Functionally characterizing obesity-susceptibility genes using CRISPR/Cas9, in vivo imaging and deep learning

There is emerging evidence that organochlorine and organophosphorus insecticide exposure may be linked to excessive weight gain and symptoms of diabetes. However, there is a lack of knowledge for other types of insecticides with potential influence on obesity and diabetes. Thus, the purpose of this investigation was to determine the role of imidacloprid, a neonicotinoid insecticide, in lipid metabolism by use of 3T3-L1 adipocytes. Imidacloprid treatment potentiated lipid accumulation in 3T3-L1 adipocytes and significantly increased expression of a key regulator of adipocyte differentiation and key regulators of lipogenesis. These results imply the involvement of imidacloprid in altered adipogenesis, resulting in increased fat accumulation. This finding is the first report of a potential link between neonicotinoid insecticide exposure and lipid accumulation in adipocytes. Further in vivo as well as epidemiological studies will be required before we can extrapolate these findings to a potential contribution of imidacloprid in human obesity.

The buffalo population is extensive in China, but its meat quality is relatively inferior. Therefore, improving meat quality should be one of the breeding goals. microRNAs (miRNAs) play an essential regulatory role in the post-transcriptional expression of genes. Some studies have reported their function regulating genes related to fat deposition and adipocyte differentiation in cattle, but there is limited reports in buffalo. We performed small RNA transcriptome sequencing of Xinyang buffalo adipose tissue between calves and adults in this study. As a result, 282 mature miRNAs were significantly differentially expressed, and co-expression analysis showed that 454 miRNAs were significantly associated with developmental stages. Target gene identification, GO (gene ontology) annotation, and KEGG analysis of miRNAs showed that miR-195, miR-192, and miR-24-3p could target key genes for lipogenesis and thus regulate adipose deposition and differentiation. Among them, miR-195 was significantly upregulated in adipose tissue and induced adipocytes of adult buffaloes, and its overexpression significantly inhibited lipid accumulation in primary adipocytes. Dual-luciferase reporter gene analysis showed that miR-195 reduced the expression of thyroid hormone response protein (THRSP) by targeting its 3′ untranslated terminal region, suggesting that miR-195 may inhibit lipid accumulation in adipocytes by regulating THRSP. The results confirmed the reliability of predictive screening of miRNAs and provided theoretical support for buffalo fattening.

Dysfunction in adipose tissue can cause serious health problems, including obesity, type-2 diabetes, and cardiovascular disease, significantly reducing human life expectancy. Differences in differentiation and lipid accumulation in adipocytes reflect their functional status, making it important to characterize adipocytes by monitoring biophysical changes during adipogenic differentiation. However, there is currently no specific cell surface marker to separate mature adipocytes from non-adipose cells based on their lipid content, and separation of mature adipocytes is challenging due to handling limitations without fixation, antibody staining, or particle conjugation. Here, we report a biomarker-free, magnetic levitation-based method to detect density changes and quantify the accumulation of lipid-rich droplets within differentiating adipogenic cells. Magnetic levitation revealed density changes within preadipocytes differentiating towards mature adipocytes, with density decreasing over time as cells accumulated lipids. We then used lipid droplets as an intracellular marker to quantify lipid accumulation in single adipocytes during adipogenesis. The significant density changes correlated with cell morphology and lipid droplet morphology within the cytoplasm. For the first time, free-floating lipid vesicle density was measured using magnetic levitation. This unique method enables efficient detection and quantification of dynamically evolving lipid droplets in cells, proving beneficial for modeling lipid storage-related diseases and drug screening applications.

The buffalo population is extensive in China, but its meat quality is relatively inferior. Therefore, improving meat quality should be one of the breeding goals. microRNAs (miRNAs) play an essential regulatory role in the post-transcriptional expression of genes. Some studies have reported their function regulating genes related to fat deposition and adipocyte differentiation in cattle, but there is limited reports in buffalo. We performed small RNA transcriptome sequencing of Xinyang buffalo adipose tissue between calves and adults in this study. As a result, 282 mature miRNAs were significantly differentially expressed, and co-expression analysis showed that 454 miRNAs were significantly associated with developmental stages. Target gene identification, GO (gene ontology) annotation, and KEGG analysis of miRNAs showed that miR-195, miR-192, and miR-24-3p could target key genes for lipogenesis and thus regulate adipose deposition and differentiation. Among them, miR-195 was significantly upregulated in adipose tissue and induced adipocytes of adult buffaloes, and its overexpression significantly inhibited lipid accumulation in primary adipocytes. Dual-luciferase reporter gene analysis showed that miR-195 reduced the expression of thyroid hormone response protein (THRSP) by targeting its 3 ' untranslated terminal region, suggesting that miR-195 may inhibit lipid accumulation in adipocytes by regulating THRSP. The results confirmed the reliability of predictive screening of miRNAs and provided theoretical support for buffalo fattening.

To explore the anti-obesity effects and the mechanism of action of Monascus pilosus(M. pilosus)-fermented black soybean (MFBS) extracts (MFBSE) and MFBS powders (MFBSP) in adipocytes and high-fat diet (HFD)-induced obese mice, respectively. Black soybean was fermented with M. pilosus, and the main constituents in MFBS were analyzed by HPLC analysis. In vitro, MFBSE were examined for anti-adipogenic effects using Oil-Red O staining. In vivo, mice were fed a normal-fat diet (NFD) control, HFD control or HFD containing 1 g/kg MFBSP for 12 weeks, and then body weight gain and tissues weight measured. Real-time PCR and western blot assay were used to determine the mechanism of anti-adipogenic effects. MFBSE inhibited lipid accumulation in 3T3-L1 adipocytes without exerting cell cytotoxicity. MFBSP treatment in HFD-fed mice significantly decreased the body weight gain compared with the HFD control mice. MFBSE and MFBSP treatment resulted in significantly lower mRNA levels of adipogenesis-related genes, such as peroxisome proliferator-activated receptor γ(PPAR γ), fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS), in adipocytes and in white adipose tissue (WAT) of HFD-induced obese mice. These results suggest that the anti-obesity effects of MFBS are elicited by regulating the expression of adipogenesis-related genes in adipocytes and WAT of HFD-induced obese mice.

Obesity is becoming a more and more serious health issue in the US and worldwide. This situation only started since a few of decades ago. It is certain that the human genomic coding has not been changed so dramatically by evolution during such short period of time. Evidence indicates that environmental factors including diets and behaviors are involved in affecting gene expressing in developing of human diseases. Since adipose tissues are significantly expanded in individuals with obesity, adipocytes were cultured in the conditions to mimic hyperlipidemia conditions that happen during the developmental process of obesity. We have observed that saturated lipid at high concentrations induces lipid accumulation in adipocytes. A few of new candidate genes that are regulated by the mimic hyperlipidemia conditions have been identified. These genes will be validated by qRT‐PCR and studied further.Grant Funding Source: Suported by startup funds from Texas A&M University‐Kingsville to YL

Excessive lipid accumulation in the body causes people to become overweight and obese, conditions that are associated with an amplified risk of serious diseases. The fungi of genus Monascus produce various secondary metabolites such as monacolins, citrinin and fungal pigments, which are water-insoluble and have inhibitory potency for the lipid accumulation in adipocytes. However, water-soluble adipogenesis inhibitors derived from Monascus-fermented products have not yet been reported. In this study, we investigated the inhibitory activity against intracellular lipid accumulation of water-soluble fractions of Monascus-fermented red mold rice (RMR) and red mold barley (RMB) on murine 3T3-L1 cells. Water soluble fractions of ten different Monascus strains were used and the inhibitory activity of their water-soluble fractions on lipid accumulation by differentiated 3T3-L1 cells was evaluated for 8 days using oil red staining. The water-soluble fraction from Monascus pilosus NBRC4507 fermented RMR cultivated at 30C for 14 days was selected since it showed comparatively the lowest relative lipid accumulation (62±1.2%), which indicated the highest inhibitory activity of lipid accumulation in adipocytes. To study the presence of monacolin and citrinin in the water soluble fractions, thin-layer chromatography was done and the results showed that the water-soluble fractions tested were free from both monacolin and citrinin. Therefore, the present study strongly suggested that the water-soluble components, except for monacolin and citrinin, in the water soluble fraction obtained from Monascus pilosus NBRC4507-fermented rice can be used as functional food material to control overweight and obesity.

TR4 has been suggested to play an important role in lipid metabolism in adipocytes. Although TR4 facilitates lipid accumulation during adipogenesis, the regulatory effect of TR4 on lipid storage in mature adipocytes remains unclear. We showed that TR4 inhibited the LXR agonist GW3965-mediated decrease of lipid accumulation in 3T3-L1 adipocytes. A reporter gene analysis revealed that TR4 suppressed LXRα transcriptional activity, although LXRα was unable to affect TR4 transcriptional activity. Moreover, adding TR4 resulted in reduced LXRα binding to the LXR responsive element in a gel shift assay. Additionally, the suppressive effect of GW3965 on perilipin expression and lipid accumulation in 3T3-L1 adipocytes was abolished by TR4 overexpression. Taken together, our data demonstrate that TR4 plays an inhibitory role in LXRα-mediated suppression of lipid accumulation in 3T3-L1 adipocytes. This TR4 protective effect is mediated, in part, by blocking the suppressive effect of GW3965 on perilipin gene expression.

Background:Excess body fat accumulation contributes to the development of metabolic disorders that can cause adverse health effects. Carnosic acid (CA), a major bioactive component of rosemary (Rosemarinus officinalis), has been suggested to possess anti-adipogenic properties. The present study was conducted to elucidate the mechanism underlying the anti-adipogenic effects of CA.Methods:3T3-L1 pre-adipocytes were treated with CA (0.1, 1, and 10 μM) from day 0 to day 8 of differentiation. On day 8, biochemical markers of lipid accumulation and the degree of fatty acid desaturation were measured.Results:Oil Red O staining results, triglyceride (TG) accumulation, and glycerol 3-phosphate dehydrogenase activity suggested that CA significantly inhibited lipid accumulation in 3T3-L1 adipocytes. CA significantly decreased mRNA expression of peroxisome proliferator-activated receptor-γ, sterol regulatory element-binding protein 1, and CCAAT/enhancer binding protein-α in a dose-dependent manner. Moreover, it decreased the ratio of both C16:1/C16:0 and C18:1/C18:0, with reduced expression of stearoyl CoA desaturase 1 mRNA and protein.Conclusions:These results suggest that CA efficiently suppressed adipogenesis in 3T3-L1 adipocytes and its action, at least in part, is associated with the downregulation of adipogenesis-related genes and the fatty acid composition of TG accumulated in adipocytes.

Aims: The objective of the present study was to evaluate the anti-obesity effects of unripe Rubus coreanus Miquel (uRC) in 3T3-L1 adipocytes and body weight, epididymal fat and perirenal fat weight, and lipid profiles in diet-induced obese (DIO) C57BL/6 mice. Methodology: The lipid accumulation in 3T3-L1 adipocytes was carried out Oil Red O staining. And uRC (50 and 100 mg/kg/day) were orally administered for 90 days from the day of feeding with high fat diet (HFD). The serum total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL)-cholesterol and low density lipoprotein(LDL)-cholesterol and glucose levels were measured using Alere cholesterol LDX system. And the serum glutamic oxaloacetic transaminase (GOT), Short Communication Jung et al; IJBcRR, 5(1): 20-26, 2015; Article no. IJBcRR.2015.003 21 glutamic pyruvic transaminase (GPT), blood urea nitrogen(BUN) and creatinine levels were measured using the respective kits. Results: Our results indicated that treatment with uRC dose-dependently inhibited lipid accumulation in 3T3-L1 adipocytes. Moreover, after oral administration for 12 weeks, uRC (50 and 100mg/kg/day) extract produced a significant decrease in the serum total cholesterol (TC), lowdensity lipoprotein (LDL) cholesterol, glucose and glutamic-oxaloacetic transaminase (GOT) levels of HFD-induced obese mice. Similarly, uRC extract elevated serum high density lipoprotein (HDL) cholesterol. These results suggest that uRC extract may be a useful resource for the management of obesity. Conclusion: These results suggest that uRC extract may be a useful resource for the management of obesity.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Obesity is a pandemic disease characterized by lipid accumulation, increased proinflammatory cytokines, and reactive oxygen species. It is associated with the development of comorbidities that lead to death. Additionally, drug treatments developed to control obesity are insufficient and have a variety of adverse effects. Thus, the search for new anti-obesity therapies is necessary. Campomanesia adamantium is a species from the Brazilian Cerrado that has the potential to treat obesity, as described by the antihyperlipidemic activity of its roots. Therefore, this study aimed to investigate the activity of the aqueous extract of C. adamantium leaves (AECa) on the control of reactive species in vitro, on lipid accumulation in adipocytes and Caenorhabditis elegans, and on the production of proinflammatory cytokines in adipocytes. The antioxidant capacity of AECa was observed by its action in scavenging DPPH• free radical, iron-reducing power, and inhibition of β-carotene bleaching. AECa reduced lipid accumulation in preadipocytes and in C. elegans. Moreover, AECa reduced the production of the proinflammatory cytokines MCP-1, TNF-α, and IL-6 in adipocytes. In summary, the antioxidant activity and the ability of AECa to reduce the accumulation of lipids and proinflammatory cytokines indicate, for the first time, the anti-obesity potential of C. adamantium leaves.

TFEB activator tanshinone IIA and derivatives derived from Salvia miltiorrhiza Bge. Attenuate hepatic steatosis and insulin resistance

The potential antidiabetic properties of green and purple tea [Camellia sinensis (L.) O Kuntze], purple tea ellagitannins, and urolithins.

This study investigated the effects and molecular mechanism of a combination of capsaicin and capsiate on promoting lipid metabolism and inducing browning in 3T3-L1 white adipocytes. The combination significantly suppressed lipid accumulation in adipocytes ( p = 0.019) and robustly improved lipid metabolic profiles, including decreased triacylglycerol (0.6703 ± 0.0385 versus 0.2849 ± 0.0188 mmol/g of protein; p < 0.001), total cholesterol (0.1282 ± 0.0241 versus 0.0651 ± 0.0178 mmol/g of protein; p = 0.003), and low-density lipoprotein cholesterol (0.0021 ± 0.0017 versus 0.0005 ± 0.0002 mmol/g of protein; p = 0.024) and increased high-density lipoprotein cholesterol (0.0162 ± 0.0141 versus 0.1002 ± 0.0167 mmol/g of protein; p = 0.012). Furthermore, this combination markedly upgraded the protein levels of cluster of differentiation 36 ( p = 0.007) and adipose triglyceride lipase ( p = 0.013) and phosphorylation of hormone-sensitive lipase at Ser660, Ser565, and Ser563 ( p < 0.001, p = 0.027, and p = 0.002, respectively), indicating increases of fatty acid transport and lipolysis. The levels of lipid metabolism regulators, phosphorylation of adenosine-monophosphate-activated protein kinases α and β ( p = 0.011, and p < 0.001, respectively), sirtuin 1 ( p = 0.004), and vanilloid transient receptor subtype I ( p = 0.014) were also increased by the combination. Moreover, the combination greatly activated the browning program in adipocytes, as demonstrated by increases in beige-specific gene and protein. Further research found that the protein levels of peroxisome proliferator-activated receptor γ (PPARγ; p = 0.001) and β3-adrenergic receptor (β3-AR; p = 0.026) were elevated by the combination, and most of the beige-specific markers were abolished by pretreatment of antagonists of PPARγ or β3-AR. In conclusion, these results indicated that a combination of capsaicin and capsiate could induce browning in white adipocytes via activation of the PPARγ/β3-AR signaling pathway, and this combination might be worth investigating as a potential cure for obesity.