Mouse Fat Cells Research Articles

AUTOMATED CELL TYPE IDENTIFICATION FOR SINGLE-CELL RNA SEQUENCING ANALYSIS OF WHOLE MOUSE KNEE JOINTS

Purpose: The use of single-cell RNA sequencing (scRNA-seq) is rapidly expanding and enables unprecedented insight into the role of tissue crosstalk in the pathogenesis and progression of osteoarthritis (OA). Our previous work describes a two-step digestion process to obtain a single cell suspension from all representative tissues in whole mouse knee joints, including cartilage, bone, fat, synovium, ligaments, tendons, meniscus, vasculature, epithelial, bone marrow, and immune cells. The whole knee digest creates a complex scRNA-seq dataset containing cells from clearly distinct tissues, many of which also have closely interrelated cell populations that require finer resolution.

DBC1, Deleted in Breast Cancer 1, Is a Nuclear STAT5A-Interacting Protein in Adipocytes, but Its Primary Effects on GLUT4 Gene Expression and Lipolysis Are STAT5 Independent

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. Although the role of STAT5A in fat cell development is well characterized, its specific roles in mature fat cells are still largely unknown. Moreover, the potential role of STAT5A in metabolic diseases, in particular type 2 diabetes, remains unclear. To further understand the function of STAT5A in adipocytes, we employed a non-biased co-immunoprecipitation and mass-spectrometry-based approach to identify novel STAT5A-interacting proteins. One of the proteins we identified was DBC1 (deleted in breast cancer 1; also known as CCAR2). DBC1 is typically localized in the nucleus and has been primarily studied in tumor cells. However, the functions of DBC1 in adipocytes are relatively unknown. Using mouse fat cells, we confirmed that there is a physical association between endogenous STAT5A and DBC1 proteins under physiological conditions in the nucleus that is not dependent upon STAT5 tyrosine phosphorylation. Knockdown of DBC1 in 3T3-L1 adipocytes using siRNA did not affect the expression of several STAT5A target genes including socs3, cish, bcl-6, socs2, and igf-1. However, we did observe increased levels glycerol and free fatty acids released from adipocytes with reduced DBC1 following TNF (tumor necrosis factor) α stimulation. Although loss of DBC1 did not alter STAT5 transcriptional activity for the genes examined, it appears to modulate TNFα-mediated lipolysis in a STAT5-independent manner. In addition, DBC1 knockdown increased GLUT4 expression in murine fat cells but did not have a profound effect on TNFα-mediated changes in gene expression. On-going studies are being performed to elucidate the function of the DBC1/STAT5A interaction and to clarify the impact of DBC1 on lipolysis in adipocytes. Disclosure A. Able: None. A.J. Richard: None. J.M. Stephens: None.

Pterostilbene Inhibits Lipogenic Activity similar to Resveratrol or Caffeine but Differently Modulates Lipolysis in Adipocytes.

The anti-obesity effects of resveratrol shown in rodents are not transposed into an efficient therapy of human obesity. Consequently, the search for molecules mimicking or surpassing resveratrol actions is ongoing. The natural phenolic compound pterostilbene exhibits beneficial health effects and has the capacity to limit fat mass in animal models. In this study, we tested whether pterostilbene modulates triacylglycerol accumulation/breakdown. Prolonged exposure to pterostilbene or resveratrol inhibited adipocyte differentiation in 3T3-F442A preadipocytes. Acute effects on lipolysis, antilipolysis and lipogenesis were determined for pterostilbene in mouse adipocytes, and compared with resveratrol. Pterostilbene was also tested on glycerol release and glucose uptake in subcutaneous human adipocytes. Dose-response analyses did not reveal a clear lipolytic effect in both species. The antilipolytic effect of insulin was improved by pterostilbene at 1-10μM in mouse fat cells only, while at 1mM, the phenolic compound was antilipolytic in human fat cells in a manner not additive to insulin. Pterostilbene dose-dependently inhibited glucose incorporation into lipids similarly to resveratrol and caffeine. However, only the former did not inhibit insulin-stimulated glucose uptake. Indeed, pterostilbene abolished the insulin lipogenic effect without inhibiting its antilipolytic action and rapid activation of glucose uptake. Pterostilbene therefore exhibits a unique panel of direct interactions with adipocytes that relies on its reported anti-obesity and antidiabetic properties. Copyright © 2017 John Wiley & Sons, Ltd.

Alternative Splicing Generates Novel Fads3 Transcript in Mice

IntroductionFatty acid desaturase (FADS) gene family consists of three genes (Fads1, Fads2 and Fads3). Fads1 and Fads2 catalyze critical steps in the biosynthesis of long chain polyunsaturated fatty acids (LCPUFA). Fads3 is the third member, found to have at least eight evolutionarily conserved alternative transcripts (AT), but with no clearly established function. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9). Our main objective is to characterize Fads3AT9, its expression in mouse tissues and in undifferentiated and differentiated fat (white and brown) cells.Methods.Total RNA obtained from mouse tissues and fat cells were reverse‐transcribed into cDNA. The resulting cDNA was used as template for PCR‐reactions.Results.Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). Fads3AT9 retains all the conserved regions characteristic of front end desaturase (cyto b5 domain and three histidine repeats). It is widely expressed in 12 different mouse tissues and fat cells, with strongest expression seen in kidney, and heart. Fads3CS had greater abundance than AT9 in all tissues.ConclusionsWidespread expression patterns in mouse tissues and fat cells suggest it may play an important role in the regulation and/or biosynthesis of LCPUFA from precursors.Funding Source:NIH R01 AT007003

Targeting a fat-accumulation gene

An enzyme that links two metabolic hubs has been found to be upregulated in the fat cells of overweight mice. Inhibition of the gene encoding this enzyme protects mice from diet-induced obesity. See Letter p.258

Distinct metabolic pathways trigger adipocyte fat accumulation induced by high-carbohydrate and high-fat diets

ObjectiveSeveral metabolic pathways may be associated with obesity development and may be differentially modulated by dietary constituents. The aim of this study was to access the adipocyte metabolic pathways that lead to lipid accumulation in fat cells of mice fed a high-carbohydrate or high-fat diet. MethodsMale Swiss mice ages 7 to 8 wk were fed a standard laboratory rodent diet—chow diet, a high-carbohydrate (HC) diet, or a high-fat (HF) diet for 8 wk. ResultsAnimals fed the HC diet exhibited a high lipogenesis rate and lipoprotein lipase activity even in a fasted state. Peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding proteins-1 mRNAs were increased in adipose tissue. The HF diet did not promote the lipogenic pathways but attenuated lipolytic activity and glucose uptake in adipocytes. ConclusionThe HC diet induces constitutive expression of lipogenic transcription factors during fasting, whereas the HF diet decreases lipolysis in adipocytes. Both pathways may contribute to increase fat stores and maintain an obese state.

Oxidation of high doses of serotonin favors lipid accumulation in mouse and human fat cells

The ingestion of serotonin-rich food (bananas, chocolate) increases 5-hydroxytryptamine (5-HT) in blood and its corresponding oxidation products in urines but without direct central consequences since the neurotransmitter does not easily cross the blood-brain barrier. However, there are numerous peripheral effects of serotonin, and recently, 5-HT aldehydic oxidation products have been demonstrated to behave as ligands of peroxisome proliferator-activated receptor γ (PPAR-γ). Since this nuclear factor manages lipid handling by adipose tissue, the response of fat cells to 5-HT exposure needed further investigation. Serotonin oxidation was studied on human adipose tissue homogenates and mouse 3T3F442A preadipocytes by fluorometric and radiometric methods. Gene expression was assessed by real-time RT-PCR in human adipocytes and in 3T3F442A after mid- and long-term exposure to 5-HT while triacylglycerols and proteins were assessed by spectrophotometry. Six-hour exposure of human adipocytes to 250 μM 5-HT increased gene expression of lipid-binding proteins, glucose carriers, and enzymes of triacylglycerol synthesis (FABP4, CD36, GLUT1, and phosphoenolpyruvate carboxykinase), as did rosiglitazone treatment. Long-term serotoninergic stimulation of cultured 3T3F442A preadipocytes by 100-250 μM 5-HT enhanced fat storage and upregulation of PPAR-γ-responsive genes, in a manner sensitive to MAO- or PPAR-γ inhibition. Our observations suggest an unpredicted peripheral effect of serotonin on adipose tissue that depends on its amine oxidation. Besides being centrally active on eating behavior, 5-HT may promote PPAR-γ activation and subsequent lipogenic effects in fat cells, raising the interest to consider its level in future diet formulations.

Heart Hormones Regulate Brown Fat

Natriuretic peptides from the heart induce brown fat thermogenesis in mouse and human fat cells.

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Methylamine but not mafenide mimics insulin-like activity of the semicarbazide-sensitive amine oxidase-substrate benzylamine on glucose tolerance and on human adipocyte metabolism

It has been reported that benzylamine reduces blood glucose in rabbits, stimulates hexose uptake, and inhibits lipolysis in mouse, rabbit, and human adipocytes. In the presence of vanadate, benzylamine is also able to improve glucose disposal in normoglycaemic and diabetic rats. Such insulin-mimicking properties are the consequence of hydrogen peroxide production during benzylamine oxidation by semicarbazide-sensitive amine oxidase (SSAO). The aim of the study was to determine whether other SSAO-substrates could share such potential antidiabetic properties. Thus, mafenide, a synthetic antimicrobial sulfonamide structurally related to benzylamine, and which has been recently reported to interact with SSAO, was tested in the above mentioned models, in parallel with methylamine, a proposed endogenous SSAO-substrate. All tested amines stimulated glucose uptake and inhibited lipolysis in rat and mouse fat cells. Methylamine and benzylamine, but not mafenide, reduced the hyperglycaemic response during a glucose tolerance test in rabbits while the three amines tested were devoid of insulin-releasing activity under both in vivo and in vitro conditions. In human adipocytes, mafenide did not stimulate glucose transport since it was not a high-affinity substrate for SSAO and generated less hydrogen peroxide than benzylamine or methylamine. Therefore, mafenide could not be considered as an antidiabetic drug despite being oxidized and exhibiting insulin-mimicking effects in rat and mouse adipocytes. By contrast, the endogenous substrate methylamine improved glucose utilization in all in vitro and in vivo models, leading to consider novel SSAO substrates as drugs with potential anti-hyperglycaemic properties.

Nonneuronal expression of Rab3A: induction during adipogenesis and association with different intracellular membranes than Rab3D.

Rab3A is a small GTP-binding protein expressed predominantly in brain and neuroendocrine cells, in which it is associated with synaptic and synaptic-like vesicles, respectively. Here we report that adult mouse fat cells and 3T3-L1 adipocytes also express Rab3A mRNA and protein. They do not express synaptophysin, an abundant protein in synaptic vesicles or synaptic-like vesicles. The amount of Rab3A mRNA and protein, like that of the highly homologous isoform Rab3D, increases severalfold during differentiation of 3T3-L1 fibroblasts into mature adipocytes. In fat cells, most Rab3D and Rab3A protein is bound to membrane, irrespective of insulin addition. Rab3A and Rab3D are localized in different subcellular compartments, since about half of the Rab3A, but none of the Rab3D, is associated with a low-density organelle(s). Rab3D and Rab3A may be involved in different pathways of regulated exocytosis in adipocytes. Moreover, in adipocytes Rab3A may define an exocytic organelle that is different from synaptic vesicles or synaptic-like microvesicles found in neuronal and endocrine cells.

A quantitative study of human fat transplants in obese nude mice and their lean littermates

Human fat cells from lean donors enlarge on transplantation into obese nude mice ( genotype ob ob nu nu ). On transplantation into lean nude mice human fat cells do not change significantly in size, even though they are much larger than mouse fat cells.

Site differences in fat cells of New Zealand obese mice—A transplantation study

Fat cells from the gonadal fat pad of New Zealand obese mice are larger than those from the subcutaneous fat pad. When fat is transplanted to a third, common site (beneath the kidney capsule), this difference in size largely disappears, suggesting it is the location of the fat cells rather than of intrinsic differences between the cells that account for their different sizes.

Effect of aging and cellularity on lipolysis in isolated mouse fat cells.

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.

Effects of thyroxine, epinephrine and cold exposure on lipolysis in genetically obese (ob/ob) mice.

SummaryTreatment of ob/ob mice with T4 24 hr prior to cold exposure did not alter plasma concentrations of glucose, insulin and FFA during cold exposure although ob/ob mice remained hyperglycemic and hyperin-sulinemic when compared to nonobese mice. FFA content of and FFA release from isolated adipocytes were significantly elevated in T4-treated obese mice after cold stress as compared to untreated obese mice. T4 treatment also produced a marked increase in epinephrine-stimulated FFA release from fat cells of obese mice in vitro.These results indicate that correction of the hypothyroid status of ob/ob mice with pharmacological doses of T4 improved the in vitro lipolytic response of fat cells, but did not alter the circulating concentrations of important energy sources for thermogenesis in vivo.The technical assistance of Ms. Teresa Pasquine is gratefully acknowledged.

Pharmacologic activity of stereoisomers of 1-substituted 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines: Inhibition of 3H-dopamine accumulation by rat brain slices and lipolytic activity with isolated mouse fat cells

A number of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines related to dopamine were studied for their inhibitory effects on accumulation of 3H-dopamine by rat brain slices and for lipolytic activity with isolated mouse fat cells. The unsubstituted 6,7-dihydroxytetrahydroisoquinoline (ED 50 7.4 × 10 −5 M) and S-(−)-salsolinol (ED 50 7.5 × 10 −5 M) were the most effective inhibitors of 3H-dopamine accumulation. R-(+)-Salsolinol, R-(+)-tetrahydropapaveroline and S-(−)-tetrahydropapaveroline were less effective inhibitors (ED 50 17–22 × 10 −5 M). S-(−)-Tetrahydropapaveroline was a very effective lipolytic agent with isolated fat cells (ED 50 2.0 × 10 −7 M), whereas the R-(+)-form was inactive up to 10 −5 M. These data illustrate the importance of the absolute configuration at the 1-position of the tetrahydroisoquinoline ring for regulating some of the pharmacologic actions of these 6,7-dihydroxylated derivatives.

Gap junctions between electrotonically coupled cells in tissue culture and in brown fat.

Although circumstantial evidence has suggested that gap junctions mediate intercellular electrotonic coupling, it has not been possible in most tissues to exclude the involvement of other, coexisting cell junctions. We have made an electron microscopic study of replicas of frozen-fractured BHK21 cells (from tissue culture) and of brown fat cells of newborn mice. Both of these cell types are known to exhibit intercellular electrical coupling. In each case, the only junctions found between the cells are small macular gap junctions (less than 1 mum in diameter) characterized by clusters of 6-nm (60 A) particles or depressions on membrane cleavage faces. Several replicas confirm the association of these particles and depressions with regions of narrowing of the intercellular space, i.e., with the sites of cell junctions. We have also determined the frequency of occurrence of gap junctions on the membrane cleavage faces of both cell types. Gap junctions occupy about 1-2% of the surface area of brown fat cells, but only 0.05% of the surface area of BHK21 cells. These observations indicate that gap junctions, when they are the only intercellular junctions present, are sufficient to account for electronic coupling between cells.

Lipolysis and α-aminoisobutyric acid uptake in isolated fat cells effects of insulin and lipolytic agents

1. 1. The relationship between lipolysis and the energy-dependent α-aminoisobutyric acid uptake by isolated mouse fat cells has been studied. 2. 2. Stimulation of lipolysis by adrenaline, ACTH or caffeine resulted in a marked inhibition of the net α-aminoisobutyric acid uptake by fat cells. The magnitude of this inhibition was proportional to that of lipolysis. 3. 3. The addition of palmitate or octanoate to the incubation medium mimicked lipolytic agents in decreasing net α-aminoisobutyric acid uptake. These effects were ascribed to an increase in intracellular free fatty acids. 4. 4. In the absence of added glucose, insulin decreased the hormone- or caffeine-induced lipolysis. Concomitantly insulin prevented the drop in α-aminoisobutyric acid uptake induced by these lipolytic agents. 5. 5. It is suggested that a decrease in energy availability and/or production occurs during lipolysis, and that it is partly responsible for the observed inhibition of α-aminoisobutyric acid uptake. In the absence of glucose, insulin is thought to prevent, via its antilipolytic action, such a decrease in energy availability and/or production, thereby preventing the drop in α-aminoisobutyric acid uptake induced by lipolytic agents.

Lipolysis and potassium accumulation in isolated fat cells effect of insulin and lipolytic agents

1. 1. The net 42K +uptake by isolated mouse fat cells has been studied. 2. 2. 42K + was found to accumulate in fat cells by an energy-requiring process that was stimulated by Na + and inhibited by ouabain at concentrations as low as 1 μM. 3. 3. Stimulation of lipolysis by adrenaline, by ACTH or by xanthine derivatives resulted in a marked decrease in 42K +uptake by fat cells. The magnitude of this inhibition was proportional to that of lipolysis. 4. 4. Insulin, even in the absence of added glucose, markedly decreased the hormone- or xanthine derivative-induced lipolysis. Concomitantly, insulin prevented the decrease in 42K + uptake induced by these lipolytic agents. 5. 5. It is suggested that a decrease in energy availability and/or production occurs during lipolysis, and that it is partly responsible for the observed decrease in net 42K + uptake. Furthermore it is thought that insulin, in the absence of added glucose, prevents such a decrease in energy availability and/or production via its antilipolytic action, thereby counteracting the drop in 42K + uptake induced by lipolytic hormones and agents.

Glycerol kinase activity in isolated fat cells of BHob mice.

Glycerol kinase activity in isolated fat cells of BHob mice.