Cultures Of Adipocytes Research Articles

E0771 and 4T1 murine breast cancer cells and interleukin 6 alter gene expression patterns but do not induce browning in cultured white adipocytes

Breast cancer remains a substantial clinical problem worldwide, and cancer-associated cachexia is a condition associated with poor prognosis in this and other malignancies. Adipose tissue is involved in the development and progression of cancer-associated cachexia, but its various roles and mechanisms of action are not completely defined, especially as it relates to breast cancer. Interleukin 6 has been implicated in several mechanisms contributing to increased breast cancer tumorigenesis, as well as a net-negative energy balance and cancer-associated cachexia via adipose tissue remodeling in other models of cancer; however, its potential role in breast cancer-associated white adipose browning has not been explored. In this study, we demonstrate localized white adipose tissue browning in a spontaneous model of murine mammary cancer. We then used an in vitro murine adipocyte culture system with the E0771 and 4T1 cell lines as models of breast cancer. We demonstrate that while the E0771 and 4T1 secretomes and cross-talk with white adipocytes alter white adipocyte mRNA expression, they do not directly induce white adipocyte browning. Additionally, we show that neither exogenous administration of interleukin 6 alone or with its soluble receptor directly induce white adipocyte browning. Together, these results demonstrate that neither the E0771 or 4T1 murine breast cancer cell lines, nor interleukin 6, directly cause browning of cultured white adipocytes. This suggests that their roles in adipose tissue remodeling are more complex and indirect in nature.

Effect of resveratrol on expression of genes involved thermogenesis in mice and humans

The present study aimed to evaluate the effects of resveratrol on FNDC5 and thermogenesis markers expression in the adipose tissue of mice and humans. Thirty-two male mice were randomly divided into four groups (n = 8) and fed with: Standard Diet; Standard Diet + Resveratrol (400 mg/kg); High-fat Diet; High-fat Diet + Resveratrol for eight weeks. Twenty male and female volunteers, aged 30–55 years, BMI ≥ 30 kg/m² were divided into two groups and treated for four weeks with 500 mg trans-resveratrol or placebo, adipose tissue biopsies were taken. Analysis of body weight, food intake, glycemic and lipid profiles, mRNA expression from tissues and primary culture of adipocytes were performed. The main results show that resveratrol improves the glycaemic and lipid profiles along with an increase in the levels of UCP1, PRDM16, PGC1α, and SIRT1. The increase in FNDC5 expression was observed in the mouse and human subcutaneous adipose tissue. The SIRT1 antagonist in adipocyte primary culture resulted in decreased FNDC5 expression. Our data suggest that improved metabolism produced by oral administration of resveratrol is, at least in part, associated with increased thermogenesis followed by high expression of UCP1, PRDM16, PGC1α and that increased FNDC5 expression in the subcutaneous adipose tissue from mice and human might be modulated by SIRT1.

Excess androgen production in subcutaneous adipose tissue of women with polycystic ovarian syndrome is not related to insulin or LH.

The purpose of this study was to investigate androgen production and the role of insulin and LH in its regulation in subcutaneous adipose tissue (SAT) of women with polycystic ovarian syndrome (PCOS). Protein and mRNA expression of androgen synthesis enzymes (Cytochrome P450 17A1 [CYP17A1] and Aldo-keto reductase 1C3 [AKR1C3]) were measured in SAT biopsies from women with PCOS, diagnosed according to the Rotterdam criteria (n=15) and healthy controls (n=15). Cultured mature adipocytes (differentiated from SAT biopsies) were treated with insulin ± phosphoinositol-3-kinase inhibitor (LY294002) or LH ± insulin. CYP17A1 and AKR1C3 mRNA expression and testosterone concentrations were measured in treated and untreated adipocyte cultures. AKR1C3 mRNA was significantly (P<0.001) greater in PCOS versus non-PCOS SAT, but CYP17A1 was not significantly different between the two groups. AKR1C3 and CYP17A1 protein expression was not significantly different in PCOS versus non-PCOS SAT. In untreated adipocyte cultures, CYP17A1, AKR1C3 and testosterone levels were significantly higher in the PCOS versus the non-PCOS groups. Addition of insulin increased AKR1C3 mRNA and testosterone levels, but not CYP17A1 mRNA in non-PCOS with no effect on PCOS adipocytes. The stimulatory effects of insulin were not inhibited by LY294002. Addition of LH increased CYP17A1, AKR1C3 and testosterone in non-PCOS adipocytes with no effect in PCOS adipocytes. In conclusion, SAT of women with PCOS produces excess androgen, which may contribute to PCOS-related hyperandrogenaemia. This SAT androgen excess is independent of obesity and is not directly stimulated by insulin or LH.

Autophagy inhibition by biotin elicits endoplasmic reticulum stress to differentially regulate adipocyte lipid and protein synthesis.

Biotin is an indispensable adipogenic agent, and its ability to coordinate carbohydrate, lipid, and amino acid metabolism sensitizes insulin signaling in adipocytes. This enables the organism to adapt and survive under nutrient stress by synthesis and storage of lipids. Biotin deficiency mimics insulin resistance with alterations in cellular intermediary metabolism. Though the mechanism of lipogenesis is well established across cell types, considering its predisposition to accumulate only lipids, it is necessary to elucidate the mechanism that minimizes the effects of biotin on adipocyte protein synthesis. In order to determine the differential metabolic phenotype by biotin, the primary cultures of adipocytes were induced to differentiate in the presence and absence of excess biotin. Serum pre-incubated with avidin was used to limit biotin availability in cultured cells. Biotin restricts cellular signaling associated with protein synthesis without altering total protein content. The decline in autophagy elicits endoplasmic reticulum stress to inhibit protein synthesis by eIF2α phosphorylation possibly via accumulation of misfolded/long-lived proteins. Furthermore, the compensatory increase in Unc51 like autophagy activating kinase 1 possibly competes with eukaryotic initiation factor 4E-binding protein 1 and ribosomal p70 S6kinase phosphorylation by mechanistic targets of rapamycin complex 1 to uncouple its effect on protein synthesis. In conclusion, autophagy inhibition by biotin uncouples protein synthesis to promote lipogenesis by eliciting endoplasmic reticulum stress and differential phosphorylation of mechanistic targets of rapamycin complex 1 substrates.

Presence of vasculature results in faster insulin response in adipocytes in vascularized adipose tissue model.

Besides being an energy storage, adipose tissue is an endocrine organ closely associated with vascular system. Human relevant in vitro models are needed to study adipose tissue and related diseases. Vasculature plays a central role in the development and inhibition of adipose tissue related diseases. Here, adipocyte culture was established from hASC (human adipose stromal cells), and a vascularized adipose tissue model was established from hASC and HUVEC (human umbilical cord vein endothelial cell) co-culture, utilizing the same differentiation procedure. Using these models together allowed analysis of the effect of vascularization on adipocytes. Adipocyte culture and Vascularized adipose tissue model were characterized on gene (adipocyte and vasculature-related), protein (von Willebrand factor, CollagenIV, CD140b and CD144, secretion of leptin, adiponectin and FABP4) and functional (triglyceride accumulation, glucose uptake and lipolysis) levels. Additionally, vascularized adipose tissue model was exposed to chemicals with known effects on adipogenesis and angiogenesis (rosiglitazone, chlorpyrifos, prochloraz, mancozeb, butylparaben, 15-deoxy-δ12,14-prostaglandin j2, bisphenol a, bis-(2-ethylhexyl) phthalate, tributyltin chloride) to compare their effects to the literature. The in vitro vascularized adipose tissue model showed presence of functional adipocytes and extensive vascular network. Adipocytes and the vasculature showed relevant gene and protein markers. Insulin induced glucose uptake, inhibited lipolysis and influenced vasculature-related genes. The results showed that vasculature led to faster insulin response in lipolysis inhibition and modulated responses to chemicals. This novel thoroughly characterized vascularized adipose tissue model is a promising new tool for studying adipose tissue as well as effect of chemicals on adipogenesis and angiogenesis in adipose tissue.

Presence of vasculature results in faster insulin response in adipocytes in vascularized adipose tissue model_suppl

Besides being an energy store, adipose tissue is an endocrine organ closely associated with the vascular system. Human relevant in vitro models are needed to study adipose tissue and related diseases. Vasculature plays a central role in the development and inhibition of adipose tissue-related diseases. Here, an adipocyte culture was established from hASC (human adipose stromal cells), and a vascularized adipose tissue model was established from hASC and HUVEC (human umbilical cord vein endothelial cells) co-culture, utilizing the same differentiation procedure. Comparing these models allowed analysis of the effect of vascularization on adipocytes. Both models were characterized on gene (adipocyte and vasculature-related), protein (von Willebrand factor, collagen IV, CD140b and CD144, secretion of leptin, adiponectin and FABP4), and functional (triglyceride accumulation, glucose uptake, and lipolysis) levels. Additionally, the vascularized adipose tissue model was exposed to chemicals with known effects on adipogenesis and angiogenesis (rosiglitazone, chlorpyrifos, prochloraz, mancozeb, butylparaben, 15-deoxy- Δ12,14-prostaglandin J2, bisphenol A, bis-(2-ethylhexyl) phthalate, tributyltin chloride) to compare their effects to the literature. The in vitro vascularized adipose tissue model demonstrated the presence of functional adipocytes and an extensive vascular network, displaying relevant gene and protein markers. Insulin induced glucose uptake, inhibited lipolysis, and influenced vasculature-related genes. The presence of vasculature led to a faster lipolysis inhibition by insulin and modulated responses to chemicals. This novel, thoroughly characterized, vascularized adipose tissue model is a promising new tool for studying adipose tissue as well as the effects of chemicals on adipogenesis and angiogenesis in adipose tissue.

Expression of gene and content of adiponectin in fatty tissue in patients with ischemic heart disease

The purpose of the study was to investigate the features of expression and adiponectin content in the adipocyte culture of subcutaneous, epicardial, and perivascular adipose tissue and the effect of various doses of rosuvastatin on these processes. 29 patients with coronary artery disease were examined. Adipocytes were isolated from the samples of SAT, EAT and PVAT which were taken during coronary artery bypass surgery, followed by cultivation in the presence of rosuvastatin and evaluation of gene expression and adiponectin concentration. Adipocytes SAT, EAT and PVAT differed in the level of adiponectin secretion and expression of its gene. On day 1 of cultivation the expression of the adiponectin gene in the EAT was 2.3 times lower than in the PVAT. On day 2 of cultivation the expression of the adiponectin gene was reduced both in the EAT and the PVAT as compared to the SAT. When rosuvastatin was added at a concentration of 1 mmol/L, adiponectin gene expression in PVAT was higher than when rosuvastatin was added at a concentration of 5 mmol/L, in the adipocyte culture of SAT effect was opposite. Thus, the adipocytes of EZhT and, to a greater extent, PAS, can be a therapeutic target for statins in the case of the pathological activation of adipose tissue.

Short-term high-fat diet affects macrophages inflammatory response, early signs of a long-term problem

Obesity is a chronic inflammatory disease that affects millions of people worldwide. Most studies observe the effects of a high-fat diet (HFD) in 10–12 weeks. This work investigated the effects induced by a HFD administered for 6 weeks on the nutritional status of mice and some aspects of the inflammatory response in mouse peritoneal macrophages. Male Swiss Webster mice, 2–3 months of age, were fed a control diet or HFD for 6 weeks. After this period, the mice were euthanized, and peritoneal macrophages were collected for immunoassays and assessment of biochemical parameters. A HFD was associated with increased cholesterol, insulin resistance, C-reactive protein (CRP), leptin, and serum resistin levels. Lipopolysaccharide (LPS)- stimulated adipocyte cultures of animals subjected to a HFD showed increased production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). However, peritoneal macrophages of the HFD group showed no changes in the levels of these cytokines. LPS-stimulated peritoneal macrophages from HFD-treated animals showed a reduction in mRNA expression of TNF-α and IL-6, as well as a decrease in expression of the transcription factor nuclear factor-kappa B (NF-kB). In conclusion, HFD treatment for 6 weeks induces similar signs to metabolic syndrome and decreases the capacity of peritoneal macrophages to develop an appropriate inflammatory response to a bacterial component.

Characterization and visualization of the liposecretion process taking place during ceiling culture of human mature adipocytes.

To investigate and further characterize the process of mature adipocyte dedifferentiation. Our hypothesis was that dedifferentiation does not involve mitosis but rather a phenomenon of liposecretion. Mature adipocytes were isolated by collagenase digestion of human adipose tissue samples. Ceiling cultures were established using our six-well plate model. Cells were treated with cytosine β-d-arabinofuranoside (AraC) or vincristine (VCR), two agents blocking cell division, and were compared with vehicle. Liposecretion events were visualized by time-lapse microscopy, with and without AraC in adipocytes transducted with a baculovirus. Microscopic analyses were performed after labeling phosphorylated histone 3 and cyclin B1 in ceiling cultures. Treatment with AraC almost entirely prevented the formation of fibroblasts up to 12 days of ceiling culture. Similar results were obtained with VCR. The antimitotic effectiveness of the treatment was confirmed in fibroblast cultures from the adipose tissue stromal-vascular fraction by proliferation assays and colony-forming unit experiments. Using time-lapse microscopy, we visualized liposecretion events in which a large lipid droplet was rapidly secreted from isolated mature adipocytes. The same phenomenon was observed with AraC. This was observed in conjunction with histone 3 phosphorylation and cyclin B1 segregation to the nucleus. Our results support the notion that dedifferentiation involves rapid secretion of the lipid droplet by the adipocytes with concomitant generation of fibroblast-like cells that subsequently proliferate to generate the dedifferentiated adipocyte population during ceiling culture. The presence of mitotic markers suggests that this process involves cell cycle progression, although cell division does not occur.

Naringenin Promotes Thermogenic Gene Expression in Human White Adipose Tissue.

ObjectiveNaringenin, a citrus flavonoid, prevents diet-induced weight gain and improves glucose and lipid metabolism in rodents. There is evidence that naringenin activates brown fat and increases energy expenditure in mice, but little is known about its effects in humans. Our goal was to examine the effects of naringenin on energy expenditure in adipose tissue.MethodsHuman white adipocyte cultures (hADSC), and subcutaneous abdominal adipose tissue (pWAT) were treated with naringenin for 7–14 days. Expression (qRT-PCR, immunoblotting) of candidate genes involved in thermogenesis and glucose metabolism was measured. Oxygen consumption rate (OCR) was measured in hADSC using a Seahorse Flux analyzer.ResultsIn hADSC, naringenin increased expression of the genes associated with thermogenesis and fat oxidation including uncoupling protein 1, adipose triglyceride lipase, and key factors associated with insulin sensitivity including glucose transporter 4, adiponectin, and carbohydrate response element binding protein (p<0.01). Similar responses were observed in pWAT. Basal, ATP-linked, maximal, and reserve OCR increased in the naringenin-treated hADSC (p<0.01).ConclusionsNaringenin increases energy expenditure in hADSC and stimulates expression of key enzymes involved in thermogenesis and insulin sensitivity in hADSC and pWAT. Naringenin may promote conversion of human white adipose tissue to a brown/beige phenotype.

Energy metabolism of white adipose tissue and insulin resistance in humans.

Insulin resistance not only occurs in obesity, but also in lipodystrophy. Although adipose tissue mass affects metabolic fluxes and participates in interorgan crosstalk, the role of energy metabolism within white adipose tissue for insulin resistance is less clear. A Medline search identified invivo studies in humans on energy and lipid metabolism in subcutaneous (SAT) and visceral adipose tissue (VAT). Studies in adipocyte cultures and transgenic animal models were included for the better understanding of the link between abnormal energy metabolism in adipose tissue and insulin resistance. The current literature indicates that higher lipolysis and lower lipogenesis in VAT compared to SAT enhance portal delivery of lipid metabolites (glycerol and fatty acids) to the liver. Thus, the lower lipolysis and higher lipogenesis in SAT favour storage of excess lipids and allow for protection of insulin-sensitive tissues from lipotoxic effects. In insulin-resistant humans, enhanced lipolysis and impaired lipogenesis in adipose tissue lead to release of cytokines and lipid metabolites, ultimately promoting insulin resistance. Adipose tissue of insulin-resistant humans also displays lower expression of proteins involved in mitochondrial function. In turn, this leads to lower availability of mitochondria-derived energy sources for lipogenesis in adipose tissue. Abnormal mitochondrial function in human white adipose tissue likely contributes to the secretion of lipid metabolites and lactate, which are linked to insulin resistance in peripheral tissues. However, the relevance of adipose tissue energy metabolism for the regulation of human insulin sensitivity remains to be further elucidated.

Enhanced ANGPTL2 expression in adipose tissues and its association with insulin resistance in obese women

Angiopoietin-like protein 2 has been proposed to be a key mediator linking obesity and insulin resistance. However, no detailed study of ANGPTL2 expression in human adipose tissues has yet been reported. To investigate the pattern and regulation of ANGPTL2 expression in human adipose tissues in obesity and its related diseases, we recruited 32 non-diabetic and 13 type 2 diabetic obese women and 32 normal-weight women. ANGPTL2 mRNA was expressed at a similar level in visceral and subcutaneous adipose tissues. Adipose tissue ANGPTL2 mRNA was much higher in obese patients. Adipose tissue ANGPTL2 mRNA and serum ANGPTL2 levels showed strong associations with metabolic parameters associated with insulin resistance. In adipose tissue, ANGPTL2 mRNA was closely correlated with the expression of genes involved in inflammation and ER stress. ANGPTL2 mRNA was principally expressed in adipocytes, and its expression was markedly higher in the adipocyte but non-adipocyte fraction of obese adipose tissues. Culture of human adipocytes under conditions mimicking the microenvironment of obese adipose tissue (especially, increased ER stress) stimulated ANGPTL2 gene expression and secretion. In addition, co-culture of adipocytes and macrophages suggested that ANGPTL2 excessively produced by adipocytes, may contribute inflammation and remodeling in obese adipose tissues, thereby promoting insulin resistance.

Adipocyte aquaporin 7 (AQP7) expression in lean children and children with obesity. Possible involvement in molecular mechanisms of childhood obesity.

Background Aquaporin 7 (AQP7), a water/glycerol transporting protein, regulates adipocyte glycerol efflux and influences lipid and glucose homeostasis. Altered AQP7 expression in adults leads to impaired glycerol dynamics, adipocyte hypertrophy and it predisposes them to obesity and diabetes. To assess its possible involvement in childhood obesity, this study investigated the expression of adipocyte AQP7 in cultured adipocytes of children. Methods Primary in vitro differentiated adipocyte cultures were developed from surgical biopsies of subcutaneous abdominal adipose tissue from 61 (46 prepubertal, 15 pubertal) lean children (body mass index [BMI] <85%) and 41 (22 prepubertal, 19 pubertal) children with obesity (BMI >95%). AQP7 expression was studied by reverse transcription polymerase chain reaction and Western immunoblotting and insulin by enzyme-linked immunosorbent assay. Results AQP7 messenger RNA (mRNA) was increased in the younger obese prepubertal (YOP) children but decreased in the obese adolescents (OA) (p=0.014) who also had increased insulin and homeostatic model assessment - insulin resistance (HOMA-IR). Lean pubertal (LP) children and YOP had increased 41 kDa AQP7 protein expression (p=0.001 and p=0.005, respectively). The OA who expressed 34 kDa AQP7 had lower triglyceride (TG) levels than those who did not express it (p=0.013). In the lean children, TG were negatively correlated with 34 kDa AQP7 (p=0.033). Conclusions The lower AQP7 mRNA expression in the OA may reflect a predisposition to adipocyte hypertrophy and metabolic dysfunction, as in the adults, whereas the YOP may be protected from this. The increased 41 kDa AQP7 protein expression in the LP may reflect the increased energy requirements of puberty for glycerol while in the YOP it may also be protective against the development of adipocyte hypertrophy.

Knockdown of LGALS12 inhibits porcine adipocyte adipogenesis via PKA-Erk1/2 signaling pathway.

Increasing intramuscular (IM) fat while concomitantly decreasing subcutaneous (SC) fat content is one major goal of pig breeding. Identifying genes involved in lipid metabolism is critical for this goal. Galectin-12 (LGALS12) has been proven to be an important regulator of fat deposition in mouse models; however, the effect and regulatory mechanisms of LGALS12 on porcine adipogenesis are still unknown. In this study, the effects of LGALS12 on fat deposition were explored with primary culture of porcine SC and IM adipocytes. Analysis of LGALS12 expression across different tissues revealed that LGALS12 was predominantly expressed in adipose tissue. The LGALS12 expression patterns across stages of adipocyte differentiation were also evaluated, with differences observed between SC and IM fat. Small interfering RNA (siRNA) of LGALS12 was designed and transfected into porcine adipocytes derived from SC and IM fat. After transfection, the expression level of LGALS12 was significantly reduced, and the number of lipid droplets was reduced in adipocytes from both SC and IM fat. Simultaneously, the levels of adipogenic markers, including PPARγ and aP2, were decreased, whereas hydrolysis markers, including adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), were increased. Furthermore, the activation of lipolysis signals, such as the phosphorylation of PKA and Erk1/2, were observed with LGALS12 knockdown in terminally differentiated adipocytes from both SC and IM sources. Taken together, these results suggest that LGALS12 knockdown can inhibit adipogenesis of porcine adipocytes by downregulating lipogenic genes and activating the PKA-Erk1/2 signaling pathway.

Promotion of lipid storage rather than of thermogenic competence by fetal versus newborn calf serum in primary cultures of brown adipocytes

ABSTRACTMuch current understanding of brown adipocyte development comes from in-vitro cell models. Serum type may affect the behavior of cultured cells and thus conclusions drawn. Here, we investigate effects of serum type (“fetal bovine” versus “newborn calf”) on responses to differentiation inducers (the PPARγ agonist rosiglitazone or the neurotransmitter norepinephrine) in cultured primary brown adipocytes. Lipid storage was enhanced by fetal versus newborn serum. However, molecular adipose conversion (Pparg2 and Fabp4 expression) was not affected by serum type. Rosiglitazone-induced (7-days) expression of thermogenic genes (i.e. Ucp1, Pgc1a, Dio2 and Elovl3) was not systematically affected by serum type. However, importantly, acute (2 h) norepinephrine-induced thermogenic gene expression was overall markedly higher (and adipose genes somewhat lower) in cells cultured in newborn serum. Thus, newborn serum promotes thermogenic competence, and the use of fetal serum in brown adipocyte cultures (as is often routine) counteracts adequate differentiation. Agents that counteract this inhibition may therefore confoundingly be ascribed genuine thermogenic competence-inducing properties.

Reprograming of Human Adipocytes to a Briter Phenotype—Enhanced Fatty Acid Oxidation and Lipid Droplet Remodeling

Converting energy storing white adipocytes to energy wasting ‘brite’ ones is now recognized to be feasible and may confer protection against obesity and its related cardiometabolic diseases. Fragments of omental and subcutaneous human adipose tissues were cultured for 7 days with insulin and dexamethasone with or without rosiglitazone (rosi) to induce britening. Rosi caused a metabolic reprograming of the adipocytes including increased rates of basal FA oxidation and the expression of mRNAs and proteins in multiple pathways that modulate FA metabolism, triacylglycerol synthesis, cellular FA trafficking, and mitochondrial oxidative capacity (PGC1a, UCP1, CIDEA, PLIN5, FABP3). Furthermore, these changes were associated with a dramatic remodeling of the surface of the adipocyte lipid droplet (LD) such that clusters of small LDs decorated with mitochondria were formed. Formation of these small LDs was blocked by inhibition of lipolysis and required FA activation and reesterification. Surprisingly, both omental and subcutaneous adipocytes were similarly ‘britened’. A similar remodeling was induced in primary cultures of newly-differentiated human adipocytes and these cells showed higher rates of basal and maximally-stimulated oxygen consumption. Consistent with transcriptome changes in both systems, lipidomics showed increased levels of unsaturated FAs, phospholipid species, and cardiolipins in brite cells. Taken together, these results identify a coordinated metabolic and structural reprogramming of mature human adipocytes toward a more oxidative phenotype. Although rosi has unacceptable clinical side effects, knowledge of the downstream effectors of this metabolic improvement may lead to development of targeted approaches to improve adipocyte and metabolic health and prevent type 2 diabetes in human obesity. Disclosure M. Lee: None. S. Jash: None. V. Puri: None. S.K. Fried: None.

CB4211 Is a Potential Treatment for Metabolic Diseases with a Novel Mechanism of Action—Sensitization of the Insulin Receptor

Metabolic dysfunction and insulin resistance are common underlying factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and many age-related diseases, including obesity and type 2 diabetes. CB4211 is a novel peptide analog of MOTS-c, a mitochondrially encoded peptide with a potential role in metabolic homeostasis. CB4211 reduces free fatty acid release from cultured adipocytes, improves NAFLD activity score (NAS) in STAM® mice, and selectively decreases fat mass in DIO mice. We investigated the mechanism of action (MOA) of CB4211 in regulating fatty acid metabolism, glucose homeostasis, and insulin sensitivity. CB4211 potentiated insulin mediated inhibition of lipolysis in isoproterenol stimulated adipocyte cultures without changing maximal response, while CB4211 alone had no effect. Inhibitors of IR auto-phosphorylation (GSK183705A) or downstream PI3K/Akt signaling pathway components (wortmannin, Akti-1/2) abolished the antilipolytic effects of insulin alone and in combination with CB4211. Further supporting sensitization of insulin signaling, CB4211 enhanced insulin mediated phosphorylation of IR, IRS-1, and Akt, without affecting IGF mediated phosphorylation of IGF-1R. Consistent with activity through IR, CB4211 potentiated insulin induced reduction in glucose production in H4-IIE cells. The acute in vivo effect of CB4211 on insulin tolerance was determined in fasted DIO mice. Administration of CB4211 with insulin enhanced insulin sensitivity, prolonging the reduction in blood glucose levels compared to insulin alone. In conclusion, CB4211 potentiates insulin effects on fatty acid metabolism and glucose homeostasis by acting at the level of IR. The observed MOA of CB4211 therefore supports its potential utility for treatment of NASH, obesity, type 2 diabetes, and other metabolic disorders. Disclosure K. Grindstaff: Employee; Self; CohBar, Inc.. R. Magnan: None. R. Shang: Employee; Self; CohBar Inc. E. Stenger: Employee; Self; CohBar, Inc.. J.S. Holland: None. D. Perez-Tilve: Research Support; Self; Novo Nordisk A/S, Cohbar. K.C. Cundy: Employee; Self; CohBar, Inc.. Stock/Shareholder; Self; CohBar, Inc..

Rosiglitazone and a β3-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture.

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β3-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β3-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β3-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β3-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β3-adrenoceptor expression and provides an improved window for detection of β3-adrenoceptor responses.

Use of 14C-glucose by primary cultures of mature rat epididymal adipocytes. Marked release of lactate and glycerol, but limited lipogenesis in the absence of external stimuli

ABSTRACTWhite adipose tissue can metabolize large amounts of glucose to glycerol and lactate. We quantitatively traced glucose label to lactate, glycerol and fats in primary cultures of mature rat epididymal adipocytes. Cells were incubated with 7/14 mM 14C-glucose for 24/48 h. Medium metabolites and the label in them and in cells' components were measured. Gene expression analysis was done using parallel incubations. Glucose concentration did not affect lactate efflux and most parameters. Glycerol efflux increased after 24 h, coinciding with arrested lipogenesis. Steady production of lactate was maintained in parallel to glycerogenesis. Changes in adipocyte metabolism were paralleled by gene expression. Glucose use for lipogenesis was minimal, and stopped (24 h-onwards) when glycerol efflux increased because of triacylglycerol turnover. Lactate steady efflux showed that anaerobic glycolysis was the main adipocyte source of energy. We can assume that adipose tissue may play a quantitatively significant effect on glycaemia, returning 3C fragments thus minimizing lipogenesis.

Cytosolic phosphoenolpyruvate carboxykinase is a response gene involved in porcine adipocyte adaptation to heat stress.

Heat stress (HS) leads to increased lipid storage and expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) in pig adipocytes. However, the importance of PCK1 activation and lipid storage in the adaptive response to HS is unknown. Therefore, in vitro experiments were conducted to investigate the effect of PCK1 inhibition with 3-mercaptopicolinic acid (3MPA) on lipid storage and adipocyte response during HS. In vitro culture of adipocytes under HS (41.0 °C) increased (P < 0.05) triacylglycerol accumulation compared with control (37.0 °C). HS increased (P < 0.05) reactive oxygen species level and 3MPA further upregulated (P < 0.05) its level. Heat shock protein 70 (HSP70) gene expression was induced (P < 0.05) by HS compared to control, and PCK1 inhibition with 3MPA attenuated (P < 0.05) its induction by HS. The endoplasmic reticulum (ER) stress markers, C/EBP homologous protein (CHOP) was also upregulated by HS and 3MPA further upregulated (P < 0.05) CHOP mRNA level. These results suggest that with inhibition of PCK1 during HS, in vitro cultured adipocytes were less able to induce adaptive responses such as upregulation of HSP70 and triglycerides, and this exacerbated ER stress during HS. Thus, PCK1 may function to alleviate ER stress that occurs during HS.