Atsushi Kosaki

Reduced Expression of the Leptin Gene (ob) by Catecholamine Through a GS Protein-Coupled Pathway in 3T3-L1 Adipocytes

Leptin, a recently identified hormone, is believed to reduce appetite and maintain body weight. The mRNA of leptin is expressed only in mature adipose cells. To clarify the regulation of leptin gene expression in adipocytes, 3T3-L1 adipocytes were treated for 16 h with various agents known to modulate lipid metabolism, and then the leptin mRNA was measured by the reverse transcription-polymerase chain reaction method. Interestingly, both norepinephrine and isoproterenol reduced the level of leptin mRNA to about 20% of that found in untreated control cells in a dose-dependent fashion. The maximum reduction occurred at 100 nmol/l of either norepinephrine or isoproterenol, and the half-maximal effect was observed at ∼ 3 nmol/l norepinephrine and ∼ 1 nmol/l isoproterenol. Propranolol reversed about 50% of the reduction by either norepinephrine or isoproterenol. In contrast, phentolamine did not inhibit the reduction by either norepinephrine or isoproterenol. Moreover, both cholera toxin and dibutyryl cAMP decreased the level of leptin mRNA to about 10% of that in control cells in a dose-dependent fashion. The maximum effect was elicited at 100 ng/ml cholera toxin and 100 micromol/l dibutyryl cAMP. The concentration producing the half-maximal effect was ∼ 1 ng/ml cholera toxin and ∼ 50 μmol/l dibutyryl cAMP. Dibutyryl cGMP, however, did not affect leptin gene expression. These results suggest that a signaling pathway that results in the activation of protein kinase A regulates leptin gene expression in 3T3-L1 adipocytes.

14-3-3β Protein Associates with Insulin Receptor Substrate 1 and Decreases Insulin-stimulated Phosphatidylinositol 3′-Kinase Activity in 3T3L1 Adipocytes

The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3β antibody at the basal condition. 100 nm insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nm wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3β fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3′-kinase (PI3K) is supposed to complex either 14-3-3β-IRS-1 or IRS-1. The 14-3-3β-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3β and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3β protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.

Regulation of glucose-transporter gene expression by insulin in cultured human fibroblasts.

To clarify the effect of insulin on glucose-transporter (GT) biosynthesis, we determined GT mRNA levels in human cultured skin fibroblasts, using HepG2 GT cDNA as a probe. Insulin specifically increased the GT mRNA level in a time- and dose-dependent manner. Time-course study demonstrated that the mRNA level peaked within 3 h of insulin (1 × 10−7 M) addition. After remaining elevated for several hours, mRNA decreased and returned to the basal level after 24 h. In the cell strains from seven normal subjects, the mean (±SE) GT mRNA level determined after 3 h of treatment with 1 × 10−7 M insulin was 164.3 ± 8.5% of the level found in untreated control cells. The insulin dose-response curve of GT mRNA levels showed that the maximum stimulation was elicited at 1 × 10−7 M, and the half-maximum stimulation occurred at ∼5 × 10−10 M. Degradation rates of GT mRNA determined in the presence of actinomycin D were not different between insulin-treated and untreated cells. These results suggest that insulin increases GT gene expression in cultured human fibroblasts.

Effects of a high-fat diet on insulin receptor kinase and the glucose transporter in rats

Abstract A high-fat (HF) diet causes insulin resistance. To characterize this type of insulin resistance, autophosphorylation of the insulin receptor (IR) in vitro and in vivo, phosphorylation of its endogenous substrates (pp190, pp175), and the amount of glucose transporters (GLUT-2, GLUT-4) were determined using muscles and liver from rats fed a HF-diet. Hyperinsulinemia and insulin resistance were observed by oral glucose tolerance test in the HF group. The levels of insulin binding to wheat germ agglutinin-purified insulin receptors from both muscles and liver were similar to those of the controls. Autophosphorylation of IR on tyrosine residues in vitro was decreased both in muscles and liver. Tyrosine-phosphorylation of both IR and an endogenous substrate (pp175) in response to insulin injection in vivo was decreased in the liver. However, a decrease in the phosphorylation of pp190 in muscles was not found. The levels of both GLUT-4 from muscles and GLUT-2 from liver in the crude membrane were comparable to those of the controls. These data suggest that insulin resistance in rats fed a HF-diet is a post-receptor defect in muscles, although tyrosine-phosphorylation of both IR and its endogenous substrate (pp175) were decreased in liver.

Insulin resistance in a boy with congenital generalized lipodystrophy

ABSTRACT: We have studied insulin resistance in a 12-year-old Japanese boy who presented with congenital generalized lipodystrophy. Oral glucose tolerance test exhibited a diabetic pattern with normal fasting plasma glucose. Results from euglycemic glucose clamp study showed decreases in both insulin sensitivity and responsiveness. Both the patients erythrocytes and Epstein-Barr virus transformed lymphocytes showed low-normal insulin binding with a slight reduction in binding affinity in the latter. Insulin binding to the cultured fibroblasts was decreased due to a lowered affinity. In addition, they displayed a rightward shift of the insulin dose-response curve for D-14C-glucose uptake with no decrease in the maximum uptake. Insulin-stimulated autophosphorylation and kinase activity of the wheat germ agglutinin purified receptors from the Epstein-Barr virus-transformed lymphocytes appeared normal. The reason for some discrepancies in insulin binding among the cells remains unknown, and we cannot formulate a conclusion as to whether or not a primary binding defect of insulin receptors exists and contributes to insulin resistance in the patient. The decrease in insulin responsiveness demonstrated in the glucose clamp study may result from a defect at the rate-limiting step in the postbinding process of insulin action, presumably a defect in the glucose transport system in muscle tissues. The defect may be secondary to changes in in vivo circumstances.

Insulin resistance in werner's syndrome

Insulin resistance in Werners syndrome (WS) was studied using the glucose clamp technique, and compared with physiologically aged and young subjects. Fasting immuno-reactive insulin (IRI) was increased in patients with Werners syndrome compared with aged and young subjects. Metabolic clearance rate (MCR) of glucose was decreased in the aged and WS. A rightward shift of the dose-response curves of insulin and MCR of glucose was observed in the aged and WS with a more pronounced shift in the latter. MCR of insulin was also decreased in WS. [125I]insulin binding to erythrocytes was similar in the three groups. These results suggest that insulin resistance associated with WS is due to a post-binding defect manifested by a rightward shift of the dose-response curve of insulin-induced glucose disposal and a decrease in insulin clearance rate.

Regulation of glucose transporter synthesis in cultured human skin fibroblasts

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.