The NAD⁺-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD⁺ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD⁺ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD⁺ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

Background: Obesity is associated with an increased risk of insulin resistance and type 2 diabetes mellitus (T2DM). However, some obese individuals maintain their insulin sensitivity and exhibit a lower risk of associated comorbidities. The underlying metabolic pathways differentiating obese insulin sensitive (OIS) and obese insulin resistant (OIR) individuals remain unclear. Methods: In this study, 107 subjects underwent untargeted metabolomics of serum samples using the Metabolon platform. Thirty-two subjects were lean controls whilst 75 subjects were obese including 20 OIS, 41 OIR, and 14 T2DM individuals. Results: Our results showed that phospholipid metabolites including choline, glycerophosphoethanolamine and glycerophosphorylcholine were significantly altered from OIS when compared with OIR and T2DM individuals. Furthermore, our data confirmed changes in metabolic markers of liver disease, vascular disease and T2DM, such as 3-hydroxymyristate, dimethylarginine and 1,5-anhydroglucitol, respectively. Conclusion: This pilot data has identified phospholipid metabolites as potential novel biomarkers of obesity-associated insulin sensitivity and confirmed the association of known metabolites with increased risk of obesity-associated insulin resistance, with possible diagnostic and therapeutic applications. Further studies are warranted to confirm these associations in prospective cohorts and to investigate their functionality.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin-resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3-L1 preadipocytes. By employing transcriptomics analyses, we discovered up-regulation of key pro-inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate-Adenosine Monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) to drive the expression of interferon β (IFNβ), C-X-C motif chemokine ligand 10 (CXCL10), and interferon signaling-related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS-STING signaling—it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway.

Exercise-associated immune response plays a crucial role in the aging process. The aim of this study is to investigate the effect of sport intensity on cytokine levels, oxidative stress markers and telomere length in aging elite athletes. In this study, 80 blood samples from consenting elite athletes were collected for anti-doping analysis at an anti-doping laboratory in Italy (FMSI). Participants were divided into three groups according to their sport intensity: low-intensity skills and power sports (LI, n = 18); moderate-intensity mixed soccer players (MI, n = 31); and high-intensity endurance sports (HI, n = 31). Participants were also divided into two age groups: less than 25 (n = 45) and above 25 years old (n = 35). Serum levels of 10 pro and anti-inflammatory cytokines and two antioxidant enzymes were compared in age and sport intensity groups and telomere lengths were measured in their respective blood samples. Tumor necrosis factor-alpha (TNF-α) was the only cytokine showing significantly higher concentration in older athletes, regardless of sport intensity. Interleukin (IL)-10 increased significantly in HI regardless of age group, whereas IL-6 concentration was higher in the older HI athletes. IL-8 showed a significant interaction with sport intensity in different age groups. Overall, significant positive correlations among levels of IL-6, IL-10, IL-8 and TNF-α were identified. The antioxidant catalase activity was positively correlated with levels of TNF-α. Telomere length increased significantly with sport intensity, especially in the younger group. HI had longer telomeres and higher levels of pro- and anti-inflammatory cytokines, suggesting less aging in HI compared to low and moderate counterparts in association with heightened immune response. Investigation of the functional significance of these associations on the health and performance of elite athletes is warranted.