Small Adipocytes Research Articles

Response to the photoperiod in the white and brown adipose tissues of Fischer 344 rats fed a standard or cafeteria diet

Researchers are identifying new factors that contribute to the obesity epidemic, with changes in the photoperiod as one promising risk factor. To study the influence of the photoperiod on adipose tissue, Fischer 344 rats were treated for 14 weeks with a long day (18 h light:6 h dark; LD) or a short day (6 h light:18 h dark; SD) and fed a standard diet (STD). Biometric measures, postprandial plasmatic parameters, gene expression in the retroperitoneal white adipose tissue (RWAT) and brown adipose tissue (BAT) and histology of the RWAT were analyzed. A second experiment with the same conditions and analysis was performed for 11 weeks with rats fed a cafeteria diet (CAF). In the STD experiment, the SD increased triglycerides and showed a tendency to reduce fat compared to the LD. In the RWAT, genes implicated in adipogenesis, lipogenesis and lipolysis were down-regulated, and the histological results showed a higher percentage of small adipocytes in the SD without changes in their total number. In the CAF experiment, lipogenesis and adipogenesis gene expression was increased in the SD, while adipocytes were smaller and their number increased. Both experiments showed in the SD a decrease in the BAT expression of lipid uptake and β-oxidation genes, while only the STD additionally showed a reduction in Ucp1 expression. In conclusion, the RWAT morphology and the expression of key genes for lipid metabolism in RWAT and BAT were influenced by the photoperiod; however, the changes observed in the RWAT were different depending on the diet.

Natural and Artificial Sweeteners and High Fat Diet Modify Differential Taste Receptors, Insulin, and TLR4-Mediated Inflammatory Pathways in Adipose Tissues of Rats.

It is difficult to know if the cause for obesity is the type of sweetener, high fat (HF) content, or the combination of sweetener and fat. The purpose of the present work was to study different types of sweeteners; in particular, steviol glycosides (SG), glucose, fructose, sucrose, brown sugar, honey, SG + sucrose (SV), and sucralose on the functionality of the adipocyte. Male Wistar rats were fed for four months with different sweeteners or sweetener with HF added. Taste receptors T1R2 and T1R3 were differentially expressed in the tongue and intestine by sweeteners and HF. The combination of fat and sweetener showed an additive effect on circulating levels of GIP and GLP-1 except for honey, SG, and brown sugar. In adipose tissue, sucrose and sucralose stimulated TLR4, and c-Jun N-terminal (JNK). The combination of HF with sweeteners increased NFκB, with the exception of SG and honey. Honey kept the insulin signaling pathway active and the smallest adipocytes in white (WAT) and brown (BAT) adipose tissue and the highest expression of adiponectin, PPARγ, and UCP-1 in BAT. The addition of HF reduced mitochondrial branched-chain amino transferase (BCAT2) branched-chain keto acid dehydrogenase E1 (BCKDH) and increased branched chain amino acids (BCAA) levels by sucrose and sucralose. Our data suggests that the consumption of particular honey maintained functional adipocytes despite the consumption of a HF diet.

MON-223 Adipose Insulin Resistance in Normal-Weight Women with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and oligo-ovulation associated with insulin resistance (IR). Adipose-IR may underlie this PCOS phenotype and could contribute to IR by impairing insulin-mediated glucose uptake and/or lipogenesis, while diminishing insulin suppression of lipolysis. The present study examined whether adipose-IR, defined as the product of fasting circulating free fatty acid and insulin levels (1), was increased in normal-weight PCOS versus age-/BMI-balanced normo-androgenic ovulatory (control) women and, if so, whether it correlated with insulin sensitivity (Si) as measured by frequently-sampled intravenous glucose tolerance testing (FSIVGTT) and/or hyperandrogenism. Ten normal-weight PCOS women (by NIH criteria) and 18 controls (19-35 years; 19-25 kg/m2) of non-Hispanic Caucasian descent underwent serum hormone/metabolic measurements, FSIVGTT, total body dual-energy x-ray absorptiometry, and subcutaneous (SC) abdominal fat biopsy. Circulating hormone/metabolite levels, adipose-IR, Si, acute insulin response to glucose (AIRg), body fat distribution and adipocyte size distribution were compared between female types using Student’s t-test. Partial correlation coefficients examined associations between adipose-IR and outcome variables, adjusting for serum androgen levels. PCOS women exhibited greater serum LH, androstenedione (A4), total testosterone (T) and free (f) T levels than controls (P<0.05, all parameters), and demonstrated trends for increased abdominal adiposity (P=0.06) and serum triglyceride (TG) levels (P=0.09) with low-normal Si values. PCOS women also had greater (P=0.007) adipose-IR than controls, with a value of 29 pM/L*mM/L providing 94% specificity (95% CI 84%-100%) and 80% sensitivity (95% CI 55%-100%) in discriminating PCOS subjects from controls (AUC=0.81, 95% CI 0.61-1.00, P<0.001). In all women combined, adipose-IR negatively correlated with Si (P=0.01) and positively correlated with serum A4 (P=0.002), total T (P=0.004), fT (P=0.001) and fasting TG (P=0.001) levels as well as with AIRg (P=0.03) and percent small SC abdominal adipocytes (P=0.01). Relationships of adipose-IR with Si (P=0.04) and with serum TG (P=0.01) levels remained after adjusting for serum total T and fT levels, respectively, while the other correlations lost significance. Thus, in normal-weight PCOS women, adipose-IR is intimately linked with dyslipidemia and is associated with reduced Si in part through hyperandrogenism. Reference: Sondergaard E, et al. JCEM 2017;102:1193. Sources of Research Support: NIH Grants and Santa Monica Bay Woman’s Club.

Cow milk does not affect adiposity in growing piglets as a model for children

The effect of milk consumption on childhood obesity is unclear and a direct demonstration of an association is needed. In the present study, we used piglets as a model for prepubertal children to determine the effect of milk on adipose tissue. Two studies were conducted: study 1 with 5-wk-old male piglets (n = 8) and study 2 with 8- to 9-wk-old male piglets (n = 12). The piglets were fed a normal growing diet and randomly assigned to receive daily either 750 mL of whole cow milk or an isocaloric maltodextrin solution (control). For approximately 12 wk, body weight, feed intake, and subcutaneous back fat thickness were determined ultrasonographically and recorded. At euthanasia, back and neck fat thicknesses were measured and samples of back fat were collected for adipose histology. In study 1, but not study 2, piglets receiving milk grew more and ate more compared with control. In study 1, both back fat and neck fat thickness were greater in the milk-fed piglets and they had a higher frequency of small adipocytes and a lower frequency of intermediate and large adipocytes compared with controls. In study 2, control pigs had a significantly greater frequency of intermediate adipocytes but the milk-fed piglets tended to have a higher frequency of the largest adipocytes. In conclusion, milk has no apparent causal or consistent effect on adipose tissue in growing piglets.

An update on immune dysregulation in obesity-related insulin resistance.

Obesity is associated with chronic low-grade inflammation of the adipose tissue (AT) that might develop into systemic inflammation, insulin resistance (IR) and an increased risk of type 2 diabetes mellitus (T2DM) in severe obese rodents and humans. In the lean state, small normal adipocytes and AT macrophages interact with each other to maintain metabolic homeostasis but during obesity, enlarged adipocytes secrete inflammatory mediators and express immune receptors to recruit immune cells and aggravate the inflammation. The better understanding of the obesity-related inflammatory milieu and the sequential events leading to IR could be helpful in designing new preventive and therapeutic strategies. The present review will discuss the cellular and molecular abnormalities participating in the pathogenesis of obesity in obese individuals as well as high-fat diet (HFD)-fed mice, a mouse model of obesity.

Adipose Insulin Resistance in Normal-Weight Women With Polycystic Ovary Syndrome.

Normal-weight women with polycystic ovary syndrome (PCOS) may have adipose tissue insulin resistance (adipose-IR). To examine whether adipose-IR and subcutaneous (SC) abdominal adipose stem cell (ASC) gene expression are altered in normal-weight women with PCOS and correlated with hyperandrogenemia and/or whole-body IR. Prospective cohort study. Academic medical center. Ten normal-weight women with PCOS and 18 control subjects matched for age and body mass index. Women underwent circulating hormone and metabolic measurements, IV glucose tolerance testing, total-body dual-energy x-ray absorptiometry, and SC abdominal fat biopsy. Adipose-IR (fasting insulin × total fatty acid levels) and SC abdominal ASC gene expression were compared between groups and correlated with clinical outcomes. Adipose-IR was greater in women with PCOS than in control subjects (P < 0.01), with 29 pmol/L × mmol/L providing 94% specificity and 80% sensitivity in discriminating the two groups (P < 0.001). Adipose-IR positively correlated with serum androgen and log of fasting triglyceride (TG) levels, percentage of small adipocytes (P < 0.01, all correlations), and acute insulin response to glucose (P < 0.05); and negatively correlated with insulin sensitivity (Si; P < 0.025) and serum adiponectin levels (P < 0.05). Adjusting for serum androgens, adipose-IR correlations with Si and log TG levels remained significant. ASC genes were differentially expressed by the two groups. Expression of functionally critical genes was associated with serum testosterone and/or fasting insulin levels. Normal-weight women with PCOS have increased adipose-IR and altered ASC gene expression related to hyperandrogenism and IR.

“Synapse-like” Connections between Adipocytes and Stem Cells: Morphological and Molecular Features of Human Adipose Tissue

Adult mesenchymal stem cells are a heterogeneous population of stem cell that is not completely defined. The importance of increasing knowledge of this cell population will allow us to understand how to use them in multiple treatments for various diseases. Adipose tissue is a rich source from which to easily obtain a great amount of stem cells. We explore and study adipose stem cells (AdSCs) from human abdominal tissue by immunohistochemical analysis, transmission (TEM) and scanning (SEM) electron microscopy. The focus of this study is on the cell population that surrounds each adipocyte. In these populations of cells are included the adipose tissue stem cells (AdSCs) with different potentiality. We observe that the putative AdSCs have an intimate relationship involving close contact with adipocytes which we define here as “synapse-like.” We show “synapse-like” connections between adipocytes and small cells to be mediated by connexin43 (Cx43). Our data suggest AdSCs constitute a heterogeneous population both in size and expression of different stem cell markers. We found that some of the AdSC attached to adipocytes are positive for Sox2, Pax6 and Nestin by immunostaining methods. TEM and SEM analysis demonstrate that small cells and adipocytes are surrounded by a compact mesh of collagen fibers that maintain physical adhesion between these cells. TEM also shows structural characteristics of putative stem cells and a presence of vesicles in synapse-like contact. SEM images moreover exhibited a large variation and quantity of cell sizes coexisting with adipocytes. Direct cell-to-cell communication could serve at least two purposes: a) as a survival strategy to maintain cells as stem cells, and b) as a source of signaling for differentiation into a new cell type.

Differential Metabolic Responses to Adipose Atrophy Associated with Cancer Cachexia and Caloric Restriction in Rats and the Effect of Rikkunshito in Cancer Cachexia.

Despite the similar phenotypes, including weight loss, reduction of food intake, and lower adiposity, associated with caloric restriction (CR) and cancer cachexia (CC), CC is a progressive wasting syndrome, while mild CR improves whole body metabolism. In the present study, we compared adipose metabolic changes in a novel rat model of CC, mild CR (70% of the food intake of control rats, which is similar to the food consumption of CC rats), and severe CR (30% of the food intake of controls). We show that CC and severe CR are associated with much smaller adipocytes with significantly lower mitochondrial DNA content; but, that mild CR is not. CC and both mild and severe CR similarly upregulated proteins involved in lipolysis. CC also downregulated proteins involved in fatty acid biosynthesis, but mild CR upregulated these. These findings suggest that CC might impair de novo fatty acid biosynthesis and reduce mitochondrial biogenesis, similar to severe CR. We also found that rikkunshito, a traditional Japanese herbal medicine, does not ameliorate the enhanced lipolysis and mitochondrial impairment, but rather, rescues de novo fatty acid biosynthesis, suggesting that rikkunshito administration might have partially similar effects to mild CR.

Mesenchymal Stem Cells Gene Signature in High-Risk Myeloma Bone Marrow Linked to Suppression of Distinct IGFBP2-Expressing Small Adipocytes

Introduction:Certain studies suggest that multiple myeloma (MM) induces expansion of bone marrow (BM) mesenchymal stem cells (MSCs), but others showed induction of MSC senescence. MM cells suppress MSC lineages such as osteoblasts, while their effects on adipocytes remain to be elucidated. Recent studies identified “regulated” adipocytes that are smaller in size than large adipocytes that are constitutively present (Scheller et al, Nature Commun 2015), and that function as an endocrine tissue and regulators of hematopoiesis (Cawthorn et al, Cell Metab 2014), suggesting that BM adipocytes are functionally heterogeneous.We established a MSC gene signature in whole bone biopsies and showed that it gradually changes in different disease stages and is associated with outcome (Schinke et al, CCR 2018).The aim of the study was to identify changes in expression of MSC genes in BM of patients with high risk MM and in focal lesions (FLs), and to elucidate whether these changes reflect altered proportion and function of MSCs and their lineages.Methods:MSC gene expression in whole bone biopsies from normal donors (n=68), and patients with MGUS/SMM (n=90) and MM (n=531) was analyzed using global gene expression profiles. Unexpanded single MSCs from normal donors (n=3; 175 single MSCs) and MM patients (n=3; 162 single MSCs) were sorted by FACSAria and expression of mesenchymal cell, proliferation and senescence markers were analyzed by qRT-PCR using Fluidigm Biomark HD. Functionally, single MSCs were tested for their ability to multiply using supportive serum and MSC-conditioned media. Cell senescence was analyzed by SA-βGal staining. IGFBP2 and adiponectin protein were detected using immunohistochemistry (IHC). Numbers of IGFBP2+ cells were analyzed in biopsies from patients with MGUS/SMM, low risk (LR) and high risk (HR) MM (10 biopsies/group). Cultured MSCs were differentiated to adipocytes by treatment with dexamethasone, insulin and indomethacin for 3-4 weeks. The effects of recombinant IGF1 and IGFBP2 on MM cell growth were performed on BM-dependent MM lines (n=3) cultured in serum-free conditions for 48 hrs.Results:We compared MSC gene expression levels in random interstitial BM biopsies of patients with LR and HR MM, and in paired biopsy samples from random BM and FLs; 41 of the 345 MSC genes were differentially expressed in both comparisons. Most overexpressed genes were related to angiogenesis and ECM, including several collagen genes (e.g., COL4A1, POSTN, and HSPG2). Several underexpressed genes were associated with adipocytes, including IGFBP2 and aldo/keto reductases.To unravel whether these differences in gene expression reflect changes in the proportion of MSCs we tested expression of genes associated with MSCs, osteoblastogenesis, adipogenesis, proliferation and senescence in unexpanded singleMSCs. MM single MSCs had significant reduction in expression of KI67 and increased expression of the senescence marker, CDKN2A/p16, whereas expression of other tested genes were modestly differentially expressed between the two groups. Functionally, unexpanded MM MSCs had increased SA-βGal expression, and only 65±8% of single MSCs divided at least once compared to 90±4% of their normal donor counterparts (p<0.03), suggesting that the in vivo MSC gene expression in MM reflects modifications in mesenchymal cell lineages that encompass most of the mesenchymal compartment in bone.Since IGFBP2 is involved in adipogenesis and bone homeostasis we traced the source of this factor using IHC. Double staining for IGFBP2 and adiponectin revealed that IGFBP2 was mainly expressed by small adipocytes. The number of IGFBP2+ cells was higher in BM biopsies from patients with MGUS/SMM than in those from patients with MM (p<0.02), and the number was lower in biopsies from patients with HR MM than with LR MM (p<0.01).Normal MSCs differentiated to adipocytes produced a high level of IGFBP2, while co-culturing MSCs with MM cells inhibits their differentiation to adipocytes by 6 folds (p<0.001) and reduced expression of IGFBP2 and adiponectin. Recombinant IGFBP2 effectively blocked IGF1-mediated growth of MM cells, indicating its role in controlling IGF1 bioavailability.Conclusions:Our data demonstrate that MM MSCs are less proliferative and that IGFBP2+ small adipocytes are a distinct mesenchymal cell population suppressed by MM, and their depletion may contribute to disease progression. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; TRM Oncology: Honoraria. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

Different Statistical Approaches to Characterization of Adipocyte Size in Offspring of Obese Rats: Effects of Maternal or Offspring Exercise Intervention

Adipocyte size (AS) shows asymmetric distribution related to current metabolic state, e.g., adipogenesis or lipolysis. We profiled AS distribution using different statistical approaches in offspring (F1) of control (C) and obese (MO) mothers (F0) with and without F0 or F1 exercise. Offspring from F0 exercise were designated CF0ex and MOF0ex. Exercised F1 of sedentary mothers were designated CF1ex and MOF1ex. F1 retroperitoneal fat cross-sectional AS was measured by median, cumulative distributions, data dispersion and extreme values based on gamma distribution modeling. F1 metabolic parameters: body weight, retroperitoneal fat, adiposity index (AI), serum leptin, triglycerides (TG) and insulin resistance index (IRI) were measured. Male and female F1 AS showed different cumulative distribution between C and MO (p < 0.0001) therefore comparisons were performed among C, CF0ex and CF1ex groups and MO, MOF0ex and MOF1ex groups. MO AI was higher than C (p < 0.05) and male MOF1ex AI lower than MO (p < 0.05). Median AS was higher in male and female MO vs. C (p < 0.05). Male and female MOF0ex and MOF1ex reduced median AS (p < 0.05). Lower AS dispersion was observed in male CF1ex and MOF1ex vs. CF0ex and MOF0ex, respectively. MO reduced small and increased large adipocyte proportions vs. C (p < 0.05); MOF0ex increased small and MOF1ex the proportion of large adipocytes vs. MO (p < 0.05). MOF0ex reduced male IRI and female TG vs. MO (p < 0.05). MOF1ex reduced male and female leptin (p < 0.05); CF1ex reduced male leptin (p < 0.05). Conclusions: several factors, diet, physical activity and gender modify AS distribution. Conventional AS distribution methods normally do not include analyzes of extreme, large and small adipocytes, which characterize different phenotypes. Maternal high fat diet affects F1 AS distribution, which was programmed during development. F0ex and F1ex have gender specific F1 beneficial effects. AS distribution characterization helps explain adipose tissue metabolic changes in different physiological conditions and will aid design of efficacious interventions to prevent and/or recuperate adverse developmental programming outcomes. Finally, precise identification of effects of specific interventions as exercise of F0 and/or F1 are needed to improve outcomes in obese women and their obesity prone offspring.

Mesenchymal stem cells gene signature in high-risk myeloma bone marrow linked to suppression of distinct IGFBP2-expressing small adipocytes.

Recent studies suggest that multiple myeloma (MM) induces proliferation and expansion of bone marrow (BM) mesenchymal stem cells (MSCs), but others showed that MM cells induce MSC senescence. To clarify the interaction between MM and MSCs, we exploited our established MSC gene signature to identify gene expression changes in myeloma MSCs and associated functional differences. Single MSCs from patients with MM had changes in expression of genes associated with cellular proliferation and senescence and a higher proportion of senescent cells and lower proliferative potential than those from age-matched healthy donors. Single MSCs from both sources heterogeneously express MSC genes associated with adipogenesis and osteoblastogenesis. We identified the gene encoding insulin-like growth factor-binding protein 2 (IGFBP2), an MSC gene commonly altered in high risk MM, as under-expressed. Morphologically, IGFBP2+ cells are underrepresented in MM BM compared to smouldering MM. Strong IGFBP2 and adiponectin co-expression was detected in a subset of small adipocytes. Co-culturing normal MSCs with myeloma cells suppressed MSC differentiation to adipocytes and osteoblasts, and reduced expression of IGFBP2 and adiponectin. Recombinant IGFBP2 blocked IGF1-mediated myeloma cell growth. Our data demonstrate that myeloma MSCs are less proliferative and that IGFBP2+ small adipocytes are a distinct mesenchymal cell population suppressed by myeloma.

Sepsis Induces Adipose Tissue Browning in Nonobese Mice But Not in Obese Mice.

Severe sepsis and septic shock are the biggest cause of mortality in critically ill patients. Obesity today is one of the world's greatest health challenges. Little is known about the extent of involvement of the white adipose tissue (WAT) in sepsis and how it is being modified by obesity. We sought to explore the involvement of the WAT in sepsis. We hypothesize that sepsis induces browning of the WAT and that obesity alters the response of WAT to sepsis. Six-week-old C57BL/6 mice were randomized to a high-fat diet to induce obesity (obese group) or control diet (nonobese group). After 6 to 11 weeks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 18, and 72 h after CLP and epididymal WAT (eWAT), inguinal WAT, and brown adipose tissue (BAT) harvested. Both types of WAT were processed for light microscopy and transmission electron microscopy to assess for morphological changes in both obese and nonobese mice. Tissues were processed for immunohistochemistry, image analyses, and molecular analyses. BATs were used as a positive control. Nonobese mice have an extensive breakdown of the unilocular lipid droplet and smaller adipocytes in WAT compared with obese mice after sepsis. Neutrophil infiltration increases in eWAT in nonobese mice after sepsis but not in obese mice. Nonobese septic mice have an increase in mitochondrial density compared with obese septic mice. Furthermore, nonobese septic mice have an increase in uncoupling protein-1 expression. Although the WAT of nonobese mice have multiple changes characteristic of browning during sepsis, these changes are markedly blunted in obesity.

Female Mice Have Higher Angiogenesis in Perigonadal Adipose Tissue Than Males in Response to High-Fat Diet.

Background: Impaired capillary growth (angiogenesis) in skeletal muscle and adipose tissue contributes to the development of metabolic disorders in obese males. This association remains unexplored in females, despite mounting evidence that endothelial cells have sex-specific transcriptional profiles. Therefore, herein we assessed whether males and females show distinct angiogenic capacities in response to diet-induced obesity.Methods: Age-matched male and female mice were fed normal chow or high-fat obesogenic diets for 16 weeks. At the end of diet period, systemic glucose disposal was assessed as well as insulin sensitivity of skeletal muscle and visceral adipose tissue. Capillary content and the expression of angiogenic regulators were also evaluated in these tissues.Results: When placed on a high-fat diet, female mice gained less weight than males and showed a metabolic phenotype similar to NC-fed mice, contrasting with the impaired whole-body glucose metabolism observed in high-fat-fed males. However, high-fat-feeding elevated serum lipid levels similarly in male and female mice. Although skeletal muscle of high-fat–fed female mice had higher insulin sensitivity than male counterparts, no sex difference was detected in muscle capillarization. Metabolic functions of perigonadal white adipose tissue (pgWAT) were retained in high-fat-fed females, as evidenced by smaller adipocytes with preserved insulin sensitivity, greater responsiveness to isoproterenol, higher expression of Adiponectin and a lower ratio of Leptin:Adiponectin mRNA. An enhanced browning phenotype was detected in HF-fed female adipocytes with upregulation of Ucp1 expression. PgWAT from high-fat-fed females also showed augmented capillary number and expression of endothelial cell markers, which was associated with elevated mRNA levels of pro-angiogenic mediators, including vascular endothelial growth factor A (Vegfa) and its receptor (Vegfr2), the Notch ligand Jagged-1 (Jag1) and Angiopoietin-2 (Angpt2).Conclusion: Taken together, our findings provide novel evidence that visceral adipose tissue of female mice display greater levels of pro-angiogenic factors and vascularity than males in response to high-fat diet. This phenotype is associated with preserved metabolic homeostasis at both tissue and systemic levels. Our study discloses that a thus-far-unappreciated sex-specific difference in the regulation of adipose angiogenesis may contribute to an individual’s susceptibility to developing adipose dysfunction and obesity-related metabolic disturbances.

Metabolic memory of dietary restriction ameliorates DNA damage and adipocyte size in mouse visceral adipose tissue.

Dietary restriction (DR) is thought to exert its beneficial effects on healthspan at least partially by a senolytic and senostatic action, i.e. by reducing frequencies of cells with markers of DNA damage and senescence in multiple tissues. Due to its importance in metabolic and inflammation regulation, fat is a prime tissue for health span determination as well as a prime target for DR. We aimed to determine here whether the beneficial effects of DR would be retained over a subsequent period of ad libitum (AL) feeding. Male mice were kept under either 40% DR or AL feeding regimes from 3 to 12 months of age and then either switched back to the opposite feeding regimen or kept in the same state for another 3 months. Visceral adipose tissue from 4 to 5 mice per group for all conditions was analysed for markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1β) using immuno-staining or qPCR. Macrophages were detected by immunohistochemistry. We found that both 9 and 12 months DR (long term) as well as 3 month (short term, mid-life onset) DR reduced the number of cells harbouring DNA damage and adipocyte size (area and perimeter) in visceral adipocytes with similar efficiency. Importantly, beneficial health markers induced by DR such as small adipocyte size and low DNA damage were maintained for at least 3 month after termination of DR, demonstrating that the previously identified 'metabolic memory' of the DR state in male mice extends to senescence markers in visceral fat.

Characterization of mouse pericardial fat: regulation by PAPP-A

Although implicated in cardiovascular disease, little is known about the fat surrounding the heart. In humans, epicardial fat is the visceral fat depot of the heart, which directly contacts the myocardium. This strategically placed fat depot is thought to produce bioactive molecules that could affect cardiac function. A major limitation in understanding the biology of epicardial fat is its restricted access in humans and its seeming absence in commonly-used experimental animal models. Although laboratory mice do not have epicardial fat per se, they do have a fat depot around the heart. In this study, we found that mouse pericardial fat has the molecular signature, small adipocyte size, and resistance to differentiation consistent with visceral fat. In addition, we show that mouse pericardial fat is regulated by pregnancy-associated plasma protein-A (PAPP-A), a key modulator of local insulin-like growth factor bioavailability. PAPP-A is highly expressed in mouse pericardial fat at levels equivalent to those in mesenteric visceral fat and 10-fold higher than in subcutaneous inguinal fat (P = .0003). Cultured pre-adipocytes isolated from pericardial fat show 2-fold increased PAPP-A secretion compared to pre-adipocytes isolated from inguinal fat. Furthermore, PAPP-A knock-out mice fed a high fat diet for 20 weeks have significantly reduced pericardial fat (by 60%; P < .0001) compared to wild-type littermates. There was no significant difference in inguinal fat between wild-type and PAPP-A knock-out mice. These data characterize a new mouse model of visceral-like pericardial fat and lay a foundation for understanding its role in human heart disease.

Adiposity and metabolic health in mice deficient in intestinal alkaline phosphatase

ABSTRACTIntestinal alkaline phosphatase 3 (AKP3) is an enzyme that was reported to play a role in lipid metabolism and to prevent high fat diet-induced metabolic syndrome in mice.To investigate a potential functional role of AKP3 in diet-induced adiposity and metabolic health, we have kept male and female wild-type or AKP3 deficient mice on a high fat diet for 15 weeks to induce obesity and compared those with mice kept on standard fat diet.Body weight as well as adipose tissue mass were statistically significantly higher upon high fat diet feeding for mice of both genders and genotypes. Female mice of either genotype kept on high fat diet gained less weight, resulting in smaller adipose tissue depots with smaller adipocytes. However, AKP3 deficiency had no significant effect on body weight gain or adipose tissue mass and did not affect adipocyte size or density. Gene expression analysis revealed no effect of the genotype on inflammatory parameters in adipose tissue, except for tumor necrosis factor alpha, which was higher in mesenteric adipose tissue of female obese mice. Plasma glucose and insulin levels were also not affected in obese AKP3 deficient mice.Overall, our data do not support a functional role of AKP3 in adipose tissue development, or insulin sensitivity.

Developmental Programming: Impact of Prenatal Testosterone Excess on Steroidal Machinery and Cell Differentiation Markers in Visceral Adipocytes of Female Sheep.

Prenatal testosterone (T)-treated female sheep manifest reduced adipocyte size and peripheral insulin resistance. The small adipocyte phenotype may reflect defects in adipogenesis and its steroidal machinery. To test whether prenatal T treatment from gestational days 30 to 90 alters the visceral adipose tissue (VAT) steroidal machinery and reduces adipocyte differentiation, we examined expression of the steroidogenic enzymes, steroid receptors, and adipocyte differentiation markers at fetal day 90 and postnatal ages 10 and 21 months. Because gestational T treatment increases fetal T and maternal insulin, the contributions of these were assessed by androgen receptor antagonist or insulin sensitizer cotreatment, either separately (at fetal day 90 and 21 months of age time points) or together (10 months of age). The effects on adipogenesis were assessed in the VAT-derived mesenchymal stem cells (AT-MSCs) from pre- and postpubertal time points to evaluate the effects of pubertal steroidal changes on adipogenesis. Our results show that VAT manifests potentially a predominant estrogenic intracrine milieu (increased aromatase and estrogen receptor α) and reduced differentiation markers at fetal day 90 and postnatal 21 months of age. These changes appear to involve both androgenic and metabolic pathways. Preliminary findings suggest that prenatal T treatment reduces adipogenesis, decreases expression of differentiation, and increases expression of commitment markers at both pre- and postpubertal time points. Together, these findings suggest that (1) increased commitment of AT-MSCs to adipocyte lineage and decreased differentiation to adipocytes may underlie the small adipocyte phenotype of prenatal T-treated females and (2) excess T-induced changes in steroidal machinery in the VAT likely participate in the programming/maintenance of this defect.

Small Proliferative Adipocytes, Newly Identified Adipose Progenitor, as the Precursors of Beige Cells

We previously identified proliferative cells in adipose tissue expressing adipocyte specific genes and preadipocyte specific genes, which were named small proliferative adipocytes (SPA). Furthermore, we found that SPA specifically express proliferin (PLF) and have superior potential for adipogenic differentiation as compared to stromal vascular cells (SVC). In this study, we investigated the role of SPA. A comparison between differentiated SPA (D-SPA) and differentiated SVC (D-SVC) revealed that UCP1 and PLF were strongly expressed in D-SPA. Therefore, we speculated that SPA might be progenitor of beige cells. The real time PCR analyses and the immunohistochemical studies revealed that beige cells associated genes, including UCP1, PPARα and PGC1α were significantly increased in D-SPA. Treatment with pioglitazone and CL316234, a specific β3-adrenergic receptor agonist, converted cultured SPA into cell clusters like beige cells which expressed UCP1 and activation of mitochondrial function confirmed by MitoRed stain. Furthermore, PLF was expressed in beige cells induced by continuous injection of CL316243. In conclusion, since D-SPA were found to express UCP1 and PLF, which were detected in beige cells, but not WAT, SPA were considered to be a progenitor of beige cells. There might be two lineages of cells in adipose tissue; adipose progenitor cells in SVC and WAT (PLF-), and SPA and beige cells (PLF+). The combination of pioglitazone and CL316423 produced an additive effect of inducing browning in SPA. Disclosure K. Taguchi: None. K. Kajita: None. M. Fuwa: None. M. Asano: None. Y. Kitada: None. T. Ikeda: None. T. Ishizuka: None. H. Morita: None.

Pancreatic Adipose Tissue in Diet-Induced Type 2 Diabetes

Obesity represents one of the most important health problems. Proper adipose tissue expandability is crucial for avoiding metabolic disorders. Furthermore, not all fat depots are functionally equal. Our previous work indicates that the expansion of the mesenteric adipose tissue (MWAT) plays a role in the intestinal barrier. Whether pancreas-associated adipose tissue (PAT) has a specific function is currently unknown. We hypothesize that storage of excess nutrients in PAT and PAT-derived adipokines (such as adiponectin) protect the pancreas against metabolic dysfunction during weight gain/obesity. An initial morphological study showed that PAT has an increased number of small, but fewer large adipocytes relative the adjacent MWAT. We compared the response to a 16-week high-fat diet (HFD)/obesity between PAT, other fat depots and pancreas. We found higher mRNA expression of inflammation markers in MWAT, inguinal (IWAT) and gonadal fat (GWAT), but not in PAT in response to HFD/obesity. HFD also downregulated the expression of insulin receptor and adiponectin in all fat depots, but not in PAT. The pancreas showed an increase in inflammatory and insulin resistance markers only after long-term (16 weeks) HFD exposure, a time point when also PAT displayed a reduction in insulin receptor and adiponectin expression. Smaller adipocytes are typically more functional than large adipocytes. Thus, these data suggest that PAT have an increased capacity for energy storage. This in turn may protect the pancreas against ectopic lipid accumulation and lipotoxicity. Moreover, our data show that PAT is protected against the deleterious effects of HFD, maintaining normal mRNA levels of adiponectin, insulin receptor and markers of inflammation. In summary, we believe that PAT protects the pancreatic beta-cells from lipotoxicity through its increased capacity for energy storage, sustained insulin sensitivity and possibly also increased insulin exposure because of its anatomical location. Disclosure B. Chanclon Garcia: None. Y. Wu: None. E. Banke Nordbeck: None. S. Musovic: None. C.S. Olofsson: None. P. Rorsman: None. I. Wernstedt Asterholm: None.

Depletion of CD2M2-Like Macrophages Promotes the Browning of the White Adipose Tissue

We have previously shown that CD2M2-like macrophages constitute a microenvironment for adipocyte progenitors (APs), in a Tgfβ-dependent manner, to retain systemic insulin sensitivity by tuning the quiescence/proliferation balance of APs to adapt to changes in nutritional status. Previously we generated CD206DTR transgenic mice and CD206-CreERT2/ Tgfβ1flox/flox (Tgfβ1 KO) mice to determine the role of CD2M2-like macrophages a role of CD206+ cell specific Tgfβ1 respectively, in the APs proliferation. Previous reports have shown that M2-like macrophages play a key role in the browning of WAT via activation of type 2 cytokines production during cold exposure. These findings revealed the relationship between the occurrence of beige adipocytes and M2-like macrophage recruitment. In order to investigate the role of CD2M2-like macrophages in the induction of browning into the WAT, we extended our study to see the browning phenomenon by using CD206DTR and Tgfβ1 KO mice. Here, we show that depletion of CD2M2-like macrophages promotes the browning of WAT. We report that cold stimulation resulted in the generation of smaller adipocytes, upregulation of UCP1 and CD137, a marker for beige progenitors, in the WAT of CD206-ablated mice. Flow cytometry analysis further confirmed enhanced beige progenitors in the WAT. Since blocking of Tgfβ is known to promote browning of the WAT. Mechanistically, we assume that enhanced browning into the WAT of CD206DTR mice might be due to the deletion of Tgfβ1 from CD206-M2-like macrophages. Taken together, we concluded that CD2M2-like macrophages induce the proliferation of beige progenitors in the WAT, which may serve as effective therapeutic tools for the prevention and treatment of obesity. Disclosure A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. I. Usui: None. K. Yagi: None. T. Nakagawa: None. T. Kado: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura &amp; Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. Y. Igarashi: None. K. Okabe: None. K. Saeki: None.