Jean Giudicelli

Methylglyoxal Impairs the Insulin Signaling Pathways Independently of the Formation of Intracellular Reactive Oxygen Species

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed α-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive α-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.

Rat liver injury after normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor

Hepatic ischemia occurs in liver transplantation, hemodynamic or cardiogenic shock, and liver resection associated with trauma or tumor. Ischemia/reperfusion (I/R) injury results in microcirculation failure followed by apoptosis and necrosis. Matrix metalloproteinases (MMPs) are involved in many physiological and pathological processes, but their expression and function during liver I/R remains poorly documented. In this study, we evaluated the expression of nine MMPs and their natural inhibitors, tissue inhibitors of MMPs (TIMPs), in a rat model of liver I/R. Analysis of MMP and TIMP expression show that although most of these genes are not constitutively expressed in the normal liver, they are induced in a specific time‐dependent manner following I/R. Stromelysin‐1, gelatinase B, and collagenase‐3 are induced during the early phase of acute liver injury associated with inflammation and increased necrosis/apoptosis, whereas gelatinase A, membrane type‐MMP, stromelysin‐3, metalloelastase, TIMP‐1, and TIMP‐2 are essentially detectable during the recovery phase of liver injury corresponding to hepatocyte regeneration. This observation suggested that MMPs and TIMPs could play both deleterious and beneficial roles following I/R. We thus tested the effect of a specific phosphinic MMP inhibitor on acute liver I/R injury. Inhibition of MMP activity was shown to significantly decrease liver injury in ischemic/reperfused liver tissue as assessed by histological studies and serum hepatic enzyme levels. We therefore propose that MMP inhibitors may be of clinical relevance in liver‐associated ischemic diseases or after liver transplantation.

Methylglyoxal impairs insulin signalling and insulin action on glucose-induced insulin secretion in the pancreatic beta cell line INS-1E

Aims/hypothesisChronic hyperglycaemia aggravates insulin resistance, at least in part, by increasing the formation of advanced glycation end-products (AGEs). Methylglyoxal (MGO) is the most reactive AGE precursor and its abnormal accumulation participates in damage in various tissues and organs. Here we investigated the ability of MGO to interfere with insulin signalling and to affect beta cell functions in the INS-1E beta cell line.MethodsINS-1E cells were incubated with MGO and then exposed to insulin or to glucose. Western blotting was used to study signalling pathways, and real-time PCR to analyse gene expression; insulin levels were determined by radioimmunoassay.ResultsNon-cytotoxic MGO concentrations inhibited insulin-induced IRS tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway activation independently from reactive oxygen species (ROS) production. Concomitantly, formation of AGE adducts on immunoprecipitated IRS was observed. Aminoguanidine reversed MGO inhibitory effects and the formation of AGE adducts on IRS. Further, the insulin- and glucose-induced expression of Ins1, Gck and Pdx1 mRNA was abolished by MGO. Finally, MGO blocked glucose-induced insulin secretion and PI3K/PKB pathway activation. These MGO effects were abolished by LiCl, which inhibits glycogen synthase kinase-3 (GSK-3).Conclusions/interpretationMGO exerted major damaging effects on INS-1E cells impairing both insulin action and secretion. An important actor in these noxious MGO effects appears to be GSK-3. In conclusion, MGO participates not only in the pathogenesis of the debilitating complications of type 2 diabetes, but also in worsening of the diabetic state by favouring beta cell failure.

Fatigue is associated with high circulating leptin levels in chronic hepatitis C.

Background and aims: Fatigue is a frequent and disabling symptom reported by patients with chronic hepatitis C (CHC). Its mechanism is poorly understood. Recent attention has focused on the role of leptin and energy expenditure in CHC. Our aims were to analyse fatigue in CHC and to determine its relationship with disease activity, resting energy expenditure (REE), circulating leptin, and tumour necrosis factor α (TNF-α). Methods: Seventy eight CHC patients, 22 healthy controls, and 13 primary biliary cirrhosis (PBC) patients underwent measurements of REE, body composition, leptin, and TNF-α. All subjects completed the fatigue impact scale (FIS) questionnaire. A liver biopsy and viral load measurements were performed in all patients. Results: Thirty eight of 78 CHC patients considered fatigue the worst or initial symptom of their disease. The fatigue score of patients was significantly higher than that of controls (53.2 (40.1) v 17.7 (16.9); p<0.0001) and was more pronounced in females (p=0.003). Leptin was increased significantly in CHC patients compared with controls (15.4 (20.7) v 6.4 (4.1) ng/ml; p<0.05). In CHC patients, the fatigue score correlated significantly with leptin corrected for fat mass (r=0.30, p=0.01). This correlation increased when the physical domain of fatigue was included (r=0.39, p=0.0009). Furthermore, a similar positive correlation was found in PBC patients (r=0.56, p=0.04). No correlation was found between fatigue and age, REE, liver function tests, viral load, or the METAVIR score in CHC patients. Conclusions: Fatigue is present in CHC patients and is more pronounced in females. The FIS questionnaire is clinically relevant and may be useful for future therapeutic trials aimed at reducing fatigue. Fatigue may be partly mediated by leptin.

INTESTINAL LACTASE-PHLORIZIN HYDROLASE (LPH) : THE TWO CATALYTIC SITES; THE ROLE OF THE PANCREAS IN PRO-LPH MATURATION

The proteolytic processing of pro‐lactase‐phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N‐terminus of ‘mature’ final, brush border lactase‐phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.

Effects of age, malnutrition and refeeding on the expression and secretion of ghrelin

BACKGROUND & AIMS Anorexia is frequent in the malnourished elderly. We studied the effects of age, nutritional status and refeeding on the expression and secretion of the orexigenic peptide ghrelin. METHODS Four groups were prospectively enrolled: 11 undernourished elderly (80+/-6 y, BMI: 17.4+/-1.9 [Mean+/-SD]), nine well-nourished elderly (76+/-9 y, 23.5+/-2.0), 10 undernourished young (26+/-6 y, 15.1+/-1.9) and 10 well-nourished young (34+/-8 y, 22.2+/-2.7). Fasting and postprandial plasma ghrelin and other hormones (every 30 min) were measured at baseline and after a 21-day enteral nutrition in malnourished patients. Gastric ghrelin mRNA levels were measured by RT-PCR at baseline in all subjects. RESULTS Ghrelin was significantly higher in undernourished (2151+/-871 ng/L) than in well-nourished (943+/-389 ng/L) adults, whereas there were no differences between undernourished (1544+/-758 ng/L) and well-nourished (1154+/-541 ng/L) elderly. Refeeding did not influence ghrelin levels. Gastric ghrelin mRNA levels were similar in all groups. CONCLUSIONS There is an absence of malnutrition-induced increase of plasma ghrelin levels in elderly subjects. This feature, post-transcriptional, may be important in the lack of adaptation of elderly subjects to malnutrition.

Glucose intolerance and hypoadiponectinemia are already present in lean patients with chronic hepatitis C infected with genotype non-3 viruses.

Objectives Steatosis and metabolic abnormalities seem to be frequent and deleterious in chronic hepatitis C. Changes in glucose homeostasis and in adiponectin levels, an adipokine with anti-inflammatory and insulin-sensitive properties, were evaluated in patients with chronic hepatitis C according to steatosis, liver fibrosis and body mass index. Methods Seventy-three patients with chronic hepatitis C (40 men, 33 women) infected with genotypes non-3 and 22 healthy controls (11 men and 11 women) were included in the study and all had a biochemical evaluation, including metabolic parameters, adiponectin measurement, and a liver biopsy. Insulin sensitivity was assessed with the HOMA 1-IR insulin resistance model. Results Steatosis was found in 65.7% of the patients and significant fibrosis (METAVIR F2–F4) was present in 28.7%. The presence of steatosis could only be predicted by fibrosis, whereas significant fibrosis could be predicted by steatosis and age. Adiponectin levels were significantly decreased (−32%) with the severity of the steatosis. Although overweight chronic hepatitis C patients (body mass index≥25 kg/m2) had insulin resistance and hypoadiponectinemia, lean chronic hepatitis C patients (body mass index<25 kg/m2) had already significantly higher glycemia and lower adiponectin levels than in controls. Conclusions This study confirms the high incidence of steatosis in patients infected by hepatitis C virus genotypes non-3, well linked to the development of fibrosis and metabolic abnormalities. Importantly, the present findings put emphasis on the early development of these metabolic abnormalities as they were already found in lean patients with chronic hepatitis C. The direct implication of hepatitis C virus is thus further stressed in the development of steatosis and insulin resistance, with or without involvement of host factors.

Effect of Caspase Inhibition on Thymic Apoptosis in Hemorrhagic Shock

In hemorrhagic shock (HS) an increased thymic apoptosis (TA) was described. The aim of this study was to evaluate the effect of administration of the caspase inhibitor N-benzyloxy-carbonil-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) during the resuscitation phase on TA, organ dysfunctions, and tumor necrosis factor (TNF)-α release in HS. Forty rats were randomly assigned to four groups: no HS/resuscitation (sham); HS/resuscitation with shed blood and normal saline (control); HS/resuscitation with shed blood and phosphate-buffered solution (PBS) (vehicle); and HS/resuscitation with shed blood and Z-VAD-FMK (inhibitor). Rats were subjected to HS by blood removal to a MAP of 35–40 mmHg. After a 1-h shock period, the animals were resuscitated according to the protocol. At 1 and 3 h after resuscitation, transaminases, creatinine, urea, lipase, TNF-α, and TA were evaluated. Our study showed that a nonlethal HS is early able to induce organ dysfunctions and increased TA. Administration of Z-VAD-FMK did not significantly decrease organ dysfunctions, while it induced a significant TNF-α release. TA was significantly reduced by Z-VAD-FMK after 1 h, but not after 3 h. Our results suggest that postinjury caspase inhibition does not attenuate organ dysfunctions, and also does not permanently reduce TA induced by HS and resuscitation in rats.