Response of multiple hormones to glucose and arginine challenge in T2DM after gastric bypass.

Purpose: In patients with type 2 diabetes mellitus (T2DM), Roux-en-Y gastric bypass (RYGB) leads to beneficial metabolic adaptations, including enhanced incretin secretion, beta-cell function, and systemic insulin sensitivity. We explored the impact of RYGB on pituitary, pancreatic, gut hormones, and cortisol responses to parenteral and enteral nutrient stimulation in patients with obesity and T2DM with repeated sampling up to 2 years after intervention. Methods: We performed exploratory post hoc analyses in a previously reported randomized trial. Levels of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH), glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), peptide YY (PYY), ACTH, insulin, and glucagon were measured in 13 patients with T2DM and obesity at four different visits: before and 4, 24, and 104 weeks after RYGB; and in three sequential conditions on the same day: fasting, intravenous arginine challenge, and OGTT. Results: RYGB surprisingly induced a rise in ACTH, cortisol, and GH levels upon an oral glucose load, together with enhanced GLP-1 and PYY responses. Fasting and post-arginine GH levels were higher after RYGB, whereas insulin, glucagon, GLP-1, GIP, and cortisol were lower. These endocrine adaptations were seen as early as 4 weeks after surgery and were maintained for up to 2 years. Conclusion: These findings indicate adaptations of glucose sensing mechanisms and responses in multiple endocrine organs after RYGB, involving the gut, pancreatic islets, the pituitary gland, the adrenals, and the brain.

ESR2 expression in subcutaneous adipose tissue is related to body fat distribution in women, and knockdown impairs preadipocyte differentiation.

Oestrogen receptor 2 (ESR2) expression has been shown to be higher in subcutaneous adipose tissue (SAT) from postmenopausal compared to premenopausal women. The functional significance of altered ESR2 expression is not fully known. This study investigates the role of ESR2 for adipose tissue lipid and glucose metabolism. SAT biopsies were obtained from 44 female subjects with or without T2D. Gene expression of ESR2 and markers of adipose function and metabolism was assessed. ESR2 knockdown was performed using CRISPR/Cas9 in preadipocytes isolated from SAT of females, and differentiation rate, lipid storage, and glucose uptake were measured.ESR2 expression was inversely correlated with measures of central obesity and expression of some fatty acid oxidation markers, and positively correlated with lipid storage and glucose transport markers. Differentiation was reduced in ESR2 knockdown preadipocytes. This corresponded to reduced expression of markers of differentiation and lipogenesis. Glucose uptake was reduced in knockdown adipocytes.Our results indicate that ESR2 deficiency in women is associated with visceral adiposity and impaired subcutaneous adipocyte differentiation as well as glucose and lipid utilization. High ESR2 expression, as seen after menopause, could be a contributing factor to SAT expansion. This may support a possible target to promote a healthy obesity phenotype.

Excess glucocorticoid exposure contributes to adipose tissue fibrosis which involves macrophage interaction with adipose precursor cells.

Chronic exposure to elevated glucocorticoid levels, as seen in patients with Cushing's syndrome, can induce adipose tissue fibrosis. Macrophages play a pivotal role in adipose tissue remodelling. We used the synthetic glucocorticoid analogue dexamethasone to address glucocorticoid effects on adipose tissue fibrosis, in particular involving macrophage to preadipocyte communication. We analysed the direct effects of dexamethasone at a supra-physiological level, 0.3 µM, on gene expression of pro-fibrotic markers in human subcutaneous adipose tissue. The effects of dexamethasone on the differentiation of human SGBS preadipocytes were assessed in the presence or absence of THP1-macrophages or macrophage-conditioned medium. We measured the expression of different pro-fibrotic factors, including α-smooth muscle actin gene (ACTA2) and protein (α-SMA). Dexamethasone increased the expression of pro-fibrotic genes, e.g. CTGF, COL6A3, FN1, in adipose tissue. Macrophages abolished preadipocyte differentiation and increased the expression of the ACTA2 gene and α-SMA protein in preadipocytes after differentiation. Exposure to dexamethasone during differentiation reduced adipogenesis in preadipocytes, and elevated the expression of pro-fibrotic genes. Moreover, dexamethasone added together with macrophages further increased ACTA2 and α-SMA expression in preadipocytes, making them more myofibroblast-like. Cells differentiated in the presence of conditioned media from macrophages pretreated with or without dexamethasone had a higher expression of profibrotic genes compared to control cells. Our data suggest that macrophages promote adipose tissue fibrosis by directly interfering with preadipocyte differentiation and stimulating gene expression of pro-fibrotic factors. Excess glucocorticoid exposure also has pro-fibrotic effect on adipose tissue, but this requires the presence of macrophages.

Evaluation of RNA Isolation Methods in Human Adipose Tissue.

OBJECTIVE: Research has shown that RNA extraction from adipose tissue (AT) is challenging because of high lipid content and low RNA quantity. We compared a traditional RNA extraction with a column-based method in human AT to evaluate RNA quantity and quality. MATERIALS AND METHODS: Human subcutaneous AT (n = 9) was collected through needle biopsy, and RNA was extracted using the phenol-chloroform traditional method and the RNeasy Lipid Tissue Mini Kit column-based method. The RNA quantity, quality, integrity, and expression of key AT genes were assessed. RESULTS: We found that the RNA quantity and integrity were reduced by 40% and 15-20%, respectively, using the column-based method compared to the traditional method, but the findings were not statistically significant. The column-based method showed a higher 260/280 ratio (~2.0) compared to the traditional method (~1.8) (P <.05), suggesting lower amounts of contaminants. The expression of AT genes was comparable between methods. CONCLUSION: The traditional extraction method provides adequate RNA yield and integrity compared to the column-based method, which is an advantage when AT specimens are small.

Role of Estrogen and Its Receptors in Adipose Tissue Glucose Metabolism in Pre- and Postmenopausal Women.

CONTEXT: Reduced estrogen levels in postmenopausal women predispose them to metabolic side effects, including insulin resistance and type 2 diabetes; however, the cellular mechanisms are not well understood. OBJECTIVE: This work aimed to study the expression of estrogen receptors in adipose tissue from pre- and postmenopausal women and the effects of estradiol (E2) on glucose uptake of adipocytes. METHODS: Subcutaneous (SAT) and visceral adipose tissue (VAT) obtained from pre- and postmenopausal women (19-51 and 46-75 years old, respectively) were used to measure gene expression of ESR1 and ESR2. SAT tissue was incubated with E2, and glucose uptake and estrogen receptor levels were measured. Polymorphisms in ESR1 and ESR2 were addressed in public databases to identify single nucleotide polymorphisms associated with metabolic traits. RESULTS: ESR2 expression was lower in pre- vs postmenopausal women, corresponding to lower ESR1:ESR2 gene expression ratio in postmenopausal women. In premenopausal women, the expression of ESR1 was higher in VAT than in SAT. In both pre- and postmenopausal women, ESR2 expression was lower in VAT than in SAT. In late, but not pre- or early postmenopausal women, E2 reduced glucose uptake and GLUT4 protein and increased expression of ESR2. ESR1 polymorphisms were associated with weight, body fat distribution, and total cholesterol, and ESR2 polymorphisms were associated with total cholesterol and triglyceride levels and with body fat percentage. CONCLUSION: E2 inhibits glucose utilization in human adipocytes in late postmenopausal women. Changes in glucose utilization over time since menopause may be explained by a lower ESR1:ESR2 ratio. This can have clinical implications on the timing of estrogen treatment in postmenopausal women.

CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation.

BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.