Narihito Yoshioka

Phosphorylation of the 6-Phosphofructo-2-Kinase/Fructose 2,6-Bisphosphatase/PFKFB3 Family of Glycolytic Regulators in Human Cancer

Purpose: Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase, which is a rate-limiting enzyme of glycolysis. The concentration of F2,6BP depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase). Four genes encoding PFK-2/FBPase have been identified and termed PFKFB1 to PFKFB4. PFKFB3 protein is expressed in high levels in human tumors in situ. The purpose of this study was to determine the role of functional interactions between the phosphorylation of PFKFB3 and activated glycolysis in human cancer cells. Experimental Design: cDNA from several human tumor cell lines and human colon carcinoma were analyzed by reverse transcription-PCR to identify different splicing variants of PFKFB3. The effect of phosphorylation of Ser461 was studied by recombinantly replacing this residue with glutamate (PFKFB3S461E). The phosphorylation of PFKFB3 protein in human cancer was determined by immunostaining using an anti-phospho-PFK-2(PFKFB3) antibody. Results: Two splicing variants of PFKFB3 are expressed in human cancer cell lines: PFKFB3-ACG and PFKFB3-AG. Quantitative, real-time PCR analysis confirmed the overexpression of PFKFB3 mRNA in colon carcinoma, with the dominant variant being the PFKFB3-ACG isoform that contains a phosphorylation site at Ser461. Forced expression of PFKFB3-ACG in COS-7 cells resulted in enhanced glycolysis. Introduction of PFKFB3-ACGS461E into COS-7 cells led to increased the lactate production and cell proliferation. Highly phosphorylated PFKFB3 protein was found in human tumor cells, vascular endothelial cells, and smooth muscle cells, as determined by immunostaining with an anti-phospho-PFK-2(PFKFB3) antibody. Conclusions: These findings support a potential role for the phosphorylation of PFKFB3 protein in the progression of cancer and angiogenesis.

The Proinflammatory Cytokine Macrophage Migration Inhibitory Factor Regulates Glucose Metabolism during Systemic Inflammation

Inflammation provokes significant abnormalities in host metabolism that result from the systemic release of cytokines. An early response of the host is hyperglycemia and resistance to the action of insulin, which progresses over time to increased glucose uptake in peripheral tissue. Although the cytokine TNF-α has been shown to exert certain catabolic effects, recent studies suggest that the metabolic actions of TNF-α occur by the downstream regulation of additional mediators, such as macrophage migration inhibitory factor (MIF). We investigated the glycemic responses of endotoxemic mice genetically deficient in MIF (MIF−/−). In contrast to wild-type mice, MIF−/− mice exhibit normal blood glucose and lactate responses following the administration of endotoxin, or TNF-α. MIF−/− mice also show markedly increased glucose uptake into white adipose tissue in vivo in the endotoxemic state. Treatment of adipocytes with MIF, or anti-MIF mAb, modulates insulin-mediated glucose transport and insulin receptor signal transduction; these effects include the phosphorylation of insulin receptor substrate-1, its association with the p85 regulatory subunit of PI3K, and the downstream phosphorylation of Akt. Genetic MIF deficiency also promotes adipogenesis, which is in accord with a downstream role for MIF in the action of TNF-α. These studies support an important role for MIF in host glucose metabolism during sepsis.

Perilipin Overexpression in White Adipose Tissue Induces a Brown Fat-Like Phenotype

Background Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model. Methodology and Principal Findings When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size. Conclusions These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.

Perilipin overexpression in mice protects against diet-induced obesity

Perilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specific aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholamine-stimulated lipolysis was decreased and glucose tolerance significantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our findings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications.

Impact of Small-Molecule Glucokinase Activator on Glucose Metabolism and β-Cell Mass

We investigated the effect of glucokinase activator (GKA) on glucose metabolism and beta-cell mass. We analyzed four mouse groups: wild-type mice and beta-cell-specific haploinsufficiency of glucokinase gene (Gck(+/-)) mice on a high-fat (HF) diet. Each genotype was also treated with GKA mixed in the HF diet. Rodent insulinoma cells and isolated islets were used to evaluate beta-cell proliferation by GKA. After 20 wk on the above diets, there were no differences in body weight, lipid profiles, and liver triglyceride content among the four groups. Glucose tolerance was improved shortly after the GKA treatment in both genotypes of mice. beta-Cell mass increased in wild-type mice compared with Gck(+/-) mice, but a further increase was not observed after the administration of GKA in both genotypes. Interestingly, GKA was able to up-regulate insulin receptor substrate-2 (Irs-2) expression in insulinoma cells and isolated islets. The administration of GKA increased 5-bromo-2-deoxyuridine (BrdU) incorporation in insulinoma cells, and 3 d administration of GKA markedly increased BrdU incorporation in mice treated with GKA in both genotypes, compared with those without GKA. In conclusion, GKA was able to chronically improve glucose metabolism for mice on the HF diet. Although chronic GKA administration failed to cause a further increase in beta-cell mass in vivo, GKA was able to increase beta cell proliferation in vitro and with a 3-d administration in vivo. This apparent discrepancy can be explained by a chronic reduction in ambient blood glucose levels by GKA treatment.

Effects of Dietary Treatment on Serum Insulin and Proinsulin Response in Newly Diagnosed NIDDM

Serum proinsulin is disproportionately elevated both in the basal state and after an oral glucose load in non-insulin dependent diabetes mellitus (NIDDM). However, there is no detailed information about the effect of glycemic control on this abnormality. We investigated the effect of glycemic control by dietary treatment on serum proinsulin level in the basal state and in response to an oral glucose load. Ten NIDDM patients (7 men and 3 women), aged 19–60 yr, with mean (± SD) body mass index of 28 ± 6 kg/m2 (range 21–42 kg/m2) and normal renal and liver function were studied. Before and after dietary therapy (25–30 kcal/kg ideal body wt), 100-g oral glucose tolerance tests were performed. Proinsulin was measured with our proinsulin-specific antiserum, which recognizes the connecting site of the B-chain of insulin and C-peptide. After dietary treatment, fasting plasma glucose decreased from 197 ± 35 to 113 ± 18 mg/dl (P < .001). Both serum insulin and proinsulin decreased (insulin from 15 ± 8 to 10 ± 4 μU/ml, P < .02; proinsulin from 31 ± 18 to 13 ± 5 pM, P < .02), and the molar ratio of proinsulin to insulin also tended to decrease (from 0.31 ± 0.08 to 0.24 ± 0.10, P < .10). Insulin response to oral glucose increased after dietary treatment, whereas proinsulin response did not change, resulting in a significant decrease in the molar ratio of the area under the curve of proinsulin to insulin after glucose load (from 0.28 ± 0.12 to 0.13 ± 0.07, P < .001). These results support the concept that glycemic control attained by calorie restriction allows recovery of disproportionate oversecretion of proinsulin in patients with NIDDM, presumably decreasing the demand for insulin secretion from β-cells.

Differences in physician and patient perceptions about insulin therapy for management of type 2 diabetes: the DAWN Japan study

Abstract Objective: To better understand patient resistance to initiation of insulin therapy, this study examined the perception gap concerning initiation of insulin therapy between individuals with type 2 diabetes and their physicians by using data from the DAWN Japan study. Methods: The DAWN Japan study is a multi-center, questionnaire-based survey, conducted between 2004–2005. Patients recommended to start insulin therapy (n = 148) answered a questionnaire by rating degree of agreement with 16 statements concerning insulin therapy on a 5-point scale (1: strongly disagree to 5: strongly agree). Ratings of 1 and 2 were categorized as ‘disagree’ with a statement, and 3, 4, and 5 as ‘agree’. Their attending physicians (n = 68) selected statements which could be associated with patient’s concerns about insulin therapy. Results: Nearly all the patients agreed with the statements ‘I don’t want to inject myself for the rest of my life’ (95%), and ‘I don’t want to be bothered with doing injections’ (90%); fewer than half agreed with ‘My friendships may suffer’ (46%), and ‘I don’t understand why insulin is necessary for me’ (45%). Estimation by the physicians and the actual perceptions patients reported differed significantly for 13 statements. Physicians seemed to particularly under-estimate the impact associated with social aspects of insulin use (e.g., ‘I don’t want to be different from others’, 55% patients vs 7% physicians). On the contrary, the statement ‘Injections are painful’ was the only concern over-estimated by the physicians. Conclusions: It was demonstrated that differences in perceptions regarding insulin therapy exist between physicians and patients, particularly in terms of social impacts. The data, obtained in 2004, may not precisely reflect the present situation, but still represents a barrier to insulin therapy widely held by patients and physicians. These results suggest that appropriate understanding of patients’ concerns about insulin therapy is important to encourage timely insulin initiation.

Step-up therapy with biphasic insulin aspart-70/30 : Sapporo 1-2-3 study

The effectiveness of BIAsp 30 step-up therapy in achieving glycemic control in Japanese patients with type 2 diabetes mellitus was investigated. Study subjects were 99 patients with type 2 diabetes mellitus aged over 20 years who were judged to require insulin therapy due to poor glucose control (HbA1c level of > or =7.5%). BIAsp 30 dosage was determined by the patients attending physician; coadministration of hypotensive agents and antilipemic agents was permitted, but OAD coadministration was limited to patients already receiving such drugs at the start of the study. Patients who did not achieve HbA1c <6.5% after 16+/-5 weeks with QD (Phase 1) were stepped up to BID (Phase 2). If patients still had not achieved HbA1c <6.5% after 16+/-5 weeks with BID, they were stepped up to TID (Phase 3). 55 of the 99 enrolled subjects completed the study and the rates of achievement of HbA1c <6.5% and HbA1c <7.0% were 45.5% and 74.5%, respectively. Of all registered subjects, 5.1% (5/99) achieved HbA1c <6.5% in QD, 19.5% (16/82) in BID, and 20.6% (7/34) in TID. Statistically significant reductions in HbA1c levels were recorded at the conclusion of each phase, with no incidents requiring intervention, indicating that BIAsp 30 step-up therapy is a safe, simple therapy that can be useful in achieving better glycemic control for Japanese patients with type 2 diabetes mellitus.

Problems in diagnosing atypical Gitelman’s syndrome presenting with normomagnesaemia

Objective  Gitelman’s syndrome, recognized as a variant of Bartter’s syndrome, is characterized by hypokalaemic metabolic alkalosis in combination with hypomagnesaemia and hypocalciuria. Overlapping biochemical features in Gitelman’s syndrome and Bartter’s syndrome has been observed. Here, we investigated the clinical, biochemical, and genetic characteristics of five, chronic, nonhypertensive and hypokalaemic Japanese patients.

HLA DR antigens in adult-onset and juvenile-onset Japanese insulin-dependent diabetic patients

In order to discover the HLA DR antigens linked to Japanese insulin-dependent diabetes (IDDM), and to relate them to the clinical features, HLA DR antigens were examined in 75 IDDM patients including 56 adult-onset cases. Among the tested HLA DR antigens, 4, w9 and w13 were significantly more frequent in IDDM (55%, 47% and 27% respectively). The relative risk was 1.71 for DR4, 2.81 for DRw9 and 4.74 for DRw13. DR2 was significantly less frequent and the relative risk was 0.14. The distribution of DR antigens did not differ between juvenile-onset and adult-onset IDDM, males and females, or cases with and without thyroid autoantibodies. Homozygotes for DRw9 were, but those for DRw13 and DR4 were not more frequent than expected by a random combination. Heterozygotes for DR4 and w9 were less frequent while other heterozygotes for high-risk antigens were as frequent as expected. 97% of IDDM had either DR4, w9 or w13. In conclusion, HLA DR4, w9 and w13 were significantly increased in patients with both juvenile- and adult-onset IDDM. There was no surplus increase in the frequency of IDDM patients with two high-risk HLA DR antigens, more than expected from random combination of each of these DR antigens. Clinical features did not differ among IDDM patients with each of these three antigens.