The prevalence and distribution of the amyloidogenic transthyretin (TTR) V122I allele in Africa.

BACKGROUND: Transthyretin (TTR) pV142I (rs76992529-A) is one of the 113 variants in the human TTR gene associated with systemic amyloidosis. It results from a G to A transition at a CG dinucleotide in the codon for amino acid 122 of the mature protein (TTR V122I). The allele frequency is 0.0173 in African Americans. METHODS: PCR-based assays to genotype 2767 DNA samples obtained from participants in genetic studies from various African populations supplemented with sequencing data from 529 samples within the 1000 Genomes Project. RESULTS: The rs76992529-A variant allele was most prevalent (allele frequency 0.0253) in the contiguous West African countries of Sierra Leone, Guinea, Ivory Coast, Burkina Faso, Ghana, and Nigeria. In other African countries, the mean allele frequency was 0.011. CONCLUSIONS: Our data are consistent with a small number of founder carriers of the amyloidogenic TTR V122I (p.Val142Ile) allele in southern West Africa, with no apparent advantage or disadvantage of an allele carrying newborn reaching adulthood. In U.S. African Americans, the allele represents a significant risk for congestive heart failure late in life. If clinical penetrance is similar in African countries with high allele frequencies, then cardiac amyloidosis could also represent a significant cause of heart disease in the elderly in those populations.

Carboxylated and uncarboxylated forms of osteocalcin directly modulate the glucose transport system and inflammation in adipocytes.

Osteocalcin is secreted by osteoblasts and improves insulin sensitivity in vivo, although mechanisms remain unclear. We tested the hypothesis that osteocalcin directly modulates cell biology in insulin-targeted peripheral tissues. In L-6 myocytes, osteocalcin stimulated glucose transport both in the absence (basal) and presence of insulin. Similarly, in primary cultured adipocytes, both carboxylated and uncarboxylated osteocalcin increased basal and insulin-stimulated glucose transport as well as insulin sensitivity. Osteocalcin also increased basal and insulin-stimulated glucose oxidation, though there was no effect on fatty acid synthesis or lipolysis. In primary-cultured adipocytes, both forms of osteocalcin suppressed secretion of tumor necrosis factor alpha into the media; however, only carboxylated osteocalcin suppressed interleukin 6 release, and neither form of osteocalcin modulated monocyte chemoattractant protein-1 secretion. Both carboxylated and uncarboxylated osteocalcin increased secretion of adiponectin and the anti-inflammatory cytokine interleukin 10. In conclusion, both carboxylated and uncarboxylated osteocalcin directly increase glucose transport in adipocytes and muscle cells, while suppressing proinflammatory cytokine secretion and stimulating interleukin 10 and adiponectin release. Thus, these results provide a mechanism for the insulin-sensitizing effects of osteocalcin and help elucidate the role that bone plays in regulating systemic metabolism.

Prostaglandin A2 enhances cellular insulin sensitivity via a mechanism that involves the orphan nuclear receptor NR4A3.

We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin's ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3.

Noggin is novel inducer of mesenchymal stem cell adipogenesis: implications for bone health and obesity.

Noggin is a glycosylated-secreted protein known so far for its inhibitory effects on bone morphogenetic protein (BMP) signaling by sequestering the BMP ligand. We report here for the first time a novel mechanism by which noggin directly induces adipogenesis of mesenchymal stem cells independently of major human adipogenic signals through C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor-γ. Evaluation of a possible mechanism for noggin-induced adipogenesis of mesenchymal stem cells identified the role of Pax-1 in mediating such differentiation. The relevance of elevated noggin levels in obesity was confirmed in a preclinical, immunocompetent mouse model of spontaneous obesity and in human patients with higher body mass index. These data clearly provide a novel role for noggin in inducing adipogenesis and possibly obesity and further indicates the potential of noggin as a therapeutic target to control obesity.

Adipocytes exhibit abnormal subcellular distribution and translocation of vesicles containing glucose transporter 4 and insulin-regulated aminopeptidase in type 2 diabetes mellitus: implications regarding defects in vesicle trafficking.

Insulin resistance in type 2 diabetes is due to impaired stimulation of the glucose transport system in muscle and fat. Different defects are operative in these two target tissues because glucose transporter 4 (GLUT 4) expression is normal in muscle but markedly reduced in fat. In muscle, GLUT 4 is redistributed to a dense membrane compartment, and insulin-mediated translocation to plasma membrane (PM) is impaired. Whether similar trafficking defects are operative in human fat is unknown. Therefore, we studied subcellular localization of GLUT4 and insulin-regulated aminopeptidase (IRAP; also referred to as vp165 or gp160), which is a constituent of GLUT4 vesicles and also translocates to PM in response to insulin. Subcutaneous fat was obtained from eight normoglycemic control subjects (body mass index, 29 +/- 2 kg/m2) and eight type 2 diabetic patients (body mass index, 30 +/- 1 kg/m2; fasting glucose, 14 +/- 1 mM). In adipocytes isolated from diabetics, the basal 3-O-methylglucose transport rate was decreased by 50% compared with controls (7.1 +/- 2.9 vs. 14.1 +/- 3.7 mmol/mm2 surface area/min), and there was no increase in response to maximal insulin (7.9 +/- 2.7 vs. 44.5 +/- 9.2 in controls). In membrane subfractions from controls, insulin led to a marked increase of IRAP in the PM from 0.103 +/- 0.04 to 1.00 +/- 0.33 relative units/mg protein, concomitant with an 18% decrease in low-density microsomes and no change in high-density microsomes (HDM). In type 2 diabetes, IRAP overall expression in adipocytes was similar to that in controls; however, two abnormalities were observed. First, in basal cells, IRAP was redistributed away from low-density microsomes, and more IRAP was recovered in HDM (1.2-fold) and PM (4.4-fold) from diabetics compared with controls. Second, IRAP recruitment to PM by maximal insulin was markedly impaired. GLUT4 was depleted in all membrane subfractions (43-67%) in diabetes, and there was no increase in PM GLUT4 in response to insulin. Type 2 diabetes did not affect the fractionation of marker enzymes. We conclude that in human adipocytes: 1) IRAP is expressed and translocates to PM in response to insulin; 2) GLUT4 depletion involves all membrane subfractions in type 2 diabetes, although cellular levels of IRAP are normal; and 3) in type 2 diabetes, IRAP accumulates in membrane vesicles cofractionating with HDM and PM under basal conditions, and insulin-mediated recruitment to PM is impaired. Therefore, in type 2 diabetes, adipocytes express defects in trafficking of GLUT4/IRAP-containing vesicles similar to those causing insulin resistance in skeletal muscle.

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance.

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.

The expression of TNF alpha by human muscle. Relationship to insulin resistance.

TNFalpha is orverexpressed in the adipose tissue of obese rodents and humans, and is associated with insulin resistance. To more closely link TNF expression with whole body insulin action, we examined the expression of TNF by muscle, which is responsible for the majority of glucose uptake in vivo. Using RT-PCR, TNF was detected in human heart, in skeletal muscle from humans and rats, and in cultured human myocytes. Using competitive RT-PCR, TNF was quantitated in the muscle biopsy specimens from 15 subjects whose insulin sensitivity had been characterized using the glucose clamp. technique. TNF expression in the insulin resistant subjects and the diabetic patients was fourfold higher than in the insulin sensitive subjects, and there was a significant inverse linear relationship between maximal glucose disposal rate and muscle TNF (r = -0.60, P < 0.02). In nine subjects, muscle cells from vastus lateralis muscle biopsies were placed into tissue culture for 4 wk, and induced to differentiate into myotubes. TNF was secreted into the medium from these cells, and cells from diabetic patients expressed threefold more TNF than cells from nondiabetic subjects. Thus, TNF is expressed in human muscle, and is expressed at a higher level in the muscle tissue and in the cultured muscle cells from insulin resistant and diabetic subjects. These data suggest another mechanism by which TNF may play an important role in human insulin resistance.

Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM.

In obesity, impaired glucose tolerance (IGT), non-insulin-dependent diabetes mellitus (NIDDM), and gestational diabetes mellitus (GDM), defects in glucose transport system activity, contribute to insulin resistance in target tissues. In adipocytes from obese and NIDDM patients, we found that pretranslational suppression of the insulin-responsive GLUT4 glucose transporter isoform is a major cause of cellular insulin resistance; however, whether this process is operative in skeletal muscle is not clear. To address this issue, we performed percutaneous biopsies of the vastus lateralis in lean and obese control subjects and in obese patients with IGT and NIDDM and open biopsies of the rectus abdominis at cesarian section in lean and obese gravidas and gravidas with GDM. GLUT4 was measured in total postnuclear membrane fractions from both muscles by immunoblot analyses. The maximally insulin-stimulated rate of in vivo glucose disposal, assessed with euglycemic glucose clamps, decreased 26% in obesity and 74% in NIDDM, reflecting diminished glucose uptake by muscle. However, in vastus lateralis, relative amounts of GLUT4 per milligram membrane protein were similar (NS) among lean (1.0 +/- 0.2) and obese (1.5 +/- 0.3) subjects and patients with IGT (1.4 +/- 0.2) and NIDDM (1.2 +/- 0.2). GLUT4 content was also unchanged when levels were normalized per wet weight, per total protein, and per DNA as an index of cell number. Levels of GLUT4 mRNA were similarly not affected by obesity, IGT, or NIDDM whether normalized per RNA or for the amount of an unrelated constitutive mRNA species. Because muscle fibers (types I and II) exhibit different capacities for insulin-mediated glucose uptake, we tested whether a change in fiber composition could cause insulin resistance without altering overall levels of GLUT4. However, we found that quantities of fiber-specific isoenzymes (phopholamban and types I and II Ca(2+)-ATPase) were similar in all subject groups. In rectus abdominis, GLUT4 content was similar in the lean, obese, and GDM gravidas whether normalized per milligram membrane protein (relative levels were 1.0 +/- 0.2, 1.3 +/- 0.1, and 1.0 +/- 0.2, respectively) or per wet weight, total protein, and DNA. We conclude that in human disease states characterized by insulin resistance, i.e., obesity, IGT, NIDDM, and GDM, GLUT4 gene expression is normal in vastus lateralis or rectus abdominis. To the extent that these muscles are representative of total muscle mass, insulin resistance in skeletal muscle may involve impaired GLUT4 function or translocation and not transporter depletion as observed in adipose tissue.

Xanthogranulomatosis in an adult: lipid analysis of xanthomas and plasma.

Xanthomatosis in the absence of hyperlipidemia is unusual but has been associated with compositional abnormalities of lipoprotein particles. An adult who developed juvenile xanthogranulomatosis in association with oral contraceptive ingestion is reported. Plasma lipids and lipoprotein electrophoresis were normal, as in a few other patients reported with this disorder. However, analysis of cutaneous xanthoma and plasma by thin-layer and gas-liquid chromatography revealed that cholesterol was the principal lipid in xanthoma and that there were no unusual sterols in plasma or tissue. Possible mechanisms of xanthoma formation are discussed. Thus juvenile xanthogranulomatosis should be considered in adults with normolipemic xanthomatosis.

PIP: This article reports the case of a 23-year-old woman with juvenile xanthogranulomatosis, an unusual normolipemic xanthomatosis most often seen in young children. Chromatographic techniques were used to analyze this patient's plasma and xanthomatous tissue for beta-sitosterol, cholestanol, and other sterols that might be present in unusual quantities. The woman had normal fasting levels of plasma cholesterol and triglyceride. The lipoprotein electrophoresis was also normal, and levels of unusual sterols, such as cholestanol and beta-sitosterol, were not increased in plasma or in the xanthomas. Analysis of xanthoma tissue revealed that the predominant lipid was cholesterol. The only medication this patient reported using was a combination oral contraceptive (OC) containing 1 mg of norethindrone and 0.035 mg of ethinyl estradiol. OC use was initiated 1 month before the onset of cutaneous symptoms. The patient refused to discontinue OC use. Since it was not possible to withdraw the drug and observe the patient for regression of the lesions, a causal association of juvenile xanthogranulomatosis with OC use can not be asserted. This case suggests that juvenile xanthogranulomatosis should be considered in adults with normolipemic xanthomatosis. Possible mechanisms for cutaneous xanthoma formation include a defect in local lipid synthesis, an abnormality in local tissue uptake and degradation of lipoproteins that may or may not be coupled with an abnormality in circulating lipoproteins, or local invasion of histiocytes that then accumulate large amounts of cholesterol because of an intrinsic cellular abnormality.