Dysmetabolic adipose tissue in obesity: morphological and functional characteristics of adipose stem cells and mature adipocytes in healthy and unhealthy obese subjects.

The way by which subcutaneous adipose tissue (SAT) expands and undergoes remodeling by storing excess lipids through expansion of adipocytes (hypertrophy) or recruitment of new precursor cells (hyperplasia) impacts the risk of developing cardiometabolic and respiratory diseases. In unhealthy obese subjects, insulin resistance, type 2 diabetes, hypertension, and obstructive sleep apnoea are typically associated with pathologic SAT remodeling characterized by adipocyte hypertrophy, as well as chronic inflammation, hypoxia, increased visceral adipose tissue (VAT), and fatty liver. In contrast, metabolically healthy obese individuals are generally associated with SAT development characterized by the presence of smaller and numerous mature adipocytes, and a lower degree of VAT inflammation and ectopic fat accumulation. The remodeling of SAT and VAT is under genetic regulation and influenced by inherent depot-specific differences of adipose tissue-derived stem cells (ASCs). ASCs have multiple functions such as cell renewal, adipogenic capacity, and angiogenic properties, and secrete a variety of bioactive molecules involved in vascular and extracellular matrix remodeling. Understanding the mechanisms regulating the proliferative and adipogenic capacity of ASCs from SAT and VAT in response to excess calorie intake has become a focus of interest over recent decades. Here, we summarize current knowledge about the biological mechanisms able to foster or impair the recruitment and adipogenic differentiation of ASCs during SAT and VAT development, which regulate body fat distribution and favorable or unfavorable metabolic responses.

Long-acting insulin analog detemir displays reduced effects on adipocyte differentiation of human subcutaneous and visceral adipose stem cells.

BACKGROUND AND AIMS: Since treatment with insulin detemir results in a lower weight gain compared to human insulin, we investigated whether detemir is associated with lower ability to promote adipogenesis and/or lipogenesis in human adipose stem cells (ASC). METHODS AND RESULTS: Human ASC isolated from both the subcutaneous and visceral adipose tissues were differentiated for 30 days in the presence of human insulin or insulin detemir. Nile Red and Oil-Red-O staining were used to quantify the rate of ASC conversion to adipocytes and lipid accumulation, respectively. mRNA expression levels of early genes, including Fos and Cebpb, as well as of lipogenic and adipogenic genes, were measured at various phases of differentiation by qRT-PCR. Activation of insulin signaling was assessed by immunoblotting. ASC isolated from subcutaneous and visceral adipose tissue were less differentiated when exposed to insulin detemir compared to human insulin, showing lower rates of adipocyte conversion, reduced triglyceride accumulation, and impaired expression of late-phase adipocyte marker genes, such as Pparg2, Slc2a4, Adipoq, and Cidec. However, no differences in activation of insulin receptor, Akt and Erk and induction of the early genes Fos and Cebpb were observed between insulin detemir and human insulin. CONCLUSION: Insulin detemir displays reduced induction of the Pparg2 adipocyte master gene and diminished effects on adipocyte differentiation and lipogenesis in human subcutaneous and visceral ASC, in spite of normal activation of proximal insulin signaling reactions. These characteristics of insulin detemir may be of potential relevance to its weight-sparing effects observed in the clinical setting.

Glucagon-like peptide-1 counteracts oxidative stress-dependent apoptosis of human cardiac progenitor cells by inhibiting the activation of the c-Jun N-terminal protein kinase signaling pathway.

Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H(2)O(2)). Phospho- and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9-39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H(2)O(2) induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H(2)O(2)-triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9-39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1.

Metabolic implications of growth hormone therapy.

GH regulates important physiological processes, including somatic growth and development, and carbohydrate and lipid metabolism. GH deficiency and GH replacement therapy exert opposite effects on body composition and fat accumulation, suggesting that GH may directly regulate adipocyte functions. Multiple studies have shown that in tissues previously deprived of GH, short-term stimulation with GH is able to mimic the actions of insulin, including stimulation of amino-acid and glucose transport, and lipogenesis. However, the antagonistic effects of GH on insulin-mediated metabolic responses are well-documented: GH excess in patients with GH-producing pituitary tumors causes hyperinsulinemia, insulin resistance, and even clinical diabetes mellitus. These apparently contradictory effects may be explained at the molecular level by the complex interplay between GH and insulin signaling. In this review, we examine the consequences of acute and chronic effects of GH on visceral fat and on metabolic responses in adipocytes, and discuss experimental data illustrating the integrated crosstalk between GH and insulin.

Associated hormonal declines in aging: DHEAS.

DHEA and its sulfate prohormone DHEAS are the most abundant circulating adrenal steroid hormones in humans. DHEA exerts its actions on peripheral target tissues either indirectly, following its conversion to androgens, estrogens or both, or directly, as a steroid hormone interacting with either a nuclear or a membrane receptor. In humans, DHEA shows a characteristic pattern of secretion throughout life. Serum DHEA concentrations decline with advancing age and vary with gender, ethnicity, and environmental factors. Epidemiological studies show an inverse relationship between plasma DHEA(S) levels in men and age-related illnesses, including cardiovascular and metabolic diseases, immune disorders, malignancies, and neurological dysfunction. This has generated great interest on the putative role of DHEA in age-associated illnesses. Administration of DHEA to rats and mice reduces visceral fat accumulation, and improves insulin resistance in experimental models of diet-induced obesity and/or Type 2 diabetes. In addition, recent studies in vitro have shown that DHEA has the capacity to improve endothelial function by increasing nitric oxide (NO) synthesis. Replacement of DHEA in patients with adrenal insufficiency has been shown to exert beneficial effects on well-being, mood, and sexuality. By contrast, in healthy individuals, the physiological age-associated decline in circulating DHEA(S) per se does not justify DHEA supplementation, since the effects of this hormone on metabolic abnormalities, endothelial function in vivo, and cardiovascular events are contradictory. However, these results do not exclude the possibility that DHEA treatment may prove beneficial in specific subgroups of elderly subjects.

Factors associated with progression to macroalbuminuria in microalbuminuric Type 1 diabetic patients: the EURODIAB Prospective Complications Study.

AIMS/HYPOTHESIS: Type 1 diabetic patients who develop microalbuminuria are clearly disadvantaged in terms of their risk of morbidity and mortality from renal and cardiovascular diseases. It is therefore important to identify potential factors that can predict progression to macroalbuminuria. METHODS: This is a 7-year follow-up study of 352 microalbuminuric Type 1 diabetic patients from 31 European centres. Risk factors at baseline were compared in patients who progressed to macroalbuminuria and in patients who remained microalbuminuric or reverted to normoalbuminuria. Risk factors and albumin excretion rate (AER) were measured centrally. RESULTS: Over 7.3 years, 13.9% of the microalbuminuric patients progressed to macroalbuminuria, 35.5% remained microalbuminuric and 50.6% reverted to normoalbuminuria. Independent baseline risk factors for progression to macroalbuminuria were HbA(1)c (7.9% vs 6.8%, p=0.004), AER (64.4 vs 44.9 microg/min, p=0.0001) and-after adjusting for diabetes duration, HbA(1)c and AER-body weight (72 vs 67 kg, p=0.05). Independent factors associated with regression to normoalbuminuria were diabetes duration (15 vs 18 years, p=0.004), AER (37.2 vs 44.9 microg/min, p=0.0001) and-after adjusting for diabetes duration, HbA(1)c and AER-waist-to-hip ratio (0.83 vs 0.86, p=0.05) and incidence of peripheral neuropathy at baseline (24% vs 38%, p=0.001). Blood pressure and smoking did not emerge as risk factors at baseline for the outcome of microalbuminuria. CONCLUSIONS/INTERPRETATION: A significant fraction of microalbuminuric Type 1 diabetic patients will progress to overt proteinuria. Patients with higher AER values, sub-optimal metabolic control, excess body fat and peripheral neuropathy may carry a particularly high risk of clinical nephropathy requiring aggressive therapeutic intervention.

Effects of streptozocin diabetes and diabetes treatment by islet transplantation on in vivo insulin signaling in rat heart.

The insulin signaling cascade was investigated in rat myocardium in vivo in the presence of streptozocin (STZ)-induced diabetes and after diabetes treatment by islet transplantation under the kidney capsule. The levels of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, insulin receptor substrate (IRS)-2, and p52(Shc) were increased in diabetic compared with control heart, whereas tyrosine phosphorylation of IRS-1 was unchanged. The amount of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the level of PI 3-kinase activity associated with IRS-2 were also elevated in diabetes, whereas no changes in IRS-1-associated PI 3-kinase were observed. Insulin-induced phosphorylation of Akt on Thr-308 was increased fivefold in diabetic heart, whereas Akt phosphorylation on Ser-473 was normal. In contrast with Akt phosphorylation, insulin-induced phosphorylation of glycogen synthase kinase (GSK)-3, a major cellular substrate of Akt, was markedly reduced in diabetes. In islet-transplanted rats, the majority of the alterations in insulin-signaling proteins found in diabetic rats were normalized, but insulin stimulation of IRS-2 tyrosine phosphorylation and association with PI 3-kinase was blunted. In conclusion, in the diabetic heart, 1) IRS-1, IRS-2, and p52(Shc) are differently altered, 2) the levels of Akt phosphorylation on Ser-473 and Thr-308, respectively, are not coordinately regulated, and 3) the increased activity of proximal-signaling proteins (i.e., IRS-2 and PI 3-kinase) is not propagated distally to GSK-3. Islet transplantation under the kidney capsule is a potentially effective therapy to correct several diabetes-induced abnormalities of insulin signaling in cardiac muscle but does not restore the responsiveness of all signaling reactions to insulin.

Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo.

GH has been shown to stimulate tyrosine phosphorylation of JAK2, several STAT proteins, insulin receptor substrate-1 (IRS-1), and SHC proteins in cultured cells. The goal of this study was to determine GH effects on protein tyrosine phosphorylation in liver and skeletal muscle of normal rats in vivo. Nonfasted male Sprague-Dawley rats (225-250 g) were injected with GH iv, and tissues were obtained after 5, 15, 30, or 60 min. At a maximally effective GH dose (1.5 mg/kg body weight), phosphotyrosine antibody immunoblots demonstrated marked stimulation of the tyrosine phosphorylation of JAK2 (maximal at 5 min) and a 95,000 Mr protein (maximal at 15 min) in both liver and skeletal muscle. The 95,000 Mr protein was recognized and immunodepleted by STAT5 antibody, but not by other STAT protein antibodies. Although basal tyrosine phosphorylation of IRS-1 and SHC was evident, GH did not stimulate tyrosine phosphorylation of either of these proteins in liver or skeletal muscle. In conclusion, GH stimulates the tyrosine phosphorylation of JAK2 and STAT5, but not IRS-1, SHC, or other STAT proteins in liver and skeletal muscle of normal rats. These results differ from findings in cultured cells and support the concept that selectivity for tyrosine kinase substrates is an important determinant of postreceptor signaling specificity in vivo.

Differential expression of the asialoglycoprotein receptor in discrete brain areas, in kidney and thyroid.

The asyaloglycoprotein receptor is a dimer formed by two polypeptide chains abundantly expressed in the liver (RHL-1 and RHL-2). Using specific primers for the two polypeptide chains we measured, by semiquantitative reverse PCR (RT-PCR), the corresponding mRNAs in different rat tissues. We found that both RHL-1 and RHL-2 mRNAs are expressed in the liver, kidney, brain and thyroid. Under the same conditions we did not detect any specific mRNA in the spleen. In the brain these sequences are expressed along a posterior-anterior gradient. Cerebellum and brainstem display the highest expression of the brain RHL-1 and RHL-2 mRNAs. Tissues and regional distribution of this receptor suggest that other body districts besides liver may participate in the clearance of serum glycoproteins.

Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy.

Hyperfunctioning thyroid adenomas are clonal neoplasms with the intrinsic capacity of growing and differentiate independently of thyroid-stimulating hormone (TSH). We analysed the mRNA encoding thyrotropin receptor of 11 adenomas obtained by fine needle aspiration biopsy (FNAB) and found 7 mutants all located in three aminoacids clustered in the sixth transmembrane domain of the receptor. These mutations were somatic and specifically present in the tumour tissue. DNA sequence revealed that 80 to 90% of the mutations can be rapidly screened and identified by restriction enzyme analysis of the amplified cDNA obtained from the FNABs. The mutation Thr->Ile was introduced in the wild type receptor and expressed in mouse fibroblasts. These cells constitutively activate the transcription of a reporter gene under the control of cyclic AMP responsive element.

P2 purinergic agonists and 12-O-tetradecanoylphorbol-13-acetate, as well as protein kinase A activators, stimulate thyroglobulin secretion in FRTL-5 cells.

We studied the role of various intracellular pathways in thyroglobulin secretion. The P2 agonists (ATP, ADP, GTP), 12-O-tetradecanoylphorbol-13-acetate (TPA), and protein kinase A activators stimulate thyroglobulin secretion in cells grown without TSH. The effects of these agents are additive. Pertussis toxin partially inhibits the effect of ATP but has no effect on the action of GTP. ATP and GTP increase cytosolic calcium (279 +/- 16% and 302 +/- 22%, respectively) while TPA and TSH (1 mU/ml) do not. Thus, both the protein kinase A and kinase C pathways regulate thyroglobulin secretion in FRTL-5 cells.