Angiotensin II type 2 receptor promotes adipocyte differentiation and restores adipocyte size in high-fat/high-fructose diet-induced insulin resistance in rats.

This study was aimed at establishing whether specific activation of angiotensin II (ANG II) type 2 receptor (AT2R) modulates adipocyte differentiation and function. In primary cultures of subcutaneous (SC) and retroperitoneal (RET) preadipocytes, both AT2R and AT1R were expressed at the mRNA and protein level. Cells were stimulated with ANG II or the AT2R agonist C21/M24, alone or in the presence of the AT1R antagonist losartan or the AT2R antagonist PD123,319. During differentiation, C21/M24 increased PPARγ expression in both RET and SC preadipocytes while the number of small lipid droplets and lipid accumulation solely increased in SC preadipocytes. In mature adipocytes, C21/M24 decreased the mean size of large lipid droplets. Upon abolishment of AT2R expression using AT2R-targeted shRNAs, expressions of AT2R, aP2, and PPARγ remained very low, and cells were unable to differentiate. In Wistar rats fed a 6-wk high-fat/high-fructose (HFHF) diet, a significant shift toward larger adipocytes was observed in RET and SC adipose tissue depots. C21/M24 treatments for 6 wk restored normal adipocyte size distribution in both these tissue depots. Moreover, C21/M24 and losartan decreased hyperinsulinemia and improved insulin sensitivity impaired by HFHF diet. A strong correlation between adipocyte size area and glucose infusion rate during euglycemic-hyperinsulinemic clamp was observed. These results indicate that AT2R is involved in early adipocyte differentiation, while in mature adipocytes and in a model of insulin resistance AT2R activation restores normal adipocyte morphology and improves insulin sensitivity.

The C-terminal domains of melanocortin-2 receptor (MC2R) accessory proteins (MRAP1) influence their localization and ACTH-induced cAMP production.

ACTH binding to the human melanocortin-2 receptor (MC2R) requires the presence of the MC2R accessory protein1 isoforms, MRAPα or MRAPß. This study evaluated the role of the isoform-specific C-terminal domains of MRAP with regard to their cellular localization, topology, interaction with MRAP2 and cAMP production. When stably expressed in HEK293/FRT cells or in B16-G4F mouse melanoma cells (an MSH receptor-deficient cell clone), MRAPα and MRAPdCT (truncated MRAP1, N-terminal only) localized mainly around the nuclear envelope and within dense intracellular endosomes, while MRAPß exhibited a strong localization at the plasma membrane, and partially with rapid recycling endosomes. MRAPß and MRAPdCT both exhibited dual-topology (N(cyto)/C(exo) and N(exo)/C(cyto)) at the plasma membrane whereas MRAPα exhibited only N(cyto)/C(exo) topology at the plasma membrane while adopting dual-topology in intracellular compartments. Both MRAPα and MRAP2 colocalized in intracellular compartments, as opposed to weak colocalization between MRAPß and MRAP2. MRAP2 and MC2R enhanced the expression of MRAP1 isoforms and vice versa. Moreover, in both HEK293/FRT and B16-G4F cells, ACTH failed to activate MC2R unless MRAP1 was present. MRAP1 expression enhanced MC2R cell-surface expression as well as concentration-dependent cAMP accumulation. In the presence of human or zebrafish MC2R, MRAPß induced the highest cAMP accumulation while MRAPdCT induced the lowest. Together, the present findings indicate that the C-terminal domains of MRAP dictate their intracellular localization in addition to regulating ACTH-induced cAMP production. These preferential localizations suggest that MRAPα is involved in MC2R targeting to the plasma membrane, while MRAPß may enhance ACTH-MC2R coupling to cAMP production.

Postprandial fatty acid uptake and adipocyte remodeling in angiotensin type 2 receptor-deficient mice fed a high-fat/high-fructose diet.

The role of the angiotensin type-2 receptor in adipose physiology remains controversial. The aim of the present study was to demonstrate whether genetic angiotensin type-2 receptor-deficiency prevents or worsens metabolic and adipose tissue morphometric changes observed following a 6-week high-fat/high-fructose diet with injection of a small dose of streptozotocin. We compared tissue uptake of nonesterified fatty acid and dietary fatty acid in wild-type and angiotensin type-2 receptor-deficient mice by using the radiotracer 14(R,S)-[(1) (8)F]-fluoro-6-thia-heptadecanoic acid in mice fed a standard or high-fat diet. Postprandial fatty acid uptake in the heart, liver, skeletal muscle, kidney and adipose tissue was increased in wild-type mice after a high-fat diet and in angiotensin type-2 receptor-deficient mice on both standard and high-fat diets. Compared to the wild-type mice, angiotensin type-2 receptor-deficient mice had a lower body weight, an increase in fasting blood glucose and a decrease in plasma insulin and leptin levels. Mice fed a high-fat diet exhibited increased adipocyte size that was prevented by angiotensin type-2 receptor-deficiency. Angiotensin type-2 receptor-deficiency abolished the early hypertrophic adipocyte remodeling induced by a high-fat diet. The small size of adipocytes in the angiotensin type-2 receptor-deficient mice reflects their inability to store lipids and explains the increase in fatty acid uptake in non-adipose tissues. In conclusion, a genetic deletion of the angiotensin type-2 receptor is associated with metabolic dysfunction of white adipose depots, and indicates that adipocyte remodeling occurs before the onset of insulin resistance in the high-fat fed mouse model.

Bioinactive ACTH causing glucocorticoid deficiency.

CONTEXT: A 4-year-old girl and a 4-month-old boy presented with hypoglycemia, normal electrolytes, low cortisol, and high ACTH. A diagnosis of primary adrenal insufficiency was made and initial treatment was with glucocorticoids and mineralocorticoids. The genes known to cause ACTH resistance were normal. Whole exome sequencing revealed that the girl was compound heterozygous for POMC mutations: one previously described null allele and one novel p.R8C mutation in the sequence encoding ACTH and α-MSH. The boy was homozygous for the p.R8C mutation. HYPOTHESIS: The p.R8C ACTH mutant is immunoreactive, but the mutant peptides, ACTH-R8C and α-MSH-R8C, are bioinactive. METHODS: Methods included whole exome sequencing, Sanger sequencing, peptide synthesis, ACTH immunoradiometric assay, hormone binding, and activation assays in cells expressing melanocortin receptors. RESULTS: ACTH-R8C was immunoreactive but failed to bind and activate cAMP production in melanocortin-2 receptor (MC2R)-expressing cells, and α-MSH-R8C failed to bind and stimulate cAMP production in MC1R- and MC4R-expressing cells. CONCLUSION: These are the first documented cases of glucocorticoid deficiency due to the secretion of an ACTH molecule that lacks biological bioactivity but conserves immunoreactivity. POMC mutations should thus be considered in patients presenting with apparent ACTH resistance. Our findings also highlight a limitation to immunoassay-based diagnostics and demonstrate the value of genetic analysis. Establishing the molecular etiology of the disorder in our patients allowed cessation of the unnecessary mineralocorticoids. Finally, discovery of this mutation indicates that in humans, the amino acid sequence His(6)Phe(7)Arg(8)Trp(9) is important not only for cAMP activation but also for ACTH binding to MC2R.

Adrenocorticotropin hormone (ACTH) effects on MAPK phosphorylation in human fasciculata cells and in embryonic kidney 293 cells expressing human melanocortin 2 receptor (MC2R) and MC2R accessory protein (MRAP)ß.

Adrenocorticotropin hormone (ACTH) exerts trophic effects on adrenocortical cells. We studied the phosphorylation of mitogen-activated proteins kinases (MAPKs) in human embryonic kidney cells stably expressing the ACTH receptor, MC2R, and its accessory protein MRAPß and in primary cultures of human adrenal fasciculata cells. ACTH induced a maximal increase in p44/p42(mapk) and of p38 MAPK phosphorylation after 5min. Neither the overexpression of wild-type arrestin2, arrestin3 or their respective dominant negative forms affected p44/p42(mapk) phosphorylation. However, preincubation with the recycling inhibitors brefeldin A and monensin attenuated both cAMP accumulation and p44/p42(mapk) phosphorylation proportionally. Cyclic AMP-related PKA inhibitors (H89, KI(6-22)) and Rp-cAMPS decreased p44/p42(mapk) phosphorylation but not ACTH-mediated cAMP production. The selective Epac1/2 activator, 8-pCPT-2'-O-MecAMP, did not modify the effect of ACTH. Thus, cAMP/PKA, but not cAMP/Epac1/2 pathways, or arrestin-coupled internalization of MC2R is involved in ACTH-induced p44/p42(mapk) phosphorylation by human MC2R. Together, ACTH binding to MC2R stimulates PKA-dependent p44/p42(mapk) phosphorylation.

Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (hek) cells: identification of Key Ser/Thr (S/T) amino acids.

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPß. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.