Overexpression of lysophospholipid acyltransferase, LPLAT10/LPCAT4/LPEAT2, in the mouse liver increases glucose-stimulated insulin secretion.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

ZFAND3 Overexpression in the Mouse Liver Improves Glucose Tolerance and Hepatic Insulin Resistance.

Genome-wide association studies have identified more than 300 loci associated with type 2 diabetes mellitus; however, the mechanisms underlying their role in type 2 diabetes mellitus susceptibility remain largely unknown. Zinc finger AN1-type domain 3 (ZFAND3), known as testis-expressed sequence 27, is a type 2 diabetes mellitus-susceptibility gene. Limited information is available regarding the physiological role of ZFAND3 in vivo. This study aimed to investigate the association between ZFAND3 and type 2 diabetes mellitus. ZFAND3 was significantly upregulated in the liver of diabetic mice compared to wild-type mice. To overexpress ZFAND3, we generated a ZFAND3-expressing adenovirus (Ad) vector using an improved Ad vector exhibiting significantly lower hepatotoxicity (Ad-ZFAND3). Glucose tolerance was significantly improved in Ad-ZFAND3-treated mice compared to the control Ad-treated mice. ZFAND3 overexpression in the mouse liver also improved insulin resistance. Furthermore, gluconeogenic gene expression was significantly lower in primary mouse hepatocytes transduced with Ad-ZFAND3 than those transduced with the control Ad vector. The present results suggest that ZFAND3 improves glucose tolerance by improving insulin resistance and suppressing gluconeogenesis, serving as a potential novel therapeutic target for type 2 diabetes mellitus.

Regulation of Blood Pressure and Phosphorylation of ß1-integrin in Renal Tissue in a Rat Model of Diabetic Nephropathy.

OBJECTIVE: Induction of hypertension by diabetic nephropathy (DN) may be dependent on increased renin secretion from juxtaglomerular cells (JGC). To reveal that the mechanisms of cell adhesion and cell motility associated with ß1-integrin phosphorylation contribute to pressure sensing in JGC, we tested the ß1-integrin phosphorylation levels in renal tissue and the relationship between ß1-integrin phosphorylation and the expression of renin. METHODS: The DN rat model was generated by intravenous injection of streptozotocin (STZ, 60 mg/kg body weight). Immunohistochemistry and an imaging analysis were performed to detect and evaluate the ß1-integrin phosphorylation levels in renal tissue. Quantitative real-time polymerase chain reaction was also performed to evaluate renin mRNA levels. RESULTS: We found that the serine-785 and threonine-788/789 sites of ß1-integrin are specifically phosphorylated in macula densa and JGC, respectively, and that changes in their expression during the progression of DN are associated with the production of renin. Phosphorylation of these ß1-integrins increased or decreased with changes of the renin expression during the progression of DN. In particular, phosphorylation of threonine-788/789 was negatively correlated with the expression of renin. CONCLUSION: These findings suggest that the phosphorylation of ß1-integrin may contribute to the regulatory mechanism of renin production in JGC.

Melatonin stimulates transcription of the rat phosphoenolpyruvate carboxykinase gene in hepatic cells.

Melatonin plays physiological roles in various critical processes, including circadian rhythms, oxidative stress defenses, anti-inflammation responses, and immunity; however, the current understanding of the role of melatonin in hepatic glucose metabolism is limited. In this study, we examined whether melatonin affects gene expression of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). We found that melatonin treatment increased PEPCK mRNA levels in rat highly differentiated hepatoma (H4IIE) cells and primary cultured hepatocytes. In addition, we found that melatonin induction was synergistically enhanced by dexamethasone, whereas it was dominantly inhibited by insulin. We also report that the effect of melatonin was blocked by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), RNA polymerase II, and protein synthesis. Furthermore, the phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) increased 15 min after melatonin treatment. We performed luciferase reporter assays to show that melatonin specifically stimulated promoter activity of the PEPCK gene. Additional reporter analysis using 5'-deleted constructs revealed that the regulatory regions responsive to melatonin mapped to two nucleotide regions, one between -467 and -398 nucleotides and the other between -128 and +69 nucleotides, of the rat PEPCK gene. Thus, we conclude that melatonin induces PEPCK gene expression via the ERK1/2 pathway at the transcriptional level, and that induction requires de novo protein synthesis.

Importance of the Evaluation of Renal Function for the Prevention of Hypoglycemia in Elderly Diabetes Patients.

OBJECTIVE: The Japan Diabetes Society and the Japan Gerontological Society Collaborative Committee recently released guidelines for the management of elderly diabetes patients. In these guidelines, patients are classified into categories I-III depending on age, cognitive function, activities of daily living (ADL), and presence or absence of multiple functional impairments. The target control value of HbA1c is set for each category. Low (< 30 mL/min/1.73 m2) estimated glomerular filtration rate (eGFR) is an independent highrisk factor for severe hypoglycemia, yet it is not included in the categorization factors. We surveyed elderly diabetes patients with normal cognitive function and ADL (Category I) who were admitted to the emergency department with severe hypoglycemia, retrospectively studied eGFR at the onset of hypoglycemic episode, and checked whether the HbA1c levels matched the guidelines. METHODS: Among 129 diabetes patients aged ≥ 65 years admitted to the Tokai University hospital for hypoglycemic emergencies, 73 had normal cognitive function and ADL. HbA1c level and eGFR at the onset of hypoglycemic attack were obtained from the medical records of these subjects. RESULTS: All subjects were prescribed anti-diabetes agents with high-risk of severe hypoglycemia, including insulin. Sixty-one patients showed eGFR ≥ 30 mL/min/1.73 m2. Among them, 31 (50.8%) had HbA1c levels below the recommended range. Among 12 patients whose eGFR < 30 mL/min/1.73 m2, 6 (50%) had HbA1c levels below the recommended range. CONCLUSION: Even with normal cognitive function and ADL, eGFR < 30 mL/min/1.73 m2 a lone i s a s trong risk factor for hypoglycemia in elderly diabetes patients. We propose that the target control HbA1c level in elderly patients with eGFR < 30 mL/min/1.73 m2 should be 7.5-8.4 %, which is equivalent to that of category III patients.

Glycemic Control After Kidney Transplantation Using Sensor-augmented Insulin Pump Therapy in a Patient with Slowly Progressive Type 1 Diabetes Mellitus.

OBJECTIVE: Patients with advanced diabetic nephropathy benefit from kidney transplantation. We report a patient who showed improved glycemic control after kidney transplantation followed by sensor-augmented pump (SAP) therapy. METHODS: The patient was a 67-year-old man on hemodialysis for diabetic nephropathy associated with slowly-progressive type 1 diabetes mellitus. He underwent living-donor kidney transplantation, followed by introduction of SAP therapy for strict glycemic control. RESULTS: SAP therapy improved glycated hemoglobin and glycated albumin levels from 7.8% and 24.2% to 7.0% and 19.2%, respectively, and reduced the frequency of hypoglycemic episodes. CONCLUSION: The case illustrates the usefulness of SAP therapy for post-kidney transplantation glycemic control.

Cranberry Attenuates Progression of Non-alcoholic Fatty Liver Disease Induced by High-Fat Diet in Mice.

Obesity is characterized by abnormal or excessive fat accumulation, which leads to the development of metabolic syndrome. Because oxidative stress is increased in obesity, antioxidants are regarded as suitable agents for preventing metabolic syndrome. Here, we examined the impact of cranberry, which contains various antioxidants, on metabolic profiles, including that during the progression of non-alcoholic fatty liver disease (NAFLD), in high-fat diet (HFD)-fed C57BL/6 mice. We observed that oxidative stress was diminished in mice that were fed HFD diets supplemented with 1 and 5% cranberry powder as compared with that in HFD-fed control mice. Notably, from 1 week after beginning the diets to the end of the study, the body weight of mice in the cranberry-treatment groups was significantly lower than that of mice in the HFD-fed control group; during the early treatment phase, cranberry suppressed the elevation of serum triglycerides; and adipocytes in the adipose tissues of cranberry-supplemented-HFD-fed mice were smaller than these cells in HFD-fed control mice. Lastly, we examined the effect of cranberry on NAFLD, which is one of the manifestations of metabolic syndrome in the liver. Histological analysis of the liver revealed that lipid-droplet formation and hepatocyte ballooning, which are key NAFLD characteristics, were both drastically decreased in cranberry-supplemented-HFD-fed mice relative to the levels in HFD-fed control mice. Our results suggest that cranberry ameliorates HFD-induced metabolic disturbances, particularly during the early treatment stage, and exhibits considerable potential for preventing the progression of NAFLD.

Detection of Autonomic Nervous System Abnormalities in Diabetic Patients by 24-hour Ambulatory Blood Pressure Monitoring.

OBJECTIVE: To determine the relationship between 24-hr blood pressure (BP) fluctuations and autonomic nervous system dysfunction in diabetic patients using non-invasive ambulatory blood-pressure monitoring (ABPM) system. METHODS: The subjects were 39 diabetic patients free of cardiovascular diseases. 24-hr BP was monitored by a non-invasive ABPM system. The relationships among 24-hr BP fluctuations and various clinical parameters relevant to diabetes and hypertension were analyzed. RESULTS: Patients were divided into the diurnal hypertension (DH, n=4), diurnal and nocturnal hypertension (DNH, n=9), normotension (N, n=14), and nocturnal hypertension (NH, n=12) groups. DH and/or NH was observed in 25 (64%) patients: 13 had DH (≥135/85 mmHg), 21 had NH (≥120/70 mmHg), and 9 had both. Furthermore, 4 patients with DH but no NH (diurnal/nocturnal+/ - ); 9 (+/+); 14 ( - / - ); and 12 ( - /+). The R-R interval coefficient of variation on the EKG (CV-RR) was significantly different among the groups (N>NH>DNH>DH). CONCLUSION: Autonomic nervous system dysfunction in diabetic patients had a negative influence on 24-hr fluctuations in BP. Monitoring nighttime hypertension and daily BP variation using ABPM diabetic is a potentially useful approach for identifying autonomic nervous system dysfunction and associated abnormal BP patterns that cannot be detected by routine check-ups.

Influence of the intensity of galvanic vestibular stimulation and cutaneous stimulation on the soleus H-reflex in healthy individuals.

Modulation of the soleus H-reflex following galvanic vestibular stimulation (GVS) has been used to evaluate vestibulospinal tract function. It is not known whether this modulation is because of vestibular stimulation and/or cutaneous stimulation, and the suitable stimulating intensity of GVS for the modulation is not established. We investigated the influence of GVS intensity and cutaneous stimulation on the soleus H-reflex in healthy adults. We applied cathodal GVS (at 1, 2, and 3 mA) or 3-mA cutaneous stimulation as a conditioning stimulation in a random order to 17 individuals in the prone position with the head facing forward, and we examined the changes in the right soleus H-reflex amplitude. We maintained the interval between the conditioning stimulation's onset and the tibial nerve stimulation evoking the soleus H-reflex constant at 100 ms. The amplitude of all conditioned H-reflexes was significantly larger than that of the unconditioned H-reflexes. The greatest facilitation of the H-reflex occurred when 3-mA GVS was applied. The degree of facilitation of the H-reflex by 3-mA GVS was significantly larger than that produced by the 3-mA cutaneous stimulation. These findings indicate that (a) the facilitation of the soleus motor neuron pool excitability by GVS is derived from both vestibular stimulation and cutaneous stimulation, and (b) the intensity of GVS affects the degree of facilitation. When this technique is used to examine vestibulospinal tract function, no less than 3 mA GVS may be appropriate as the conditioning stimulation.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside stimulates the rat enhancer of split- and hairy-related protein-2 gene via atypical protein kinase C lambda.

The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is thePEPCKgene. In this study, we examined an issue of whether theSHARP-2gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKCλ). In addition, when a dominant negative form of aPKCλ was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKCλ. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of theSHARP-2gene is mediated at transcription level by a PI 3-K/aPKCλ pathway.