carefully removed, flash-frozen, and prepared for immunoblot analysis of the proteins. RESULTS: Cyp2E1 levels increased significantly in response to ethanol consumption. Cyp2E1 levels were further elevated in ethanol-fed CTRP3 transgenic overexpressing mice. Cyp2E1 levels in CTRP3 transgenic mice were nearly twice that of wild type ethanol-fed mice. CONCLUSIONS: The results of the experiment show a significant increase in Cyp2E1 in mice which overexpress CTRP3. This upregulation of Cyp2E1 with CTRP3 overexpression could explain the mechanism for reduced hepatic lipid accumulation in ethanol-fed CTRP3 transgenic mice.

Alcohol use can negatively impact financial, cognitive, and psychiatric aspects of human life. In the brain, alcohol can have many devastating effects. Alcohol is a well-known cytotoxic agent that can cause specific brain pathology in humans; however, the exact biological mechanisms are not well-elucidated. Animal models are invaluable tools to investigate potential novel treatments in a substance abuse model. Mice studies can be used to screen for negative outcomes prior to human trials. We hypothesize that the C1q tumor necrosis factor-related protein, CTRP3, overexpression in mice reduces neuroinflammation from ethanol consumption that has been coupled with a high fat diet when compared to control mice. The CTRP family of proteins are adipokines and CTRP3 specifically influences cell viability, metabolism, and peripheral inflammation levels. Antibody specific immunoblotting is used to probe protein expression changes in neuroinflammatory markers in mouse cerebellum brain tissue in an overexpression mouse model of CTRP3 when compared to high-fat ethanol exposed mice and baseline control mice. The two proteins examined are MAG and GFAP. Myelin associated glycoprotein, or MAG, is a protein expressed by oligodendrocytes that mediate axonal growth and myelin interactions with neurons in the brain. Oligodendrocytes are extremely sensitive to oxidative stress to which cognitive deficits in ethanol exposure is thought to be attributed. Glial fibrillary acidic protein, or GFAP, is a marker of astrocyte reactivity. Astrocytes are cells in the brain that are responsible for environmental stabilization and actively participate in neurotransmission. Currently, GFAP alterations in ethanol-exposed animals are dose and age dependent. We chose to use young adult mice where GFAP reactiveness is increased during chronic ethanol exposure. The proposed studies are essential in determining CTRP3’s relationship to detrimental neuroinflammatory effects of alcohol and high fat diet in mice. The data obtained from these studies will provide compelling evidence for future clinical trials to investigate CTRP3 as a therapeutic agent in people with a high fat diet that use alcohol chronically.

Adipose tissue secretes many important biologically active proteins called adipokines. A subset of adipokines, called C1q tumor necrosis factor (TNF) related proteins (CTRPs), play a key role in metabolism, inflammation, and cell signaling. C1q TNF Related Protein 3 (CTRP3) increases hepatic fatty acid oxidation, decreases inflammation, and aids in cardiovascular recovery following a myocardial infarction. However, the mechanisms behind CTRP3’s protective effects on organ systems are unknown. This exploratory study aims to analyze the circulating oligomeric state of CTRP3 and the circadian regulation of CTRP3 to help understand the role of CTRP3 in preventing disease. METHODS: For analysis of the oligomeric state of CTRP3 non-fasting mouse serum was collected from high fat fed hyper-glycemic mice or low fat fed normoglycemic mice and was separated by size exclusion filtration. For analysis of the circadian regulation of CTRP3 serum samples were collected from mice at 4 different time points (2 dark cycle and 2 light cycle) throughout the day and circulating CTRP3 levels were analyzed by immunoblot analysis. RESULTS: In both high fat and low fat fed mice CTRP3 was found to circulate in both >300 kDa oligomers and >100kDa oligomers, with no detectable amount of CTRP3 less 100 kDa. Interestingly, although there was no difference in the total amount of CTRP3 between the high fat and low fat fed mice there was a higher abundance of CTRP3 >300 kDa in the high fat fed and a greater abundance of CTRP3 found 100-300 kDa. Additionally, we found that serum CTRP3 levels vary greatly throughout a 24-hour time-period within each mouse, but no consensus circadian pattern was observed. CONCLUSION: In vitro mammalian produced recombinant CTRP3 protein was found to exist as trimer, hexamer, and high molecule weight. This is the first study to indicate that CTRP3 circulates in different oligomeric states in vivo, and this is also the first study to observe a difference in the oligomeric state of CTRP3 related to metabolic state. Combined these findings indicate that oligomeric state of CTRP3 may be more metabolically relevant than total amount of circulating CTRP3. In addition, our finding of a high variability of CTRP3 within the same mouse at different times throughout the day indicates that is not regulated by circadian rhythms but is susceptible to variability due to some unknown regulatory factor. These findings have identified novel unknown aspects of CTRP3, which require further research to understand the role of CTRP3 in human health and disease.

The primary function of the testis is twofold: 1, it is responsible for production of testosterone and 2, it is responsible for spermatogenesis. Previous studies in alcohol fed mice have shown that chronic alcohol consumption causes reduced sperm counts and testicular lesions. CTRP3 is a novel adipokine which has been shown to promote follicular proliferation and reduce apoptosis in granulosa cells in the ovaries. Both folliculogenesis and spermatogenesis occur via the process of meiosis and therefore have some similarities. Since CTRP3 has been shown to be involved in folliculogenesis it would be reasonable to assume that it will play a role in spermatogenesis. CTRP3 has been shown to have protective properties in some organs in alcohol fed mice. This study was designed to determine if CTRP3 conveyed protective properties to the testicular tissue in chronic alcohol fed mice by comparing testicular morphology across 4 treatment groups: wild-type control mice, wild-type mice on a high alcohol diet, CTRP3 over expressing mice, and CTRP3 over expressing alcohol fed mice. To date this study indicates that alcohol did decrease germ cells due to apoptosis in the wild-type mice. Our study indicates that apoptosis of germ cells increased the intercellular space in seminiferous tubules and separated spermatogenic cells in the wild type mice. The CTRP3 mice do not show as aggressive results, indicating that CTRP3 may be playing a protective role. At this time, only a small number of tissues from the study have been analyzed so these results should be considered to be preliminary.

Effect of Sepsis on Circulating CTRP3 Levels Adam Musick, Madison Shipley, Fei Tu, Chuanfu Li, Valentin Yakubenko, and Jonathan M. Peterson. Adipose tissue is an active endocrine organ which secrets several pro- and anti- inflammatory mediators, collectively called adipokines. Our previous work has identified a novel anti-inflammatory adipokine called C1q TNF Related Protein 3 (CTRP3). Others have previously demonstrated that localized overexpression of CTRP3 protects myocardial tissue from lipopolysaccharide (LPS)-induced sepsis, further supporting the potential protective role of CTRP3. However, endogenous regulation of CTRP3 in response to a sepsis event has not been explored. Further, other adipokines have been identified as diagnostic/prognostic biomarkers for critically ill patients. Therefore, the purpose of this project was to determine the effects of a sepsis event on the circulating CTRP3 levels. METHODS: Gonadal adipose tissue and serum were collected 8 hours after induction of the cecal-puncture and ligation (CLP) model of sepsis or sham control mice. The circulating levels of CTRP3 were quantified by immunoblot analysis. The transcription levels of CTRP3 in adipose tissue were measured by Real-Time PCR. In addition, to explore a potential mechanism for a protective role of CTRP3, thioglycollate-induced peritoneal macrophages were isolated and binding of recombinant CTRP3 was determined by imaging flow cytometry. RESULTS: Circulating CTRP3 levels decreased by approximately 90% compared to sham mice. However, adipose tissue transcription levels of CTRP3 was not difference between CLP and sham mice. Further, imaging flow cytometry demonstrated that CTRP3 binds directly to isolated macrophages. CONCLUSION: The overserved reduction in circulating CTRP3 protein levels and the absence of changes to the CTRP3 transcription, indicate that during sepsis CTRP3 is actively removed from the blood. As CTRP3 binds directly to macrophages and has been previously shown to attenuate LPS-induced macrophage activation these data could indicate that under normal conditions CTRP3 combines with active macrophages to help suppress cytokine overexpression. However, it appears that during sepsis the endogenous CTRP3 levels are quickly depleted. Combine these data support future research to determine if circulating CTRP3 levels are a biomarker indicative of sepsis prognosis and to determine if increasing the circulating levels of CTRP3 could reduce the cytokine storm associated to a sepsis event. Further, as we have demonstrated CTRP3 binds directly to macrophages, future studies are also needed to explore the potential anti-inflammatory mechanism of CTRP3 action on macrophages.

Alcoholic fatty liver disease (AFLD) is a significant public health concern. Excessive alcohol (ethanol) consumption causes liver cell damage and death, which results in eventual failure of the liver and death. AFLD is the number one cause of liver-related mortality in the United States. Our lab works with the novel protein C1q TNF Related Protein 3 (CTRP3), which inhibits non-alcoholic fatty liver disease, however the effects on AFLD are unknown. Therefore, the purpose of this experiment is to determine if CTRP3 prevents ethanol-induced liver cell death. The H4IIE rat hepatoma cell line was chosen for experimentation as a cell culture model of liver tissue. To determine a suitable alcohol level H4IIE cells were treated with 50, 100, and 200 mmol of ethanol for 18-24 hours. Trypan Blue was used to identify the dead/damaged cells, as only dead/damaged cells will be stained blue with this protocol. We observed that 100 mmol of ethanol consistently induced ~10% mortality rate in these cells. Next, we tested the ability of CTRP3 to reduce ethanol-induced mortality. We added purified CTRP3 protein to the cell media along with the 100 mmol ethanol treatment. The addition of CTRP3 reduced the amount of alcohol-induced cell death/damage in the H4IIE cell line by approximately 60%. Our next goal was to determine how CTRP3 reduces ethanol-induced death. The Akt signaling pathway is a well-known inhibitor of cell death. Therefore, to determine if CTRP3 attenuated ethanol-induced cell damage/death through activation of the Akt signaling pathway, another set of cells was treated with 100 mmol of ethanol and CTRP3 (with or without insulin). Western blots were used to compare the amount of active Akt (phosphorylated) in the CTRP3-treated and non-treated cells. A Western blot utilizes an electric current to separate denatured protein samples on a SDS-page gel, separating the proteins based on size. The smaller the protein the faster it migrates across the gel. The proteins are then transferred to a membrane for analysis, through exposure to commercial antigens and chemiluminescence imaging. There was no change in the amount of total or active Akt between the samples treated with or without CTRP3. We conclude that CTRP3 protects liver cells from ethanol-induced damage/death, but not through activation of the Akt pathway.

Abstract available in The FASEB Journal.