Robert L. Oliver

Proopiomelanocortin (POMC), the ACTH/ melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin‐1 receptor (MC‐1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melano‐cytes in culture. Much lower levels of α‐MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and α‐MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone conver‐tases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and α‐MSH, and this was enhanced in response to corti‐cotrophin‐releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably trans‐fected with the MC‐1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, α‐MSH, and β‐MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC‐1R highlight the importance of POMC processing as a key regulatory event in the skin.—Rousseau, K., Kauser, S., Pritchard, L. E., Warhurst, A., Oliver, R. L., Slominski, A., Wei, E. T., Thody, A. J., Tobin, D. J., White, A. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and mela‐nocytes and stimulates melanogenesis. FASEB J. 21, 1844–1856 (2007)

Characterisation of ACTH related peptides in ectopic Cushing's Syndrome

Adrenocorticotrophin (ACTH) is derived by cleavage from the precursor, pro-opiomelanocortin (POMC), and depending on the degree of processing by the tissue or tumor, there is the potential for a number of ACTH-related peptides to be secreted from POMC expressing cells. Previous chromatographic approaches have indicated the presence of high molecular weight forms of ACTH in the human peripheral circulation. However a quantitative assessment of the degree of processing requires two-site immunoradiometric assays which distinguish ACTH precursors and ACTH. Using this approach, we have previously identified the precursors of ACTH (POMC and proACTH) in the circulation of normal subjects in the range 5–40 pmol/l, which suggests that processing in the normal pituitary cell is incomplete.This study aimed to examine the extent of POMC processing by tumors that give rise to Cushings Syndrome as a means of evaluating its usefulness as a diagnostic marker. In a retrospective analysis of 86 patients with Cushings Syndrome, 34/35 patients with pituitary tumors had low levels of ACTH precursors (below 100 pmol/l) and the mean ratio of ACTH precursors:ACTH was 5:1 which indicates that these tumors do process POMC to ACTH relatively efficiently. In ectopic Cushings Syndrome, it is unlikely that the extra-pituitary tumor cells, process POMC as efficiently. Therefore increased prevalence of ACTH precursors in the circulation would be expected and this was substantiated by the large excess of ACTH precursors (139–18,000 pmol/l) in the circulation of the 51 patients with the ectopic ACTH Syndrome.The diagnostic accuracy of the measurement of ACTH precursors was then prospectively compared with a group of 62 patients undergoing the current “gold standard” test of inferior petrosal sinus sampling (IPSS). All those patients with ACTH precursors below a diagnostic cut-off of 100 pmol/l were subsequently shown to have pituitary tumors, whereas levels of >100 pmol/l were seen in the four patients with ectopic tumors. In comparison the IPSS had a specificity of 100% but a sensitivity of 93% and for these false negative results the ACTH precursors proved diagnostically useful. Therefore measurement of ACTH precursors offers a simple non-invasive diagnostic test for the differential diagnosis of Cushings Syndrome which compares favourably with IPSS.

Mutations in the Amino-Terminal Region of Proopiomelanocortin (POMC) in Patients with Early-Onset Obesity Impair POMC Sorting to the Regulated Secretory Pathway

CONTEXT Mutations in the proopiomelanocortin (POMC) gene that impair the synthesis or structure of POMC-derived peptides predispose to human obesity. OBJECTIVE Our objective was to identify and characterize novel mutations in the POMC gene found in patients with early-onset obesity. DESIGN AND PATIENTS The POMC gene was screened in 500 patients with severe early-onset obesity. The biosynthesis, processing, sorting, and secretion of wild-type POMC and two newly identified POMC mutants was studied using metabolic labeling, Western blotting, and immunoassay analysis of lysates and conditioned media of transiently transfected beta-TC3 cells. RESULTS Two novel heterozygous missense mutations in POMC (C28F and L37F) were identified in unrelated probands with early-onset obesity and their overweight or obese family members. Both mutations lie in a region of the N terminus of POMC that has been suggested to be involved in its sorting to the regulated secretory pathway. Metabolic labeling studies indicate that whereas the mutations do not reduce intracellular levels of POMC, both mutations (C28F>L37F) impair the ability of POMC to be processed to generate bioactive products. Studies of the secretion of POMC products suggest, particularly with C28F, that the impaired propeptide processing of these mutations results, at least in part, from a mistargeting of mutant POMC to the constitutive rather than the regulated secretory pathway. CONCLUSION These mutations in patients with early-onset obesity represent a novel molecular mechanism of human POMC deficiency whereby naturally occurring mutations in its N-terminal sequence impair the ability of POMC to enter the trafficking pathway in which serial propeptide processing normally occurs.

IGFBP2 is a biomarker for predicting longitudinal deterioration in renal function in type 2 diabetes

Objective Insulin-like growth factors are implicated in the development of diabetic nephropathy. IGF-binding protein 2 (IGFBP2) and IGF2 are expressed in the kidney, but their associations with diabetic nephropathy are unclear. We therefore tested the hypothesis that circulating levels of IGF2 and IGFBP2 predict longitudinal renal function in individuals with type 2 diabetes. Design and methods IGFBP2 and IGF2 measurements were performed in 436 individuals (263 males) with type 2 diabetes. Linear mixed-effect regression analysis was used to model the relationship between plasma IGFBP2 concentration and longitudinal changes in estimated glomerular filtration rate (eGFR) over an 8-year period. Analyses were also performed for IGF1, IGF2, IGFBP1 and IGFBP3 concentrations as predictors of longitudinal renal outcomes. Results High IGFBP2 concentration at baseline was associated with a decreased eGFR over an 8-year period (β=−0.02, (95% confidence interval −0.03 to −0.01), P<0.001). High IGFBP1, IGFBP2 and IGFBP3 were also associated with low baseline eGFR concentration. Conclusion This study demonstrates that IGFBP2 is a predictor of longitudinal deterioration of renal function in type 2 diabetes.

Do immunoassays differentially detect different acidity glycoforms of FSH

The possibility of the carbohydrate residues of glycoproteins affecting their recognition in immunoassays is an important and unresolved issue. This study looked for evidence of differential recognition of FSH glycoform preparations, of variable isoelectric point (pI) and known molarity, using three routine assays employing different antibody configurations.

Insulin-like growth factor-II and insulin-like growth factor binding protein-2 prospectively predict longitudinal elevation of HDL-cholesterol in type 2 diabetes.

Introduction Associations of insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-2 (IGFBP-2) with cardiovascular risk have been inadequately studied. We hypothesized that IGF-II and IGFBP-2 associate with longitudinal trends in lipid profiles in type 2 diabetes patients. Subjects and methods Four hundred and eighty nine subjects with type 2 diabetes (age 27–87 years) from the Salford Diabetes Cohort were studied. Longitudinal clinical information was extracted for an eight-year period (2002–2009) from an integrated electronic dataset of primary care and hospital data. Results There were 294 male subjects and mean age was 62.9 years. At baseline, IGF-II concentration was 602 ng/mL. HDL cholesterol at baseline was associated with log-IGF-II concentration in a model adjusted for age, gender, baseline body-mass index (BMI), estimated glomerular filtration rate (eGFR) and lipid-lowering therapy. IGFBP-1 and IGFBP-2 were associated with high HDL-cholesterol. A higher circulating IGF-II concentration at baseline was also associated with longitudinal increase in HDL-cholesterol in mixed-effects regression analyses independent of IGF-I, IGFBP-1, IGFBP-2, IGFBP-3, age, gender, eGFR, BMI and lipid-lowering therapy. Log-transformed baseline concentrations of IGFBP-1 and IGFBP-2 were also associated with longitudinal elevation in HDL-cholesterol. No association was observed for IGF-II or IGFBP-2 with longitudinal LDL cholesterol trends. Conclusion Our analyses based on ‘real world’ data demonstrate that higher baseline IGF-II and IGFBP-2 predict increased HDL concentration over time, implicating IGF-II in modulation of circulating HDL-cholesterol concentrations.

Atorvastatin administration is associated with dose-related changes in IGF bioavailability

OBJECTIVE IGF levels, their binding proteins (IGFBPs) and high-dose statin therapy have been linked to the development of diabetes. We aimed to identify whether atorvastatin caused dose-related changes in IGF proteins. DESIGN AND METHODS We measured IGF1, IGF2, IGFBP1 and IGFBP3 concentrations at baseline, 6 and 12 months in Protection Against Nephropathy in Diabetes with Atorvastatin trial participants with type 2 diabetes randomised to 10 mg (n=59) vs 80 mg (n=60) of atorvastatin (n=119; mean (S.D.): age 64 (10) years; 83% male; HbA1c 61 (10) mmol/mol; blood pressure 131/73 mmHg). RESULTS Atorvastatin was associated with overall reductions in circulating IGF1, IGF2 and IGFBP3 concentrations (P<0.05 for all changes). The adjusted mean (95% CI) between-group differences that indicate dose-related changes in IGF proteins were not significant for IGF1: -3 (-21 to 14) ng/ml; IGF2: -23 (-65 to 18) ng/ml and IGFBP3: -0.34 (-0.71 to 0.03) μg/ml, negative values indicating numerically greater lowering with high dose. The IGFBP1 concentration did not change with atorvastatin therapy overall but the adjusted mean (95% CI) between-group difference indicating a dose-related change in log IGFBP1 was highly significant -0.41 (-0.69 to 0.13, P=0.004). CONCLUSION IGF1, IGF2 and IGFBP3 concentrations decreased following atorvastatin therapy. A differential effect of low- vs high-dose atorvastatin on IGFBP1 concentrations was observed with likely implications for IGF bioavailability. The dose-related differential impact of atorvastatin treatment on concentration of IGF proteins merits investigation as a mechanism to explain the worsening of glucose tolerance with statin therapy.

Troglitazone regulates anaplerosis via a pull/push affect on glutamate dehydrogenase mediated glutamate deamination in kidney-derived epithelial cells; implications for the Warburg effect.

Mitochondrial Krebs cycle keto acid pool depends upon input from pyruvate and glutamate to maintain homeostasis. We studied the affect of glucose-derived pyruvate removal on compensatory input from glutamine-derived glutamate by accelerated glutamate metabolism via glutamate dehydrogenase (GDH). In glutamine minus glucose media (Gln-Glc), NH4+ production increased 41% without an increase in glutamine uptake consistent with accelerated glutamate metabolism via GDH. Alanine production dropped 40% consistent with a shift of glutamate from alanine aminotransferase (ALT) to GDH. Troglitazone (TRO) added to the Gln-Glc media further enhanced glutamate metabolism via GDH at the expense of glutamate metabolism via ALT since alanine production dropped an additional 70%. TRO reduced cell glutamate content 30% while increasing lactate production 5-fold consistent with blocking of cytosolic pyruvate formed from mitochondrial malate from reentering the cycle and maintaining keto acid pool homeostasis. Consequently mitochondrial keto acid pool deficit pulls glutamate via GDH into the cycle. Additionally TRO reduced cytosolic pH which effectively pushes glutamate via GDH, rather than merely shifting glutamate from ALT to GDH. Providing intramitochondrial pyruvate in the form of methyl pyruvate reduced glutamate metabolism via GDH and elevated glutamate metabolism via ALT to control levels restoring acid-base balance. Our findings are consistent with TRO regulation of anaplerosis dependent upon dual pull (cycle keto-acid deficit)/push (cytosolic acidosis) mechanisms.