Successful treatment of myxedema coma with a combination of levothyroxine and liothyronine.

Myxedema coma is a rare endocrine emergency resulting from the decompensation of severe hypothyroidism, which is associated with a high mortality rate. It is characterized by the deterioration of mental status, hypothermia, hypotension, hyponatremia, and hypoventilation. Early disease diagnosis and advancements in intensive supportive care have reduced the mortality rate. Besides intensive supportive care, appropriate management of the underlying thyroid hormone deficiency is essential. However, as the disease is rare and unrecognized, evidence-based treatment of myxedema has not yet been established in many countries. An 84-year-old Japanese man with a history of Hashimoto's thyroiditis was referred to our hospital. On arrival, conscious disturbance, hypothermia, hypotension, and hypoventilation were observed. He had discontinued thyroid hormone replacement therapy for a year. He was diagnosed with myxedema coma. Immediately, he received intensive supportive care and a combination therapy of 200 µg levothyroxine and 50 µg liothyronine until the fifth hospital day. Subsequently, monotherapy with levothyroxine was continued at a dose of 150 µg daily. The thyroid hormone level reached the normal range a few days later, and cardiovascular disease did not develop during hospitalization. This case demonstrated the efficacy of the combination of levothyroxine and liothyronine in treating myxedema coma.

Endogenous GIP ameliorates impairment of insulin secretion in proglucagon-deficient mice under moderate beta cell damage induced by streptozotocin.

AIMS/HYPOTHESIS: The action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored. METHODS: In this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)-namely glucagon gene knockout (GcgKO) mice-administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg-Gipr double knockout (DKO) mice. RESULTS: GcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets. CONCLUSIONS/INTERPRETATION: These results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival.

Identification of physical and psychosocial problems associated with diabetic nephropathy using the International Classification of Functioning, Disability and Health Core Set for Diabetes Mellitus.

BACKGROUND: We previously demonstrated validation of the Comprehensive International Classification of Functioning, Disability and Health Core Set for Diabetes Mellitus (ICF-CS for DM) in patients with diabetic nephropathy (DMN). The objective of the present study was to identify differences in experience of physical and psychosocial problems between DMN patients with and without hemodialysis (HD), and diabetes patients without nephropathy using the ICF-CS for DM. METHODS: A total of 302 diabetes outpatients (men, 68 %; mean age, 62 years) were interviewed using four components of the ICF-CS for DM including "Body functions", "Body structures", "Activities and participation", and "Environmental factors". RESULTS: The mean number of categories in which difficulty was experienced in the four components was significantly greater in DMN patients with HD followed by DMN patients without HD, and diabetes patients without nephropathy (23.9 vs. 18.0 vs. 13.1, respectively). Multivariate logistic regression models revealed that, compared with diabetes patients without nephropathy, diabetes patients with nephropathy were more likely to have difficulty with physical problems and social activities and participation. Among DMN patients, dialysis patients were found to have larger numbers of problems, and face difficulty with employment status after adjusting for sex, age, type, and duration of diabetes. CONCLUSION: The results of this study using the ICF-CS for DM identified the areas for improvement among physical and psychosocial problems in DMN patients with and without HD in contrast to diabetes patients without nephropathy.

Validation of the Comprehensive International Classification of Functioning, Disability and Health (ICF) Core Set for Diabetes Mellitus in patients with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DMN) is the most common cause of end-stage renal disease. Progression of DMN leads to impairment of physical activity, restriction of daily activities, and diminished social participation. Therefore, the precise assessment of the physical and psychosocial problems of DMN patients is important. The objective of this study was to validate the Comprehensive International Classification of Functioning, Disability and Health Core Set for Diabetes Mellitus (ICF-CS for DM) from the perspective of DMN patients. METHODS: A total of 176 DMN outpatients were interviewed using the ICF-CS for DM. Content and construct validity were evaluated. Patients were divided into 2 groups: DMN patients without hemodialysis (HD) (non-HD group) and DMN patients undergoing HD (HD group). Content validity was evaluated based on the frequency of patients who had a problem in each category. For construct validity, the patients were divided into two groups based on DM duration and hemoglobin A1C levels. RESULTS: Content validity evaluation revealed 58 categories reported as problem categories: 39 categories in the non-HD group and 50 categories in the HD group. Construct validity evaluation showed that longer DM duration and poor glycemic control contributes to increased problems. CONCLUSIONS: Content and construct validity of the ICF-CS for DM was supported from the DMN patients' perspective. Some categories of the "Environmental factors" component need further studies to be appropriate.

A Case of Hereditary Coproporphyria in which the Patient's Course Improved after the Discontinuation of Givosiran.

Hereditary coproporphyria (HCP) is caused by a partial deficiency of coproporphyrinogen oxidase during heme biosynthesis. Givosiran is approved for the treatment of acute hepatic porphyria. We herein report the case of a 47-year-old woman with HCP. Monthly givosiran administration improved her subjective symptoms and reduced her δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) levels to the normal range. However, givosiran was discontinued after six months due to a decreased renal function. The patient's ALA and PBG levels remained within the normal ranges, and her HCP-related symptoms resolved more than 2 years after the discontinuation of givosiran.

Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes.

AIMS/INTRODUCTION: Glucagon plays an essential role in hepatic glucogenesis by enhancing glycogen breakdown, inducing gluconeogenesis, and suppressing glycogenesis. Moreover, glucagon increases cyclic adenosine monophosphate (cAMP) levels, thereby activating protein kinase A (PKA) and cAMP guanine nucleotide exchange factor (also known as Epac). Although the function of PKA in the liver has been studied extensively, the function of hepatic Epac is poorly understood. The aim of this study was to elucidate the role of Epac in mediating the action of glucagon on the hepatocytes. MATERIALS AND METHODS: Epac mRNA and protein expression, localization, and activity in the hepatocytes were analyzed by reverse transcription polymerase chain reaction, western blotting, immunofluorescence staining, and Rap1 activity assay, respectively. Additionally, we investigated the effects of an Epac-specific activator, 8-CPT, and an Epac-specific inhibitor, ESI-05, on glycogen metabolism in isolated rat hepatocytes. Further mechanisms of glycogen metabolism were evaluated by examining glucokinase (GK) translocation and mRNA expression of gluconeogenic enzymes. RESULTS: Epac2, but not Epac1, was predominantly expressed in the liver. Moreover, 8-CPT inhibited glycogen accumulation and GK translocation and enhanced the mRNA expression of gluconeogenic enzymes. ESI-05 failed to reverse glucagon-induced suppression of glycogen storage and partially inhibited glucagon-induced GK translocation and the mRNA expression of gluconeogenic enzymes. CONCLUSIONS: Epac signaling plays a role in mediating the glucogenic action of glucagon in the hepatocytes.

Dipeptidyl peptidase-4 inhibitor anagliptin facilitates restoration of dextran sulfate sodium-induced colitis.

OBJECTIVE. Inflammatory bowel disease (IBD) is a chronic debilitating disease associated with severe damage to the intestinal mucosa. Glucagon-like peptide-2 (GLP-2) is a potent and specific gastrointestinal growth factor. GLP-2 released from enteroendocrine cells is inactivated by dipeptidyl peptidase-4 (DPP-4). The aim of this study was to examine whether the DPP-4 inhibitor anagliptin improves experimental murine colitis. MATERIAL AND METHODS. Male C57BL/6 mice aged 8 weeks were exposed to 1.5% dextran sulfate sodium (DSS) in drinking water for 7 days to induce experimental colitis. Anagliptin (0.1% in diet) was administrated from 2 days before the beginning of DSS to 7 days after the end of DSS. Changes in body weight and disease activity index were evaluated daily. Histological colitis severity, cellular proliferation and gene expression were determined in colonic tissues. RESULTS. Treatment with anagliptin clearly improved body weight loss and disease activity index in the recovery phase. Histological score in the DSS + anagliptin group at day 14 was significantly lower than that in the DSS alone group. Treatment with anagliptin increased the Ki67-positive rate at days 10 and 14, and tended to increase insulin-like growth factor-1 mRNA expression in the DSS + anagliptin group. CONCLUSION. In this model of experimental colitis, the DPP-4 inhibitor anagliptin facilitated the restoration of mucosal damage, thereby resulting in the acceleration of healing. These findings suggest a new and novel therapeutic approach for the treatment of IBD.

Time-dependent changes in proinflammatory and neurotrophic responses of microglia and astrocytes in a rat model of osmotic demyelination syndrome.

Osmotic demyelination syndrome (ODS) is a serious demyelinating disease in the central nervous system usually caused by rapid correction of hyponatremia. In an animal model of ODS, we previously reported microglial accumulation expressing proinflammatory cytokines. Microglia and astrocytes secreting proinflammatory cytokines and neurotrophic factors are reported to be involved in the pathogenesis of demyelinative diseases. Therefore, to clarify the role of microglial and astrocytic function in ODS, we examined the time-dependent changes in distribution, morphology, proliferation, and mRNA/protein expression of proinflammatory cytokines, neurotrophic factors, and matrix metalloproteinase (MMP) in microglia and astrocytes 2 days (early phase) and 5 days (late phase) after the rapid correction of hyponatremia in ODS rats. The number of microglia time dependently increased at demyelinative lesion sites, proliferated, and expressed tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase, and MMP2, 9, and 12 at the early phase. Microglia also expressed leukemia inhibitory factor (a neurotrophic factor) and phagocytosed myelin debris at the late phase. The number of astrocytes time dependently increased around demyelinative lesions, extended processes to lesions, proliferated, and expressed nerve growth factor and glial cell line-derived neurotrophic factor at the late phase. Moreover, treatment with infliximab, a monoclonal antibody against TNF-α, significantly attenuated neurological impairments. Our results suggest that the role of microglia in ODS is time dependently shifted from detrimental to protective and that astrocytes play a protective role at the late phase. Modulation of excessive proinflammatory responses in microglia during the early phase after rapid correction may represent a therapeutic target for ODS.

Normal plasma apoB48 despite the virtual absence of apoB100 in a compound heterozygote with novel mutations in the MTTP gene.

"Normotriglyceridemic abetalipoproteinemia (ABL)" was originally described as a clinical entity distinct from either ABL or hypobetalipoproteinemia. Subsequent studies identified mutations in APOB gene which encoded truncated apoB longer than apoB48. Therefore, "Normotriglyceridemic ABL" can be a subtype of homozygous familial hypobetalipoproteinemia. Here, we report an atypical female case of ABL who was initially diagnosed with "normotriglyceridemic ABL", because she had normal plasma apoB48 despite the virtual absence of apoB100 and low plasma TG level. Next generation sequencing revealed that she was a compound heterozygote of two novel MTTP mutations: nonsense (p.Q272X) and missense (p.G709R). We speculate that p.G709R might confer residual triglyceride transfer activity of MTTP preferentially in the intestinal epithelium to the hepatocytes, allowing production of apoB48. Together, "normotriglyceridemic ABL" may be a heterogenous disorder which is caused by specific mutations in either APOB or MTTP gene.

Investigation of d-allulose effects on high-sucrose diet-induced insulin resistance via hyperinsulinemic-euglycemic clamps in rats.

d-Allulose, a C-3 epimer of d-fructose, is a rare sugar that has no calories. Although d-allulose has been reported to have several health benefits, such as anti-obesity and anti-diabetic effects, there have been no reports evaluating the effects of d-allulose on insulin resistance using a hyperinsulinemic-euglycemic clamp (HE-clamp). Therefore, we investigated the effects of d-allulose on a high-sucrose diet (HSD)-induced insulin resistance model. Wistar rats were randomly divided into three dietary groups: HSD containing 5% cellulose (HSC), 5% d-allulose (HSA), and a commercial diet. The insulin tolerance test (ITT) and HE-clamp were performed after administration of the diets for 4 and 7 weeks. After 7 weeks, the muscle and adipose tissues of rats were obtained to analyze Akt signaling via western blotting, and plasma adipocytokine levels were measured. ITT revealed that d-allulose ameliorated systemic insulin resistance. Furthermore, the results of the 2-step HE-clamp procedure indicated that d-allulose reversed systemic and muscular insulin resistance. d-Allulose reversed the insulin-induced suppression of Akt phosphorylation in the soleus muscle and epididymal fat tissues and reduced plasma TNF-α levels. This study is the first to show that d-allulose improves systemic and muscle insulin sensitivity in conscious rats.

Activation of vasopressin neurons leads to phenotype progression in a mouse model for familial neurohypophysial diabetes insipidus.

Familial neurohypophysial diabetes insipidus (FNDI) is a rare disease that is inherited in an autosomal dominant manner. In a previous study, we made a mouse model for FNDI, which showed progressive polyuria accompanied by inclusion bodies in the arginine vasopressin (AVP) neurons formed by aggregates in the endoplasmic reticulum. The present study was conducted to determine whether the activities of AVP neurons are related to the phenotype progression in the FNDI model. In the first experiment, female heterozygous mice were administered either desmopressin (dDAVP) or a vehicle (control) subcutaneously with osmotic minipumps for 30 days. The dDAVP treatment significantly decreased the urine volume, AVP mRNA expression, and inclusion bodies in the AVP neurons. Urine volume in the dDAVP group remained significantly less than the control for 14 days even after the minipumps were removed. In the second experiment, the males were fed either a 0.2% Na or 2.0% Na diet for 6 mo. Urine AVP excretion was significantly increased in the 2.0% Na group compared with the 0.2% Na group for the first 2 mo but gradually decreased thereafter. Throughout the experiments, urine volume increased progressively in the 2.0% Na group but not in the 0.2% Na group. Immunohistochemical analyses revealed that inclusion bodies in the AVP cells had significantly increased in the 2.0% Na compared with the 0.2% Na group. These data demonstrated that activation of AVP neurons could accelerate the aggregate formation as well as the progression of the polyuria in the FNDI model mice.

Retroperitoneal solitary fibrous tumor-induced hypoglycemia associated with high molecular weight insulin-like growth factor II.

A man, aged 65 years, presented with frequent episodes of hypoglycemia and unconsciousness. Hypoglycemia was accompanied by undetectable serum insulin and C-peptide levels and a high serum insulin-like growth factor (IGF)-II level. He was found to have a retroperitoneal solitary fibrous tumor. He underwent successful resection of the tumor and had no hypoglycemic episodes after the operation. Immunohistochemical analysis revealed positive immunostaining for IGF-II in tumor cells. The presence of the high-molecular-weight form of IGF-II in the patient's serum was confirmed by immunoblotting, which suggests that his hypoglycemia was due to an increase in the plasma level of IGF-II secreted by the tumor.

Up-regulation of surfactant protein production in a mouse model of secondary pulmonary alveolar proteinosis.

Although Pneumocystis infection might be one of the causes of secondary pulmonary alveolar proteinosis (PAP), the mechanism of its pathogenesis is uncertain. We analyzed a mouse model of secondary PAP resulting from Pneumocystis infection using mice deficient in CD40 (CD40KO), and evaluated the mechanism of the pathogenesis of secondary PAP from the viewpoint of surfactant-associated protein (SP) homeostasis, the overproduction of SP by type II alveolar epithelial cells, and the phagocytic function of alveolar macrophages (AMs). The effect of CD40 on SP production was also investigated in vitro using the H441 cell line, which has a phenotype similar to type II alveolar epithelial cells and primary alveolar epithelial cells. After long-term exposure to ovalbumin, CD40KO mice showed Pneumocystis infection and accumulation of surfactants in the alveoli (ApCD40KO). The amounts of SP production were up-regulated in ApCD40KO mice compared with wild-type mice treated using the same procedure. On the other hand, AMs from ApCD40KO mice did not show either phagocytic dysfunction or down-regulation of PU.1 expression. Furthermore, the stimulation of CD40-CD40 ligand (CD154) pathway regulated the production of SPs in H441 cells or primary alveolar epithelial cells. These results suggested that CD40KO mice could be one of the models useful for developing secondary PAP resulting from Pneumocystis infection. Surfactant accumulation was due to the overproduction in our model of secondary PAP. The CD40-CD154 interaction plays an important role in the regulation of surfactant-associated protein production.

Glucocorticoids increase neuropeptide Y and agouti-related peptide gene expression via adenosine monophosphate-activated protein kinase signaling in the arcuate nucleus of rats.

Recent studies suggest that the AMP-activated protein kinase (AMPK) signaling in the hypothalamus is the master regulator of energy balance. We reported in previous studies that glucocorticoids play a permissive role in the regulation of orexigenic neuropeptide Y (Npy) gene expression in the arcuate nucleus. In this study, we examined whether any cross talk occurs between glucocorticoids and AMPK signaling in the hypothalamus to regulate Npy as well as agouti-related peptide (Agrp) gene expression in the arcuate nucleus. In the hypothalamic organotypic cultures, the addition to the medium of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside, increased phosphorylated AMPK (p-AMPK) as well as phosphorylated acetyl-coenzyme A carboxylase (p-ACC) in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The incubation with dexamethasone (DEX) also activated AMPK signaling in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The addition of the AMPK inhibitor compound C to the medium, which blocked increases of p-AMPK and p-ACC by DEX, significantly attenuated Npy and Agrp gene expression stimulated by DEX. Furthermore, p-AMPK and p-ACC levels in the arcuate nucleus were significantly decreased in adrenalectomized rats compared with sham-operated rats, and a replacement of glucocorticoids reversed the AMPK signaling in adrenalectomized rats. Thus, our data demonstrated that glucocorticoids up-regulate the Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling, suggesting that the activation of the hypothalamic APMK signaling by glucocorticoids might be essential to the energy homeostasis.

Novel treatment for lithium-induced nephrogenic diabetes insipidus rat model using the Sendai-virus vector carrying aquaporin 2 gene.

Congenital nephrogenic diabetes insipidus (NDI) is a chronic disorder involving polyuria and polydipsia that results from unresponsiveness of the renal collecting ducts to the antidiuretic hormone vasopressin. Either of the genetic defects in vasopressin V2 receptor or the water channel aquaporin 2 (AQP2) cause the disease, which interfere the water reabsorption at the epithelium of the collecting duct. An unconscious state including a perioperative situation can be life threatening because of the difficulty to regulate their water balance. The Sendai virus (SeV) vector system deleting fusion protein (F) gene (SeV/DeltaF) is considered most suitable because of the short replication cycle and nontransmissible character. An animal model for NDI with reduced AQP2 by lithium chloride was used to develop the therapy. When the SeV/DeltaF vector carrying a human AQP2 gene (AQP2-SeV/DeltaF) was administered retrogradely via ureter to renal pelvis, AQP2 was expressed in the renal collecting duct to reduce urine output and water intake by up to 40%. In combination with the retorograde administration to pelvis, this system could be the cornerstone for the applicable therapies on not only NDI patients but also other diseases associate with the medullary collecting duct.

Direct and indirect modulation of neuropeptide Y gene expression in response to hypoglycemia in rat arcuate nucleus.

Expression of neuropeptide Y (Npy) heteronuclear (hn) RNA, an indicator of gene transcription, was significantly increased in the arcuate nucleus of rats 30min after insulin injection. Npy hnRNA levels were also increased significantly in response to hypoglycemia in rats in which the hypothalamus was deafferentated, although the absolute levels were significantly lower than in sham-operated rats. Direct effects of lowering glucose levels on Npy gene expression were also confirmed in hypothalamic organotypic cultures. Thus, Npy gene transcription in the arcuate nucleus increases rapidly in response to hypoglycemia, and both direct and indirect inputs are involved in the rapid upregulation.

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5).

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.

Peripherally administered baclofen reduced food intake and body weight in db/db as well as diet-induced obese mice.

Peripheral administration of baclofen significantly reduced food intake and body weight increase in both diabetic (db/db) and diet-induced obese mice for 5 weeks, whereas it had no significant effects on energy balance in their lean control mice. Despite the decreased body weight, neuropeptide Y expression in the arcuate nucleus was significantly decreased, whereas pro-opiomelanocortin expression was significantly increased by baclofen treatment. These data demonstrate that the inhibitory effects of baclofen on body weight in the obese mice were mediated via the arcuate nucleus at least partially, and suggest that GABA(B) agonists could be a new therapeutic reagent for obesity.

Central administration of melanocortin agonist increased insulin sensitivity in diet-induced obese rats.

In this study, we examined the effects of intracerebroventricular administration of melanotan II (MTII), a melanocortin agonist, on insulin sensitivity in diet-induced obese (DIO) rats. Although MTII treatment significantly decreased food intake and body weight for 10 days, there was no significant difference in body weight between MTII and pair-fed groups. The insulin tolerance test showed that insulin sensitivity was significantly improved in the MTII group compared to the pair-fed group. Furthermore, MTII treatment increased the number of small-sized adipocytes in epididymal white adipose tissues, suggesting that MTII increased insulin sensitivity through action on the white adipose tissues in DIO rats.

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A.

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.