bluuebunny/crossref_metadata_2025 huggingface dataset:
Counterregulatory Hormone Responses to Hypoglycemia in the Elderly Patient with Diabetes
Authors: Meneilly Graydon S , Cheung Elsie , Tuokko Holly
Published: September 2013

In patients with non-insulin-dependent diabetes mellitus (NIDDM), the risk of severe or fatal hypoglycemia associated with the use of oral agents or insulin increases exponentially with age. We conducted this study with the hypothesis that this increased susceptibility to hypoglycemia is caused by alterations in release of counterregulatory hormones and psychomotor performance during hypoglycemia. Ten healthy nonobese elderly subjects (74 ± 1 years of age; body mass index, 24.5 ± 0.6 kg/m2) and 10 nonobese elderly NIDDM subjects (72 ± 1 years of age; body mass index, 25.6 ± 0.9 kg/m2) underwent two hyperinsulinemic glucose clamp studies (insulin infusion, 60 mU·m₋2 · min₋1). In the control study, glucose was maintained at 5 mM for 5 h; in the hypoglycemic study, glucose was kept at 5 mM for 1 h and then lowered in a stepwise fashion to 4.4, 3.8, 3.3, and 2.8 mM in each subsequent hour. At regular intervals in each study, neuropsychological tests were performed, counterregulatory hormones were measured, and a hypoglycemic symptom questionnaire was administered. At a glucose level of 2.8 mM, NIDDM patients had reduced incremental glucagon (normal subjects, 114 ± 18 ng/l; NIDDM subjects, 63 ± 9 ng/l; P < 0.05) and growth hormone responses (normal subjects, 13.8 ± 1.0 micrograms/l; NIDDM subjects, 7.0 ± 2.0 micrograms/l; P < 0.01) and increased epinephrine (normal subjects, 925 ± 198 pM; NIDDM subjects, 4175 ± 824 pM; P < 0.001) and cortisol responses (normal subjects, 291 ± 49 nM; NIDDM subjects, 524 ± 92 mM; P < 0.05). Symptom scores were similar in both groups at all levels of glycemia. at all levels of glycemia. At a glucose level of 2.8 mM, NIDDM patients had a greater impairment on the simple (normal subjects, ₋ 10 ± 24 ms; NIDDM subjects, 108 ± 18 ms; P < 0.001) and choice (normal subjects, 20 ± 18 ms; NIDDM subjects, 111 ± 18 ms; P < 0.01) reaction time tests. We conclude that the elderly NIDDM patients have altered release of counterregulatory hormones and altered psychomotor performance during hypoglycemia.
Isolated Low HDL Cholesterol: An Insulin-Resistant State
Authors: Karhapää Pauli , Malkki Mari , Laakso Markku
Published: September 2013

High levels of very-low-density lipoprotein (VLDL) triglycerides (TGs) and low levels of high-density lipoprotein (HDL) cholesterol have been found to be associated with insulin resistance. However, direct evidence that patients with isolated low HDL cholesterol are insulin resistant is still lacking. Therefore, we investigated the degree of insulin resistance and intracellular metabolism of glucose by the euglycemic glucose clamp technique and indirect calorimetry in three groups of subjects with normal glucose tolerance: 17 male control subjects with normolipidemia, 12 male patients with isolated low HDL cholesterol (low HDL group), and 10 male patients with low HDL cholesterol and hypertriglyceridemia (low HDL/high TG group). Fasting, 1-h, and 2-h glucose levels did not differ between the groups in an oral glucose tolerance test (OGTT). In contrast, insulin levels during an OGTT were significantly higher in the low HDL group than in the control group (fasting insulin: 85 ± 11 vs. 50 ± 6 pM, P = 0.005; 1-h insulin: 622 ± 92 vs. 394 ± 64 pM, P = 0.004; and 2-h insulin: 343 ± 73 vs. 194 ± 40 pM, P = 0.006). Similarly, insulin levels were also higher in the low HDL/high TG group than in the control group (fasting insulin: 82 ± 14 pM, P = 0.037; 1-h insulin: 795 ± 179 pM, P = 0.063; and 2-h insulin: 488 ± 145 pM, P = 0.040). During the euglycemic hyperinsulinemic clamp, the rates of whole body glucose uptake were similarly reduced in the low HDL group (46.5 ± 2.8 μmol · kg−1 · min−1, P = 0.003) and in the low HDL/high TG group (45.1 ± 5.4 μmol · kg−1 · min−1, P = 0.021) compared with those in the control group (58.8 ± 2.5 μmol · kg−1 · min−1). The rates of glucose oxidation during the euglycemic clamp were lower in the low HDL patients than in control subjects, but the differences were not statistically significant (low HDL group: 17.3 ± 0.9 μmol · kg−1 · min−1 P = 0.092; low HDL/high TG group: 17.5 ±1.9 μmol · kg−1 · min−1 P = 0.098; and control group: 21.0 ±1.5 junol · kg−1 · min−1). The rates of glucose nonoxidation were reduced in the low HDL group (29.3 ± 2.5 μmol · kg−1 · min−1, P = 0.042) and in the low HDL/high TG group (27.6 ± 5.0 μmol · kg−1 · min−1, P = 0.056) compared with those in the control group (37.7 ± 2.6 μmol · · kg−1 · min−1), although the latter difference was not statistically significant. We conclude that patients with low HDL cholesterol are insulin resistant independently of TG levels.
Potential Autoantigens In IDDM: Expression of Carboxypeptidase-H and Insulin But Not Glutamate Decarboxylase on the β-Cell Surface
Authors: Aguilar-Diosdado Manuel , Parkinson David , Corbett John A , Kwon Guim , Marshall Connie A , Gingerich Ron L , Santiago Julio V , McDaniel Michael L
Published: September 2013

Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent diabetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cells and suggested that cell-surface insulin was derived during exocytosis by the fusion of insulin secretory granules with the β-cell plasma membrane. These findings predict that insulin and other secretory granule-derived proteins such as the putative autoantigen CP-H may be colocalized with insulin at specific sites of exocytosis on the β-cell surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with antibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was essentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the β-cell surface. Colocalization of CP-H and insulin on the cell surface of β-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of β-cells positive for both of these autoantigens increased twofold with increases in insulin secretion. In contrast, islet cells failed to reveal cell-surface staining for GAD65, another putative autoantigen in IDDM, under either basal or insulin stimulatory conditions or following exposure of islet cells to the cytokines interleukin-1β, tumor necrosis factor-α, and recombinant human interferon-γ. These results demonstrate that the insulin secretory granule–derived proteins, insulin and CP-H, colocalize on the cell surface of β-cells during exocytosis and in this manner could be recognized by components of the immune system under certain disease settings. These findings further delineate a cellular mechanism whereby the functional activity of the pancreatic (β-cell, i.e., resting versus actively secreting, may correlate with cell-surface localization of antigens and raises the possibility that other unidentified granule derived antigens also may colocalize at sites of exocytosis on the β-cell membrane.
β2-Microglobulin–Deficient NOD Mice Do Not Develop Insulitis or Diabetes
Authors: Wicker Linda S , Leiter Edward H , Todd John A , Renjilian Robert J , Peterson Erin , Fischer Paul A , Podolin Patricia L , Zijlstra Maarten , Jaenisch Rudolf , Peterson Laurence B
Published: September 2013

The role of CD8+ T-cells in the development of diabetes in the nonobese diabetic (NOD) mouse remains controversial. Although it is widely agreed that class II-restricted CD4+ T-cells are essential for the development of diabetes in the NOD model, some studies have suggested that CD8+ T-cells are not required for β-cell destruction. To assess the contribution of CD8+ T-cells to diabetes, we have developed a class of NOD mouse that lacks expression of βxs2-microglobulin (NOD-B2mnull). NOD-B2mnull mice, which lack both class I expression and CD8+ T-cells in the periphery, not only failed to develop diabetes but were completely devoid of insulitis. These results demonstrate an essential role for CD8+ T-cells in the initiation of the autoimmune response to β-cells in the NOD mouse.
Major Histocompatibility Complex Class I-Deficient NOD-B2mnull Mice are Diabetes and Insulitis Resistant
Authors: Serreze David V , Leiter Edward H , Christianson Gregory J , Greiner Dale , Roopenian Derry C
Published: September 2013

Specific allelic combinations within the class II region of the major histocompatibility complex (MHC) represent a major genetic component for susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM) in humans. We produced and used a stock of NOD/Lt mice congenic for a functionally inactivated β2-microglobulin (B2mnull) locus to assess whether there was an absolute requirement for MHC class I expression and/or CD8+ T-cells in diabetogenesis. These NOD-B2mnull mice do not express cell surface MHC class I molecules or produce detectable levels of CD8+ T-cells and are diabetes and insulitis resistant. Previous results from transgenic mouse models indicated that intracellular accumulation of MHC class I molecules negatively affects pancreatic β-cell function and can result in the development of nonautoimmune insulin-dependent diabetes mellitus (IDDM). MHC class I molecules have been shown to accumulate intracellularly in the presence of a disrupted B2m locus, but this mutation does not negatively affect plasma insulin levels in either NOD/Lt mice or in those of a mixed 129 and C57BL/6 genetic background. Interestingly, 14% of the male mice in this mixed background did develop hyperinsulinemia (> 1,500 pM) independent of the disrupted B2m locus, suggesting that these mice could conceivably develop insulin-resistant diabetes. However, none of these mice became diabetic at up to 22 months of age. Thus, elimination of cell surface MHC class I expression with a disrupted B2m gene blocks autoimmune diabetes in NOD/Lt mice, without engendering a separate, distinct form of glucose intolerance.
Saturated fatty Acid–Induced Insulin Resistance in Rat Adipocytes
Authors: Hunnicutt John W , Hardy Robert W , Williford Jodie , McDonald Jay M
Published: September 2013

Palmitate has been shown to stimulate glucose transport, translocation of GLUT4 and insulin receptor autophosphorylation in isolated rat adipocytes (Biochem Biophys Res Commun 177:343–49, 1991). Here we further characterize the ability of short-term treatment with free fatty acids to stimulate glucose transport in isolated rat adipocytes and demonstrate that prolonged treatment induces insulin resistance. Treatment of adipocytes for 15 min with 1 mM myristate (14:0), palmitate (16:0), or stearate (18:0) stimulates glucose transport by 119 ± 33, 89 ± 29, and 114 ± 30%, respectively. In contrast, oleate (cis 18:1), 1), elaidate (trans 18:1), and linoleate (cis 18:2) do not stimulate glucose transport. Palmitate stimulates glucose transport in a concentration-dependent manner, demonstrating saturation at 1 mM and half-maximal stimulation at 0.25–0.5 mM. Prolonged treatment (4 h) of rat adipocytes with 1 mM palmitate induces insulin resistance. After a 4-h preincubation with palmitate (1 mM), insulin stimulates glucose transport in rat adipocytes by 4.4-fold ± 0.8, vs. 8.8-fold ± 0.8 in controls (n = 3). Palmitate-induced resistant cells demonstrated a 40% inhibition in maximal insulin responsiveness with little change in insulin sensitivity. Insulin binding is only slightly decreased (8%) in palmitate-pretreated cells. These studies indicate that saturated fatty acids stimulate glucose transport acutely and on prolonged exposure induce insulin resistance via a post-insulin binding defect. The underlying molecular mechanisms of insulin resistance induced by prolonged treatment with saturated fatty acids may now be investigated using this unique cellular model.
Skewed T-Cell Receptor Usage and Junctional Heterogeneity Among Isletitis αβ and γδ T-cells in Human IDDM
Authors: Santamaria Pere , Lewis Cinthia , Jessurun Jose , Sutherland David E R , Barbosa Jose J
Published: September 2013

Because of anatomical limitations, molecular characterization of islet-inflammatory T-cells in human insulin-dependent diabetes mellitus (IDDM) has remained elusive. We have isolated isletitis T-cells from pancreas graft biopsies of two patients (syngeneic and allogeneic, respectively) shortly after onset of recurrent IDDM and have characterized their repertoire by sequencing T-cell receptor (TcR)-specific cDNAs. Histopathological analysis of the grafts revealed selective β-cell loss and isletitis characteristic of recurrent disease with no evidence of chronic inflammation or rejection. Most of the in vivo-activated isletitis T-cells were CD8+TcRαβ+ and CD4−CD8−TcRγδ+ in both patients. Comparison of the different TcRα,β,γ, and δ sequences revealed V(D)J junctional heterogeneity but skewed TcR usage within patients. Eighth of 13 different isletitis TcRβ sequences (19 of 26 cDNAs) from the syngeneic graft of patient 1 were Vβ3+, as opposed to only 1 of 31 peripheral TcRβ sequences (1 of 31 cDNAs) (61.5 vs. 3.2%, P < 0.0001). Of the 19 different isletitis TcRα clonotypes of this patient (24 of 42 cDNAs), 5 were Vα14+. The isletitis TcRβ clonotypes of the human leukocyte antigen-identical allogeneic graft of patient 2 showed selective Jβ, but not Vβ, gene usage. Two of three predominant isletitis clonotypes of patient 2 were Vα22+ (19 of 28 cDNAs) and the other (5 of 28 cDNAs) was also Vα14+. As opposed to peripherl γδ T-cell, which usedall Vγ and Vδ gene families, isletitis γδ T-cells only used VγI, Vγ2, Vδ1, and Vδ2 (patient 1) or VγI, Vγ2, Vγ3, and Vδ1 (patient 2) genes. These data are compatible with preferential recruitment of certain CD8+ and CD4−CD8− T-cells to islets on the basis of TcR-V and/or J gene usage, which varies among patients, but favors a response against multiple target major histocompatibility complex/peptide complexes.
Immunoregulatory and Cytokine Imbalances in the Pathogenesis of IDDM: Therapeutic Intervention by Immunostimulation?
Authors: Rabinovitch Alex
Published: September 2013

The autoimmune response that leads to destruction of pancreatic islet β-cells and insulin-dependent diabetes mellitus (IDDM) has a genetic basis; however, environmental factors can exert profound modulating effects on the genetic predisposition to this autoimmune response. Recent studies in animal models for human IDDM, the genetically diabetes-prone NOD mouse and BB rat, have revealed that microbial agents—including certain viruses and extracts of bacteria, fungi, and mycobacteria—often have a protective action against diabetes development. Many of these microbial preparations are immune adjuvants, which are agents that stimulate the immune system. The protective effects of these agents against diabetes appear to involve perturbations in the production of cytokines, which are polypeptides produced by and acting on cells of the immune system. Thus, recent studies in NOD mice suggest that the islet β-cell-directed autoimmune response may be mediated by a T-helper 1 (Th1) subset of T-cells producing the cytokines interleukin-2 (IL-2) and interferon-γ. These studies also suggest that the diabetes-protective effects of administering microbial agents, adjuvants, and a β-cell autoantigen (GAD65 [glutamic acid decarboxylase]) may result from activation of a Th2 subset of T-cells that produce the cytokines IL-4 and IL-10 and consequently downregulate the Th1-cell-mediated autoimmune response. The clinical implication of these findings is that the autoimmune response leading to islet β-cell destruction and IDDM may be amenable to prevention or suppression by therapeutic interventions aimed at stimulating the host's own immunoregulatory mechanisms.
Progressive Decline in Renal Function in Diabetic Patients With and Without Albuminuria
Authors: Tsalamandris Con , Allen Terri J , Gilbert Richard E , Sinha Ashim , Panagiotopoulos Sianna , Cooper Mark E , Jerums George
Published: September 2013

This study describes patterns of progression of albuminuria and renal function in a subgroup of 40 patients from a total cohort of 211 diabetic patients (118 type I, 93 type II) followed over a period of 8–14 years. Forty patients (18 with type I diabetes, 22 with type II diabetes) showed progressive increases in albumin excretion rate (AER) and/or decreases in creatinine clearance (CC) during the study period. Of these, AER alone increased in 15 patients, AER increased and CC decreased in 13 patients, and CC alone decreased in 12 patients, with a similar distribution of type I and type II diabetic patients in each group. Of the 28 patients who showed an increase in albuminuria, AER increased at an annual rate of 30–40%, resulting in a 4- to 8-fold increase in AER to > 20 μg/min during the study. Of the 25 patients who showed a decrease in renal function, CC decreased at an annual rate of 4–5 ml/min, resulting in an approximate halving of CC to < 90 ml/min during the study. The rate of fall in CC was not related to the presence or absence of concomitant increases in albuminuria. However, a significant preponderance of women in the group showed a decline in CC alone. The decline in CC was associated with an increase in plasma creatinine as well as a progressive decrease in urinary creatinine excretion, but the underlying mechanisms remain unexplained. These data support the concept that a subgroup of diabetic patients may show a decline in renal function in the absence of significant increases in AER. Additional functional and structural data will be needed to determine if these patients have diabetic nephropathy. However, this study does suggest that albuminuria alone may not predict renal functional changes in all diabetic patients.
Glycation, Glycoxidation, and Cross-Linking of Collagen by Glucose: Kinetics, Mechanisms, and Inhibition of Late Stages of the Maillard Reaction
Authors: Fu Min-Xin , Wells-Knecht Kevin J , Blackledge James A , Lyons Thorpe J , Thorpe Suzanne R , Baynes John W
Published: September 2013

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products Nε-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.
Increased Catalytic Activity of Glucokinase in Isolated Islets From Hyperinsulinemic Rats
Authors: Chen Chuan , Bumbalo Lisa , Leahy Jack L
Published: September 2013

The high Km glucose phosphorylation enzyme glucokinase is believed to be the α-cell glucose sensor, i.e., the site in glucose metabolism that determines the sensitivity and specificity of glucose-induced insulin secretion. We investigated the regulation of this enzyme by measuring glucokinase Vmax and protein levels in isolated islets from hyperinsulinemic rats. Rats were infused for 48 h with 2 ml/h of 20% glucose, 50% glucose, or 0.45% NaCl (control rats). At the end of the infusion, 20% glucose-infused rats were normoglycemic and hyperinsulinemic (2.3-fold rise in basal plasma insulin level). Their islets had a 2.3-fold increase in insulin secretion at 8.3 mM glucose (51 ± 10% of capacity vs. 22 ± 5% in NaCl rats, P < 0.03), a 75% increase in glucokinase Vmax and little if any increase in glucokinase protein level (111 ± 3% of control). The rats infused with 50% glucose had marked hyperglycemia and higher basal plasma insulin levels. Their islets were maximally stimulated by 8.3 mM glucose in combination with a 270% increase in glucokinase Vmax and a 69 ± 11% increase in glucokinase protein level. Hexokinase Vmax was also doubled. Thus, compensatory increases in β-cell glucose phosphorylation are a key mechanism for adaptive hyperinsulinemia. Our results show two types of regulation for the β-cell high Km phosphorylation enzyme, glucokinase. The content of glucokinase protein is controlled by the plasma glucose level. Variable catalytic activity of this protein was also observed in this study.
Compensation in Pancreatic β-Cell Function in Subjects With Glucokinase Mutations
Authors: Sturis Jeppe , Kurland Irwin J , Byrne Marie M , Mosekilde Erik , Froguel Philippe , Pilkis Simon J , Bell Graeme I , Polonsky Kenneth S
Published: September 2013

The relationship between the in vivo insulin secretory responsiveness of the pancreatic β-cell to glucose and the flux of glucose through the enzyme glucokinase was investigated in six subjects with heterozygous glucokinase mutations and in six matched control subjects. This was done by combining data published previously on the in vivo dose-response relationships between glucose and insulin secretion and on the in vitro enzymatic properties of wild-type and mutant forms of glucokinase. The flux of glucose through glucokinase (GK flux) in these subjects was estimated using a model based on the approximate Michaelis-Menten kinetics of wild-type and mutant forms of the enzyme. In two subjects with glucokinase mutations, which resulted in only a small reduction in enzymatic activity, the decrease in insulin secretion was directly proportional to the decrease in GK flux predicted using a Michaelis-Menten model for both mutant and wild-type glucokinase. However, in four subjects with glucokinase mutations, which resulted in severe reductions in enzymatic activity, insulin secretion was reduced compared with control subjects but less than predicted. This latter result implies the existence of a compensatory change in the β-cells of such subjects, which results in a relative increase in insulin secretory response. We propose modifications to the simple model relating glucose concentration and GK flux, including glucose-induced overexpression of the normal allele and a role of glucokinase regulatory protein. The modifications take into account the possibility that the degree of compensation may be directly related to the severity of the mutation.
Decreased Adrenal Medullary Catecholamine Release in Spontaneously Diabetic BB-Wistar Rats: Role of Hypoglycemia
Authors: Wilke Russell A , Hillard Cecilia J
Published: September 2013

We have demonstrated previously that spontaneously diabetic BB-Wistar rats exhibit decreased adrenal medullary catecholamine secretion in response to splanchnic nerve terminal stimulation. We hypothesized that this abnormality is caused by changes in the sensitivity of the adrenomedullary chromaffin cells to acetylcholine (ACh). To study this hypothesis, we isolated adrenal glands from control and spontaneously diabetic BB-Wistar rats, perfused them with ACh, and measured catecholamine secretion. Adrenal catecholamine release in response to ACh was significantly decreased at 2, 8, and 16 weeks after the onset of diabetes compared with age-matched, nondiabetic control rats. Catecholamine release in response to perfusion with 20 mM K+ was the same in adrenals from diabetic and control rats. The decreased responsiveness of diabetic rat adrenals to perfusion with ACh was significantly correlated with a decrease in the release of catecholamines in response to splanchnic nerve stimulation. A similar defect in catecholamine secretion was also seen in adrenals harvested from nondiabetic BB-Wistar rats following a 3-h period of acute hypoglycemia; however, the adrenal response to potassium was also decreased as was the catecholamine content of the adrenal. Conversely, nondiabetic BB-Wistar rats made diabetic with streptozocin (STZ) and maintained in a hyperglycemic state did not exhibit catecholamine hyposecretion 2 weeks after STZ administration. Collectively, the data describe decreased adrenomedullary response to cholinergic stimulation in spontaneously diabetic rats as early as 2 weeks after the onset of diabetes and that a similar, although more severe, hyposecretion occurs after acute, severe hypoglycemia.
cAMP-Independent Synergistic Effects of Insulin and Dexamethasone on Fructose 2,6-Bisphosphate Metabolism in H4IIE Cells
Authors: Vargas Alberto M , Sola Maria M , Lange Alex J , Poveda Gracia , Pilkis Simon J
Published: September 2013

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased liver 6PF-2-K/Fru-2,6-P2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.
Increased Skeletal Muscle Capillary Density Precedes Diabetes Development in Men With Impaired Glucose Tolerance: A 15-Year Follow-Up
Authors: Eriksson Karl-F , Saltin Bengt , Lindgärde Folke
Published: September 2013

Gastrocnemius muscle morphology, metabolic potential, and capillarization were analyzed in 48-year-old men with regard to subsequent development of non-insulin-dependent diabetes mellitus (NIDDM) in 29 subjects with impaired glucose tolerance (IGT) and in 38 control subjects. Over a 15-year period, although participating in an intervention program, 13 of the IGT subjects developed diabetes, but none of the control subjects developed diabetes. In view of their poor aerobic capacity, lack of physical fitness, and reduced glycolytic and oxidative enzymes, these 13 subjects manifested an unexpectedly high number of capillaries around all types of muscle fibers, especially type IIb fibers, as predictors of their progression to diabetes. Moreover, the number of capillaries per muscle fiber and the 2-h insulin value in the oral glucose tolerance test were highly correlated (r = 0.82, P < 0.005), whereas no correlation was found among IGT subjects who remained nondiabetic and in the control group. With body mass index and the 2-h glucose concentration included in a regression model, 68% of the variation in the number of capillaries per muscle fiber was explained (P < 0.05), with the 2-h insulin value independently accounting for 33%. These findings may suggest that the increased circulating insulin concentrations in IGT subjects have a capillary proliferative effect, perhaps to compensate for reduced capillary insulin diffusion and metabolic capacity of the muscle.
More Direct Evidence for a Malonyl-CoA–Carnitine Palmitoyltransferase I Interaction as a Key Event in Pancreatic β-Cell Signaling
Authors: Chen Songyvan , Ogawa Atsushi , Ohneda Makoto , Unger Roger H , Foster Daniel W , McGarry J Denis
Published: September 2013

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802–5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose → malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic β-cell. They also raise the possibility that the tested CPT I inhibitors act, in part, by generating non-metabolizable acyl-CoA species that mimic the effects of natural acyl-CoAs in triggering insulin release.
Acute Antihyperglycemic Mechanisms of Metformin in NIDDM: Evidence for Suppression of Lipid Oxidation and Hepatic Glucose Production
Authors: Perriello Gabriele , Misericordia Paolo , Volpi Elena , Santucci Antonella , Santucci Carala , Ferrannini Eleuterio , Ventura Mariarosa M , Santeusanio Fausto , Brunetti Paolo , Bolli Geremia B
Published: September 2013

To establish the antihyperglycemic mechanisms of metformin in non-insulin-dependent diabetes mellitus (NIDDM) independently of the long-term, aspecific effects of removal of glucotoxicity, 21 NIDDM subjects (14 obese, 7 nonobese) were studied on two separate occasions, with an isoglycemic (plasma glucose ∼9 mM) hyperinsulinemic (two-step insulin infusion, 2 h each, at the rate of 4 and 40 mU · m−2 · min−1) clamp combined with [3−3H]glucose infusion and indirect calorimetry, after administration of either metformin (500 mg per os, at –5 and –1 h before the clamp) or placebo. Compared with placebo, hepatic glucose production (HGP) decreased ∼30% more after metformin (from 469 ± 50 to 330 ± 54 μmol/min), but glucose uptake did not increase. Metformin suppressed free fatty acids (FFAs) by ∼17% (from 0.42 ± 0.04 to 0.35 ± 0.04 mM) and lipid oxidation by ∼25% (from 4.5 ± 0.4 to 3.4 ± 0.4 μmol · kg−1 · min−1) and increased glucose oxidation by ∼ 16% (from 16.2 ± 1.4 to 19.3 ± 1.3 μmol.kg−1 · min−1) compared with placebo (P < 0.05), but did not affect nonoxidative glucose metabolism, protein oxidation, or total energy expenditure. Suppression of FFA and lipid oxidation after metformin correlated with suppression of HGP (r = 0.70 and r = 0.51, P < 0.001). The effects of metformin in obese and nonobese subjects were no different. We conclude that the specific, antihyperglycemic effects of metformin in the clinical condition of hyperglycemia in NIDDM are primarily due to suppression of HGP, not stimulation of glucose uptake, and are mediated, at least in part, by suppression of FFA and lipid oxidation.
Hepatocyte Growth Factor/Scatter Factor Has Insulinotropic Activity in Human Fetal Pancreatic Cells
Authors: Otonkoski Timo , Beattie Gillian M , Rubin Jeffrey S , Lopez Ana D , Baird Andrew , Hayek Alberto
Published: September 2013

Fetal mesenchyme-derived factors are likely to play an important role in pancreatic islet development and growth. We have used primary cultures of human fetal pancreatic tissue to identify growth factors that have morphogenic, mitogenic, and insulinotropic activity. The formation of islet-like cell clusters (ICCs) during a 6-day culture was stimulated two- to threefold by hepatocyte growth factor/scatter factor (HGF/SF) basic fibroblast growth factor (FGF)-2, and to a lesser extent by keratinocyte growth factor (FGF-7) and insulin-like growth factor-II (IGF-II). In contrast, transforming growth factor-β (TGF-β) had a strong inhibitory effect. The ICCs formed during HGF/SF stimulation consisted mainly of epithelial cells, whereas FGF-2-induced ICCs were predominantly nonepithelial. Furthermore, although both FGF-2 and HGF/SF increased the total insulin content of the cultures, only HGF/SF increased the insulin content per DNA. Quantitatively, HGF/SF stimulated a 2.3-fold increase in the proportion of insulin-positive cells and a 3-fold higher number of replicating β-cells. Blocking of the IGF-I receptor inhibited ICC formation but did not affect their insulin content. Immunoneutralizing TGF-β resulted in increased cell growth and insulin content, indicating the presence of an endogenous inhibitory TGF-β activity in the model system. Our results suggest that HGF/SF may be an important component of the fetal mesenchyme-derived factors responsible for pancreatic islet development. HGF/SF also may prove valuable for supporting the in vitro growth of islet cells.
High Level of Concordance Between Assays for Glutamic Acid Decarboxylase Antibodies: The First International Glutamic Acid Decarboxylase Antibody Workshop
Authors: Schmidli Robert S , Colman Peter G , Bonifacio Ezio , Franco Bottazzo Gian , Harrison Leonard C
Published: September 2013

Glutamic acid decarboxylase antibodies (GADAbs) are being increasingly used in clinical and research programs for the prediction and classification of insulin-dependent diabetes mellitus (IDDM). A number of different assay formats for the measurement of GADAbs have been reported, but the degree of concordance between assays is unknown. In this study, GADAbs were measured on 16 coded sera in 34 assays to examine concordance between GADAb assays and establish the feasibility of an international GADAb standard of measurement unit. The 16 lyophilized coded samples consisted of sera from healthy control subjects (n = 2), IDDM patients (n = 3), a patient with polyendocrine autoimmunity (n = 1), and duplicate dilutions of plasmapheresis serum from a patient with stiff-man syndrome (SMS). A high level of concordance was found in the ranking of GADAb levels (P = 0.99, Friedman's test) in the samples. Thirteen (38%) assays could reproducibly distinguish dilutions of SMS serum and detect GADAbs in all IDDM and polyendocrine autoimmunity sera tested. Although assessed on only four samples, disease specificity was 100% in 29 assays. The majority of assays that immunoprecipitated radiolabeled GAD gave high results for sensitivity and specificity. Enzyme-linked immunosorbent assays and assays using immunofluorescence were generally less sensitive. Several assays, in particular those measuring GAD enzymatic activity immunoprecipitated in fluid phase from rat brain homogenate, showed a prozone-like phenomenon in the SMS dilution curve. Interpolation of results from a standard curve into workshop units resulted in relatively low scatter in samples with lower levels of GADAbs. Hence, the use of an international reference serum to enable comparison of results between laboratories appears feasible. A second serum exchange is currently in progress to explore differences in sensitivity and specificity using larger numbers of disease and control sera.
High Glucose Attenuates Peptide Agonist-Evoked Increases in Cytosolic Free [Ca2+] in Rat Aortic Smooth Muscle Cells
Authors: Sandirasegarane Lakshman , Herman Robert J , Gopalakrishnan Venkat
Published: September 2013

Incubation of cultured rat aortic smooth muscle cells (ASMCs) in a medium containing high glucose concentrations (25 mM) did not affect the basal cytosolic free calcium ([Ca2+]i) but led to significant reductions in peak [Ca2+]i response evoked by arginine vasopressin, angiotensin II, and endothelin-1 (ET-1). This was observed in both the presence and absence of extracellular Ca2+. Maintenance of rat ASMCs in a medium containing mannose (an osmotic control for high glucose) did not affect either the basal or peptide agonist-evoked increase in [Ca2+]i. However, pretreatment with either the nonselective protein kinase C (PKC) inhibitor staurosporine or the selective PKC inhibitor 2,6-diamino-N-([1-(1-oxotridecyl)-2 piperidinyl] methyl) hexanamide reversed the attenuating effect of high glucose on peak [Ca2+]i response evoked by ET-1. Also, short-term incubation of ASMCs with the active phorbol ester, phorbol 12-myristate 13-acetate, led to a reduction in peak [Ca2+]i response to all three agonists, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate, which does not activate PKC, had no such effect. Although high-glucose treatment of rat ASMCs led to significant reductions in the maximal number of binding sites to the extent of 39% of [125I]ET-1 specific binding, no significant differences in the affinity (Kd ∼110 pM) characteristics were evident between control and high-glucose treatment groups. It is proposed that incubation of rat ASMCs with high glucose enhances the de novo synthesis of diacylglycerol and activates membrane-bound PKC and that this, in turn, impairs agonist-mediated intracellular Ca2+ mobilization.
High Glucose Concentration Causes a Decrease in Mesangium Degradation: A Factor in the Pathogenesis of Diabetic Nephropathy
Authors: McLennan Susan V , Fisher Elizabeth J , Yue Dennis K , Turtle John R
Published: September 2013

Mesangium enlargement is a constant feature of diabetic nephropathy and is likely to be important in the pathogenesis of this diabetic complication. Whether decreased degradation of mesangium plays any role in causing the enlargement is uncertain. We developed a system of preparing radioactively labeled mesangium matrix from mesangial cell cultures to be used as substrates for studies of mesangium degradation. Degradation is commenced by growing mesangial cells on the labeled matrix and monitored by the release of radioactivity into the culture medium. High glucose concentration (30 mM), whether present 1) when the matrix is being made or 2) when the degradation is taking place, reduces the rate of mesangium degradation. The second but not the first of these two phenomena was abolished by aminoguanidine. Phorbol 12-myristate 13-acetate, added in a manner to antagonize the action of protein kinase C, inhibited mesangium degradation and was not able to nullify the effect of high glucose. Thus it appears unlikely that a high glucose concentration inhibits mesangium degradation by increasing mesangial cell protein kinase C activity. We conclude that decreased degradation of mesangium as a result of hyperglycemia may play a role in causing the mesangium enlargement that occurs in diabetic nephropathy.
The Role of Liver Glucosensors in the Integrated Sympathetic Response Induced by Deep Hypoglycemia in Dogs
Authors: Hamilton-Wessler Marianthe , Bergman Richard N , Halter Jeffrey B , Watanabe Richard M , Donovan Casey M
Published: September 2013

The significance of the portohepatic glucosensors for counterregulation in deep hypoglycemia (i.e., glycemia < 2.8 mM) was studied in chronically cannulated male mongrel dogs in the conscious state. A total of 16 experiments were carried out on 6 dogs using the liver clamp technique under hyperinsulinemic conditions (insulin infusion, 39 pmol · min−1 · kg−1, 0–150 min). The level of glycemia presented to the liver was made to differ from the systemic arterial glucose level via portal glucose infusion. Tracer-determined rates of glucose clearance and hepatic glucose output (HGO) were assessed using D-[3-3H]glucose (0.26 μCi · min−1). Three protocols were used. In protocol I, liver clamp, systemic hypoglycemia at 2.60 ± 0.09 mM, and liver glycemia at 3.86 ± 0.05 mM were achieved with portal glucose infusion (28.2 ± 3.0 μmol · min−1 · kg−1). For protocol II, glucose was infused peripherally (18.2 ± 4.3 μmol · min−1 · kg−1), while systemic and liver glycemia were sustained at deep hypoglycemia, 2.50 ± 0.08 mM. In protocol III, via peripheral glucose infusion (62.9 ± 5.8 μmol · min−1 · kg−1), systemic and liver glycemia were maintained at a level matched to the liver glycemia during protocol I (3.98 ± 0.05 mM, P > 0.10). When compared with protocols I and III, the catecholamine response above basal was significantly greater during protocol II with liver and systemic deep hypoglycemia (7.30 ± 1.51 and 2.89 ± 0.5 nM for epinephrine and norepinephrine, respectively, P < 0.005). These values reflect net increases in the catecholamine responses of 100% and 85% for epinephrine and norepinephrine when compared with protocol I. Glucose clearance was similar among protocols (13.03 ± 1.26 ml · min−1 · kg−1 P > 0.10). HGO was essentially unchanged from basal during protocol II but suppressed during protocols I and III (P < 0.05). These results are consistent with the hypothesis that portohepatic glucosensors play a significant role in eliciting the sympathoadrenal response to deep hypoglycemia.
Intracellular Mechanisms Involved in D-Glucose–Mediated Amplification of Agonist-Induced Ca2+ Response and EDRF Formation in Vascular Endothelial Cells
Authors: Wascher Thomas C , Toplak Hermann , Krejs Güunter J , Simecek Sabine , Kukovetz Walter R , Graier Wolfgang F
Published: September 2013

Prolonged treatment of vascular endothelial cells with pathologically high D-glucose amplifies autacoid-induced Ca2+ mobilization and thus formation of nitric oxide. This study investigated the Ca2+ source for the change in endothelial Ca2+ response on agonist stimulation. Pretreatment with high D-glucose (44 vs. 5 mM) enhanced release of intracellular Ca2+ by bradykinin as a result of a 2.0-fold increased formation of inositol 1,4,5-trisphosphate. High D-glucose also amplified Ca2+ influx (2.0-fold). In high D-glucose preincubated cells, stimulation with bradykinin significantly increased transplasmalemmal 45Ca2+ flux (3.2-fold) and caused a 2.0-fold increase in permeability to Mn2+, a surrogate for endothelial plasma membrane Ca2+ channels. A significant 2.0-fold increase occurred in the maximal slope, suggesting a higher rate of Mn2+ (Ca2+) influx. Ca2+ influx, stimulated by an inositol phosphate-independent depletion of intracellular Ca2+ stores with 2,5-di-(tert-butyl)-hydroquinone was also significantly increased 2.4-fold by high D-glucose, with no effect on intracellular Ca2+ release. D-glucose failed to modulate resting or stimulated cAMP levels. We suggest that prolonged exposure to pathologically high D-glucose increases formation of inositol polyphosphates, thus increasing Ca2+ release. Ca2+ entry is increased by amplification of unknown signal transduction mechanisms triggered by Ca2+ store depletion.
Dexamethasone-Induced Hyperglycemia in Obese Avy/a (Viable Yellow) Female Mice Entails Preferential Induction of a Hepatic Estrogen Sulfotransferase
Authors: Gill Anne M , Leiter Edward H , Powell Jerry G , Chapman Harry D , Yen Terence T
Published: September 2013

Sex steroid sulfotransferases (ST) sulfurylate and thus inactivate estrogens or androgens, producing an androgenized or estrogenized state in the liver. The expression of diabetes in a number of animal models is sexually dimorphic and has been associated with steroidal states. Although the viable yellow (Avy) mutation produces an insulin-resistant obesity syndrome in mice of both sexes, only males develop chronic hyperglycemia. Hyperglycemia was rapidly induced in Avy/a females by dexamethasone (dex). This treatment completely suppressed both endogenous plasma corticosterone and hepatic corticosterone-binding globulin (CBG) mRNA within 24 h. Hyperglycemia in dex-implanted Avy/a females was accompanied by aberrant shifts in hepatic androgen/estrogen balance. This was effected by induction of estrogen sulfotransferase (EST) mRNA together with a > 10-fold increase in enzymatic activity. Similar dex-induced increases in androgen ST or phenol ST were not observed. Prior implantation of estrogen prevented development of hyperglycemia. The time-dependent spontaneous reversal of dex-induced hyperglycemia correlated with re-expression of CBG mRNA transcripts and reduced levels of EST transcripts and enzyme activity. Although dex-induced hyperglycemia was limited to Avy/a females, dex elicited hyperinsulinemia in lean α/α control mice of both sexes and exacerbated constitutive hyperinsulinemia in Avy/a males and females. In summary, dex-induced hyperglycemia in Avy/a females was associated with increased catabolism of hepatic estrogens mediated by induction of EST.
Toxicity of Mildly Modified Low-Density Lipoproteins to Cultured Retinal Capillary Endothelial Cells and Pericytes
Authors: Lyons Timothy J , Li Wei , Wells-Knecht Mary C , Jokl Rudolf
Published: September 2013

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37°C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P < 0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal < glycated ≤ minimally oxidized < glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.