Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metast... more Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade ...
Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue e... more Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photo-encapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for co-encapsulation of DNA and bone cells and examined whether co-encapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), co-encapsulated with bone tumor cells, was capable of transfecting the cells and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the co-encapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen-treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.
2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytoto... more 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2-ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2-ME on cultured osteoclasts. 2-ME (2 microM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co-cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M-CSF)). The 2-ME-mediated effect was ligand specific; 2-hydroxyestradiol (2-OHE), the immediate precursor to 2-ME, exhibited less cytotoxicity; and 2-methoxyestrone (2-MEOE1) the estrone analog of 2-ME, was not cytotoxic. Co-treatment with ICI 182,780 did not antagonize 2-ME, suggesting that the cytotoxicity was not estrogen receptor-dependent. 2-ME-induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2-ME-mediated decrease in cell survival was partially inhibited by anti-lymphotoxin(LT)beta antibodies, suggesting that 2-ME-dependent effects involve LTbeta. These results suggest that 2-ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone.
Severe hypoglycaemia with brain dysfunction limits intensified therapy in patients with insulin-d... more Severe hypoglycaemia with brain dysfunction limits intensified therapy in patients with insulin-dependent diabetes mellitus, despite evidence that such therapy reduces the risk of chronic complications of the disease. We have investigated the effect of infusing lactate (a potential non-glucose fuel for brain metabolism) on protective, symptomatic neurohumoral responses and on brain function during hypoglycaemia in seven healthy men. Elevation of lactate (within a physiological range) substantially diminished catecholamines, growth hormone, cortisol, and symptomatic responses to hypoglycaemia and lowered the glucose level at which these responses began. Glucagon responses were unaffected. Lactate was also associated with a significant lowering of the glucose level at which brain function deteriorated, suggesting that brain function was protected during the hypoglycaemia. The defect in counter-regulation is similar to that seen in hypoglycaemia-prone diabetic patients. Initiation of the protective responses to hypoglycaemia (except glucagon) can be delayed by supporting metabolism with an alternative metabolic fuel. Cerebral cortical dysfunction of severe hypoglycaemia is also delayed. Our demonstration that higher brain function can be protected during hypoglycaemia may have therapeutic potential.
Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol ... more Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor beta1 (TGF-beta1) mRNA levels and decreases in TGF-beta2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal con... more Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal control to normalize gene expression data based on the belief that this gene is constitutively expressed. However, GAPDH mRNA levels increased by more than 2.5-fold in tibiae of hindlimb unloaded female rats compared to L32 mRNA levels. Similarly, GAPDH mRNA levels increased compared to 18S ribosomal RNA. Treatment with growth hormone and alcohol show no disparity in GAPDH mRNA levels whereas in some experiments, parathyroid hormone and 17beta-estradiol increased GAPDH mRNA levels. Taken together, these findings indicate that it is essential to demonstrate that GAPDH expression is not altered prior to using the gene as an internal control.
... Dr Mabin illustrates, however, that this policy can be difficult to implement: should we real... more ... Dr Mabin illustrates, however, that this policy can be difficult to implement: should we really cancel an advertisement for an anticoagulant because an article on recent advances in orthopaedics ... (18 February.) 3 Maran A, Lomas J, Archibald HR, Macdonald I, Gale E, Amiel S ...
To investigate the possibility of restoring awareness; symptomatic, cognitive, and hormonal respo... more To investigate the possibility of restoring awareness; symptomatic, cognitive, and hormonal responses to controlled hypoglycaemia were studied in insulin-dependent diabetic patients with long disease duration (6 with good glycaemic control and 6 with poor control) before and after ...
A clinically important task in diabetes management is the prevention of hypo/hyperglycemic events... more A clinically important task in diabetes management is the prevention of hypo/hyperglycemic events. The availability of continuous glucose monitoring (CGM) devices allow to develop new strategies, but new problems have also emerged. In this contribution, we discuss three major challenges which, in practical real time CGM applications, should be dealt with: filtering to enhance the signal-to-noise ratio, ahead-of-time prediction of glucose concentration, and generation of hypo/hyper-alerts. For all these challenges, some techniques, with a different degree of sophistication, have been proposed recently in the literature, but several issues remain open.
OBJECTIVE—To evaluate the accuracy of a new subcutaneous glucose sensor (Glucoday; A. Menarini Di... more OBJECTIVE—To evaluate the accuracy of a new subcutaneous glucose sensor (Glucoday; A. Menarini Diagnostics) compared with venous blood glucose measurement in type 1 and type 2 diabetic patients. RESEARCH DESIGN—A multicenter study was performed in 70 diabetic patients. A microdialysis fiber was inserted subcutaneously into the periumbelical region and perfused with a buffer solution. Glucose concentrations in the dialysate were then measured every 3 min by the glucose sensor over a 24-h period, during which nine venous blood samples were also collected throughout the day. RESULTS—Both the insertion of the fiber and the wearing of the device were well tolerated by the patients. Subcutaneous glucose levels were well correlated with venous glucose measurements (r = 0.9, P < 0.001) over a wide range (40–400 mg/dl) for up to 24 h, with a single-point calibration. An analysis of 381 data pairs showed a linear relationship between the GlucoDay and serial venous blood glucose levels, and 97% of the data fell in the A and B regions of the error grid analysis. Percentage bias between the GlucoDay and the blood venous levels was −2.0% in the hypoglycemic range (<70 mg/dl), 6.9% in the euglycemic range (70–180 mg/dl), and 11.2% in the hyperglycemic range (>180 mg/dl). CONCLUSIONS—The GlucoDay system demonstrated high reliability and reported values that closely agreed with venous blood glucose measurements. The system was well tolerated and thus constitutes a relatively easy method to monitor glucose excursions in diabetic patients.
Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2&#39... more Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2',5'-linked oligoadenylates, known as 2-5A, is one pathway of interferon action. Unaided uptake into HeLa cells of 2-5A linked to an antisense oligonucleotide resulted in the selective ablation of messenger RNA for the double-stranded RNA (dsRNA)-dependent protein kinase PKR. Similarly, purified, recombinant human 2-5A-dependent ribonuclease was induced to selectively cleave PKR messenger RNA. Cells depleted of PKR activity were unresponsive to activation of nuclear factor-kappa B (NF-kappa B) by the dsRNA poly(I):poly(C), which provides direct evidence that PKR is a transducer for the dsRNA signaling of NF-kappa B.
Aims The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily inje... more Aims The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) with glargine is uncertain. In this randomized cross-over study, we compared CSII and MDI with glargine in patients with Type 1 diabetes well controlled with CSII. The primary end-point was glucose variability.Methods Thirty-nine patients [38.1 ± 9.3 years old (mean ± sd), diabetes duration 16.6 ± 8.2 years, glycated haemoglobin (HbA1c) 7.6 ± 0.8%], already on CSII for at least 6 months, were randomly assigned to CSII with lispro or MDI with lispro and glargine. After 4 months they were switched to the alternative treatment. During the last month of each treatment blood glucose variability was analysed using glucose standard deviation, mean amplitude of glycaemic excursions (MAGE), lability index and average daily risk range (ADRR). As secondary end-points we analysed blood glucose profile, HbA1c, number of episodes of hypo- and hyperglycaemia, lipid profile, free fatty aci...
Exercise is a cornerstone of diabetes therapy in type 1 diabetes mellitus (DMT1) patients. The ty... more Exercise is a cornerstone of diabetes therapy in type 1 diabetes mellitus (DMT1) patients. The type of exercise is important in determining the propensity to hypoglycemia. We assessed, by continuous glucose monitoring (CGM), the glucose profiles during and in the following 20h after a session of two different types of exercise. Eight male volunteers with well-controlled DMT1 were studied. They underwent 30min of both intermittent high-intensity exercise (IHE) and moderate-intensity exercise (MOD) in random order. Expired air was recorded during exercise, while metabolic and hormonal determinations were performed before and for 120 min after exercises. The CGM system and activity monitor were applied for the subsequent 20h. Blood glucose level declined during both type of exercise. At 150 min following the start of exercise, plasma glucose content was slightly higher after IHE. No changes were observed in plasma insulin concentration. A significant increase of norepinephrine concentration was noticed during IHE. Between midnight and 6:00 a.m. the glucose levels were significantly lower after IHE than those observed after MOD (area under the curve, 23.3 ± 3 vs. 16 ± 3 mg/dL/420 min [P = 0.04]; mean glycemia at 3 a.m., 225 ± 31 vs. 147 ± 17 mg/dL [P<0.05]). The number of hypoglycemic episodes after IHE was higher than that observed after MOD (seven vs. two [P<0.05]). We demonstrate that (1) CGM is a useful approach in DMT1 patients who undergo an exercise program and (2) IHE is associated with delayed nocturnal hypoglycemia.
Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metast... more Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade ...
Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue e... more Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photo-encapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for co-encapsulation of DNA and bone cells and examined whether co-encapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), co-encapsulated with bone tumor cells, was capable of transfecting the cells and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the co-encapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen-treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.
2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytoto... more 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2-ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2-ME on cultured osteoclasts. 2-ME (2 microM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co-cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M-CSF)). The 2-ME-mediated effect was ligand specific; 2-hydroxyestradiol (2-OHE), the immediate precursor to 2-ME, exhibited less cytotoxicity; and 2-methoxyestrone (2-MEOE1) the estrone analog of 2-ME, was not cytotoxic. Co-treatment with ICI 182,780 did not antagonize 2-ME, suggesting that the cytotoxicity was not estrogen receptor-dependent. 2-ME-induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2-ME-mediated decrease in cell survival was partially inhibited by anti-lymphotoxin(LT)beta antibodies, suggesting that 2-ME-dependent effects involve LTbeta. These results suggest that 2-ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone.
Severe hypoglycaemia with brain dysfunction limits intensified therapy in patients with insulin-d... more Severe hypoglycaemia with brain dysfunction limits intensified therapy in patients with insulin-dependent diabetes mellitus, despite evidence that such therapy reduces the risk of chronic complications of the disease. We have investigated the effect of infusing lactate (a potential non-glucose fuel for brain metabolism) on protective, symptomatic neurohumoral responses and on brain function during hypoglycaemia in seven healthy men. Elevation of lactate (within a physiological range) substantially diminished catecholamines, growth hormone, cortisol, and symptomatic responses to hypoglycaemia and lowered the glucose level at which these responses began. Glucagon responses were unaffected. Lactate was also associated with a significant lowering of the glucose level at which brain function deteriorated, suggesting that brain function was protected during the hypoglycaemia. The defect in counter-regulation is similar to that seen in hypoglycaemia-prone diabetic patients. Initiation of the protective responses to hypoglycaemia (except glucagon) can be delayed by supporting metabolism with an alternative metabolic fuel. Cerebral cortical dysfunction of severe hypoglycaemia is also delayed. Our demonstration that higher brain function can be protected during hypoglycaemia may have therapeutic potential.
Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol ... more Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor beta1 (TGF-beta1) mRNA levels and decreases in TGF-beta2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal con... more Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels are commonly used as an internal control to normalize gene expression data based on the belief that this gene is constitutively expressed. However, GAPDH mRNA levels increased by more than 2.5-fold in tibiae of hindlimb unloaded female rats compared to L32 mRNA levels. Similarly, GAPDH mRNA levels increased compared to 18S ribosomal RNA. Treatment with growth hormone and alcohol show no disparity in GAPDH mRNA levels whereas in some experiments, parathyroid hormone and 17beta-estradiol increased GAPDH mRNA levels. Taken together, these findings indicate that it is essential to demonstrate that GAPDH expression is not altered prior to using the gene as an internal control.
... Dr Mabin illustrates, however, that this policy can be difficult to implement: should we real... more ... Dr Mabin illustrates, however, that this policy can be difficult to implement: should we really cancel an advertisement for an anticoagulant because an article on recent advances in orthopaedics ... (18 February.) 3 Maran A, Lomas J, Archibald HR, Macdonald I, Gale E, Amiel S ...
To investigate the possibility of restoring awareness; symptomatic, cognitive, and hormonal respo... more To investigate the possibility of restoring awareness; symptomatic, cognitive, and hormonal responses to controlled hypoglycaemia were studied in insulin-dependent diabetic patients with long disease duration (6 with good glycaemic control and 6 with poor control) before and after ...
A clinically important task in diabetes management is the prevention of hypo/hyperglycemic events... more A clinically important task in diabetes management is the prevention of hypo/hyperglycemic events. The availability of continuous glucose monitoring (CGM) devices allow to develop new strategies, but new problems have also emerged. In this contribution, we discuss three major challenges which, in practical real time CGM applications, should be dealt with: filtering to enhance the signal-to-noise ratio, ahead-of-time prediction of glucose concentration, and generation of hypo/hyper-alerts. For all these challenges, some techniques, with a different degree of sophistication, have been proposed recently in the literature, but several issues remain open.
OBJECTIVE—To evaluate the accuracy of a new subcutaneous glucose sensor (Glucoday; A. Menarini Di... more OBJECTIVE—To evaluate the accuracy of a new subcutaneous glucose sensor (Glucoday; A. Menarini Diagnostics) compared with venous blood glucose measurement in type 1 and type 2 diabetic patients. RESEARCH DESIGN—A multicenter study was performed in 70 diabetic patients. A microdialysis fiber was inserted subcutaneously into the periumbelical region and perfused with a buffer solution. Glucose concentrations in the dialysate were then measured every 3 min by the glucose sensor over a 24-h period, during which nine venous blood samples were also collected throughout the day. RESULTS—Both the insertion of the fiber and the wearing of the device were well tolerated by the patients. Subcutaneous glucose levels were well correlated with venous glucose measurements (r = 0.9, P < 0.001) over a wide range (40–400 mg/dl) for up to 24 h, with a single-point calibration. An analysis of 381 data pairs showed a linear relationship between the GlucoDay and serial venous blood glucose levels, and 97% of the data fell in the A and B regions of the error grid analysis. Percentage bias between the GlucoDay and the blood venous levels was −2.0% in the hypoglycemic range (<70 mg/dl), 6.9% in the euglycemic range (70–180 mg/dl), and 11.2% in the hyperglycemic range (>180 mg/dl). CONCLUSIONS—The GlucoDay system demonstrated high reliability and reported values that closely agreed with venous blood glucose measurements. The system was well tolerated and thus constitutes a relatively easy method to monitor glucose excursions in diabetic patients.
Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2&#39... more Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2',5'-linked oligoadenylates, known as 2-5A, is one pathway of interferon action. Unaided uptake into HeLa cells of 2-5A linked to an antisense oligonucleotide resulted in the selective ablation of messenger RNA for the double-stranded RNA (dsRNA)-dependent protein kinase PKR. Similarly, purified, recombinant human 2-5A-dependent ribonuclease was induced to selectively cleave PKR messenger RNA. Cells depleted of PKR activity were unresponsive to activation of nuclear factor-kappa B (NF-kappa B) by the dsRNA poly(I):poly(C), which provides direct evidence that PKR is a transducer for the dsRNA signaling of NF-kappa B.
Aims The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily inje... more Aims The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) with glargine is uncertain. In this randomized cross-over study, we compared CSII and MDI with glargine in patients with Type 1 diabetes well controlled with CSII. The primary end-point was glucose variability.Methods Thirty-nine patients [38.1 ± 9.3 years old (mean ± sd), diabetes duration 16.6 ± 8.2 years, glycated haemoglobin (HbA1c) 7.6 ± 0.8%], already on CSII for at least 6 months, were randomly assigned to CSII with lispro or MDI with lispro and glargine. After 4 months they were switched to the alternative treatment. During the last month of each treatment blood glucose variability was analysed using glucose standard deviation, mean amplitude of glycaemic excursions (MAGE), lability index and average daily risk range (ADRR). As secondary end-points we analysed blood glucose profile, HbA1c, number of episodes of hypo- and hyperglycaemia, lipid profile, free fatty aci...
Exercise is a cornerstone of diabetes therapy in type 1 diabetes mellitus (DMT1) patients. The ty... more Exercise is a cornerstone of diabetes therapy in type 1 diabetes mellitus (DMT1) patients. The type of exercise is important in determining the propensity to hypoglycemia. We assessed, by continuous glucose monitoring (CGM), the glucose profiles during and in the following 20h after a session of two different types of exercise. Eight male volunteers with well-controlled DMT1 were studied. They underwent 30min of both intermittent high-intensity exercise (IHE) and moderate-intensity exercise (MOD) in random order. Expired air was recorded during exercise, while metabolic and hormonal determinations were performed before and for 120 min after exercises. The CGM system and activity monitor were applied for the subsequent 20h. Blood glucose level declined during both type of exercise. At 150 min following the start of exercise, plasma glucose content was slightly higher after IHE. No changes were observed in plasma insulin concentration. A significant increase of norepinephrine concentration was noticed during IHE. Between midnight and 6:00 a.m. the glucose levels were significantly lower after IHE than those observed after MOD (area under the curve, 23.3 ± 3 vs. 16 ± 3 mg/dL/420 min [P = 0.04]; mean glycemia at 3 a.m., 225 ± 31 vs. 147 ± 17 mg/dL [P<0.05]). The number of hypoglycemic episodes after IHE was higher than that observed after MOD (seven vs. two [P<0.05]). We demonstrate that (1) CGM is a useful approach in DMT1 patients who undergo an exercise program and (2) IHE is associated with delayed nocturnal hypoglycemia.
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