Heikki A. Koistinen

Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes

BACKGROUND The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. METHODS We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. RESULTS Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). CONCLUSIONS Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).

Interleukin-6 Directly Increases Glucose Metabolism in Resting Human Skeletal Muscle

Interleukin (IL)-6 is a proinflammatory cytokine shown to modify insulin sensitivity. Elevated plasma levels of IL-6 are observed in insulin-resistant states. Interestingly, plasma IL-6 levels also increase during exercise, with skeletal muscle being the predominant source. Thus, IL-6 has also been suggested to promote insulin-mediated glucose utilization. In this study, we determined the direct effects of IL-6 on glucose transport and signal transduction in human skeletal muscle. Skeletal muscle strips were prepared from vastus lateralis biopsies obtained from 22 healthy men. Muscle strips were incubated with or without IL-6 (120 ng/ml). We found that IL-6 increased glucose transport in human skeletal muscle 1.3-fold (P < 0.05). A 30-min pre-exposure to IL-6 did not affect insulin-stimulated glucose transport. IL-6 also increased skeletal muscle glucose incorporation into glycogen, as well as glucose oxidation (1.5- and 1.3-fold, respectively; P < 0.05). IL-6 increased phosphorylation of STAT3 (signal transducer and activator of transcription 3; P < 0.05), AMP-activated protein kinase (P = 0.063), and p38 mitogen-activated protein kinase (P < 0.05) and reduced phosphorylation of S6 ribosomal protein (P < 0.05). In contrast, phosphorylation of protein kinase B/Akt, AS160 (Akt substrate of 160 kDa), and GSK3α/β (glycogen synthase kinase 3α/β) as well as insulin receptor substrate 1–associated phosphatidylinositol 3-kinase activity remained unaltered. In conclusion, acute IL-6 exposure increases glucose metabolism in resting human skeletal muscle. Insulin-stimulated glucose transport and insulin signaling were unchanged after IL-6 exposure.

Regulation of glucose transport in human skeletal muscle

Glucose transport, the rate limiting step in glucose metabolism in skeletal muscle, is mediated by insulin-sensitive glucose transporter 4 (GLUT4) and can be activated in skeletal muscle by two separate and distinct signalling pathways: one stimulated by insulin and the second by muscle contractions. Skeletal muscle is the principal tissue responsible for insulin-stimulated glucose disposal and thus the major site of peripheral insulin resistance. Impaired glucose transport in skeletal muscle leads to impaired whole body glucose uptake, and contributes to the pathogenesis of Type 2 diabetes mellitus. A combination of genetic and environmental factors is likely to contribute to the pathogenesis of Type 2 diabetes mellitus; however, the primary defect is still unknown. Intense efforts are underway to define the molecular mechanisms that regulate glucose metabolism in insulin sensitive tissues. This review will present our current understanding of mechanisms regulating glucose transport in skeletal muscle in humans. Elucidation of the pathways involved in the regulation of glucose homeostasis will offer insight into the pathogenesis of insulin resistance and Type 2 diabetes mellitus and may lead to the identification of biochemical entry points for drug intervention to improve glucose homeostasis.

Plasma Acylation Stimulating Protein Concentration and Subcutaneous Adipose Tissue C3 mRNA Expression in Nondiabetic and Type 2 Diabetic Men

Abstract—We studied the effect of an oral fat load on plasma acylation stimulating protein (ASP) concentrations in 9 lean healthy (age 59±2 years, body mass index[BMI] 23.2±0.4 kg/m2; both mean±SEM), 9 obese nondiabetic (58±2 years, BMI 29.4±0.5 kg/m2), and 12 type 2 diabetic (60±2 years, BMI 29.6±1.0 kg/m2) men. Because ASP is a cleavage product of complement protein C3 (C3adesArg) and its secretion is regulated by insulin, we also examined the subcutaneous adipose tissue expression of C3 mRNA before and after a 240-minute euglycemic hyperinsulinemic clamp in a subgroup of these men. Plasma ASP concentration and adipose tissue C3 mRNA expression were higher in the obese groups than in the lean men. Plasma ASP concentration did not change significantly after the fat load. Fasting plasma ASP concentration and C3 mRNA expression were correlated negatively with insulin sensitivity and positively with the magnitude of postprandial lipemia in nondiabetic but not in type 2 diabetic men. The expression of C3 mRNA was not regulated by insulin. These data suggest that ASP is associated with whole-body glucose and lipid metabolism in nondiabetic individuals, whereas metabolic disturbances in diabetes may overcome the regulatory role of ASP in lipid and glucose metabolism.

Leptin during and after preeclamptic or normal pregnancy: its relation to serum insulin and insulin sensitivity.

Hyperleptinemia may be part of the insulin resistance syndrome. We studied serum leptin in preeclampsia, which is an insulin-resistant state, and sought associations between leptin and insulin or insulin sensitivity during and after pregnancy. Twenty-two proteinuric preeclamptic women and 16 normotensive controls were studied during the third trimester. Leptin was higher in preeclampsia (mean +/- SE, 34.6 +/- 3.9 v 20.0 +/- 3.3 microg/L, P = .002) and correlated directly with the level of proteinuria (r = .47, P = .03) and normal pregnancy (r = .52, P = .04), whereas insulin sensitivity as assessed by an intravenous glucose tolerance test showed no relationship to leptin. Leptin was 19.0 +/- 3.6 microg/L in 14 preeclamptic women and 10.1 +/- 2.0 microg/L (P = .11) in 11 controls 3 months after delivery. Leptin correlated directly with insulin both in preeclamptic puerperal women (r = .63, P = .02) and in controls (r = .81, P = .003). Leptin and insulin sensitivity correlated only in preeclamptic puerperal women (r = -.59, P = .02). In conclusion, (1) serum leptin is elevated in preeclampsia, (2) insulin is an important determinant of serum leptin in preeclamptic and normotensive women both during pregnancy and in the puerperium, and (3) hyperleptinemia may be part of the insulin resistance syndrome also in women with prior preeclampsia.

Insulin signaling and glucose transport in skeletal muscle from first-degree relatives of type 2 diabetic patients.

Aberrant insulin signaling and glucose metabolism in skeletal muscle from type 2 diabetic patients may arise from genetic defects and an altered metabolic milieu. We determined insulin action on signal transduction and glucose transport in isolated vastus lateralis skeletal muscle from normal glucose-tolerant first-degree relatives of type 2 diabetic patients (n = 8, 41 ± 3 years, BMI 25.1 ± 0.8 kg/m2) and healthy control subjects (n = 9, 40 ± 2 years, BMI 23.4 ± 0.7 kg/m2) with no family history of diabetes. Basal and submaximal insulin-stimulated (0.6 and 1.2 nmol/l) glucose transport was comparable between groups, whereas the maximal response (120 nmol/l) was 38% lower (P < 0.05) in the relatives. Insulin increased phosphorylation of Akt and Akt substrate of 160 kDa (AS160) in a dose-dependent manner, with comparable responses between groups. AS160 phosphorylation and glucose transport were positively correlated in control subjects (R2 = 0.97, P = 0.01) but not relatives (R2 = 0.46, P = 0.32). mRNA of key transcriptional factors and coregulators of mitochondrial biogenesis were also determined. Skeletal muscle mRNA expression of peroxisome proliferator–activated receptor (PPAR) γ coactivator (PGC)-1α, PGC-1β, PPARδ, nuclear respiratory factor-1, and uncoupling protein-3 was comparable between first-degree relatives and control subjects. In conclusion, the uncoupling of insulin action on Akt/AS160 signaling and glucose transport implicates defective GLUT4 trafficking as an early event in the pathogenesis of type 2 diabetes.

Molecular Mechanisms of Skeletal Muscle Insulin Resistance in Type 2 Diabetes

Type 2 (non-insulin-dependent) diabetes mellitus afflicts millions of people worldwide and is one of the main causes of morbidity and mortality. Current therapeutic agents to treat Type 2 diabetes are insufficient and thus, newer approaches are desperately needed. Type 2 diabetes is manifested by progressive metabolic impairments in tissues such as skeletal muscle, adipose tissue and liver, such that these tissues become less responsive to insulin. Skeletal muscle is quantitatively the most important tissue involved in maintaining glucose homeostasis under insulin-stimulated conditions, and is a major site of insulin resistance in Type 2 diabetic patients. At the cellular level, glucose transport into skeletal muscle is the rate-limiting step for whole body glucose uptake and a primary site of insulin resistance in Type 2 diabetes. Thus, skeletal muscle is a key insulin target tissue that harbours intrinsic defects that impinges upon whole body glucose homeostasis. Here, we review the current knowledge of signalling events that regulate glucose transport in human skeletal muscle.

Insulin-independent glucose transport regulates insulin sensitivity

The glucose transport proteins (GLUT1 and GLUT4) facilitate glucose transport into insulin‐sensitive cells. GLUT1 is insulin‐independent and is widely distributed in different tissues. GLUT4 is insulin‐dependent and is responsible for the majority of glucose transport into muscle and adipose cells in anabolic conditions. We suggest the hypothesis that insulin resistance is dependent on whether glucose is entering through GLUT1 or GLUT4 and on the two functional compartments of glucose 6‐phosphate formation within the cell. Glucose entering the muscle cell through GLUT4 and phosphorylated by hexokinase II is mainly directed to glycogen synthesis and glycolysis. If glucose is entering through GLUT1 and phosphorylated by hexokinase I, the glucose 6‐phosphate so formed is available for all metabolic pathways, including the hexosamine pathway. Hexosamines have a negative feedback effect on GLUT4, and reduced GLUT4 activity decreases insulin‐mediated glucose uptake. Thus, insulin‐independent glucose transport through GLUT1 can meet the basal needs of the muscle cell. If glucose entrance through GLUT1 and the activation of the hexosamine pathway is abundant, it can decrease the insulin‐mediated glucose transport through GLUT4 leading to insulin resistance.

The effect of exercise on leptin concentration in healthy men and in type 1 diabetic patients.

PURPOSE Leptin is a recently discovered hormone that appears as a regulator of energy balance. It is important to know whether leptin concentrations are changed under conditions of altered energy homeostasis. Consequently, we examined the effects of exercise with fasting and exercise with feeding on circulating leptin concentrations in healthy men and in type 1 diabetic patients with normal body weight and well controlled diabetes. METHODS Leptin concentrations were determined with radioimmunoassay. RESULTS During a 3-h cycle ergometer exercise with fasting, leptin decreased by 42% (P < 0.01) in nine healthy men and by 23% (P = 0.05) in eight male type 1 diabetic patients. Leptin fell equally by 12% (P < 0.03) both in nine healthy men and in eight male type 1 diabetic patients who were studied as a resting control group. The absolute fall in leptin in healthy men was similar in the exercise and resting control groups (0.8 +/- 0.1 microgram.L-1 vs 0.8 +/- 0.2 microgram.L-1). However, due to lower leptin concentration before the exercise, the relative decrease (42%) was greater than during the resting control study (12%, P < 0.005). This difference was not seen in the diabetic patients. Fasting leptin concentration correlated positively with BMI (r = 0.75, P < 0.001) and fasting insulin (r = 0.71, P < 0.01) in healthy men as well as with insulin level (r = 0.54, p < 0.05) in type 1 diabetic patients. When exercise was performed with feeding, and this was associated with a significant rise in serum cortisol level (marathon run, 14 healthy men and 7 type 1 diabetic patients), leptin concentration did not change significantly. CONCLUSIONS 1) During morning hours, leptin decreases both in healthy men and in type 1 diabetic patients, reflecting a diurnal variation of leptin concentration and the effect of fasting on leptin concentration. 2) The fall in leptin during morning hours is augmented by physical exercise in healthy men. 3) If exercise is performed with feeding and associated with a rise in serum cortisol level, leptin concentration remains unchanged. These data suggest that although exercise may reduce circulating leptin levels, the effect is small and can be counterbalanced by feeding or a rise in serum cortisol concentration.

Kinetics of GLUT4 Trafficking in Rat and Human Skeletal Muscle

OBJECTIVE In skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity. RESEARCH DESIGN AND METHODS Using a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 ± 11 years and BMI 25.8 ± 0.8 kg/m2). RESULTS In rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min−1, respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min−1, respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle. CONCLUSIONS Insulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle.