Real-World Clinical Profile of Patients Prescribed Evolocumab in Japan.

BACKGROUND: Real-world utilization data for evolocumab, the first proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor to be introduced in Japan in 2016, to date are limited. This study aimed to clarify the current real-world patient user profiles of evolocumab based on large-scale health claims data.Methods and Results: This retrospective database study examined patients from a health administrative database (MDV database) who initiated evolocumab between April 2016 (baseline) and November 2021. Characteristics and clinical profiles of this patient population are described. In all, 4,022 patients were included in the final analysis. Most evolocumab prescriptions occurred in the outpatient setting (3,170; 78.82%), and 940 patients (23.37%) had a recent diagnosis of familial hypercholesterolemia. Common recent atherosclerotic cardiovascular disease events at baseline included myocardial infarction (1,633; 40.60%), unstable angina (561; 13.95%), and ischemic stroke (408; 10.14%). Comorbidity diseases included hypertension (2,504; 62.26%), heart failure (1,750; 43.51%), diabetes (1,199; 29.81%), and chronic kidney disease (297; 7.38%). Among the lipid-lowering regimens concomitant with evolocumab, ezetimibe+statin was used most frequently (1,281; 31.85%), followed by no concomitant lipid-lowering regimen (1,190; 29.59%), statin (950; 23.62%), and ezetimibe (601; 14.94%). The median evolocumab treatment duration for all patients was 260 days (interquartile range 57-575 days). CONCLUSIONS: This study provides real-world insights into evolocumab utilization in Japan for optimizing patient care and adherence to guideline-based therapies to better address hypercholesterolemia in Japan.

Fasiglifam/TAK-875, a Selective GPR40 Agonist, Improves Hyperglycemia in Rats Unresponsive to Sulfonylureas and Acts Additively with Sulfonylureas.

Sulfonylureas (SUs) are widely used insulin secretagogues, but they have adverse effects including hypoglycemia and secondary failure. Fasiglifam/TAK-875, a selective GPR40 agonist, enhances glucose-stimulated insulin secretion and improves hyperglycemia. In the present study, we compared the in vivo glucose-lowering effects of fasiglifam with SUs. The risk of secondary failure of fasiglifam and the efficacy in rats desensitized to SUs were also evaluated. Moreover, we assessed whether fasiglifam was effective when combined with SUs. In diabetic neonatally streptozotocin-induced rats 1.5 days after birth (N-STZ-1.5), oral administrations of fasiglifam (3-30 mg/kg) dose dependently improved glucose tolerance; the effect was greater than that of glibenclamide at maximal effective doses (glucose AUC: fasiglifam, -37.6%; glibenclamide, -12.3%). Although the glucose-lowering effects of glibenclamide (10 mg/kg/day) were completely diminished in N-STZ-1.5 rats after 4 weeks of treatment, effects were maintained in rats receiving fasiglifam (10 mg/kg/day), even after 15 weeks. Fasiglifam (3-10 mg/kg) was still effective in two models desensitized to SUs: 15-week glibenclamide-treated N-STZ-1.5 rats and aged Zucker diabetic fatty (ZDF) rats. Acute administration of fasiglifam (3 mg/kg) and glimepiride (10 mg/kg) in combination additively decreased glucose AUC (fasiglifam, -25.3%; glimepiride, -20.0%; combination, -43.1%). Although glimepiride (10 mg/kg) decreased plasma glucose below normal in nonfasted control rats, fasiglifam (3 mg/kg) maintained normoglycemia, and no further exaggeration of hypoglycemia was observed with combination treatment. These results indicate that GPR40 agonists could be more effective and durable than SUs. Our results also provide new insights into GPR40 pharmacology and rationale for the use of GPR40 agonists in diabetic patients with SU failure.

Inhibition of gastric inhibitory polypeptide signaling prevents obesity.

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.

Evolocumab Effects on Lipoproteins, Measured by High-Performance Liquid Chromatography.

AIMS: Profiling of lipoproteins can predict risk of cardiovascular disease; gel permeation high-performance liquid chromatography (HPLC) improves prediction accuracy by providing detailed data for specific lipoprotein subclasses. This study applied HPLC to examine the effects of evolocumab, which effectively treats hyperlipidemia and mixed dyslipidemia, on lipoprotein subclasses, specifically the number and size of lipoprotein particles. METHODS: This post-hoc analysis used patient blood samples from YUKAWA-2, a phase 3 trial evaluating the efficacy of evolocumab in Japanese adult patients with hyperlipidemia or mixed dyslipidemia and at high risk for cardiovascular disease. We used HPLC to assess observed values and percent change from baseline in cholesterol and triglyceride (TG) concentrations, number of particles in lipoprotein subclasses to week 12, and mean observed values and mean percent change from baseline in variables to weeks 10 and 12. HPLC was also compared with conventional methods in assessing low-density lipoprotein (LDL) cholesterol (LDL-C) values. RESULTS: Data for all 404 patients were analyzed. Evolocumab significantly decreased cholesterol and TG concentrations, and total particle count, in very low-density lipoprotein (VLDL) and LDL subclasses. Particle size increased slightly in LDL, high-density lipoprotein (HDL), and VLDL, but data varied widely. At very low LDL-C, HPLC measurements were higher than those from conventional methods. CONCLUSION: This research used HPLC to assess the effects of evolocumab in 20 lipid subclasses. By lowering lipid content and improving the lipid profile, evolocumab may reduce atherogenicity. This reduction is better quantified by HPLC than by conventional methods in the very low LDL-C range.

Inhibition of GIP signaling modulates adiponectin levels under high-fat diet in mice.

Gastric inhibitory polypeptide (GIP) is an incretin and directly promotes fat accumulation in adipocytes. Inhibition of GIP signaling prevents onset of obesity and increases fat oxidation in peripheral tissues under high-fat diet (HFD), but the mechanism is unknown. In the present study, we investigated the effects of inhibition of GIP signaling on adiponectin levels after 3 weeks of HFD by comparing wild-type (WT) mice and GIP receptor-deficient (Gipr(-/-)) mice. In HFD-fed Gipr(-/-) mice, fat oxidation was significantly increased and adiponectin mRNA levels in white adipose tissue and plasma adiponectin levels were significantly increased compared to those in HFD-fed WT mice. In addition, the PPARalpha mRNA level was increased and the ACC mRNA level was decreased in skeletal muscle of HFD-fed Gipr(-/-) mice compared with those in HFD-fed WT mice. These results indicate that inhibition of GIP signaling increases adiponectin levels, resulting in increased fat oxidation in peripheral tissues under HFD.

Genetic inactivation of GIP signaling reverses aging-associated insulin resistance through body composition changes.

Aging is associated with increased fat mass and decreased lean mass, which is strongly associated with the development of insulin resistance. Gastric inhibitory polypeptide (GIP) is known to promote efficient storage of ingested nutrients into adipose tissue; we examined aging-associated changes in body composition using 10-week-old and 50-week-old wild-type (WT) and GIP receptor knockout (Gipr-/-) mice on a normal diet, which show no difference in body weight. We found that Gipr-/- mice showed significantly reduced fat mass without reduction of lean mass or food intake, while WT mice showed increased fat mass and decreased lean mass associated with aging. Moreover, aged Gipr-/- mice showed improved insulin sensitivity, which is associated with amelioration in glucose tolerance, higher plasma adiponectin levels, and increased spontaneous physical activity. We therefore conclude that genetic inactivation of GIP signaling can prevent the development of aging-associated insulin resistance through body composition changes.

Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter.

Mutations in the hepatocyte nuclear factor (HNF)-1alpha gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1alpha via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1alpha expression plasmid into a pancreatic beta-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1alpha enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1alpha binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1alpha (amino acids 391-540) to interact with both the NH(2)-terminal region (amino acids 180-662) and the COOH-terminal region (amino acids 1,818-2,079) of p300. These findings demonstrated that HNF-1alpha binds to the 5'-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1alpha. In addition, these results provided new insight into the regulatory function of HNF-1alpha by suggesting a molecular basis for human GLUT2 gene expression.

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice.

OBJECTIVE: ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, K(ATP) channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of K(ATP) channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in K(ATP) channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. METHODS: Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. RESULTS: Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15 min (345+/-10 mg/dl vs 294+/-20 mg/dl, P<0.05) and 30 min (453+/-20 mg/dl vs 381+/-26 mg/dl, P<0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. CONCLUSION: GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in K(ATP) channel-deficient mice.

The role of the TSC-22 (-396) A/G variant in the development of diabetic nephropathy.

TSC-22 is a leucine zipper transcriptional factor and expression of the TSC-22 gene is highly induced by TGF-beta treatment. We estimated the frequency of the -396 A/G polymorphism of the TSC-22 gene with an Alu I-Restriction fragment length polymorphism (RFLP) method in 498 Japanese subjects with type 2 diabetes mellitus. We also determined the promoter activity. The diabetic patients with the AA genotype had a significantly higher incidence of the diabetic nephropathy (vs. the AG genotype, P<0.05, odds ratio: 1.95; 95% confidence intervals 1.14-3.33). There was no significant difference in the promoter activity between the fragments with -396A and -396G. These findings suggest that the TSC-22 gene (-396) A allele is associated with an increasing risk of the diabetic nephropathy.

[Regenerative medicine of the pancreas].

Insulin has been used generally in treatment of diabetic patients with absolute insulin deficiency since its discovery. However, while normal pancreatic beta-cells continually adjust insulin secretion in response to varying blood glucose levels, insulin administration cannot maintain blood glucose levels within a physiological range that protects from the development of various diabetic complications. It is possible to achieve normoglycemia in absolute insulin insufficiency by transplantation of pancreas or pancreatic islets, but the approach is impractical especially because of the shortage of transplantable pancreases and islets. For this reason, the transplantation of pancreatic beta-cells or islets generated from stem cells has become the more promising therapeutic approach to normoglycemia. In this article, recent progress of regenerative medicine of the pancreas is reviewed.

A novel antidiabetic drug, fasiglifam/TAK-875, acts as an ago-allosteric modulator of FFAR1.

Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic ß cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca(2+) influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca(2+) influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.

Tracing phenotypic reversibility of pancreatic ß-cells in vitro.

UNLABELLED: Aims/Introduction: Studies have suggested that pancreatic ß-cells undergo dedifferentiation during proliferation in vitro. However, due to limitations of the methodologies used, the question remains whether such dedifferentiated cells can redifferentiate into ß-cells. MATERIALS AND METHODS: We have established a method for cell tracing in combination with fluorescence-activated cell sorter (FACS). Using this method, mouse pancreatic ß-cells labeled with green fluorescent protein (GFP) under the control of the insulin promoter are collected by FACS. These ß-cells can be traced and characterized throughout the culture process, even when insulin becomes undetectable, because the cells are also marked with monomeric red fluorescent protein (mRFP) driven by the CAG promoter. RESULTS: When cultured with fetal mouse pancreatic cells, FACS sorted ß-cells lost GFP expression, but retained mRFP expression. The cells also lost expressions of genes characteristic of the ß-cell phenotype, such as Pdx1 and glucokinase, indicating dedifferentiation. More than 30% of such dedifferentiated pancreatic ß-cells were detected in S or G2/M phase. Furthermore, these dedifferentiated cells redifferentiated into insulin-expressing cells on cultivation with a MEK1/2 inhibitor. CONCLUSIONS: Our data provide direct evidence that pre-existing ß-cells can undergo dedifferentiation and redifferentiation in vitro, their phenotype is reversible and that dedifferentiation in ß-cells is associated with progression of the cell cycle. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00051.x, 2010).

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes.

We recently found that pancreatic acinar cells isolated from normal adult mouse can transdifferentiate into insulin-secreting cells in vitro. Using two different animal models of type 1 diabetes, we show here that insulin-secreting cells can also be generated from pancreatic acinar cells of rodents in the diabetic state with absolute insulin deficiency. When pancreatic acinar cells of streptozotocin-treated mice were cultured in suspension in the presence of epidermal growth factor and nicotinamide under low-serum condition, expressions of insulin genes gradually increased. In addition, expressions of other pancreatic hormones, including glucagon, somatostatin, and pancreatic polypeptide, were also induced. Analysis by the Cre/loxP-based direct cell lineage tracing system revealed that these newly made cells originated from amylase-expressing pancreatic acinar cells. Insulin secretion from the newly made cells was significantly stimulated by high glucose and other secretagogues. In addition, insulin-secreting cells were generated from pancreatic acinar cells of Komeda diabetes-prone rats, another animal model of type 1 diabetes. The present study demonstrates that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of rodents in the diabetic state and further suggests that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of type 1 diabetes.

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

Gastric inhibitory polypeptide modulates adiposity and fat oxidation under diminished insulin action.

Gut hormone gastric inhibitory polypeptide (GIP) stimulates insulin secretion from pancreatic beta-cells upon ingestion of nutrients. Inhibition of GIP signaling prevents the onset of obesity and consequent insulin resistance induced by high-fat diet. In this study, we investigated the role of GIP in accumulation of triglycerides into adipocytes and in fat oxidation peripherally using insulin receptor substrate (IRS)-1-deficient mice and revealed that IRS-1(-/-)GIPR(-/-) mice exhibited both reduced adiposity and ameliorated insulin resistance. Furthermore, increased gene expression of CD36 and UCP2 in liver, and increased expression and enzyme activity of 3-hydroxyacyl-CoA dehydrogenase in skeletal muscle of IRS-1(-/-)GIPR(-/-) mice might contribute to the lower respiratory quotient and the higher fat oxidation in light phase. These results suggest that GIP plays a crucial role in switching from fat oxidation to fat accumulation under the diminished insulin action as a potential target for secondary prevention of insulin resistance.

Glucose-dependent insulinotropic polypeptide receptor null mice exhibit compensatory changes in the enteroinsular axis.

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression.