Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells.

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [(3)H]D-chiro-inositol (DCI) by 159-fold. [(3)H]myo-Inositol uptake increased by 37-fold. In contrast, [(14)C]D-glucose, [(14)C]2-deoxy-D-glucose, or [(14)C]3-O-methyl-D-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K(m) of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 microM, respectively, whereas glucose competed for DCI uptake with a K(i) of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 microM for 24 h) increased [(3)H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.

Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele.

Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70-80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob(38.9/38.9)) mice were crossbred with Apobec-1 knockout (Apobec-1(-/-)) mice or apoB-100-only (Apob(100/100)) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice compared with those of the respective Apobec-1(-/-)/Apob(+/+) and Apob(100/100) control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele.

Amino terminal 38.9% of apolipoprotein B-100 is sufficient to support cholesterol-rich lipoprotein production and atherosclerosis.

OBJECTIVE: Carboxyl terminal truncation of apolipoprotein (apo)B-100 and apoB-48 impairs their capacity for triglyceride transport, but the ability of the resultant truncated apoB to transport cholesterol and to support atherosclerosis has not been adequately studied. The atherogenicity of apoB-38.9 was determined in this study by using our apoB-38.9-only (Apob38.9/38.9) mice. METHODS AND RESULTS: ApoB-38.9-lipoproteins (Lp-B38.9) circulate at very low levels in Apob38.9/38.9 mice as small LDLs or HDLs. Disruption of apoE gene in these mice caused accumulation of large amounts of betaVLDL-like LpB-38.9 in plasma. These betaVLDL particles were more enriched with cholesteryl esters but poor in triglycerides compared with the apoB-48-betaVLDL of the apoB-wild-type/apoE-null (Apob+/+/Apoe-/-) mice. Likewise, apoB-38.9-VLDL secreted by cultured Apob38.9/38.9 mouse hepatocytes also had higher ratios of total cholesterol to triglycerides than apoB-48-VLDL secreted by the apoB-48-only hepatocytes. Thus, despite its impaired triglyceride-transporting capacity, apoB-38.9 has a relatively intact capacity for cholesterol transport. Spontaneous aortic atherosclerotic lesions were examined in apoB-38.9-only/apoE-null (Apob38.9/38.9/Apoe-/-) mice at ages 9 and 13 months. Extensive lesions were found in the Apob38.9/38.9/Apoe-/- mice as well as in their Apob+/38.9/Apoe-/- and Apob+/+/Apoe-/- littermates. CONCLUSIONS: Deleting the C-terminal 20% from apoB-48 does not impair its ability to transport cholesterol and to support atherosclerosis, thus narrowing the "atherogenic region" of apoB.

Hypobetalipoproteinemic mice with a targeted apolipoprotein (Apo) B-27.6-specifying mutation: in vivo evidence for an important role of amino acids 1254-1744 of ApoB in lipid transport and metabolism of the apoB-containing lipoprotein.

Carboxyl-terminal deletion of apoB-100 may impair its triglyceride (TG)-transporting capability and alter its catabolism. Here, we compare our newly generated apoB gene (Apob)-targeted apoB-27.6-bearing mice to our previously reported apoB-38.9 mice to understand further the relationship between the size of a truncated apoB variant and its function/metabolism in vivo. The apoB-27.6-specifying mutation produces a premature stop codon six amino acids (aa) downstream of the last codon of mouse Apob exon 24 (corresponding to aa 1254 of human apoB-100). ApoB-27.6 transcripts were 3- and 5-fold more abundant than apoB wild type and apoB-38.9 transcripts in the liver. Likewise, hepatic secretion rates of apoB-27.6 were 7-fold higher than those of apoB-48 and apoB-38.9. In contrast, apoB-27.6 heterozygotes (Apob(27.6/+)) had lower hepatic TG secretion rates and higher liver TG contents than both apoB-38.9 heterozygotes (Apob(38.9/+)) and apoB wild type mice (Apob(+/+)). ApoB-27.6 was secreted by Apob(27.6/+) hepatocytes as dense high density lipoprotein particles. Moreover, despite its high secretion rates, apoB-27.6 was barely detectable in plasma. Disruption of apoE gene in Apob(38.9/+) and Apob(27.6/+) dramatically increased plasma levels of apoB-38.9 as well as apoB-48 but caused no change in plasma apoB-27.6 concentrations. Finally, the birth rate of apoB-27.6 homozygotes (Apob(27.6/27.6)) from intercrosses of Apob(27.6/+) was 7-fold lower than that of Apob(38.9/38.9) from Apob(38.9/+) intercrosses (1.8% versus 12%). Crossbreeding of Apob(27.6/27.6) and Apob(38.9/38.9) produced viable Apob(27.6/38.9) offspring, but Apob(27.6/27.6) intercrosses produced no offspring. Together, these results demonstrate in vivo that the apoB-27.6-apoB-38.9 peptide segment (aa 1254-1744) plays a critical role, not only in supporting hepatic TG-secretion and in modulating catabolism of apoB-containing lipoproteins, but also in normal mouse embryonic development.