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Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity-related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non-coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN(adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAINknockdown (KD) increases KLF5 and interleukin-8 (IL-8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL-8, via linc-ADAINKD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitroand in vivo. KD of linc-ADAINin human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity-induced adipose tissue remodeling. linc-ADAINis an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Treatment with PPARγ agonists in vivo improves human adipocyte metabolism, but the cellular mechanisms and possible depot differences in responsiveness to their effects are poorly understood. To examine the ex vivo metabolic effects of rosiglitazone (Rosi), we cultured explants of human visceral (omental) and abdominal subcutaneous adipose tissues for 7 days. Rosi increased mRNA levels of transcriptional regulators of brite/beige adipocytes (PGC1α, PRDM16), triglyceride synthesis (GPAT3, DGAT1), and lipolysis (ATGL) similarly in adipose tissues from both depots. In parallel, Rosi increased key modulators of FA oxidation (UCP1, FABP3, PLIN5 protein), rates of FA oxidation, and protein levels of electron transport complexes, suggesting an enhanced respiratory capacity as confirmed in newly differentiated adipocytes. Rosi led to the formation of small lipid droplets (SLDs) around the adipocyte central lipid droplet; each SLD was decorated with redistributed mitochondria that colocalized with PLIN5. SLD maintenance required lipolysis and FA reesterification. Rosi thus coordinated a structural and metabolic remodeling in adipocytes from both visceral and subcutaneous depots that enhanced oxidative capacity. Selective targeting of these cellular mechanisms to improve adipocyte FA handling may provide a new approach to treat metabolic complications of obesity and diabetes.

Direct comparisons between types of dietary carbohydrate in terms of cardiometabolic risk indicators are limited. This study was designed to compare the effects of an isocaloric exchange of simple, refined, and unrefined carbohydrates on serum cardiometabolic risk indicators, adipose tissue inflammatory markers, and peripheral blood mononuclear cell (PBMC) fractional cholesterol efflux.

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The glucocorticoid-induced leucine zipper (GILZ), a primary target of glucocorticoids, is expressed in human adipocytes, but its importance in adipocyte function is unknown. Because TNFα is increased in obese adipose tissue and antagonizes a number of glucocorticoid actions, we investigated the interplay of these pathways. GILZ knockdown increased and GILZ overexpression decreased interleukin-6 (IL-6) and leptin mRNA and protein secretion. GILZ knockdown increased the magnitude of the glucocorticoid effect on leptin secretion, but did not affect the glucocorticoid suppression of IL-6. Although GILZ silencing decreased adiponectin mRNA levels, it did not affect the amount of adiponectin secreted. GILZ negatively modulated pro-inflammatory signaling pathways, blocking basal and TNFα-stimulated (1 h) p65 nuclear factor κB nuclear translocation and transcriptional activity by binding to p65 in the cytoplasm. GILZ silencing increased basal ERK1/2 and JNK phosphorylation, and decreased MAPK phosphatase-1 protein levels. Longer term TNFα (4 h or 24 h) treatment decreased GILZ expression in human adipocytes. Furthermore, adipose tissue GILZ mRNA levels were reduced in proportion to the degree of obesity and expression of inflammatory markers. Overall, these results suggest that GILZ antagonizes the pro-inflammatory effects of TNFα in human adipocytes, and its downregulation in obesity may contribute to adipose inflammation and dysregulated adipokine production, and thereby systemic metabolism.

Early postnatal exposures to sex steroids have been well recognized to modulate predisposition to diseases of adulthood. There is a complex interplay between timing, duration and dose of endocrine exposures through environmental or dietary sources that may alter the sensitivity of target tissues to the exogenous stimuli. In this study, we determined the metabolic and reproductive programming effects of a single developmentally entrained pulse of testosterone (T) given to female mice in early postnatal period. CD-1 female mice pups were injected with either 5 μg of T enanthate (TE) or vehicle (control [CON] group) within 24 hours after birth and followed to adult age. A total of 66% of T-treated mice exhibited irregular cycling, anovulatory phenotype, and significantly higher ovarian weights than vehicle-treated mice. Longitudinal nuclear magnetic resonance measurements revealed that TE group had greater body weight, whole-body lean, and fat mass than the CON group. Adipose tissue cellularity analysis in TE group revealed a trend toward higher size and number than their littermate CONs. The brown adipose tissue of TE mice exhibited white fat infiltration with down-regulation of several markers, including uncoupling protein 1 (UCP-1), cell death-inducing DNA fragmentation factor, α-subunit-like effector A, bone morphogenetic protein 7 as well as brown adipose tissue differentiation-related transcription regulators. T-injected mice were also more insulin resistant than CON mice. These reproductive and metabolic reprogramming effects were not observed in animals exposed to TE at 3 and 6 weeks of age. Collectively, these data suggest that sustained reproductive and metabolic alterations may result in female mice from a transient exposure to T during a narrow postnatal developmental window.

Recent studies demonstrate that adipose tissue undergoes a continuous process of remodeling that is pathologically accelerated in the obese state. Contrary to earlier dogma, adipocytes die and are replaced by newly differentiated ones. This review will summarize recent advances of our knowledge of the mechanisms that regulate adipose tissue remodeling and highlight the influences of obesity, depot, and sex, as well as the relevance of rodent models to humans.

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of ‘basal’ [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of 'basal’ [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.

The mechanisms of the increased serum leptin in response to feeding are poorly understood. Therefore, we used metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover in adipose tissue from 14 h-starved compared with fed 12–14 week old rats. Starvation decreased serum leptin (–47 ± 7%), adipose tissue leptin content (–32 ± 5%), and leptin secretion during 3 h of incubation (–65 ± 12%). Starvation did not affect leptin mRNA levels but decreased rates of leptin biosynthesis by tissue fragments, as determined by [35S]methionine/cysteine incorporation into immunoprecipitable leptin. Insulin in vitro did not acutely increase leptin biosynthesis or rates of 125I-leptin degradation. Pulse-chase studies showed that in adipose tissue from fed but not starved rats, insulin accelerated the secretion of [35S]leptin by ∼2-fold after 30 and 60 min of chase. Degradation of newly synthesized leptin was slower in adipose tissue of starved than fed rats (half-lives of 50 and 150 min, respectively). Inhibitor experiments showed that both lysosomes and proteosomes contributed to leptin degradation. In conclusion, feeding compared with starvation influences leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion. The insulin-stimulated release of leptin from a preformed intracellular leptin pool may contribute to increases in serum leptin levels after meals.

The mechanisms of the increased serum leptin in response to feeding are poorly understood. Therefore, we used metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover in adipose tissue from 14 h-starved compared with fed 12–14 week old rats. Starvation decreased serum leptin (−47 ± 7%), adipose tissue leptin content (−32 ± 5%), and leptin secretion during 3 h of incubation (−65 ± 12%). Starvation did not affect leptin mRNA levels but decreased rates of leptin biosynthesis by tissue fragments, as determined by [35S]methionine/cysteine incorporation into immunoprecipitable leptin. Insulin in vitro did not acutely increase leptin biosynthesis or rates of 125I-leptin degradation. Pulse-chase studies showed that in adipose tissue from fed but not starved rats, insulin accelerated the secretion of [35S]leptin by ∼2-fold after 30 and 60 min of chase. Degradation of newly synthesized leptin was slower in adipose tissue of starved than fed rats (half-lives of 50 and 150 min, respectively). Inhibitor experiments showed that both lysosomes and proteosomes contributed to leptin degradation. In conclusion, feeding compared with starvation influences leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion. The insulin-stimulated release of leptin from a preformed intracellular leptin pool may contribute to increases in serum leptin levels after meals.

We have previously shown that exposure of C57BL/6J mice to intermittent hypoxia (IH) leads to 1) hypertriglyceridemia due to upregulation of pathways of lipid biosynthesis, including sterol regulatory element binding protein (SREBP)-1 and stearoyl CoA desaturase (SCD)-1; and 2) hypercholesterolemia due to impaired cholesterol uptake. The goal of the present study was to examine whether hypoxia-inducible factor (HIF)-1 is implicated in changes in lipid metabolism induced by IH. Lean HIF-1α (Hif1a)+/−mice, which are heterozygous for a null allele at the locus encoding the HIF-1α subunit, and their wild-type (WT) Hif1a+/+littermates were exposed to IH or control conditions for 5 days. IH increased fasting blood glucose, serum total cholesterol, and high-density lipoprotein-cholesterol, phospholipids, triglycerides (TG), and leptin in mice of both genotypes, whereas serum insulin and interleukin-6 were elevated only in WT mice. The impact of IH on serum TG levels in WT mice was significantly greater than that in Hif1a+/−mice (95 ± 9 vs. 66 ± 6 mg/dl, P< 0.05), whereas cholesterol and glucose levels were affected independently of genotype. Under hypoxic conditions, mRNA and protein levels of SREBP cleavage-activating protein (SCAP) and SCD-1 and protein levels of nuclear isoform of SREBP-1 in the liver were induced to significantly higher levels in WT mice than in Hif1a+/−mice. We conclude that 1) the effect of IH on serum TG levels is mediated through HIF-1, 2) HIF-1 may impact on posttranscriptional regulation of SREBP-1, and 3) the effect of IH on serum cholesterol levels was not altered by partial HIF-1α deficiency.

Objective: Perilipins are phosphoproteins that are localized to the surface of triacylglycerol droplets within adipocytes where they regulate the rate of lipolysis. We sought to determine the effects of severe obesity and depot [omental (Om) vs. subcutaneous (Sc)] on perilipin expression in the adipose tissue of individuals. Research Methods and Procedures: Samples of Om and Sc adipose tissues obtained at surgery from severely obese subjects and fat aspirations from nonobese subjects were analyzed for perilipin protein and mRNA levels by Northern and Western analysis. Results: Perilipin A (periA) was the major perilipin expressed in adipose tissues. periA mRNA relative abundance was significantly lower in Sc adipose tissue from severely obese compared to that from nonobese subjects. Western blotting of adipose tissue extracts showed that periA protein levels expressed relative to tissue protein or fat cell surface area were significantly lower (∼ −40%) in abdominal Sc adipose tissue from severely obese compared to that from nonobese subjects. However, the calculated mass of perilipin per fat cell did not differ between the two groups. Perilipin mRNA levels were higher in Sc compared to Om adipose tissue from obese individuals (p< 0.025; n= 26; 17 women, 9 men); however, periA protein levels did not differ. In addition, perilipin protein, but not mRNA, levels were higher in Sc adipose tissue from obese men than from women (p< 0.025). Discussion: Variations in perilipin expression may contribute to the higher basal lipolytic rates observed in obese compared to nonobese individuals and in obese women compared to obese men.

The current study aimed to compare the effects of treatment (2 months) with thiazolidinediones (TZDs) and placebo on glucose and fat metabolism in patients with type 2 diabetes (T2DM) in a crossover design. Eight patients received placebo (2 months) followed by TZD (2 months). Two-stage (1.5 and 6.0 pmol/kg min) hyperinsulinemic-euglycemic clamps were performed in all 8 patients pre- and post-placebo and post-TZD (post-placebo = pre-TZD). We determined rates of glucose disappearance (GRd), glycolysis (GLS), glycogen synthesis (GS) (all with 3-3H glucose), carbohydrate (CHO) oxidation (indirect calorimetry), endogenous glucose production (EGP), free fatty acid (FFA) release (2H5glycerol), and oxidation (indirect calorimetry) and re-esterification, as well as plasma adiponectin and leptin concentrations, and fat cell size and number (determined in upper thigh biopsy samples). Placebo treatment had no effects on any of the measured parameters. TZD treatment improved insulin-stimulated glucose uptake (ISGU) from 17.1 to 26.4 μmol/kg min (P< .01) and insulin-stimulated GS from 4.8 to 13.4 μmol/kg min (P< 0.03), potentiated insulin-induced suppression of lipolysis from 4.3 to 2.3 μmol/kg min (P< .03) and FFA re-esterification from 1.9 to 1.0 μmol/kg min (P< .02), increased plasma adiponectin levels from 2.7 to 7.2 μg/mL (P< .05), and decreased plasma leptin levels from 30.8 to 23.4 ng/mL (P< .02). In addition, TZD tended to increase the number of small adipocytes (<50 μm) in subcutaneous adipose tissue. We conclude that TZDs have multiple actions and that many, but perhaps not all, can be accounted for by their action on adipose tissue.

Objective:We have previously shown that morning administration of dexamethasone in combination with food induces a doubling of serum leptin levels starting at 7 hours after dexamethasone administration, with a maximum effect at 10 hours, the latest time point that we have studied. However, dexamethasone given in the absence of food had no effect on serum leptin at 10 hours. The present experiment was undertaken to determine the duration of the effect of dexamethasone on 24‐hour serum leptin under fasted and fed conditions in humans. Research Methods and Procedures:Six healthy non‐obese male volunteers were studied under the following four conditions: 1) dexamethasone (2 mg intravenously, given at 0900 hours) with fasting; 2) dexamethasone with food (1700 kcal, 55% carbohydrate, 15% protein, and 30% fat, given in one meal 2 hours after dexamethasone administration at 1100 hours); 3) saline with food (same meal); 4) saline with fasting. Serum leptin, glucose, insulin, and cortisol were monitored every 30 minutes for 24 hours. Results:1) Under the fasting condition, dexamethasone increased leptin nocturnal secretion between 2100 and 2400 hours. 2) A single meal (1700 kcal) at 1100 hours increased nocturnal leptin secretion when compared with the fasting condition. The peak increase of leptin was 123% over baseline between 2100 and 2400 hours, 10 to 14 hours after the meal. 3) In the fed + dexamethasone condition, leptin levels increased from baseline starting 8 hours after dexamethasone injection, reached a maximum increase of 260% between 2100 and 2400 hours, then decreased thereafter, remaining elevated compared to baseline for 16 hours. There was a correlation between 24‐hour leptin secretion and insulin secretion after a single morning meal. Discussion:A single bolus of dexamethasone, given before a single large meal, produces a delayed (6‐hour) but long‐lasting increase in serum leptin (over 16 hours). Under fasted conditions, dexamethasone does not increase daytime leptin but does increase leptin during the night.

RICCI, MATTHEW R. AND SUSAN K. FRIED. Isoproterenol decreases leptin expression in adipose tissue of obese humans. Obes Res. Objective: We investigated the effects of the non‐selective β‐adrenergic agonist, isoproterenol (Iso), on leptin expression in human adipose tissue. Research Methods and Procedures: Subcutaneous (SQ) and omental adipose (OM) tissue taken during surgery from 12 morbidly obese subjects (10 women and 2 men) were cultured for up to 24 hours with insulin (7 nM) and/or dexamethasone (25 nM), a synthetic glucocorticoid, in the presence or absence of isoproterenol (10 μM). Adipose tissue was also acutely incubated for 3 hours in media alone with or without isoproterenol. Leptin secretion and leptin mRNA abundance were measured. Results: Iso acutely decreased leptin release by −30% (vs. no hormone controls) in fragments of OM and SQ adipose tissue. In 24‐hour culture, addition of Iso (in the presence of insulin) resulted in lower leptin accumulation in the medium (−20–30%) and leptin mRNA levels (−40–50%) from both tissue depots. Culture with insulin and dexamethasone increased leptin expression vs. insulin alone. Addition of Iso with insulin and dexamethasone decreased media leptin (−40–60%) and leptin mRNA levels were lower (−65%) in Iso‐treated adipose tissue from both depots after 24 hours. Iso effects were not detectable after 5 hours of culture. Discussion: We conclude that stimulation of β‐adrenergic receptors may modulate leptin expression in human adipose tissue by two mechanisms: an acute effect on leptin release and a longer‐term antagonism of stimulatory effects of insulin and dexamethasone on leptin mRNA expression. These mechanisms may contribute to the decline in serum leptin that occurs during fasting.

A previously undescribed single missense mutation (C→G) was detected within exon 5 of the LPL gene in two members of an Italian family affected with type 1 hyperlipoproteinemia. This mutation causes a highly conservative amino acid replacement (Asp→Glu) at position 180 of the mature LPL protein resulting in a virtual absence of LPL enzyme activity and LPL enzyme mass in postheparin plasma. Adipose tissue mRNA concentrations and mRNA sizes were not affected. Both patients were homozygous for the mutation, whereas the parents were heterozygous. Comparison of the expression of the mutated cDNA and the wildtype cDNA in cos-7 cells revealed proper transcription and translation of the mutated clone into an immunologically detectable protein. The mutated LPL protein was secreted from the cells in a manner similar to that of wildtype LPL and bound to heparin-Sepharose with identical properties. However, the mutated enzyme, in contrast to wildtype LPL, exhibited no detectable lipolytic activity against a triglyceride substrate. Our results demonstrate that even a highly conservative amino acid replacement outside the proposed active site of LPL is incompatible with proper enzyme function.

Intraabdominal fat in humans is located in two major depots, the omental and mesenteric. We compared basal and stimulated lipolysis in adipose tissue from these two depots and the subcutaneous abdominal depot of obese women and men. Omental fat cells of women are smaller and have lower rates of basal lipolysis than in men. Basal Iipolysis rates are significantly higher in subcutaneous than intraabdominal adipose tissues of both genders. In men, the incremental lipolytic response to norepinephrine is significantly greater in both intraabdominal fat depots than in the subcutaneous fat, while in women tlie response of tlie mesenteric is lower than tlie omental. In women, but not men, responsiveness to tlie beta agonist isoproterenol is also increased in omental tissue. Thus, in women, omental and mesenteric adipose tissues show distinctly different metabolic properties which may moderate the impact of intraabdominal obesity.

The effect of insulin on the regulation of lipoprotein lipase (LPL) activity in isolated adipocytes from rats in various nutritional states was studied. Because LPL is secreted from adipocytes, possible insulin effects on LPL activity were assessed as 1) total cellular LPL activity, 2) LPL activity spontaneously secreted during incubations and 3) LPL secretion during perifusion of adipocytes preincubated with or without insulin. Incubation with insulin for 2 h produced no increase in LPL activity associated with adipocytes isolated from 5- to 6-wk-old fasted rats but increased by 83% the activity of LPL secreted into the incubation medium. Insulin pretreatment increased by 230% the capacity of the cells to secrete LPL activity when perifused in the presence of 5% fasted human serum. In adipocytes from young rats killed in the fed state, preincubation with insulin caused a small increase in cell-associated LPL activity (+21%) but no increase in LPL activity spontaneously secreted into the incubation medium. Similar to results in fasted rats, insulinpretreated cells showed a 101% increase in their ability to secrete LPL activity during a perifusion. In contrast, larger adipocytes from 3-mo-old rats were totally unresponsive to insulin effects on both cellular and secreted LPL. However, 3 d of fasting followed by 9 d of refeeding restored responsiveness to insulin effects on both cellular and secreted LPL activity. Thus, variations in cellular responsiveness to insulin effects on LPL activity may play an important role in regulating nutrition-induced changes in LPL activity. Moreover, a major action of insulin on adipocyte LPL activity is to increase the cellular capacity for LPL secretion. This may be of importance in determining its functional activity at its extracellular site of action.

As a consequence of their increased total adipose tissue mass, morbidly obese patients have many more fat cells than individuals of normal weight and the cells are enlarged compared to normal. Contrary to earlier beliefs, fat cell numbers can increase throughout adult life. Once formed, fat cells do not undergo involution. Thus, it seems that an individual who has reached the morbidly obese state maintains an increased amount of body fat that limits his/her ability to achieve and maintain commonly accepted levels of “ideal” body weight.

We studied the effects of a 3-day fast and 20 days of refeeding on lipoprotein lipase activity (LPL) in the interscapular brown adipose tissue (BAT) of sexually mature male rats. Rats were refed either ad libitum or restricted quantities (75 or 50% of normal ad libitum food intake). BAT-LPL, expressed on a per depot basis, decreased with fasting and returned to control values by day 3 of refeeding ad libitum. In contrast, refeeding restricted quantities for 5 to 20 days limited regain of body weight and increased BAT-LPL significantly above the values observed in rats refed ad libitum. These adaptations in BAT-LPL activity differ markedly from those observed in rat white adipose tissue or heart muscle and may serve to increase the supply of fatty acids from circulating lipoproteins for BAT metabolic needs and maintenance of thermogenesis during undernutrition.

We studied lipoprotein activity in white adipose tissue of male rats during a 3-day fast and up to 21 days of refeeding. Changes in LPL were considered in relation to concomitant changes in fat cell size. During fasting fat cell size decreased and LPL activity declined markedly (80%). With ad libitum refeeding LPL activity returned to control values by 3 or 5 days of refeeding, increased above control values (60–100%) by 10 days of refeeding and returned to control values by 20 days of refeeding. The normalization and overshoot of LPL activity preceded and accompanied increases in fat cell size, wich occurred between days 5 and 10 of refeeding; LPL activity returned to control values only after fat cell size also returned to control values. Mild food restriction (approximately 25%) during refeeding allowed LPL activity to return to control values but reduced the magnitude and duration of the overshoot. Severe food restriction (approximately 50%) during refeeding delayed the return of LPL activity to control values until day 20 of refeeding. These data support a role for LPL in restoration of adipocyte lipid stores during refeeding. Food restriction during refeeding influences LPL and may modulate the rate of lipid accumulation by adipocytes during refeeding.