Fat storage in pancreas and in insulin-sensitive tissues in pathogenesis of type 2 diabetes

Obesity is associated with increased storage of lipids in nonadipose tissues like skeletal muscle, liver, and pancreatic beta cells. These lipids constitute a continuous source of long-chain fatty acyl CoA (LC-CoA) and derived metabolites like diacylglycerol and ceramide, acting as signalling molecules on protein kinases activities (in particular, the family of PKCs), ion channel, gene expression, and protein acylation. In skeletal muscle, the increase in LC-CoA and diacylglycerol translocates and activates specific protein kinase C (PKC) isoforms, which will phosphorylate IRS-1 on serine, preventing its phosphorylation on tyrosine and association with PI3 kinase. This interrupts the insulin signalling pathway leading to the stimulation of glucose transport. In pancreatic beta cells, short-term excess of fatty acids or LC-CoA activates PKC and also directly stimulates insulin exocytosis. Long-term exposure to free fatty acids (FFA) leads to an increased basal and blunted glucose-stimulated insulin secretion by affecting gene expression, increase in K(ATP) channel activity, and uncoupling of the mitochondria. In addition, the saturated FFA palmitate increases cell death by apoptosis via increase in ceramide synthesis.     

UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues

Summary Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc β-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using ³H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2–4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14–36-fold higher in submandibular gland and 5–18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 ± 0.3 nmol/mg · min, which was not different from that in normal subjects (3.3 ± 0.4 nmol/mg · min). A 180-min intravenous insulin infusion (40 mU/m²· min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions. [Diabetologia (1997) 40: 76–81]     

Superoxide and nitric oxide production by Kupffer cells in rats with obstructive jaundice: effect of internal and external drainage

BACKGROUND AND AIMS: The role of Kupffer cells in obstructive jaundice (OJ) has not been fully understood. The aims of the present study were to measure superoxide and nitric oxide (NO) production by Kupffer cells in experimental OJ in rats and to investigate the response to internal and external biliary drainage. METHODS: Eighty male Sprague-Dawley rats were assigned to four groups: sham operation, OJ, and internal and external biliary drainage. Kupffer cells were isolated on day 7 in the sham operation and OJ group, and on day 7 after drainage procedures. Cells were cultured with or without lipopolysaccharide (LPS). Superoxide production was quantified in cultured Kupffer cells at 2 h and 48 h, respectively, after cell isolation using the superoxide dismutase inhibitable ferricytochrome c reduction method. Nitrite production in cell culture supernatants was measured 48 h later using Greiss reagents. RESULTS: Without LPS stimulation, Kupffer cells produced comparable superoxide and nitrite in each group (P > 0.05). With LPS stimulation, Kupffer cells in the OJ group produced significantly higher superoxide anions than the other groups (P = 0.006). Nitrite production was significantly increased in the OJ group and external biliary drainage group compared to rats in the sham operation and internal drainage groups (P < 0.01). CONCLUSIONS: Kupffer cells from rats with OJ produce great amounts of endotoxin-mediated oxidants. Both internal and external biliary drainage can decrease the elevated superoxide production. Internal drainage is superior to external drainage for reversing the distortional capacity of NO production by Kupffer cells.     

Similarities and differences in early retinal phenotypes in hypertension and diabetes

The use of retinal photography in clinical practice and research has substantially increased the knowledge about the epidemiology, natural history and significance of diabetic and hypertensive retinopathy. Early retinopathy signs, including retinal microaneurysms, blot hemorrhages, cotton-wool spots and hard exudates, are common vascular abnormalities found in middle-aged to older people with diabetes and hypertension. The presence of these early retinopathy signs is associated with an increased risk of systemic vascular diseases, such as stroke, cognitive impairment, coronary heart disease, heart failure and nephropathy. These retinopathy lesions may therefore be considered as biomarkers of systemic microvascular processes caused by diabetes and hypertension. Nevertheless, whereas the interest in retinopathy assessment continues to grow, a core concept remains undefined: what is the relative importance and contribution of diabetes and hypertension in the development of early retinopathy signs? The answer of this fundamental question holds the key to better understanding of the systemic associations of early hypertensive and diabetic retinopathy. In this review, we summarize the similarities and differences of early retinopathy signs seen in diabetes and hypertension, and discuss the conceptual relevance from epidemiological, pathophysiological, and clinical perspectives.     

Superoxide and respiratory coupling in mitochondria of insulin-deficient diabetic rats

Mitochondrial reactive oxygen species have been implicated in both diabetic complications and the progression of the underlying diabetic state. However, it is not clear whether mitochondria of diabetic origin are intrinsically altered to generate excess reactive oxygen species independent of the surrounding diabetic milieu. Mitochondria were isolated from gastrocnemius, heart, and liver of 2-wk and 2-month streptozotocin diabetic rats and controls. We rigidly quantified mitochondrial superoxide, respiration and ATP production, respiratory coupling, the expression of several proteins with antioxidant properties, and the redox state of glutathione. Both fluorescent assessment and electron paramagnetic spectroscopy revealed that superoxide production was unchanged or reduced in the 2-month diabetic mitochondria compared with controls. Kinetic analysis of the proton leak showed that diabetic heart and muscle mitochondria were actually more coupled compared with control despite an approximate 2- to 4-fold increase in uncoupling protein-3 content. Adenine nucleotide translocator type 1 expression was reduced by approximately 50% in diabetic muscle mitochondria. Catalase was significantly up-regulated in muscle and heart tissue and in heart mitochondria, whereas glutathione peroxidase expression was increased in liver mitochondria of diabetic rats. We conclude that gastrocnemius, heart, and liver mitochondria of streptozotocin diabetic rats are not irrevocably altered toward excess superoxide production either by complex I or complex III. Moreover, gastrocnemius and heart mitochondria demonstrate increased, not decreased, respiratory coupling. Mitochondria of insulin-deficient diabetic rats do show signs of adaptation to antecedent oxidative stress manifested as tissue-specific enzyme and uncoupling protein expression but remain remarkably robust with respect to superoxide production.     

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells

Objective. To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. Methods. The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. Results. Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. Conclusions. Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.     

糖尿病急性心肌梗死早期病死率性别差异的探讨

目的 探讨性别对糖尿病急性心肌梗死(AMI)早期病死率的影响及其原因。资料与方法 分析181例AMI:男95例,年龄(67.86±11.18)岁,其中糖尿病50例;女86例,年龄(75.08±6.78)岁,其中糖尿病46例。对男女患者住院30d病死率、年龄、血糖、血脂、从症状出现至就诊时间、溶栓及介入治疗进行比较。结果 女性AMI病死率高于男性(27.91％vs 12.63％),糖尿病AMI病死率高于非糖尿病(25.00％vs 14.12％),糖尿病AMI女性病死率高于男性(36.96％vs 14.00％),女性糖尿病AMI年龄较大,空腹血糖、甘油三酯高于男性,出现症状至入院时间较长,溶栓治疗与介入治疗较少,以上因素进行多因素回归分析发现性别、出现症状至入院时间与糖尿病急性心肌梗死早期病死率有关。结论 女性急性心肌梗死病死率高于男性,尤其是糖尿病急性心肌梗死。     

Superoxide anion (O2-) production by polymorphonuclear leukocytes in insulin dependent diabetes mellitus (IDDM)]

The superoxide anion (O2-) production in polymorphonuclear leukocytes stimulated by phorbol myristate acetate in IDDM and non-insulin dependent diabetes mellitus (NIDDM) was determined by the method of Johnston et al, compared with that of each age matched controls. And the correlation between O2- production and hemoglobin (Hb) A1 and A1c value was investigated. The O2- production in IDDM was 24.4 +/- 7.4 (mean +/- SD, n mol per 4 X 10(5) cells) at 10 min. and 51.4 +/- 8.7 at 30 min., in NIDDM each 31.6 +/- 9.3, 60.2 +/- 14.4, and in controls each 40.5 +/- 4.2, 72.4 +/- 3.1. O2- production in IDDM was significantly lower than that in NIDDM (p less than 0.001 at 10 min. and p less than 0.01 30 min.) and controls (p less than 0.001 at 10 and 30 min.). O2- production at 10 and 30 min. possessed a negative correlation with Hba1 and A1c value (HbA1: p less than 0.01 at 10 min. p less than 0.05 at 30 min., HbA1c: p less than 0.01 at 10 and 30 min.). These findings suggest that impaired O2- production might be one of the factors accounting for depressed bactericidal activity of polymorphonuclear leukocytes in IDDM, and that a protracted hyperglycemia might shed some effect on O2- production.     

The mechanism of the prolonged stimulatory effect of corticotrophin on pregnenolone production by guinea-pig adrenocortical mitochondria

The postulated prolonged stimulatory influence of ACTH on the adrenocortical mitochondrial synthesis of pregnenolone in response to ACTH was studied in adrenal mitochondria isolated from control guinea-pigs and from animals treated s.c. with 100 micrograms ACTH(1-24) twice daily on the day before the animals were killed. The animals from both groups were injected with 100 micrograms ACTH s.c. 30 min before killing. The mitochondrial production of pregnenolone (expressed in nmol per mg mitochondrial protein after 10-min incubation) increased from 1.52 +/- 0.46 (S.E.M.) in the control group to 4.50 +/- 0.59 for mitochondria from ACTH-treated animals, despite a similar free cholesterol content in the mitochondria, even when determined after a previous in-vivo treatment with aminoglutethimide to block further metabolism of cholesterol into pregnenolone. In addition, in the presence of an excess of exogenous cholesterol (100 mumol/l), the production of pregnenolone remained higher for mitochondria from ACTH-treated animals. In contrast, when the calcium concentration in the incubation medium was raised to 1 mmol/l, with subsequent enhancement in pregnenolone synthesis, the mitochondrial pregnenolone production became similar for both groups (8.28 +/- 1.11 nmol in the ACTH-treated group and 9.55 +/- 1.90 nmol in the control group), even in the presence of 100 mumol cholesterol/l (13.5 +/- 1.80 nmol in ACTH-treated animals and 14.8 +/- 1.93 nmol in controls). Cycloheximide treatment administered on the day before the animals were killed was without any effect on pregnenolone production in control animals (3.51 +/- 0.43 nmol before and 3.65 +/- 0.63 nmol after cycloheximide treatment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate oxidation and energy production by Guerin epithelioma mitochondria.

The mitochondria from Guerin epithelioma and normal rat liver were isolated and their energy production abilities were compared. Both mitochondrial preparations were of the same degree of purity. The percentage of mitochondrial protein in tumour homogenates was three times lower than in rat liver. Guerin epithelioma mitochondria characterized a high respiratory control ratio with succinate and low with NAD dependent substrates in the presence of ADP. The ADP-ATP exchange rate was five times lower in tumour than in rat liver. Energy dependent potassium ions accumulation was about 45% of that found in rat liver mitochondria. The uptake of respiratory substrates was similar in tumour and liver mitochondria. These results indicate on the low efficiency of oxidative phosphorylation in the mitochondria of Guerin epithelioma.     

Analysis of gene expression profiles in insulin-sensitive tissues from pre-diabetic and diabetic Zucker diabetic fatty rats

Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats. These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.     

Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver

Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis).     

In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin

To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3-kinase activity in adipose tissue only 2.0-fold (P < 0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P < 0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 microg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.     

Glucose metabolism in lean patients with mild type 2 diabetes mellitus: evidence for insulin-sensitive and insulin-resistant variants

Obesity and type 2 diabetes mellitus (DM2) are 2 closely related syndromes, with obesity occurring in 70% to 80% of DM2 patients. Both syndromes are characterized by insulin resistance (IR). However, the metabolic characteristics of lean DM2 patients are not clearly defined, a fact attributed to the heterogeneity of the diabetes syndrome. Our objective was to study glucose metabolism in lean DM2 patients, in terms both of the basal and the insulin-stimulated states, and particularly, to investigate whether 2 subpopulations of diabetic patients are identifiable on the basis of degree of IR. Sixteen nonobese (body mass index [BMI] less than 27 kg. m(-2)) DM2 subjects with light to moderate fasting hyperglycemia were studied. Ten healthy subjects were used as a control group, with no family history of DM2 and matched by age, sex, and BMI in the diabetic group. All participants underwent an intravenous glucose tolerance test with frequent sampling over 180 minutes. Insulin sensitivity (IS) and glucose effectiveness at zero insulin (GEZI) were calculated using Bergman's minimal model. Non-insulin-mediated glucose uptakes (NIMGU) and insulin-mediated glucose uptakes (IMGU) were calculated for the basal (F) and insulin-stimulated states at 11.1 mmol/L of glucose (11.1). The beta-cell function was calculated via the acute insulin response to glucose (AIRg). Clustering techniques were used to identify subpopulations of DM2 patients on the basis of insulin sensitivity. The group of DM2 patients was characterized by both IR (IS index, 6.23 +/- 4.68 v 12.75 +/- 7.74 x 10(-5). min(-1). (pmol. L(-1))(-1), P <.01) and insulin secretion abnormalities (AIRg, 336 +/- 456 v 1,912 +/- 1,293 pmol/L. min, P <.0001), but showed similar values for GEZI (0.011 +/- 0.005 v 0.011 +/- 0.007 min(-1), not significant [NS]) in comparison to the control group. For the basal state, no differences were found between the DM2 patients and control subjects for NIMGU(F) (0.13 +/- 0.07 v 0.08 +/- 0.05 mmol/kg. min, NS) or for IMGU(F) (0.05 +/- 0.04 v 0.05 +/- 0.02 mmol/kg. min, NS). For the insulin-stimulated state, the DM2 patients showed a reduction of approximately 50% in the IMGU(11.1) value (0.20 +/- 0.17 v 0.38 +/- 0.24 mmol/kg. min, P <.05), but no significant differences were found for NIMGU(11.1) (0.19 +/- 0.09 v 0.20 +/- 0.12 mmol/kg. min, NS) in relation to the control group. Using the clustering technique, it was possible to identify 2 subpopulations of DM2 patients, a DM-IS group (n = 6) that was insulin sensitive (IS index, 11.70 +/- 2.40 x 10(-5). min(-1). (pmol. L(-1))(-1)) and a DM-IR group (n = 10) that was insulin resistant (IS index, 3.02 +/- 1.60 x 10(-5). min(-1). (pmol. L(-1))(-1)). The DM-IS group was characterized by an absence of IR, diminished GEZI, and a reduction in AIRg; whereas the DM-IR group was characterized by IR and a reduction in AIRg, but normal GEZI. We conclude that (1) as a group, DM2 patients are characterized by IR and beta-cell dysfunction, but normal NIMGU; (2) two subpopulations of DM2 patients can be identified on the basis of insulin sensitivity, with the DM-IS group further characterized by diminished GEZI; and finally, (3) deterioration in the pancreatic response to glucose stimulus is a sine qua non condition for a profound alteration in glucose metabolism in DM2 patients.