Glucokinase (GK) catalyses the key regulatory step in glucose-stimulated insulin secretion. Correspondingly, hetero- and homozygous mutations in human GCK cause maturity-onset diabetes of the young (GCK-MODY) and permanent neonatal diabetes mellitus (PNDM), respectively. To explore the possible utility of glucokinase activators (GKA) and of glucagon–like receptor-1 (GLP-1) agonists in these diseases, we have developed a novel hypomorphic Gck allele in mice encoding an aberrantly-spliced mRNA. In islets from homozygous knock-in (GckKI/KI) mice, GK immunoreactivity was reduced by >85%, and glucose-stimulated insulin secretion eliminated. Homozygous GckKI/KI mice displayed frank diabetes (fasting blood glucose >18 mmol/L; HbA1c ~108 mmol/mol), ketosis and nephropathy. Heterozygous GckKI/+ mice were glucose intolerant (HbA1c ~37 mmol/mol). Abnormal glucose-stimulated Ca2+ dynamics in GckKI/+ islets were completely reversed by the GKA, dorzagliatin, which was largely inactive in homozygous GckKI/KI mouse islets. The GLP-1 receptor agonist exendin-4 improved glucose tolerance in male GckKI/+ mice, an action potentiated by dorzagliatin. Sex-dependent additive effects of these agents were also observed on insulin secretion in vitro. Similar additive effects of the drugs were observed in obese hyperglycemic db/db mice. Combined treatment with GKA and incretin mimetics may thus be useful in GCK-MODY and in more common forms of type 2 diabetes.

Adipose tissue androgen turnover, dictated at least in part by the enzymes aldo-keto reductase 1 C type 2 (AKR1C2) and aldo-keto reductase 1 C type 3 (AKR1C3), has been linked to abdominal obesity. Recently, we investigated a single-nucleotide polymorphism (SNP) named rs28571858, that might increase AKR1C2 and AKR1C3 expression in human adipose tissue. Here, we studied the impact of rs28571848 on adipose tissue function and cardiometabolic health in bariatric surgery candidates. We genotyped a sample of 2,776 bariatric surgery candidates and retrospectively obtained anthropometry, blood lipid and glucose profiles, menopausal status, and medication use. In a subsample of 135 individuals (62% women, age 42 yr, body mass index of 51 kg/m2), we additionally assessed AKR1C2 and AKR1C3 expression in whole tissue by RT-qPCR. Features of adipose tissue dysfunction, such as mean adipocyte diameter and pericellular fibrosis, were assessed by histological staining and semiautomated image analysis. Finally, adipose tissue AKR1C family enzyme activity was measured by fluorimetry. The rs28571848 SNP affected AKR1C3 expression in both subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in women only, while not altering AKR1C2 expression in either men or women. Individuals carrying the minor allele exhibited increased VAT AKR1C activity compared to those with the wildtype genotype. Analysis of blood lipid profile in the whole cohort revealed that "TT" carriers had elevated total cholesterol, low-density lipoprotein-cholesterol, and indices of insulin resistance. The rs28571858 SNP increased adipose tissue AKR1C3 expression and activity in women. Increased AKR1C3 may contribute to an adipose tissue milieu that prompts lipogenesis, adversely affecting cardiometabolic health by disrupting lipid homeostasis and insulin sensitivity.NEW & NOTEWORTHY rs28571858 has a minor allele frequency of 18.7% in a cohort of 2,776 residents of Quebec, Canada. rs28571858 is associated with adipose tissue AKR1C3 expression and activity in women. Minor allele homozygote carriers have increased total cholesterol, LDL-cholesterol, and indices of insulin resistance.

Introduction: Adipocyte hypertrophy is an important marker of adipose tissue dysfunction which significantly correlates with cardiometabolic risk factors. Fat cell size increases with adiposity and then plateaus at body mass index (BMI) values higher than 30 kg/m2. It is unknown whether fat cell size still associates with markers of dysmetabolism in severe obesity. Our objectives were to examine the associations between adipocyte diameter and markers of cardiometabolic health in a large sample of participants with severe obesity while adjusting for age, BMI, and waist circumference. Methods: Biopsy samples of liver as well as abdominal subcutaneous and omental adipose tissues were obtained from 337 bariatric surgery patients. Evaluation of histological liver characteristics (steatosis, steatohepatitis, portal and lobular inflammation, hepatocellular ballooning, and hepatic fibrosis) was performed by specialized pathologists. Adipocyte diameters were measured automatically using microscopy imaging. Lipid-lipoprotein profile, glucose-insulin homeostasis, and adipokine levels were measured from blood samples. Results: Omental fat cell size was significantly larger with the presence of metabolic dysfunction-associated steatotic liver disease, metabolic dysfunction-associated steatohepatitis, liver fibrosis and lobular inflammation, as well as the presence and severity of portal inflammation. When stratified by sex, omental adipocytes were larger in women with liver injury, but not in men. Conclusion: In participants with severe obesity, associations between adipocyte hypertrophy and cardiometabolic risk factors are mostly seen in the omental fat compartment, and especially in women.

The aim of this study was to create and validate a high-throughput method based on open-source software for the measurement of adipocyte diameters in white adipose tissue histological sections. Human omental and subcutaneous adipose tissue samples collected during bariatric surgery were used to prepare haematoxylin and eosin-stained histological slides. Adipocyte diameters were measured both manually and with an automated procedure created using ImageJ. Comparative analysis of our automated method with the manual measurement and associations of the mean adipocyte diameters with cardiometabolic markers were used to validate our method. A total of 377 adipose samples (190 participants) were included in the analysis. Pearson correlation of mean adipocyte diameters showed a strong linear relationship between methods (r = 0.87, p < 0.0001). Omental adipocyte diameters of both methods were significantly associated with the same markers of cardiometabolic risk (fasting concentrations of TG, HDL-Chol, homoeostasis model assessment of insulin resistance, and visceral adiposity index values) with no significant differences between methods. There were also no significant differences between the manual and automated method regarding the correlations between mean subcutaneous adipocyte diameters and anthropometric or metabolic markers. In conclusion, we have created and validated a rapid automated method to measure adipocyte diameters from whole-slide adipose tissue images.

DFAT cells represent an attractive source of stem cells in tissue engineering and in the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT;mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing of mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes . However, the population of DFAT cells is heterogenous.

BackgroundDedifferentiated fat (DFAT) cells have been used in regenerative medicine due to their multipotent potential. According to the literature, the process of adipocyte dedifferentiation is characterized by liposecretion which results in a fibroblastlike, proliferating cell population, with increased expression of genes related to cell cycle. A number of pathways have been implicated in the process, but the role of the cell cycle in adipocyte dedifferentiation has yet to be investigated. Here we characterize the process of liposecretion, the cellular features of DFAT cells and the role of the cell cycle.MethodsPrimary adipocytes and adipocyte-derived pluripotent cells (APC) were isolated from human adipose tissue and mature adipocytes were dedifferentiated in ceiling culture. The intracellular organization of DFAT and APC were compared using transmission electron microscopy (TEM), and the changes of intracellular lipid content over time were tracked with Oil Red O. Finally, we tested whether liposecretion is a cell cycle-dependent phenomenon by cultivating mature adipocytes in ceiling culture with or without four different inhibitors of the cell cycle (AraC, Irinotecan, Vincristine and RO-3306).ResultsDFAT cells were enriched in intracellular lipids, which are stored in small lipid droplets. In addition, liposecretion, which characterizes mature adipocyte dedifferentiation, is characterized by the rapid secretion of a large lipid droplet that is coated by a membrane. This phenomenon seems to be hindered by the presence of cyclin dependent kinase 1 (CDK1) inhibitor RO-3306.ConclusionBoth human adipose tissue depots undergo dedifferentiation in vitro, but visceral adipose tissue DFAT cells retain more lipids than subcutaneous-derived DFAT cells. Liposecretion is characterized by the rapid ejection of a membrane-wrapped lipid droplet. This phenomenon is dependent on CDK1 and likely relies on the presence of integrin-mediated cellular adherence.

Context Body fat distribution is a risk factor for obesity-associated comorbidities, and adipose tissue dysfunction plays a role in this association. In humans, there is a sex difference in body fat distribution, and steroid hormones are known to regulate several cellular processes within adipose tissue. Objective Our aim was to investigate if intra-adipose steroid concentration and expression or activity of steroidogenic enzymes were associated with features of adipose tissue dysfunction in individuals with severe obesity. Methods Samples from 40 bariatric candidates (31 women, 9 men) were included in the study. Visceral (VAT) and subcutaneous adipose tissue (SAT) were collected during surgery. Adipose tissue morphology was measured by a combination of histological staining and semi-automated quantification. Following extraction, intra-adipose and plasma steroid concentrations were determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Aromatase activity was estimated using product over substrate ratio, while AKR1C2 activity was measured directly by fluorogenic probe. Gene expression was measured by quantitative PCR. Results VAT aromatase activity was positively associated with VAT adipocyte hypertrophy (P value(adj) < 0.01) and negatively with plasma high-density lipoprotein (HDL)-cholesterol (P value(adj) < 0.01), while SAT aromatase activity predicted dyslipidemia in women even after adjustment for waist circumference, age, and hormonal contraceptive use. We additionally compared women with high and low visceral adiposity index (VAI) and found that VAT excess is characterized by adipose tissue dysfunction, increased androgen catabolism mirrored by increased AKR1C2 activity, and higher aromatase expression and activity indices. Conclusion In women, increased androgen catabolism or aromatization is associated with visceral adiposity and adipose tissue dysfunction.

Abstract Background Adipose tissue is known to play an active role in androgen turnover, as illustrated by the presence of enzymes of the aldo-keto reductase 1C family such as AKR1C2 and AKR1C3, respectively coding for 3α-hydroxysteroid dehydrogenase type 3 and 17β-hydroxysteroid dehydrogenase type 5. Our group demonstrated that these enzymes are mainly expressed in mature adipocytes and that in women, trunk fat percentage is positively associated with adipose tissue mRNA abundance of both AKR1C2 and AKR1C3. In this context, our aim was to assess whether abdominal adipose tissue activity of AKR1C2 relates to excess visceral adiposity index (VAI) in women. Methods AKR1C2 activity was measured in visceral (VAT) and subcutaneous adipose tissue (SAT) of 31 women (age: 39 ± 7; BMI: 51 ± 6) undergoing bariatric surgery. Adipose tissue homogenate activity was measured by fluorimetry in a 12-hour kinetic experiment using the chemical compound cumberone, a competitive substrate in the inactivation of 5a-dihydrotestosterone to 3α-androstanediol by AKR1C2. Visceral adipose tissue excess was quantified using the previously published VAI equation and using the age-specific cut-offs for women, who were categorized into either high VAI or low VAI. Other markers of adipose tissue dysfunction included diacylglycerol acyltransferase 2 (DGAT2) and glutathione peroxidase 3 (GPX3) mRNA abundance, adipocyte size and pericellular fibrosis. Results Our technique allowed us to detect significant AKR1C2 activity in SAT (13.5±5.7 fluorescence units (FU)/min) and VAT (8.3±4.8 FU/min). Using the VAI equation, we identified 18 women with high VAI and 13 with low VAI. Women with high VAI were characterized by significantly higher VAT AKR1C2 activity when compared to women with low VAI (9.5 ± 4.4 vs. 6.4± 3.6 FU/min, p<0. 05). AKR1C2 mRNA abundance in either VAT or SAT was not significantly different as a function of VAI. Women with high VAI also had larger VAT adipocyte diameter and lower mRNA abundance of GPX3 in both adipose tissue depots compared to women with low VAI (p<0. 05 for all). No differences were seen in DGAT2 mRNA abundance, pericellular fibrosis or SAT adipocyte diameter as a function of VAI. Conclusion In women with severe obesity, excess visceral adiposity as indicated by a high VAI, is associated with higher AKR1C2 activity in VAT only. Similarly, adipose tissues of women with high VAI displayed features of adipose tissue dysfunction such as increased VAT adipocyte diameter and decreased GPX3expression in comparison to women with low VAI. Taken together our results confirm the link between adipose AKR1C2 and excess visceral adiposity. Presentation: No date and time listed

Weight loss is key to controlling the increasing prevalence of metabolic syndrome (MS) and its components, i.e., central obesity, hypertension, prediabetes and dyslipidaemia. The goals of our study were two-fold. First, we characterised the relationships between eating duration, unprocessed and processed food consumption and metabolic health. During 4 weeks of observation, 213 adults used a smartphone application to record food and drink consumption, which was annotated for food processing levels following the NOVA classification. Low consumption of unprocessed food and low physical activity showed significant associations with multiple MS components. Second, in a pragmatic randomised controlled trial, we compared the metabolic benefits of 12 h time-restricted eating (TRE) to standard dietary advice (SDA) in 54 adults with an eating duration > 14 h and at least one MS component. After 6 months, those randomised to TRE lost 1.6% of initial body weight (SD 2.9, p = 0.01), compared to the absence of weight loss with SDA (−1.1%, SD 3.5, p = 0.19). There was no significant difference in weight loss between TRE and SDA (between-group difference −0.88%, 95% confidence interval −3.1 to 1.3, p = 0.43). Our results show the potential of smartphone records to predict metabolic health and highlight that further research is needed to improve individual responses to TRE such as a shorter eating window or its actual clock time.

OBJECTIVE:Both the cortisol awakening response (CAR; corresponding to the state measurement) and hair cortisol concentration (HCC; corresponding to the trait measurement) are considered reliable markers of hypothalamus-pituitary-adrenal axis activity. Because cortisol has long been associated with adiposity, this systematic review and meta-analysis aims to summarize and compare the literature around CAR and HCC and their association with obesity or fat distribution indices. METHODS:The PubMed, Web of Science (Web of Science Core Collection and Medline), EBSCO Information Services, Embase, and PsycNET databases were searched, and full-text articles investigating the association between CAR or HCC and markers of adiposity in humans were included. Meta-analyses were then performed to compare studies associating CAR or HCC with BMI (a marker of general adiposity) and waist circumference (a marker of fat distribution). RESULTS:The results of this review highlight inconsistencies in cortisol sampling and CAR computation, which makes comparisons between studies difficult. It was found that adiposity indices are not associated with CAR but that they correlate significantly and positively with HCC. The subgroup analysis hinted to possible age differences in the magnitude of the association between HCC and BMI. CONCLUSIONS:Trait rather than state measurement of the hypothalamus-pituitary-adrenal axis activity is associated with increased general and abdominal adiposity in humans.

Abstract Background: There is a large body of evidence linking obesity to the HPA axis and cortisol secretion or metabolism. Early work hypothesized that obesity onset may be associated with over-activation of the HPA axis, causing an extenuation of the system. The cortisol awakening response (CAR) has been suggested to be a reliable marker of HPA axis activity and has already been examined in samples of individuals with obesity. Our objective was to compare individuals showing a morning cortisol peak (CAR responders) from those who did not (non-responders) and identify possible metabolic or psychological differences among these two groups. Our hypothesis was that the two groups differed in the level of anxiety and aspects of their personality rather than in their metabolic profile. Methods: CAR response was determined using a baseline to peak cut-off of 2.5 nM. Nine CAR non-responder women awaiting bariatric surgery (BMI: 50.8 ± 4.6 kg/m2) were compared to 9 sex- and age-matched CAR responders (BMI: 48.1 ± 5.9 kg/m2). Participants collected salivary cortisol upon awakening as well as 15, 30 minutes after and responded to psychological questionnaires that measured anxiety (State and trait anxiety inventory questionnaire) and personality traits (Temperament and character inventory). Results: Non-responders were all non-diabetic women aged 37 ± 8 years. There was no significant difference between CAR responders and non-responders in terms of BMI or waist circumference. No difference was found in metabolic variables such as glycaemia or the lipid profile. As expected, non-responders had a significantly lower CAR AUCi when compared to responders (p<0.001). However there was no difference in awakening cortisol concentration. Despite our hypothesis, no significant difference was found in general level of anxiety between the two groups. Finally, we analyzed aspects of human personality. We found that CAR responders scored significantly higher in character traits such as self-directedness (p=0.02), cooperativeness (p=0.03) and self-transcendence (p<0.01). Conclusion: The CAR differences in women with severe obesity are not associated with adiposity. Our data show that non-responders exhibit traits related to reduced self-determination and responsibility but also lower level of self-consciousness and helpfulness, which could be associated with a reduction in patient compliance and possibly less weight loss after surgery.

Background: Changes in androgen dynamics within adipose tissue have been proposed as modulators of body fat accumulation. In this context, AKR1C2 likely plays a significant role by inactivating 5 alpha-dihydrotestosterone. Aim: To characterize AKR1C2 expression patterns across adipose depots and cell populations and to provide insight into the link with body fat distribution and genetic regulation. Methods: We used RNA sequencing data from severely obese patients to assess patterns of AKR1C2 and AKR1C3 expression in abdominal adipose tissue depots and cell fractions. We additionally used data from 856 women to assess AKR1C2 heritability and to link its expression in adipose tissue with body fat distribution. Further, we used public resources to study AKR1C2 genetic regulation as well as reference epigenome data for regulatory element profiling and functional interpretation of genetic data. Results: We found that mature adipocytes and adipocyte-committed adipocyte progenitor cells (APCs) had enriched expression of AKR1C2. We found adipose tissue AKR1C2 and AKR1C3 expression to be significantly and positively associated with percentage trunk fat mass in women. We identified strong genetic regulation of AKR1C2 by rs28571848 and rs34477787 located on the binding sites of two nuclear transcription factors, namely retinoid acid-related orphan receptor alpha and the glucocorticoid receptor. Conclusion: We confirm the link between AKR1C2, adipogenic differentiation and adipose tissue distribution. We provide insight into genetic regulation of AKR1C2 by identifying regulatory variants mapping to binding sites for the glucocorticoid receptor and retinoid acid-related orphan receptor alpha which may in part mediate the effect of AKR1C2 expression on body fat distribution.

Abstract Background: Excess visceral adipose tissue accumulation on anatomical structures such as the greater omentum and mesentery are strong predictors of obesity-associated comorbidities (1). High glucocorticoid levels have been associated with body fat distribution and preferential visceral fat accumulation as well as features of the metabolic syndrome (MetS) (2). These effects are thought to be mediated by the glucocorticoid receptor, a nuclear receptor showing affinity for both glucocorticoids and mineralocorticoids. In this study, we examined plasma concentrations of glucocorticoids and mineralocorticoids in women with or without the MetS. In addition, we assessed the ability of these steroids to predict fat accumulation and features of the MetS. Methods: In a sample of 49 women (age 47 ± 4.99 years; BMI 26.4 ± 4.70 kg/m2), plasma concentrations of cortisol, 11-deoxycortisol, cortisone, aldosterone, corticosterone and 11-deoxycorticosterone were analyzed by electrospray ionization-liquid chromatography-tandem mass spectroscopy (ESI-LC-MS/MS). Metabolic parameters were assessed to establish the presence of the MetS using NCEP-III criteria. Subcutaneous and visceral adipocyte cell size was measured by histomorphometry. Results: We found HDL-triglycerides to be positively associated with levels of 11-deoxycorticosterone, 11-deoxycortisol, corticosterone, cortisone and cortisol (p<0.05 for all). 11-deoxycorticosterone concentration was also negatively associated with waist circumference (-0.294, p<0.05), LDL-cholesterol and LDL-triglyceride content (-0.264 and -0.362, p<0.05) whereas cortisone level was positively associated with fasting glucose (0.3, p<0.05). Our model including mineralocorticoids predicted systolic blood pressure (R2=0.303), while the one including glucocorticoids predicted HDL-cholesterol (R2=0.495). In addition, as expected, we found that women with the MetS were characterized by significantly higher percentage body fat and displayed subcutaneous and visceral adipocyte hypertrophy (p<0.05). Interestingly, women with the MetS also showed a trend for lower plasma cortisol concentrations (p=0.07). Conclusion: Our data suggest that glucocorticoids and mineralocorticoids are associated with individual components of the MetS in women. (1) Tchernof et al.(2013), Physiol Rev, 93(1); (2) Constantinopoulos et al., (2015), Eur J Endocrinol, 172(1)

This review addresses the impact of bariatric surgery on the endocrine aspects of white adipose tissue, muscle and the liver. We describe literature supporting the notion that adipokines, myokines and hepatokines likely act in concert and drive many of the long-term metabolic improvements following surgery. Circulating adiponectin is increased while secretion of pro-inflammatory interleukins (1, 6 and 8) decreases, alongside leptin secretion. The metabolic improvements observed in the muscle might relate to reduction of myokines contributing to insulin resistance (including myostatin, brain-derived neurotrophic factor and fibroblast growth factor-21). Subject to exception, hepatokine secretion is generally increased (such as insulin-like growth factor-binding protein 2, adropin and sex hormone-binding globulin). In conclusion, bariatric surgery restores metabolic functions by enhancing the time-dependent secretion of anti-inflammatory, insulin-sensitizing and antilipemic factors. Further research is needed to understand the molecular mechanisms by which these factors may trigger the remission of obesity-related comorbidities following bariatric surgery.

It is largely believed that after undergoing differentiation, adipocytes can no longer divide. Yet, using ceiling culture, it was demonstrated in vitro that some adipocytes are able to regain proliferative abilities by becoming fibroblast-like cells called dedifferentiated adipocytes. Mature adipocytes are abundant, can be easily isolated, and represent a homogenous cell population. Because of these advantageous characteristics, dedifferentiated adipocytes are clinically attractive in tissue engineering as a potential treatment resource for conditions such as type 2 diabetes, cardiac and kidney diseases, as well as autoimmune diseases. The aim of this review article is to summarize current knowledge on adipocyte dedifferentiation by accurately describing dedifferentiated adipocyte characteristics such as morphological appearance, gene expression, antigen signature, pluripotency, and functionality. Current hypotheses possibly explaining the biological mechanisms and cellular reprogramming of the dedifferentiation process are summarized. Dedifferentiated adipocytes show a stem cell-like antigen profile and genome signature which add to their proliferative capacities and their ability to re-differentiate into diverse cell lineages. The dedifferentiation process likely involves liposecretion, that is, the rapid secretion of the cell's lipid droplet. Dedifferentiated adipocytes may allow development of new uses in tissue engineering.

ObjectiveTo 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. MethodsMature 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 -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. ResultsTreatment 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. ConclusionOur 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.