Oleg Varlamov

Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity.

Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulfn. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity–diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Sex-Specific Differences in Lipid and Glucose Metabolism

Energy metabolism in humans is tuned to distinct sex-specific functions that potentially reflect the unique requirements in females for gestation and lactation, whereas male metabolism may represent a default state. These differences are the consequence of the action of sex chromosomes and sex-specific hormones, including estrogens and progesterone in females and androgens in males. In humans, sex-specific specialization is associated with distinct body-fat distribution and energy substrate-utilization patterns; i.e., females store more lipids and have higher whole-body insulin sensitivity than males, while males tend to oxidize more lipids than females. These patterns are influenced by the menstrual phase in females, and by nutritional status and exercise intensity in both sexes. This minireview focuses on sex-specific mechanisms in lipid and glucose metabolism and their regulation by sex hormones, with a primary emphasis on studies in humans and the most relevant pre-clinical model of human physiology, non-human primates.

Hypoxia-induced inflammatory cytokine secretion in human adipose tissue stromovascular cells

AimsHypoxia has been implicated as a cause of adipose tissue inflammation in obesity, although the inflammatory response of human adipose tissue to hypoxia is not well understood. The goal of this study was to define in vitro inflammatory responses of human adipose tissue to hypoxia and identify molecular mechanisms of hypoxia-induced inflammation.MethodsThe inflammatory milieu and responses of visceral (VAT) and subcutaneous (SAT) adipose tissue explants and purified stromovascular cells (SVFs) from obese and lean humans were studied in an in vitro hypoxic culture system using quantitative real-time PCR, ELISA, western blotting, immunofluorescence microscopy, flow cytometry and immunohistochemistry.ResultsHuman adipose tissue in obesity demonstrates an increased leucocyte infiltrate that is greater in VAT than SAT and involves macrophages, T cells and natural killer (NK) cells. Hypoxic culture regulates inflammatory cytokine secretion and transcription of metabolic stress response genes in human adipose tissue SVF. Adipocyte diameter is increased and adipose tissue capillary density is decreased in obese participants. Inhibition of c-Jun terminal kinase (JNK) or p38 significantly attenuates hypoxia-induced SVF inflammatory responses. Hypoxia induces phosphorylation of p38 in adipose tissue.ConclusionsHuman adipose tissue in obesity is characterised by a depot-specific inflammatory cell infiltrate that involves not only macrophages, but also T cells and NK cells. Hypoxia induces inflammatory cytokine secretion by human adipose tissue SVF, the primary source of which is adipose tissue macrophages. These data implicate p38 in the regulation of hypoxia-induced inflammation and suggest that alterations in adipocyte diameter and adipose tissue capillary density may be potential underlying causes of adipose tissue hypoxia.

Localization of metallocarboxypeptidase D in atT-20 cells: Potential role in prohormone processing

Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in thetrans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.

Beta-cell lines derived from transgenic Cpe(fat)/Cpe(fat) mice are defective in carboxypeptidase E and proinsulin processing.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing. Cell lines derived from the pancreatic beta-cells of Cpe(fat)/Cpe(fat) mice were generated by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe(fat) mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.

Androgen Effects on Adipose Tissue Architecture and Function in Nonhuman Primates

The differential association of hypoandrogenism in men and hyperandrogenism in women with insulin resistance and obesity suggests that androgens may exert sex-specific effects on adipose and other tissues, although the underlying mechanisms remain poorly understood. Moreover, recent studies also suggest that rodents and humans may respond differently to androgen imbalance. To achieve better insight into clinically relevant sex-specific mechanisms of androgen action, we used nonhuman primates to investigate the direct effects of gonadectomy and hormone replacement on white adipose tissue. We also employed a novel ex vivo approach that provides a convenient framework for understanding of adipose tissue physiology under a controlled tissue culture environment. In vivo androgen deprivation of males did not result in overt obesity or insulin resistance but did induce the appearance of very small, multilocular white adipocytes. Testosterone replacement restored normal cell size and a unilocular phenotype and stimulated adipogenic gene transcription and improved insulin sensitivity of male adipose tissue. Ex vivo studies demonstrated sex-specific effects of androgens on adipocyte function. Female adipose tissue treated with androgens displayed elevated basal but reduced insulin-dependent fatty acid uptake. Androgen-stimulated basal uptake was greater in adipose tissue of ovariectomized females than in adipose tissue of intact females and ovariectomized females replaced with estrogen and progesterone in vivo. Collectively, these data demonstrate that androgens are essential for normal adipogenesis in males and can impair essential adipocyte functions in females, thus strengthening the experimental basis for sex-specific effects of androgens in adipose tissue.

Single-cell analysis of insulin-regulated fatty acid uptake in adipocytes

Increased body fat correlates with the enlargement of average fat cell size and reduced adipose tissue insulin sensitivity. It is currently unclear whether adipocytes, as they accumulate more triglycerides and grow in size, gradually become less insulin sensitive or whether obesity-related factors independently cause both the enlargement of adipocyte size and reduced adipose tissue insulin sensitivity. In the first instance, large and small adipocytes in the same tissue would exhibit differences in insulin sensitivity, whereas, in the second instance, adipocyte size per se would not necessarily correlate with insulin response. To analyze the effect of adipocyte size on insulin sensitivity, we employed a new single-cell imaging assay that resolves fatty acid uptake and insulin response in single adipocytes in subcutaneous adipose tissue explants. Here, we report that subcutaneous adipocytes are heterogeneous in size and intrinsic insulin sensitivity. Whereas smaller adipocytes respond to insulin by increasing lipid uptake, adipocytes with cell diameters larger than 80-100 microm are insulin resistant. We propose that, when cell size approaches a critical boundary, adipocytes lose insulin-dependent fatty acid transport. This negative feedback mechanism may protect adipocytes from lipid overload and restrict further expansion of adipose tissue, which leads to obesity and metabolic complications.

Biosynthesis and Packaging of Carboxypeptidase D into Nascent Secretory Vesicles in Pituitary Cell Lines

Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2–3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention.

Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking*

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT‐20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C‐terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C‐terminal region functions in TGN‐retention; removal of 10 amino acids from the C‐terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half‐life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10‐residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half‐life than either the wild type sequence or the EDE mutant. Adaptor protein‐1 and ‐2 bind to most of the deletion mutants, the EDE point mutant, and the CK2‐phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C‐terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins. J. Cell. Biochem. 85: 101–111, 2002.

Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity.

Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.