Persistently high glucose levels in young children with type 1 diabetes.

OBJECTIVES: The aim of the study was to characterize glucose levels and variability in young children with type 1 diabetes (T1D). METHODS: A total of 144 children of 4-10 yr old diagnosed with T1D prior to age 8 were recruited at five DirecNet centers. Participants used a continuous glucose monitor (CGM) every 3 months during an 18-month study. Among the 144 participants, 135 (mean age 7.0 yr, 47% female) had a minimum of 48 h of CGM data at more than five of seven visits and were included in analyses. CGM metrics for different times of day were analyzed. RESULTS: Mean hemoglobin A1c (HbA1c) at the beginning and end of the study was 7.9% (63 mmol/mol). Fifty percent of participants had glucose levels >180 mg/dL (10.0 mmol/L) for >12 h/d and >250 mg/dL (13.9 mmol/L) for >6 h/d. Median time <70 mg/dL (3.9 mmol/L) was 66 min/d and <60 mg/dL (3.3 mmol/L) was 39 min/d. Mean amplitude of glycemic excursions (MAGE) was lowest overnight (00:00-06:00 hours). The percent of CGM values 71-180 mg/dL (3.9-10.0 mmol/L) and the overall mean glucose correlated with HbA1c at all visits. There were no differences in CGM mean glucose or coefficient of variation between the age groups of 4 and <6, 6 and <8, and 8 and <10. CONCLUSIONS: Suboptimal glycemic control is common in young children with T1D as reflected by glucose levels in the hyperglycemic range for much of the day. New approaches to reduce postprandial glycemic excursions and increase time in the normal range for glucose in young children with T1D are critically needed. Glycemic targets in this age range should be revisited.

Dopamine during alpha- or beta-adrenergic blockade in man. Hormonal, metabolic, and cardiovascular effects.

We studied the contribution of alpha- and beta-adrenergic receptor activation to the cardiovascular, metabolic, and hormonal effects of dopamine. At a concentration of 1.5 mug/kg.min, the infusion of dopamine in 12 normal volunteers was associated with a transient but significant rise in pulse rate, which was prevented by propranolol. Venous plasma glucose did not change throughout the experiments, and a mild increase in plasma free fatty acid levels observed during the administration of dopamine alone was antagonized by propranolol. In contrast, neither the beta-adrenergic blocker, propranolol, nor the alpha-adrenergic blocker, phentolamine, was effective in inhibiting the dopamine-induced rise in plasma glucagon (from 82+/-9 to 128+/-14 pg/ml; P < 0.005) and serum insulin (from 7.5+/-1 to 13+/-1.5 muU/ml; P < 0.005) or its suppression of plasma prolactin (from 8.5+/-1 to 5.2+/-0.8 ng/ml; P < 0.001). Although serum growth hormone levels did not change during the infusion of dopamine alone, an obvious rise occurred in three subjects during the combined infusion of propranolol and dopamine. Whereas some metabolic and cardiovascular effects of dopamine are mediated through adrenergic mechanisms, these observations indicate that this is not the case for the effects of this catecholamine on glucagon, insulin, and prolactin secretion, and thus provide further support for the theory of a specific dopaminergic sensitivity of these hormonal systems in man.

Effects of physiologic levels of glucagon and growth hormone on human carbohydrate and lipid metabolism. Studies involving administration of exogenous hormone during suppression of endogenous hormone secretion with somatostatin.

To study the individual effects of glucagon and growth hormone on human carbohydrate and lipid metabolism, endogenous secretion of both hormones was simultaneously suppressed with somatostatin and physiologic circulating levels of one or the other hormone were reproduced by exogenous infusion. The interaction of these hormones with insulin was evaluated by performing these studies in juvenile-onset, insulin-deficient diabetic subjects both during infusion of insulin and after its withdrawal. Infusion of glucagon (1 ng/kg-min) during suppression of its endogenous secretion with somatostatin produced circulating hormone levels of approximately 200 pg/ml. When glucagon was infused along with insulin, plasma glucose levels rose from 94 +/- 8 to 126 +/- 12 mg/100 ml over 1 h (P less than 0.01); growth hormone, beta-hydroxy-butyrate, alanine, FFA, and glycerol levels did not change. When insulin was withdrawn, plasma glucose, beta-hydroxybutyrate, FFA, and glycerol all rose to higher levels (P less than 0.01) than those observed under similar conditions when somatostatin alone had been infused to suppress glucagon secretion. Thus, under appropriate conditions, physiologic levels of glucagon can stimulate lipolysis and cause hyperketonemia and hyperglycemia in man; insulin antagonizes the lipolytic and ketogenic effects of glucagon more effectively than the hyperglycemic effect. Infusion of growth hormone (1 mug/kg-h) during suppression of its endogenous secretion with somastostatin produced circulating hormone levels of approximately 6 ng/ml. When growth hormone was administered along with insulin, no effects were observed. After insulin was withdrawn, plasma beta-hydroxybutyrate, glycerol, and FFA all rose to higher levels (P less than 0.01) than those observed during infusion of somatostatin alone when growth hormone secretion was suppressed; no difference in plasma glucose, alanine, and glucagon levels was evident. Thus, under appropriate conditions, physiologic levels of growth hormone can augment lipolysis and ketonemia in man, but these actions are ordinarily not apparent in the presence of physiologic levels of insulin.

Defective glucose counterregulation after subcutaneous insulin in noninsulin-dependent diabetes mellitus. Paradoxical suppression of glucose utilization and lack of compensatory increase in glucose production, roles of insulin resistance, abnormal neuroendocrine responses, and islet paracrine interactions.

To characterize glucose counterregulatory mechanisms in patients with noninsulin-dependent diabetes mellitus (NIDDM) and to test the hypothesis that the increase in glucagon secretion during hypoglycemia occurs primarily via a paracrine islet A-B cell interaction, we examined the effects of a subcutaneously injected therapeutic dose of insulin (0.15 U/kg) on plasma glucose kinetics, rates of glucose production and utilization, and their relationships to changes in the circulating concentrations of neuroendocrine glucoregulatory factors (glucagon, epinephrine, norepinephrine, growth hormone, and cortisol), as well as to changes in endogenous insulin secretion in 13 nonobese NIDDM patients with no clinical evidence of autonomic neuropathy. Compared with 11 age-weight matched nondiabetic volunteers in whom euglycemia was restored primarily by a compensatory increase in glucose production, in the diabetics there was no compensatory increase in glucose production (basal 2.08 +/- 0.04----1.79 +/- 0.07 mg/kg per min at 21/2 h in diabetics vs. basal 2.06 +/- 0.04----2.32 +/- 0.11 mg/kg per min at 21/2 h in nondiabetics, P less than 0.01) despite the fact that plasma insulin concentrations were similar in both groups (peak values 22 +/- 2 vs. 23 +/- 2 microU/ml in diabetics and nondiabetics, respectively). This abnormality in glucose production was nearly completely compensated for by a paradoxical decrease in glucose utilization after injection of insulin (basal 2.11 +/- 0.03----1.86 +/- 0.06 mg/kg per min at 21/2 h in diabetics vs. basal 2.08 +/- 0.04----2.39 +/- 0.11 mg/kg per min at 21/2 h nondiabetics, P less than 0.01), which could not be accounted for by differences in plasma glucose concentrations; the net result was a modest prolongation of hypoglycemia. Plasma glucagon (area under the curve [AUC] above base line, 12 +/- 3 vs. 23 +/- 3 mg/ml X 12 h in nondiabetics, P less than 0.05), cortisol (AUC 2.2 +/- 0.5 vs. 4.0 +/- 0.7 mg/dl X 12 h in nondiabetics, P less than 0.05), and growth hormone (AUC 1.6 +/- 0.4 vs. 2.9 +/- 0.4 micrograms/ml X 12 h in nondiabetics, P less than 0.05) responses in the diabetics were decreased 50% while their plasma norepinephrine responses (AUC 49 +/- 12 vs. 21 +/- 5 ng/ml X 12 h in nondiabetics, P less than 0.05) were increased twofold (P less than 0.05) and their plasma epinephrine responses were similar to those of the nondiabetics (AUC 106 +/- 17 vs. 112 +/- 10 ng/ml X 12 h in nondiabetics). In both groups of subjects, increases in plasma glucagon were inversely correlated with plasma glucose concentrations (r = -0.80 in both groups, P less than 0.01) and suppression of endogenous insulin secretion (r = -0.57 in nondiabe

Studies on the mechanism of epinephrine-induced hyperglycemia in man. Evidence for participation of pancreatic glucagon secretion.

In man, epinephrine induces increases in plasma levels of glucagon, a lipolytic and hyperglycemic hormone. To determine glucagon's contribution to this hyperglycemia and lipolysis, the effects of inhibition of pancreatic alpha-cell responses to epinephrine were investigated with somatostatin and adrenergic receptor blockade. To avoid ambiguities that might result from concomitant changes in endogenous insulin secretion, these studies were performed in juvenile-type, insulin-deficient diabetic subjects. Compared with normal subjects, the diabetics had excessive glucagon responses to epinephrine, which had been infused to attain circulating levels within the range found in man in severe stress. Both somatostatin and propranolol completely prevented glucagon responses and diminished the glycemic response to epinephrine by 40 to 50 per cent. Free fatty acid responses to epinephrine were completely prevented by propranolol but unaffected with somatostatin. Phentolamine had no effect on glucose, free fatty acid, or glucagon responses to epinephrine. These studies demonstrate that epinephrine, via a beta-adrenergic receptor mechanism, causes excessive plasma glucagon elevation in human diabetes mellitus and indicate that this hyperglucagonemia participates in the hyperglycemic, but not the lipolytic, response to epinephrine. Catecholamine-induced hyperglucagonemia may thus provide an additional explantation for the deterioration in carbohydrate tolerance associated with stress.

Plasma cyclic nucleotide levels in juvenile-onset diabetes.

In patients with juvenile-onset diabetes, plasma concentrations of 3',5'-adenosine cyclic monophosphate (cAMP) were significantly lower than those of norman subjects [16 +/- 4 and 24 +/- 7 pmol per milliliter (p less than 0.025), respectively] as determined in this laboratory; whereas there were essentially no differences in plasma levels of 3',5'-guanosine cyclic monophosphate (cGMP). Because cAMP inhibits cell growth and cGMP stimulates it, these findings may represent an important factor in the atherosclerotic and obliterative angiopathies of diabetic individuals. We observed that cyclic nucleotide values were the same whether or not the subjects were receiving insulin. Those given insulin plus enough glucose to maintain hyperglycemia revealed modest elevations in cyclic nucleotide levels. Thus, the ratio of cAMP to cGMP, abnormally low in juvenile-onset diabetes, is relatively independent of short-term variations in plasma levels of either glucose of insulin.

Effects of acute insulin withdrawal and administration on plasma glucagon responses to intravenous arginine in insulin-dependent diabetic subjects.

To assess further the role of insulin in the abnormal alpha-cell dysfunction found in human diabetes mellitus, the effects of acute insulin withdrawal and administration on plasma glucagon responses to intravenous arginine were studied in eight insulin-dependent diabetic subjects. Arginine infusions (30 gm. over 30 minutes) were performed during and at one and four hours after discontinuation of a 14-hour insulin infusion (1.5 U. per hour), which had rendered the subjects euglycemic, and on another occasion before and one and four hours into a five-hour infusion of insulin (1.5 U. per hour). During the last hour of the 14-hour infusion, glucagon responses to arginine (area under the curve, nanograms per milliliter per minute) were similar to those found in normal subjects (10.3 +/- 0.8 vs. 9.0 +/- 0.8, respectively). After discontinuation of the insulin infusions, glucagon responses increased progressively (p less than 0.01) to values (16.8 +/- 1.2) that exceeded those of normal subjects by four hours (p less than 0.01). These were similar to results found in the same subjects studied when their diabetes was in less than optimal control (14.9 +/- 1.3). Infusion of insulin under these conditions progressively decreased glucagon responses to arginine to values (9.6 +/- 0.8; p less than 0.01) that, at four hours, were similar to those of normal subjects and to values found at the end of the 14-hour infusion of insulin in the same diabetic individuals. These results demonstrate a rapid effect of insulin on glucagon responses to arginine and suggest that the abnormal responses seen in diabetes mellitus are the immediate result of insulin deficiency. Since abnormal glucagon responses to glucose in diabetes are not as readily corrected by insulin, the mechanisms underlying the abnormal responses to these two stimuli may differ.

Discordance of diabetic microangiopathy in identical twins.

In a pair of 19-year-old monozygotic twin girls, one developed insulin-dependent, ketosis-prone diabetes at the age of three and has required insulin for the past 16 years. Her identical twin has maintained normal oral and intravenous glucose tolerance with normal insulin release and glucagon suppression. An unequivocal hypertrophy of the muscle capillary basement membrane (1,800 +/- 148 A) was dound in the diabetic twin, while a normal thickness of 1,149 +/- 62 A was present in her nondiabetic sister. Follow-up of the present subjects and data from other discordant identical twins who have reached adulthood could determine whether muscle capillary basement membrane hypertrophy is an independent marker of genetic diabetes in adults. Discordance of diabetic microangiopathy in a pair of monozygotic twins has important implications regarding the influence of heredity and environment on diabetic microangiopathy.

The effect of chronic oral antidiabetic therapy on insulin and glucagon responses to a meal.

Nineteen maturity-onset diabetic patients receiving oral hypoglycemic therapy in a university diabetes clinic completed a study to assess the efficacy of the oral agents and to determine their effects on pancreatic islet hormone secretion. All patients were receiving sulfonylureas, and seven were also receiving phenformin. The subjects were studied as outpatients in the clinic setting on four different occasions with collections of a baseline blood sample before a standard breakfast and a second sampling two hours postprandially, twice while on their prescribed medication and twice after having been withdrawn from the medication. The values obtained during the two studies on the two studies off medications were reproducible for each subject. Analysis of the results by paired differences revealed that mean 24-hour urine glucose values deteriorated significantly (p less than 0.005) after oral antidiabetic therapy was withdrawn; similarly, mean plasma glucose values, both at baseline and two hours postprandially, rose significantly (p less than 0.001) when subjects were off medication. Baseline serum insulin values were not changed, but postprandial levels were significantly higher on oral agents (p less than 0.005). Plasma immunoreactive glucagon was significantly lower both at baseline (p less than 0.02) and postprandially (p less than 0.005) when the subjects were on their antidiabetic medications. During the trial off medication, 16 patients became symptomatic, with three of these developing symptoms severe enough to require hospitalization. It is apparent from this study that oral hypoglycemic medications can play a role in controlling symptoms in maturity-onset diabetic patients and that the beneficial effect of these agents on hyperglycemia may, in part, be explained by their stimulation of endogenous insulin secretion and partial suppression of endogenous glucagon.

Plasma glucagon and alanine responses to acute insulin deficiency in man.

Plasma glucagon levels rose within 1 hr after withdrawal of insulin in 7 juvenile-type diabetics previously kept normoglycemic by prolonged intravenous infusions of insulin. These changes preceded subsequent elevations in plasma alanine levels. Individual rises in plasma glucagon were correlated with elevations in plasma glucose, beta-hydroxybutyrate, free fatty acid, and glycerol levels, suggesting that glucagon may play an important role in the development of diabetic ketoacidosis in man.

Differential effects of L-dopa and apomorphine on glucagon secretion in man: evidence against central dopaminergic stimulation of glucagon.

In 6 normal subjects, L-dopa (500 mg PO) and apomorphine (0.6 mg sc) increased circulating growth hormone and suppressed prolactin levels in a parallel and quantitatively similar fashion, but only L-dopa induced a rise in plasma glucagon, glucose, and insulin levels. The failure of apomorphine to affect glucagon secretion, despite a substantial effect on growth hormone and prolactin, was also observed in insulin-dependent diabetics known to exhibit A-cell hyperresponsiveness to various stimuli. In view of the highly dissimilar molecular and pharmacologic characteristics of L-dopa and apomorphine, these data do not exclude a local dopaminergic effect of L-dopa at the pancreatic level, but strongly militate against a central dopaminergic pathway for glucagon stimulation.

Differential effects of hyperglycemia and hyperinsulinemia on leucine rate of appearance in normal humans.

To determine the independent effects of insulin and glucose on the rate of appearance of leucine in humans, five normal volunteers were studied using a pancreatic clamp with sequential insulin infusions of 1.8, 3.6 and 7.2 pmol kg/min-1. On each of three separate occasions, the plasma glucose level was maintained at 5.0, 9.4 or 14 mM. The rate of appearance of leucine was similar at each insulin infusion rate regardless of the ambient glucose concentration or the glucose infusion rate. These studies demonstrate that during insulin and glucose infusion, insulin, but neither the plasma glucose concentration nor the rate of glucose utilization, primarily regulates leucine flux (a reflection of endogenous whole body proteolysis) in normal humans.

Normalization of fasting hyperglucagonemia and excessive glucagon responses to intravenous arginine in human diabetes mellitus by prolonged infusion of insulin.

Infusion of insulin (1 U/hr) for 14 hr suppressed basal glucagon levels and normalized previously excessive glucagon responses to arginine in juvenile-onset, insulin-dependent diabetic subjects, indicating that abnormal pancreatic alpha-cell function in human juvenile-onset diabetes mellitus may be a consequence of insulin lack.

Mechanism of hyperglycemia and response to treatment with an inhibitor of fatty acid oxidation in a patient with insulin resistance due to antiinsulin receptor antibodies.

Severe hyperglycemia and insulin resistance due to antiinsulin receptor antibodies developed over a period of 3 months in a 50-yr-old insulin-requiring diabetic patient. The hyperglycemia resulted from overproduction of glucose due to excessive rates of glycogenolysis and gluconeogenesis rather than decreased glucose utilization. Treatment with methyl-2-tetradecylglycidate, an inhibitor of fatty acid oxidation, resulted in a decrease in plasma glucose concentration. This was associated with a decrease in the rate of glucose production due to decreases in both gluconeogenesis and glycogenolysis rates, as well as an increase in the respiratory quotient. Plasma glucose concentrations continued to respond to the drug for the next 2 months until the sudden development of terminal hypoglycemia. The hypoglycemic action of the drug is consistent with the existence of an insulin-independent effect of fatty acid oxidation on glucose metabolism in man.

Sustained reductions in oxipurinol renal clearance during a restricted diet.

The renal clearance of oxipurinol, the major metabolite of allopurinol, was studied in six healthy subjects during normal and restricted (low protein and low calorie) diets. A 600 mg oral dose of allopurinol was administered after 7 days of a normal diet (100 mg protein/day) and again after 2 and 4 weeks of a restricted diet (19 gm protein/day). The renal clearance of oxipurinol was reduced from 19.6 +/- 1.5 ml/min during the normal diet to 10.9 +/- 0.8 and 12.0 +/- 0.9 ml/min (both P less than 0.001) during the restricted diet at 2 and 4 weeks, respectively. These changes in oxipurinol renal clearance paralleled changes in uric acid renal clearance. Furthermore, the plasma oxipurinol half-life was increased from 27.0 +/- 1.7 hours during the normal diet to 51.1 +/- 4.3 and 45.7 +/- 3.7 hours (both P less than 0.001) during the restricted diet at 2 and 4 weeks, respectively. We conclude that dietary protein and calorie restriction cause a sustained reduction in the elimination of oxipurinol.

Renal clearances of oxypurinol and inulin on an isocaloric, low-protein diet.

In previous studies a low-calorie, low-protein diet caused a sustained reduction in both oxypurinol and uric acid renal clearances (CLR). With the hypothesis that the decrease in CLR was due to the low-protein and not the low-caloric content of the diet, we studied the CLR of oxypurinol, uric acid, creatinine, and inulin in normal subjects during isocaloric (2600 calories per 70 kg per day), normal-protein (150 gm per day), and low-protein (19 gm per day) diets. There were three major findings: (1) the CLR of oxypurinol declined from 26.6 +/- 1.8 ml/min on the normal-protein diet to 13.5 +/- 1.4 ml/min on the isocaloric low-protein diet (p less than 0.05); (2) the CLR of inulin and creatinine fell 14% and 20%, respectively, on the low-protein diet compared with the normal-protein diet (both p less than 0.05); and (3) there was a diurnal variation in the CLR of oxypurinol. We conclude that the decreased CLR of oxypurinol was the result of the reduced protein content of the diet and the CLR of both inulin and creatinine were decreased on the low-protein diet.

Diabetes and fructose metabolism.

The clinical aspects of fructose supplementation in the diets of individuals with diabetes should focus on the balance between beneficial effects and possible side effects. Fructose supplementation in diabetes mellitus was advocated before insulin was discovered. Fructose elicits a lower glucose and insulin response in healthy individuals and in individuals with diabetes. The use of fructose as a sweetener in the diets of diabetics has been debated repeatedly. Short-term studies have now shown that substitution of fructose for sucrose in the diets of individuals with diabetes improves glycemic control and does not appear to have substantial side effects. In balanced diets, reasonable amounts of fructose supplementation do not affect lipoprotein metabolism or result in gastrointestinal symptoms. Long-term studies are still needed to ascertain that long-term fructose supplementation has a sustained beneficial effect in diabetes and is devoid of deleterious side effects.

Augmentation of protein degradation by L-triiodothyronine in uremia.

To ascertain if excessive protein catabolism is a feature of uremia, we determined leucine flux and nitrogen balance in 11 stable chronic dialysis patients and in 7 normal subjects. Leucine flux was determined during primed constant infusion of 2H3 and 15N leucine. Nitrogen balance was determined by measurement of nitrogen in the food, dialysate, and urine, and in the dialysis patients by correcting for the changing urea nitrogen pool. To assess if thyroid hormone adversely affects protein metabolism, the above-mentioned studies were done once in the basal state and once after a 7-day course of L-triiodothyronine (T3) treatment. Leucine carbon flux (mumol/kg/min) was 1.22 +/- 0.05 in the controls and 1.40 +/- 0.09 in the renal patients in the basal state (P = NS). Following T3 treatment, leucine carbon flux was increased to 1.40 +/- 0.05 and 1.72 +/- 0.09, respectively, in the controls and the renal patients (P less than .05). Fractional increment of the leucine carbon flux was 14% +/- 3% in the controls and 23% +/- 9% in the renal patients (P less than .05). The leucine nitrogen flux (mumol/kg/min) was 2.10 +/- 0.15 in the controls and 2.54 +/- 0.23 in the renal patients in the basal state (P = NS), and increased to 2.48 +/- 0.14 and 3.44 +/- 0.22, respectively, in controls and renal patients after T3 administration (P less than .05). Fractional increment of leucine nitrogen flux was 19.5% +/- 4.3% in the controls and 36.4% +/- 5.0% in the renal patients (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of local sympathetic blockade on forearm blood flow and glucose uptake during hypoglycemia.

During hypoglycemia, hepatic glucose production increases and peripheral glucose utilization decreases. Systemic beta-adrenergic blockade during hypoglycemia increases peripheral glucose utilization. To explore the local effects of increased alpha- and beta-adrenergic activity on skeletal muscle glucose utilization, we measured arterial and venous plasma glucose concentrations, forearm blood flow (FBF), and forearm glucose uptake (FGU) during a hyperinsulinemic (40 mU/m2/min) stepped-hypoglycemic clamp with intrabrachial artery infusion of saline, phentolamine, propranolol, or combined phentolamine and propranolol. A control study was also performed with a euglycemic clamp and intraarterial saline. During hypoglycemia with saline and phentolamine, there were significant increases in FBF (130% +/- 38% and 180% +/- 35%, respectively) and FGU (120% +/- 51% and 230% +/- 150%, respectively). During hypoglycemia with propranolol and phentolamine + propranolol, FBF remained constant. FGU during hypoglycemia with propranolol was not different versus hypoglycemia with saline. No differences were found in these studies for forearm lactate output (FLO) or venous free fatty acid concentrations. These results demonstrate that local, as opposed to systemic, blockade during hypoglycemia does not alter peripheral glucose utilization.

Reduced renal clearance of oxypurinol during a 400 calorie protein-free diet.

A decrease in dietary protein intake lowers the clearance of a number of substances excreted principally by the kidney including uric acid and oxypurinol, the major metabolite of allopurinol. We studied the kinetics of uric acid and oxypurinol in seven healthy volunteers on a normal protein diet (2600 calories; 100 g protein) followed by a 400 calorie, protein-free diet. A 600 mg dose of allopurinol was given orally after 6 days of the normal protein diet and again after 2 days of the 400 calorie, protein-free diet. Two major findings emerged: first, the renal clearance of oxypurinol was reduced from 21.2 +/- 1.9 ml/min during the normal protein diet to 12.3 +/- 1.2 ml/min (P less than .05) during the 400 calorie, protein-free diet, and second, there was a striking diurnal difference in oxypurinol renal clearance with a 41% decrease in the oxypurinol clearance at night (8 PM to 8 AM) versus day (8 AM to 8 PM) on the 400 calorie, protein-free diet.