Increment of plasma glucose by exogenous glucagon is associated with present and future renal function in type 2 diabetes:a retrospective study from glucagon stimulation test.

BACKGROUND: Glucagon stimulation test (GST) is often employed to assess the insulin reserve of the pancreatic beta cells in diabetic subjects. The clinical significance of the increment of plasma glucose (Δglucose) by exogenous glucagon during GST has not been elucidated. We investigated the relationship between Δglucose and clinical parameters including the liver and renal function in type 2 diabetic subjects, since we hypothesized that Δglucose is associated with the liver and renal function reflecting the capacity for gluconeogenesis in the organs. METHODS: A total of 209 subjects with type 2 diabetes who underwent GST during admission were included in this cross-sectional study. We defined the difference between plasma glucose at fasting and 6 min after intravenous injection of 1 mg glucagon as Δglucose. We assessed correlations between Δglucose and clinical parameters such as diabetic duration, BMI, HbA1c, beta cell function, serum free fatty acids (FFA) which is known to stimulate gluconeogenesis, liver function, the indices of liver function, renal function, and urinary albumin excretion (UAE). RESULTS: In correlation analysis, Δglucose positively correlated to FFA and estimated glomerular filtration rate (eGFR), but inversely to serum creatinine and cystatin C, although Δglucose showed no correlation with both liver function and the indices of residual liver function. Multiple regression analysis revealed that Δglucose was an independent determinant for the eGFR after 1 year, equally BMI, HbA1c, serum lipids, and UAE, which are known as the predictors for the development of chronic kidney disease. CONCLUSION: Our results suggest that Δglucose during GST might be related to gluconeogenesis in the kidney and could be the determinant of future renal function in type 2 diabetes.

Pancreatic glucose-dependent insulinotropic polypeptide (GIP) (1-30) expression is upregulated in diabetes and PEGylated GIP(1-30) can suppress the progression of low-dose-STZ-induced hyperglycaemia in mice.

AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released from gut K cells. While the predominant form is GIP(1-42), a shorter form, GIP(1-30), is produced by pancreatic alpha cells and promotes insulin secretion in a paracrine manner. Here, we elucidated whether GIP(1-30) expression is modulated in mouse models of diabetes. We then investigated whether PEGylated GIP(1-30) can improve islet function and morphology as well as suppress the progression to hyperglycaemia in mice treated with low-dose streptozotocin (LD-STZ). METHODS: We examined pancreatic GIP immunoreactivity in rodent diabetic models. We synthesised [D-Ala(2)]GIP(1-30) and modified the C-terminus with polyethylene glycol (PEG) to produce a dipeptidyl peptidase-4 (DPP-4)-resistant long-acting GIP analogue, [D-Ala(2)]GIP(1-30)-PEG. We performed i.p.GTT and immunohistochemical analysis in non-diabetic and LD-STZ diabetic mice, with or without administration of [D-Ala(2)]GIP(1-30)-PEG. RESULTS: Pancreatic GIP expression was concomitantly enhanced with alpha cell expansion in rodent models of diabetes. Treatment with DPP-4 inhibitor decreased both the GIP- and glucagon-positive areas and preserved the insulin-positive area in LD-STZ diabetic mice. Body weight was not affected by [D-Ala(2)]GIP(1-30)-PEG in LD-STZ or non-diabetic mice. Treatment with GIP significantly ameliorated chronic hyperglycaemia and improved glucose excursions in LD-STZ mice. Treatment with GIP also reduced alpha cell expansion in the islets and suppressed plasma glucagon levels compared with non-treated LD-STZ mice. Additionally, [D-Ala(2)]GIP(1-30)-PEG preserved beta cell area via inhibition of apoptosis in LD-STZ mice. CONCLUSIONS/INTERPRETATION: Our data suggest that GIP(1-30) expression is upregulated in diabetes, and PEGylated GIP(1-30) can suppress the progression to STZ-induced hyperglycaemia by inhibiting beta cell apoptosis and alpha cell expansion.

Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells.

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12 cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

Implication of intracellular localization of transcriptional repressor PLZF in thyroid neoplasms.

BACKGROUND: Promyelocytic leukaemia zinc finger (PLZF) is a transcriptional repressor that was originally isolated from a patient with promyelocytic leukaemia. PLZF also affects key elements for cell cycle progression, such as cyclin A, and can affect the tumourigenicity of various cancers. Thus far, the behaviour of PLZF in thyroid carcinoma remains unclear. METHODS: We analysed the expression profile of PLZF in different types of benign and malignant thyroid lesions as well as in normal thyroid tissue. Specifically, we examined PLZF expression in normal thyroid (N; n = 4), adenomatous lesion (AL; n = 5), follicular adenoma (FA; n = 2), papillary thyroid carcinoma (PTC; n = 20), and anaplastic thyroid carcinoma (ATC; n = 3) samples. PLZF expression was estimated by western blotting and immunohistochemical (IHC) staining. RESULTS: PLZF was expressed in all samples of thyroid lesions examined. In N, AL, and FA, PLZF was mainly localized in the nucleus. In contrast, in PTC and ATC, PLZF was mainly expressed in the cytosol with high intensity. In more detail, the cytoplasmic IHC scores in PTC with capsular invasion (CI) and lymph node (LN) metastasis were higher than those in PTC without CI and LN metastasis. CONCLUSIONS: PLZF shows different subcellular localizations among PTC, ATC, and other thyroid lesions. Furthermore, high cytoplasmic expression of PLZF may be correlated with CI and LN metastasis in thyroid carcinoma. The present report is the first to describe the implications of intracellular PLZF expression in thyroid carcinomas.

A natural Nrf2 activator glucoraphanin improves hepatic steatosis in high-fat diet-induced obese male mice associated with AMPK activation.

Genetic and pharmacological activation of the transcription factor nuclear factor, erythroid derived 2, like 2 (Nrf2) alleviates high-fat diet (HFD)-induced obesity in mice; however, synthetic Nrf2 activators are not clinically available due to safety concerns. Dietary glucoraphanin (GR), a naturally occurring compound found in cruciferous vegetables that activates Nrf2 and induces its target antioxidant genes. We previously demonstrated that GR increased thermogenesis and mitigated HFD-induced obesity in lean healthy mice. In this study, we investigated the therapeutic effects of GR on pre-existing obesity and associated metabolic disorders, such as hepatic steatosis, with or without low-fat dietary intervention. Eight-week-old male C57BL/6J mice were fed an HFD for 9 weeks to induce obesity. Subsequently, these obese mice were fed either the HFD or a normal chow diet, supplemented with or without GR, for an additional 11 weeks. GR supplementation did not decrease the body weight of HFD-fed mice; however, it significantly reduced plasma alanine aminotransferase and aspartate aminotransferase levels and hepatic triglyceride accumulation. These improvements in liver damage by GR were associated with decreased expression levels of fatty acid synthesis genes and proinflammatory chemokine genes, suppressed c-Jun N-terminal kinase activation, and reduced proinflammatory phenotypes of macrophages in the liver. Moreover, metabolome analysis identified increased hepatic levels of adenosine 5'-monophosphate (AMP) in HFD-GR mice compared with those in HFD mice, which agreed with increased phosphorylation levels of AMP-activated protein kinase. Our results show that GR may have a therapeutic potential for treating obesity-associated hepatic steatosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-023-00658-6.

Efficacy of Alogliptin/Metformin Fixed-Dose Combination Tablets and Vildagliptin/Metformin Fixed-Dose Combination Tablets on Glycemic Control in Real-World Clinical Practice for the Patients with Type 2 Diabetes: A Multicenter, Open-Label, Randomized, Parallel Group, Comparative Trial.

Background: This study was aimed to compare the efficacy of two combination tablets of dipeptidyl peptidase-4 (DPP-4) inhibitors and metformin with different dosages, alogliptin/metformin (AM) and vildagliptin/metformin (VM), on glycemic control in patients with type 2 diabetes (T2D). Methods: This was a prospective, multicenter, open-label, randomized, parallel group, comparative trial. After a run-in period of treatment with metformin alone, a total of 59 Japanese outpatients with T2D, aged 20-79 years with glycated hemoglobin (HbA1c) levels of 6.5%-10% were randomly assigned to 12-week AM treatment, alogliptin 25 mg/metformin 500 mg combination tablet orally once a day, or VM treatment, vildagliptin 50 mg/metformin 250 mg combination tablet orally twice a day. The primary endpoints were the changes in HbA1c and fasting plasma glucose (FPG) levels from baseline to week 12 between the two groups. Blinded intermittently scanned continuous glucose monitoring (isCGM) was performed between weeks 10 and 12. The incidence of adverse events during the study was also evaluated. Results: In all, 52 participants were analyzed. Significant decreases in HbA1c and FPG levels from baseline to week 12 were observed in both treatment groups. However, there were no significant differences between the AM and VM groups in the change in HbA1c level (-0.3% and -0.4%, P = 0.309) or the FPG level (-9.0 and -15.0 mg/dL, P = 0.789). The isCGM revealed that both treatments achieved the recommended glycemic target range. No adverse events, such as severe hypoglycemia, were observed in either group. Conclusions: We concluded that there were no significant differences in the efficacy of two combination tablets of DPP-4 inhibitors and metformin with different dosages on glycemic control in patients with T2D.

A Successful Case of Hepatocellular Carcinoma Treated with Atezolizumab Plus Bevacizumab with Multisystem Immune-related Adverse Events.

A 63-year-old man with hepatitis C was treated with atezolizumab plus bevacizumab for unresectable diffuse hepatocellular carcinoma (HCC). After four cycles of atezolizumab plus bevacizumab, the diffuse HCC markedly shrank; however, he complained of general fatigue, loss of appetite, and slight loss of muscle strength in the lower legs. He was diagnosed with isolated adrenocorticotropic hormone deficiency (IAD), hypothyroidism, and myopathy, suggesting multisystem immune-related adverse events (irAEs). After administration of hydrocortisone, the clinical symptoms rapidly disappeared. Patients with multisystem irAEs can have favorable outcomes; thus, to continue immune-checkpoint inhibitors therapy, a correct diagnosis and management of multisystem irAEs are important.

A maternal high-fat diet induces fetal origins of NASH-HCC in mice.

Maternal overnutrition affects offspring susceptibility to nonalcoholic steatohepatitis (NASH). Male offspring from high-fat diet (HFD)-fed dams developed a severe form of NASH, leading to highly vascular tumor formation. The cancer/testis antigen HORMA domain containing protein 1 (HORMAD1), one of 146 upregulated differentially expressed genes in fetal livers from HFD-fed dams, was overexpressed with hypoxia-inducible factor 1 alpha (HIF-1alpha) in hepatoblasts and in NASH-based hepatocellular carcinoma (HCC) in offspring from HFD-fed dams at 15 weeks old. Hypoxia substantially increased Hormad1 expression in primary mouse hepatocytes. Despite the presence of three putative hypoxia response elements within the mouse Hormad1 gene, the Hif-1alpha siRNA only slightly decreased hypoxia-induced Hormad1 mRNA expression. In contrast, N-acetylcysteine, but not rotenone, inhibited hypoxia-induced Hormad1 expression, indicating its dependency on nonmitochondrial reactive oxygen species production. Synchrotron-based phase-contrast micro-CT of the fetuses from HFD-fed dams showed significant enlargement of the liver accompanied by a consistent size of the umbilical vein, which may cause hypoxia in the fetal liver. Based on these findings, a maternal HFD induces fetal origins of NASH/HCC via hypoxia, and HORMAD1 is a potential therapeutic target for NASH/HCC.

A case of insulinoma diagnosed postpartum with hypoglycemic symptoms that were masked during pregnancy.

The diagnosis of insulinoma in perinatal women can be difficult, as hypoglycemic symptoms may be masked by pregnancy-associated insulin resistance. In addition, when multiple insulinomas are observed, it is necessary to consider the possibility not only of MEN1, but also of insulinomatosis.

High glucose activates HIF-1-mediated signal transduction in glomerular mesangial cells through a carbohydrate response element binding protein.

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1alpha (HIF-1alpha) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1alpha has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1alpha expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1alpha and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1alpha and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1alpha or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1alpha mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy.

Hypoxia-inducible factor-1α is the therapeutic target of the SGLT2 inhibitor for diabetic nephropathy.

Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.

Impacts of Diabetes and an SGLT2 Inhibitor on the Glomerular Number and Volume in db/db Mice, as Estimated by Synchrotron Radiation Micro-CT at SPring-8.

BACKGROUND: Recent large-scale clinical studies demonstrate that sodium-glucose cotransporter 2 (SGLT2) inhibitors protect the diabetic kidney. However, clinical and animal studies have not shown the changes of the total glomeruli in the whole kidney treated with SGLT2 inhibitors. METHODS: We performed computed tomography (CT) imaging on mice using synchrotron radiation to investigate the impact of luseogliflozin, a SGLT2 inhibitor, on the number and volume of glomeruli in the whole kidney. FINDINGS: We did not observe a significant difference in the total glomerular number (Nglom) among mice. Luseogliflozin redistributed the number of glomeruli in different regions, accompanied by the normalization of diabetes-augmented renal volume (Vkidney). Diabetic db/db mice had a larger glomerular volume in the mid-cortex than did control db/m mice, and luseogliflozin increased the glomerular volume in all renal cortical zones of the whole kidney in db/db mice. According to the multivariate regression analysis, hemoglobin A1c level was the most relevant determinant of Vkidney, not Nglom or mean glomerular volume (Vglom), indicating that hyperglycemia induced renal (tubular) hypertrophy, but not glomerular enlargement. Luseogliflozin increased hypoxia in the juxtamedullary region, sustained upregulated renal renin expression and plasma renin activity, and failed to decrease albuminuria by downregulating megalin in db/db mice. INTERPRETATION: Based on our findings, SGLT2 inhibitors may alter glomerular distribution and size in addition to their glucose-lowering effects, presumably by affecting oxygen metabolism and humoral factors. FUND: Funding for this research was provided by The Japan Society for the Promotion of Science, the Japan Diabetes Foundation, and Asahikawa Medical University.

Dipeptidyl peptidase-4 inhibitor treatment induces a greater increase in plasma levels of bioactive GIP than GLP-1 in non-diabetic subjects.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) possess multiple bioactive isoforms that are rendered non-insulinotropic by the enzyme dipeptidyl peptidase-4 (DPP-4). Recently, some ELISA kits have been developed to specifically measure "active" GIP and GLP-1, but it is unclear if these kits can accurately quantify all bioactive forms. Therefore, it remains uncertain to what extent treatment with a DPP-4 inhibitor boosts levels of biologically active GIP and GLP-1. Thus, we evaluated our novel receptor-mediated incretin bioassays in comparison to commercially available ELISA kits using plasma samples from healthy subjects before and after DPP-4 inhibitor administration. METHODS: We utilized cell lines stably co-transfected with human GIP or GLP-1 receptors and a cAMP-inducible luciferase expression construct for the bioassays and commercially available ELISA kits. Assays were tested with synthetic GIP and GLP-1 receptor agonists and plasma samples collected from subjects during a 75 g oral glucose tolerance test (OGTT) performed before or following 3-day administration of a DPP-4 inhibitor. RESULTS: A GIP isoform GIP(1-30)NH2 increased luciferase activity similarly to GIP(1-42) in the GIP bioassay but was not detectable by either a total or active GIP ELISA kit. During an OGTT, total GIP levels measured by ELISA rapidly increased from 0 min to 15 min, subsequently reaching a peak of 59.2 ± 8.3 pmol/l at 120 min. In contrast, active GIP levels measured by the bioassay peaked at 15 min (43.4 ± 6.4 pmol/l) and then progressively diminished at all subsequent time points. Strikingly, at 15 min, active GIP levels as determined by the bioassay reached levels approximately 20-fold higher after the DPP-4 inhibitor treatment, while total and active GIP levels determined by ELISA were increased just 1.5 and 2.1-fold, respectively. In the absence of DPP-4 inhibition, total GLP-1 levels measured by ELISA gradually increased up to 90 min, reaching 23.5 ± 2.4 pmol/l, and active GLP-1 levels determined by the bioassay did not show any apparent peak. Following administration of a DPP-4 inhibitor there was an observable peak of active GLP-1 levels as determined by the bioassay at 15 min after oral glucose load, reaching 11.0 ± 0.62 pmol/l, 1.4-fold greater than levels obtained without DPP-4 inhibitor treatment. In contrast, total GLP-1 levels determined by ELISA were decreased after DPP-4 inhibitor treatment. CONCLUSION: Our results using bioassays indicate that there is a greater increase in plasma levels of bioactive GIP than GLP-1 in subjects treated with DPP-4 inhibitors, which may be unappreciated using conventional ELISAs.

Alternative form of glucose-dependent insulinotropic polypepide and its physiology.

Glucose-dependent insulinotropic polypepide (GIP) was first extracted from porcine gut mucosa and identified as "incretin" decades ago. Though early studies have shown the possible GIP isoforms by gel filtration profiles from porcine or human intestinal extracts analyzed by radioimmunoassay (RIA), GIP is currently believed to consist of 42 amino acids (GIP1-42), which are released from gut K-cells and promote postprandial insulin release. In fact, GIP1-42 is usually processed from proGIP by the action of prohormone convertase (PC) 1/3 in the gut. GIP expression is occasionally found in the intestinal glucagon-like peptide-1-secreting cells, suggesting gene expression of both GIP and proglucagon can co-exist in identical cells. However, GIP1-42 immunoreactivity is rarely found in α-cells or other pancreatic endocrine cells of wild-type mammals. Interestingly, we found that short-form GIP1-30 is expressed in and released from pancreatic α-cells and a subset of enteroendocrine cells through proGIP processing by PC2. GIP1-30 is also insulinotropic and modulates glucose-stimulated insulin secretion in a paracrine manner. It is also suggested that short-form GIP1-30 possibly plays a crucial role for the islet development. It has not been well elucidated whether expression of GIP1-30 is modulated in the diabetic status, and whether GIP1-30 might have therapeutic potentials. Our preliminary data suggest that short-form GIP1-30 might play important roles in glucose metabolism.

Role of IGFBP7 in Diabetic Nephropathy: TGF-ß1 Induces IGFBP7 via Smad2/4 in Human Renal Proximal Tubular Epithelial Cells.

Tubular injury is one of the important determinants of progressive renal failure in diabetic nephropathy (DN), and TGF-ß1 has been implicated in the pathogenesis of tubulointerstitial disease that characterizes proteinuric renal disease. The aim of this study was to identify novel therapeutic target molecules that play a role in the tubule damage of DN. We used an LC-MS/MS-based proteomic technique and human renal proximal epithelial cells (HRPTECs). Urine samples from Japanese patients with type 2 diabetes (n = 46) were used to quantify the candidate protein. Several proteins in HRPTECs in cultured media were observed to be driven by TGF-ß1, one of which was 33-kDa IGFBP7, which is a member of IGFBP family. TGF-ß1 up-regulated the expressions of IGFBP7 mRNA and protein in a dose- and time-dependent fashion via Smad2 and 4, but not MAPK pathways in HRPTECs. In addition, the knockdown of IGFBP7 restored the TGF-ß1-induced epithelial to mesenchymal transition (EMT). In the immunohistochemical analysis, IGFBP7 was localized to the cytoplasm of tubular cells but not that of glomerular cells in diabetic kidney. Urinary IGFBP7 levels were significantly higher in the patients with macroalbuminuria and were correlated with age (r = 0.308, p = 0.037), eGFR (r = -0.376, p = 0.01), urinary ß2-microglobulin (r = 0.385, p = 0.008), and urinary N-acetyl-beta-D-glucosaminidase (NAG) (r = 0.502, p = 0.000). A multivariate regression analysis identified urinary NAG and age as determinants associated with urinary IGFBP7 levels. In conclusion, our data suggest that TGF-ß1 enhances IGFBP7 via Smad2/4 pathways, and that IGFBP7 might be involved in the TGF-ß1-induced tubular injury in DN.

High glucose induces platelet-derived growth factor-C via carbohydrate response element-binding protein in glomerular mesangial cells.

Persistent high concentration of glucose causes cellular stress and damage in diabetes via derangement of gene expressions. We previously reported high glucose activates hypoxia-inducible factor-1αand downstream gene expression in mesangial cells, leading to an extracellular matrix expansion in the glomeruli. A glucose-responsive transcription factor carbohydrate response element-binding protein (ChREBP) is a key mediator for such perturbation of gene regulation. To provide insight into glucose-mediated gene regulation in mesangial cells, we performed chromatin immunoprecipitation followed byDNAmicroarray analysis and identified platelet-derived growth factor-C (PDGF-C) as a novel target gene of ChREBP In streptozotocin-induced diabetic mice, glomerular cells showed a significant increase inPDGF-C expression; the ratio ofPDGF-C-positive cells to the total number glomerular cells demonstrated more than threefold increase when compared with control animals. In cultured human mesangial cells, high glucose enhanced expression ofPDGF-C protein by 1.9-fold. Knock-down of ChREBPabrogated this induction response. UpregulatedPDGF-C contributed to the production of typeIVand typeVIcollagen, possibly via an autocrine mechanism. Interestingly, urinaryPDGF-C levels in diabetic model mice were significantly elevated in a fashion similar to urinary albumin. Taken together, we hypothesize that a high glucose-mediated induction ofPDGF-C via ChREBPin mesangial cells contributes to the development of glomerular mesangial expansion in diabetes, which may provide a platform for novel predictive and therapeutic strategies for diabetic nephropathy.

Sodium-Glucose Cotransporter 2 Inhibitor and a Low Carbohydrate Diet Affect Gluconeogenesis and Glycogen Content Differently in the Kidney and the Liver of Non-Diabetic Mice.

A low carbohydrate diet (LCHD) as well as sodium glucose cotransporter 2 inhibitors (SGLT2i) may reduce glucose utilization and improve metabolic disorders. However, it is not clear how different or similar the effects of LCHD and SGLT2i are on metabolic parameters such as insulin sensitivity, fat accumulation, and especially gluconeogenesis in the kidney and the liver. We conducted an 8-week study using non-diabetic mice, which were fed ad-libitum with LCHD or a normal carbohydrate diet (NCHD) and treated with/without the SGLT-2 inhibitor, ipragliflozin. We compared metabolic parameters, gene expression for transcripts related to glucose and fat metabolism, and glycogen content in the kidney and the liver among the groups. SGLT2i but not LCHD improved glucose excursion after an oral glucose load compared to NCHD, although all groups presented comparable non-fasted glycemia. Both the LCHD and SGLT2i treatments increased calorie-intake, whereas only the LCHD increased body weight compared to the NCHD, epididimal fat mass and developed insulin resistance. Gene expression of certain gluconeogenic enzymes was simultaneously upregulated in the kidney of SGLT2i treated group, as well as in the liver of the LCHD treated group. The SGLT2i treated groups showed markedly lower glycogen content in the liver, but induced glycogen accumulation in the kidney. We conclude that LCHD induces deleterious metabolic changes in the non-diabetic mice. Our results suggest that SGLT2i induced gluconeogenesis mainly in the kidney, whereas for LCHD it was predominantly in the liver.

Decreased expression of retinoid X receptor isoforms in human thyroid carcinomas.

Retinoid X receptors (RXRs) are ligand-inducible transcription factors that belong to the superfamily of nuclear hormone receptors. Because RXRs heterodimerize with thyroid hormone receptor, retinoic acid receptor, vitamin D(3) receptor, and peroxisome proliferator-activated receptor, they play central roles in regulating a number of signaling pathways. To understand the roles of RXRs in human thyroid carcinogenesis, we have investigated the immunohistochemical expression of RXRs in normal and neoplastic thyroid tissues. Whereas nontumorous human thyroid cells exhibited distinct nuclear staining for the RXRs, thyroid carcinomas showed decreased nuclear expression of all three RXR isoforms. In particular, some thyroid carcinoma cells showed intense RXR-alpha cytoplasmic staining accompanied by decreased immunoreactivity in their nuclei. This subcellular localization of RXR-alpha was confirmed by Western blot analysis, which showed both lower nuclear expression levels of RXR-alpha and a cytosolic presence of RXR-related protein in neoplastic regions. We present here, for the first time, the histological distribution of each RXR protein (alpha, beta, and gamma) in human thyroid follicular cells. In addition, we found that the nuclear expression of RXRs was lower in thyroid carcinomas than in normal tissue. The differential expressions of these RXRs in thyroid carcinomas might be implicated in the pathogenesis of thyroid cancers.

Thyroid-stimulating hormone induces interleukin-18 gene expression in FRTL-5 cells: immunohistochemical detection of interleukin-18 in autoimmune thyroid disease.

Interleukin (IL)-18 is a cloned cytokine that was identified originally as a factor having potent interferon (IFN)-gamma-inducing activity on Kupffer cells. First, we analyzed IL-18 gene expression by reverse transcription-polymerase chain reaction (RT-PCR) in rat thyroid FRTL-5 cells and human thyroid tissue samples. The expression of IL-18 mRNA in FRTL-5 cells was enhanced by thryoid-stimulating hormone (TSH) in a dose-dependent manner. 8-Bromo-cyclic adenosine monophosphate (cAMP) also increased in IL-18 mRNA levels. Furthermore, TGCT clones that exhibited an increase in intracellular cAMP accumulation showed an increased IL-18 mRNA signal when compared to controls. Taken together, these data suggested that the effect of TSH on IL-18 gene expression was mediated by activating protein kinase A. Treatment of FRTL-5 cells with the antithyroid drug, methimazole (MMI), suppressed this stimulatory action of TSH on IL-18 gene expression. Next, we examined IL-18 expression in human thyroid tissue derived from patients with autoimmune thyroid diseases (ATD). RT-PCR and immunohistology demonstrated that human thyroid follicular cells expressed IL-18. Especially in thyroid tissue from a patient with Hashimoto's thyroiditis, expression was more diffuse and extensive, generally observed in close relation to a lymphocytic infiltrate. Also, IL-18 protein was distributed in the same follicles that express Fas-L and HLA-DR. This study is the first to demonstrate the detection of IL-18 in the thyroid gland. The frequent expression of IL-18 in thyrocytes suggests that IL-18 itself might be a secreted immunomodulator in ATD.