Intensive glucose control and macrovascular outcomes in type 2 diabetes.

AIMS/HYPOTHESIS: Improved glucose control in type 2 diabetes is known to reduce the risk of microvascular events. There is, however, continuing uncertainty about its impact on macrovascular disease. The aim of these analyses was to generate more precise estimates of the effects of more-intensive, compared with less-intensive, glucose control on the risk of major cardiovascular events amongst patients with type 2 diabetes. METHODS: A prospectively planned group-level meta-analysis in which characteristics of trials to be included, outcomes of interest, analyses and subgroup definitions were all pre-specified. RESULTS: A total of 27,049 participants and 2,370 major vascular events contributed to the meta-analyses. Allocation to more-intensive, compared with less-intensive, glucose control reduced the risk of major cardiovascular events by 9% (HR 0.91, 95% CI 0.84-0.99), primarily because of a 15% reduced risk of myocardial infarction (HR 0.85, 95% CI 0.76-0.94). Mortality was not decreased, with non-significant HRs of 1.04 for all-cause mortality (95% CI 0.90-1.20) and 1.10 for cardiovascular death (95% CI 0.84-1.42). Intensively treated participants had significantly more major hypoglycaemic events (HR 2.48, 95% CI 1.91-3.21). Exploratory subgroup analyses suggested the possibility of a differential effect for major cardiovascular events in participants with and without macrovascular disease (HR 1.00, 95% CI 0.89-1.13, vs HR 0.84, 95% CI 0.74-0.94, respectively; interaction p = 0.04). CONCLUSIONS/INTERPRETATION: Targeting more-intensive glucose lowering modestly reduced major macrovascular events and increased major hypoglycaemia over 4.4 years in persons with type 2 diabetes. The analyses suggest that glucose-lowering regimens should be tailored to the individual.

Glycaemic separation and risk factor control in the Veterans Affairs Diabetes Trial: an interim report.

OBJECTIVE: The Veterans Affairs Diabetes Trial (VADT) will assess the effect of intensive (INT) vs improved standard (STD) glycaemic control on major cardiovascular (CV) events, treating other risk factors equally in both arms. Four-year results of main metabolic parameters are presented. RESEARCH DESIGN AND METHODS: VADT is a 7.5 years prospective randomized study of 1791 patients, 20 centres, of men and women of age 60.5 +/- 8.7 years, diagnosed for 11.5 +/- 7.5 years. Their body mass index (BMI) at baseline was 31 +/- 4 kg/m(2) and mean A1C 9.4 +/- 1.5% after maximum dose of oral agents or insulin treatment. Step treatment consists of glimepiride or metformin, rosiglitazone, insulin and other agents; A1C goals are 8-9% in STD and <6% in INT. Lifestyle, blood pressure and lipids are treated uniformly in both arms. RESULTS: A1C improved in both arms. INT kept median A1C <7% all years, A1C separation is 1.5-1.7%. From year 1 to 4, mean blood pressure is <129/74 mmHg, similar throughout. Median LDL-C was <97 mg/dl by year 1 and triglycerides 150 or less by 2 years. Triglycerides were lower in INT (12-16 mg/dl; p < 0.01). By 4 years, 88% are on lipid-lowering agents and 93% are on antiplatelet/anticoagulant agents. BMI is higher in INT every year (0.9-1.6 kg/m(2); p < 0.01). CONCLUSION: VADT is maintaining the expected A1C in both STD and INT, and LDL-C, triglycerides and blood pressure are at target. The trial is continuing to June 2008. It will be the first long-term completed type 2 diabetes study of the role of glycaemia on CV disease with modern treatments.

Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells.

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. 125I-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with 3H-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 microm. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 microm. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 microm nelfinavir decreased 125I-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 microm nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.

Factors affecting diabetes knowledge in Type 2 diabetic veterans.

AIMS/HYPOTHESIS: To describe the clinical, psychological and social factors affecting diabetes knowledge of veterans with established Type 2 diabetes. METHODS: We conducted an observational study of 284 insulin-treated veterans with stable Type 2 diabetes. All subjects completed the University of Michigan Diabetes Research and Training Centre Knowledge Test, the Diabetes Care Profile, the Mini-Mental State Examination, the Geriatric Depression Scale, and the Diabetes Family Behaviour Checklist. Stepwise multiple linear regression was used to develop a model for the diabetes knowledge score based upon clinical and psychosocial variables. RESULTS: One hundred eighty subjects were evaluated in a derivation set. The mean age +/- SD was 65.4+/-9.6 years, 94% were men, and 36% were members of a minority group. Performance on the diabetes knowledge test was poor (64.9+/-15.3% correct). Self-perceived understanding of all management objectives explained only 6% of the variance in the knowledge scores. Multivariate analysis showed that age, years of schooling, duration of treatment, cognitive function, sex, and level of depression were independent determinants of the knowledge score. When the model was applied to 104 subjects in a validation set, there was a strong correlation between observed and predicted scores (r=0.537; p<0.001). CONCLUSIONS/INTERPRETATION: Stable, insulin-treated veterans have major deficiencies in diabetes knowledge that could impair their ability to provide self-care. A multivariate model comprised of demographic variables and psychosocial profiling can identify patients who have limited diabetes knowledge and be used to assess individual barriers to ongoing diabetes education.

Insulin inhibition of the proteasome is dependent on degradation of insulin by insulin-degrading enzyme.

A consequence of insulin-dependent diabetes mellitus is the loss of lean muscle mass as a result of accelerated proteolysis by the proteasome. Insulin inhibition of proteasomal activity requires interaction with insulin-degrading enzyme (IDE), but it is unclear if proteasome inhibition is dependent merely on insulin-NIDE binding or if degradation of insulin by IDE is required. To test the hypothesis that degradation by IDE is required for proteasome inhibition, a panel of insulin analogues with variable susceptibility to degradation by IDE binding was used to assess effects on the proteasome. The analogues used were [Lys(B28), Pro(B29)]-insulin (lispro), [Asp(B10)]-insulin (Asp(B10)) and [Glu(B4), Gln(B16), Phe(B17)]-insulin (EQF). Lispro was as effective as insulin at inhibition of degradation of iodine-125 ((125)I)-labeled insulin, but Asp(B10) and EQF were somewhat more effective. All agents inhibited cross-linking of (125)I-insulin to IDE, suggesting that all were capable of IDE binding. In contrast, although insulin and lispro were readily degraded by IDE, Asp(B10) was degraded more slowly, and EQF degradation was undetectable. Both insulin and lispro inhibited the proteasome, but Asp(B10) was less effective, and EQF had little effect. In summary, despite effective IDE binding, EQF was poorly degraded by IDE, and was ineffective at proteasome inhibition. These data suggest that insulin inhibition of proteasome activity is dependent on degradation by IDE. The mechanism of proteasome inhibition may be the generation of inhibitory fragments of insulin, or by displacement of IDE from the proteasome.

Insulin inhibition of protein degradation in cells expressing wild-type and mutant insulin receptors.

The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.

Advances in the treatment of diabetes-insulin analogues.

Insulin analogues are molecules derived by modifying the structure of the human insulin molecule, resulting in altered physico-chemical, biological and pharmacodynamic properties. The pharmacokinetic characteristics of the previously available rapid-, intermediate-, and long-acting preparations of human insulin make it almost impossible to achieve sustained normoglycemia. All currently available analogues have been shown to have a more physiological time-action profile with either a shorter onset and shorter duration of action (insulin lispro and insulin aspart) or a more constant effect lasting at least 24 hours (insulin glargine). These advantages in the time-action profiles have been shown to improve various surrogate parameters (e.g., postprandial blood glucose concentrations) in a number of randomized controlled trials. Insulin analogues also represent a unique tool to unravel structure-function relationships in insulin biochemistry and insulin action. Data on the currently available, currently tested and currently being developed analogs are reviewed.

Recombinant DNA technology in the treatment of diabetes: insulin analogs.

After more than half a century of treating diabetics with animal insulins, recombinant DNA technologies and advanced protein chemistry made human insulin preparations available in the early 1980s. As the next step, over the last decade, insulin analogs were constructed by changing the structure of the native protein with the goal of improving the therapeutic properties of it, because the pharmacokinetic characteristics of rapid-, intermediate-, and long-acting preparations of human insulin make it almost impossible to achieve sustained normoglycemia. The first clinically available insulin analog, lispro, confirmed the hopes by showing that improved glycemic control can be achieved without an increase in hypoglycemic events. Two new insulin analogs, insulin glargine and insulin aspart, have recently been approved for clinical use in the United States, and several other analogs are being intensively tested. Thus, it appears that a rapid acceleration of basic and clinical research in this arena will be seen, which will have direct significance to both patients and their physicians. The introduction of new short-acting analogs and the development of the first truly long-acting analogs and the development of analogs with increased stability, less variability, and perhaps selective action, will help to develop more individualized treatment strategies targeted to specific patient characteristics and to achieve further improvements in glycemic control. Data on the currently available and tested analogs, as well as data on those currently being developed, are reviewed.

Hyperglycemia and cardiovascular disease.

Diabetes is associated with many microvascular and macrovascular complications. Hyperglycemia plays a pivotal role in the development of microvascular complications, but the actual effect of hyperglycemia on the development and progression of macrovascular complications remains unclear and even somewhat controversial, particularly in type 2 diabetes. Macrovascular complications are increased in individuals with type 2 diabetes long before there is significant hyperglycemia, and in many, but not all, studies a clear association of glucose elevations and macrovascular complications cannot be shown. The complicated nature of metabolic abnormalities in type 2 diabetes and the relative role of these associated conditions in the development of macrovascular disease make definitive conclusions somewhat difficult. In spite of these considerations, there are certain aspects of hyperglycemia associated with macrovascular disease, particularly elevations of postprandial glucoses, and a number of basic mechanisms to explain these associations that could lead to the development of cardiovascular disease. Some of these basic abnormalities include activation of the sorbital pathway, oxidative stress, advanced glycation endproducts (AGE), and AGE precursors. These changes can result in many abnormalities, such as endothelial dysfunction, alteration of protein function, increased cytokine production, and glycosylation of structural proteins. These considerations suggest that hyperglycemia may play an important, but as yet not clearly defined, role in clinical macrovascular disease. Pursuant to this, several major multisite studies are currently underway to clarify the role of hyperglycemia in cardiovascular disease in type 2 diabetes.

Insulin pumps and glucose regulation.

With recent technologic advances there has been a resurgence of interest in implantable insulin infusion devices. A satisfactory closed loop system has been elusive. Open loop systems include the pump, delivery catheter, and patient pump communicator. Several such systems are currently undergoing clinical investigation. Implantable pumps can be placed with minimal morbidity. Insulin underdelivery is the most frequent long-term problem. Several recent studies suggest that implantable pumps can safely and effectively maintain good glucose control. The development of a satisfactory implantable closed loop system will be the next step in this technology.

Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatocytes.

Inhibition by insulin of long chain fatty acid oxidation in mitochondria is mediated in part by elevating malonyl-CoA levels, which inhibit carnitine palmitoyl-transferase I. Whether insulin alters peroxisomal oxidation has not been studied. We present data which show that insulin inhibits the oxidation of palmitic acid by peroxisomes (IC(50) = 8.5 x 10(-11) M) at hormone concentrations 100-fold less than those needed for mitochondrial inhibition (IC(50) = 1.3 x 10(-8) M). We used a purified peroxisome preparation to study the mechanism of insulin action. Insulin had a direct effect in the peroxisome preparations to decrease oxygen consumption, fatty acyl-CoA oxidizing system activity and acyl-CoA oxidase by approximately 40%, 30% and 15%, respectively. Since insulin degrading enzyme (IDE) is an insulin-binding protein known to be in peroxisomes, we studied the effect of an inhibitory anti-IDE antibody on the ability of insulin to inhibit the fatty acyl-CoA oxidizing system. The antibody eliminated the inhibitory effect of insulin. We conclude that insulin inhibits peroxisomal fatty acid oxidation by a mechanism requiring IDE.

Characterization of the inhibition of protein degradation by insulin in L6 cells.

In muscle cells, protein degradation occurs by lysosomal and nonlysosomal mechanisms but the mechanism by which insulin inhibits protein degradation is not well understood. Using cultured L6 myotubes, the effect of insulin on muscle cell protein degradation was examined. Cells were labeled for 18 h with [3H]leucine or [3H]tyrosine and protein degradation measured by release of TCA-soluble radioactivity. Incubation with insulin for 0.5, 1, 2, or 3 h produced 0, 6, 12, and 13% inhibition, respectively, at 10(-7) M. If the cells were incubated for 3 h prior to the addition of insulin to remove short-lived proteins, the effect of insulin was enhanced, producing 26% inhibition. Very long-lived protein degradation (cells labeled for 48 h, chased for 24 h before the addition of insulin) was only inhibited 17% by insulin. This was due to serum starvation during the chase since the addition of serum to the chase medium produced a subsequent inhibition of 38% by insulin. Thus insulin had a greater effect on the degradation of longer-lived proteins. Use of inhibitors suggested that insulin requires internalization and degradation to produce inhibition of protein degradation and acts through both the proteasome and lysosomes. There appears to be no interaction with the calpains.

Insulin and analogue effects on protein degradation in different cell types. Dissociation between binding and activity.

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

Degradation of amylin by insulin-degrading enzyme.

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.

Insulin inhibits the ubiquitin-dependent degrading activity of the 26S proteasome.

A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 approximately 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.

Genetically engineered insulin analogs: diabetes in the new millenium.

Tight glucose control is essential to minimize complications in diabetic patients. However, the pharmacokinetic characteristics of the currently available rapid-, intermediate-, and long-acting preparations of human insulin make it almost impossible to achieve sustained normoglycemia. Until recently, improvements in insulin formulations were seriously limited as advances were only achieved in insulin purity, species, and characteristics of the retarding agent. The availability of molecular genetic techniques opened new windows to create insulin analogs by changing the structure of the native protein and to improve the therapeutic properties. The first clinically available insulin analog, Lispro, confirmed the hopes by showing that improved glycemic control can be achieved without an increase in hypoglycemic events. This requires, however, optimal basal insulin replacement, either by multiple daily injections of neutral protein Hagedorn (NPH) insulin or by insulin pump. Evidence suggests that short-acting insulin analogs would be better matched by a true basal insulin than by the erratically absorbed and rather short-acting NPH insulin. Therefore, future availability of long-acting analogs raises the hope to realize the true potential benefits of the currently available short-acting analog, Lispro, and of those still awaiting approval. The introduction of new short-acting and the first truly long-acting analogs, the development of analogs with increased stability, less variability and perhaps selective action will help to develop more individualized treatment strategies targeted to specific patient characteristics and to achieve further improvements in glycemic control.

Hepatitis A-induced diabetes mellitus, acute renal failure, and liver failure.

A 38-year-old otherwise healthy man presented with hepatic failure (aspartate aminotransferase of 7212 U/L, alanine aminotransferase of 6629 U/L, total and direct bilirubin of 10.7 mg/dL) and acute renal failure (creatinine of 11.6 mg/dL and blood urea nitrogen of 42 mg/dL), which required hemodialysis when the creatinine increased to 21 mg/dL, with a blood urea nitrogen of 115 mg/dL, and the patient became oliguric. On admission, this patient also had a lipase of 1833 U/L, amylase of 211 U/L, glucose of 210 mg/dL, and reactive IgM antibody for acute hepatitis A. The hepatitis and acute renal failure resolved in 3 months, but this patient continues to have type II diabetes mellitus 7 years after the hepatitis A infection. This case illustrates that hepatitis A infection may be severe with liver failure, acute renal failure, and permanent diabetes mellitus as sequale of this infection.

Differences in the cellular processing of AspB10 human insulin compared with human insulin and LysB28ProB29 human insulin.

Cellular metabolism studies were performed comparing human insulin with two rapid-acting analogs, LysB28ProB29 insulin (LysPro) and AspB10 insulin (B10-Asp). B10-Asp bound to isolated hepatocytes at 37 degrees C to a greater extent than LysPro or native insulin, which were equivalent. The rate of degradation was similar for the three materials, resulting in a significant reduction in the degraded/bound ratio for the B10 analog. The processing of membrane-bound material was examined by incubating cells with hormone at 4 degrees C, removing unbound insulin, and incubating the cells at 37 degrees C. Again, binding was greater for B10-Asp versus LysPro or native insulin, with a reduction in the degraded/bound ratio. Hormone internalization and processing was examined by an acid wash of cells incubated with 125I(A14)-labeled hormone to remove surface-bound materials. The processing rate was slower for B10-Asp versus LysPro or native insulin. Cell extraction and examination on molecular-sieve chromatography confirmed that B10-Asp was processed at a slower rate than either LysPro or native insulin. Intact B10-Asp was found in the cell after 4 hours, whereas all native insulin and LysPro were degraded by 90 to 120 minutes. B10-Asp also caused a greater incorporation of thymidine into DNA in cultured cells than LysPro or native insulin, which were similar. These data show that the cellular processing of LysPro is essentially identical to that of native insulin. However, B10-Asp has markedly different properties and is processed much more slowly. The prolonged cell residence time of B10-Asp could contribute to its greater effects on cell growth and mitogenesis.