Comparison of cold crystalloid and colloid infusions for induction of therapeutic hypothermia in a porcine model of cardiac arrest

Introduction

Large-volume cold intravenous infusion of crystalloids has been used for induction of therapeutic hypothermia after cardiac arrest. However, the effectiveness of cold colloids has not been evaluated. Therefore, we performed an experimental study to investigate the cooling effect of cold normal saline compared to colloid solution in a porcine model of ventricular fibrillation.

Methods

Ventricular fibrillation was induced for 15 minutes in 22 anesthetized domestic pigs. After spontaneous circulation was restored, the animals were randomized to receive either 45 ml/kg of 1°C cold normal saline (Group A, 9 animals); or 45 ml/kg of 1°C cold colloid solution (Voluven®, 6% hydroxyethyl starch 130/0.4 in 0.9% NaCl) during 20 minutes (Group B, 9 animals); or to undergo no cooling intervention (Group C, 4 animals). Then, the animals were observed for 90 minutes. Cerebral, rectal, intramuscular, pulmonary artery, and subcutaneous fat body temperatures (BT) were recorded. In the mechanical ex-vivo sub study we added a same amount of cold normal saline or colloid into the bath of normal saline and calculated the area under the curve (AUC) for induced temperature changes.

Results

Animals treated with cold fluids achieved a significant decrease of BT at all measurement sites, whereas there was a consistent significant spontaneous increase in group C. At the time of completion of infusion, greater decrease in pulmonary artery BT and cerebral BT in group A compared to group B was detected (−2.1 ± 0.3 vs. -1.6 ± 0.2°C, and −1.7 ± 0.4 vs. -1.1 ± 0.3°C, p < 0.05, respectively). AUC analysis of the decrease of cerebral BT revealed a more vigorous cooling effect in group A compared to group B (−91 ± 22 vs. -68 ± 23°C/min, p = 0.046). In the mechanical sub study, AUC analysis of the induced temperature decrease of cooled solution revealed that addition of normal saline led to more intense cooling than colloid solution (−7155 ± 647 vs. -5733 ± 636°C/min, p = 0.008).

Conclusions

Intravenous infusion of cold normal saline resulted in more intense decrease of cerebral and pulmonary artery BT than colloid infusion in this porcine model of cardiac arrest. This difference is at least partially related to the various specific heat capacities of the coolants.     

Arterial Stiffness Is Associated With Cardiovascular,Renal, Retinal,and Autonomic Disease in Type 1 Diabetes

OBJECTIVE

In patients with type 1 diabetes, we investigated the association between arterial stiffness and diabetes complications.

RESEARCH DESIGN AND METHODS

This was a cross-sectional study including 676 Caucasian patients with type 1 diabetes (374 [55%] men, aged 54 ± 13 years [mean ± SD]) and 51 nondiabetic controls (28 [55%] men, aged 47 ± 13 years). Aortic pulse wave velocity (PWV) was measured with SphygmoCor (AtCor Medical, Sydney, Australia) for 635 patients and all 51 controls.

RESULTS

PWVs (mean ± SD) in patients and controls were 10.4 ± 3.4 and 7.6 ± 1.9 m/s, respectively (P < 0.001). After multivariate adjustment, PWV correlated with age, diabetes duration, urinary albumin excretion rate, heart rate, and blood pressure (P < 0.05 for all). ANCOVA was used for comparisons between groups and adjusted for gender, age, estimated glomerular filtration rate, heart rate, HbA_1c, and 24-h mean arterial pressure. PWVs in normoalbuminuric, microalbuminuric, and macroalbuminuric patients were 9.5 ± 3.2, 11.0 ± 3.6, and 11.4 ± 3.0 m/s, respectively (adjusted P < 0.001). PWV in patients with previous cardiovascular disease, versus patients without, was 12.1 ± 3.5 vs. 10.0 ± 3.2 m/s, respectively (adjusted P < 0.001). PWVs in patients with high (≥140/90 mmHg) versus intermediate (130–40/80–89 mmHg) and low (<130/80 mmHg) blood pressure were 11.8 ± 3.6, 10.0 ± 3.0, and 9.8 ± 3.3 m/s, respectively (adjusted P < 0.001). Furthermore, PWV increased with increasing degree of retinopathy: 8.0 ± 2.5 m/s (nil), 10.0 ± 2.8 m/s (simplex), 12.1 ± 3.5 m/s (proliferative), and 12.7 ± 2.4 m/s (blind), respectively (adjusted P < 0.001). Finally, PWV increased with abnormal heart rate variability: 11.5 ± 3.3 m/s vs. 10.1 ± 3.1 m/s (borderline) and 8.1 ± 2.1 m/s (normal) (adjusted P = 0.027).

CONCLUSIONS

Arterial stiffness increased with presence and duration of type 1 diabetes. Furthermore, PWV increased with all the investigated diabetes complications (cardiovascular, renal, retinal, and autonomic disease) independently of other risk factors.Arterial stiffness predicts cardiovascular disease (CVD) events in the general population (1), in hypertension (2,3), and in diabetes (4,5). A recent meta-analysis by Vlachopoulos et al. (6) showed pulse wave velocity (PWV) to predict both CVD and all-cause mortality. The gold standard for measurement of arterial stiffness is aortic PWV measurements (7,8), which have shown to be reproducible in the general population (9) and in patients with chronic kidney disease (10).In hypertension, PWV is a marker of subclinical organ damage. Recent studies have shown PWV to be predictive of future changes in systolic blood pressure (SBP) development of hypertension (11) to potentially improve CVD risk scoring (12,13), and also to be higher among patients less responsive to antihypertensive medication (AHT) (14).Furthermore, in patients with end-stage renal disease, elevated PWV is an independent predictor of all-cause mortality (15), and lowering PWV by AHT reduces the mortality independently of the blood pressure (BP) reduction (16).In type 1 diabetes, PWV has been shown to be associated with cerebral microvascular disease, cardiac (17) and renal function (18), and in type 2 diabetes with microalbuminuria (19). These associations are suggestive of PWV being related to vascular dysfunction (20) and large artery alterations, thereby possibly engaging in the development of kidney impairment (21). Arterial stiffness also is associated with autonomic neuropathy in type 1 diabetes (22,23) and retinopathy in type 2 diabetes (24), further illustrating the association between arterial stiffness and microvascular disease.The current study is the first in a relatively large and diverse group of patients with type 1 diabetes to investigate the association between PWV and a range of diabetes complications, including albuminuria, CVD, elevated BP, retinopathy, and autonomic neuropathy.     

Enhanced Absorption of Insulin Aspart as the Result of a Dispersed Injection Strategy Tested in a Randomized Trial in Type 1 Diabetic Patients

OBJECTIVE

We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo.

RESEARCH DESIGN AND METHODS

Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide–negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration.

RESULTS

The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08).

CONCLUSIONS

A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.Fast-acting insulin analogs have been developed to avoid postprandial glucose peaks (1,2). Some studies suggest that postprandial hyperglycemia can contribute to elevated levels of hemoglobin A_1c (3,4) and lead to the development of short- and long-term diabetes complications (5,6). Although currently available fast-acting insulin analogs have been designed for a better match with meal-induced glucose excursions, insulin absorption and insulin action still lag behind (7,8). Even bolus administration of fast-acting insulin analogs immediately before meals does not completely avoid postprandial glucose peaks. Modern fast-acting insulin analogs still only insufficiently mimic physiological insulin profiles; however, their effect could be further improved by accelerating insulin absorption from the injection site into the vascular system.Accelerated insulin absorption in response to an increased blood flow has been described for heated injection sites (9) or coadministered adjuvants such as hyaluronidase (10–12) but also for a larger distribution of the subcutaneous insulin depot achieved with a modified injection strategy. Human insulin absorption has been tested with a “sprinkler needle” that has 14 holes in its walls and a sealed tip, thus dispersing the insulin bolus at the injection site. With the sprinkler needle, insulin was absorbed more rapidly and glucose levels were less raised relative to a regular injection needle (13). A dispersed insulin bolus should have an increased surface-to-volume ratio and could further contribute to even faster insulin absorption of modern already fast-acting insulin analogs.The aim of our study was to test whether the absorption rate of a fast-acting insulin analog (insulin aspart) could be further accelerated through the dispersion of a single predefined insulin bolus into nine separate insulin injections. We compared the two different injection strategies ex vivo by using microfocus computed tomography (micro-CT) to assess the increase in the surface-to-volume ratio and in vivo by assessing the pharmacokinetic and pharmacodynamic response in a clinical trial.     

Metabolic Effects of Chronic Cannabis Smoking

OBJECTIVE

We examined if chronic cannabis smoking is associated with hepatic steatosis, insulin resistance, reduced β-cell function, or dyslipidemia in healthy individuals.

RESEARCH DESIGN AND METHODS

In a cross-sectional, case-control study, we studied cannabis smokers (n = 30; women, 12; men, 18; 27 ± 8 years) and control subjects (n = 30) matched for age, sex, ethnicity, and BMI (27 ± 6). Abdominal fat depots and intrahepatic fat content were quantified by magnetic resonance imaging and proton magnetic resonance spectroscopy, respectively. Insulin-sensitivity indices and various aspects of β-cell function were derived from oral glucose tolerance tests (OGTT).

RESULTS

Self-reported cannabis use was: 9.5 (2–38) years; joints/day: 6 (3–30) [median (range)]. Carbohydrate intake and percent calories from carbohydrates, but not total energy intake, were significantly higher in cannabis smokers. There were no group differences in percent total body fat, or hepatic fat, but cannabis smokers had a higher percent abdominal visceral fat (18 ± 9 vs. 12 ± 5%; P = 0.004). Cannabis smokers had lower plasma HDL cholesterol (49 ± 14 vs. 55 ± 13 mg/dL; P = 0.02), but fasting levels of glucose, insulin, total cholesterol, LDL cholesterol, triglycerides, or free fatty acids (FFA) were not different. Adipocyte insulin resistance index and percent FFA suppression during an OGTT was lower (P < 0.05) in cannabis smokers. However, oral glucose insulin sensitivity index, measures of β-cell function, or incretin concentrations did not differ between the groups.

CONCLUSIONS

Chronic cannabis smoking was associated with visceral adiposity and adipose tissue insulin resistance but not with hepatic steatosis, insulin insensitivity, impaired pancreatic β-cell function, or glucose intolerance.Cannabinoid receptors (CB₁R and CB₂R) and their endogenous ligands, the endocannabinoids, play an important role in regulating energy balance, appetite, insulin sensitivity, pancreatic β-cell function, and lipid metabolism (1–4). Endocannabinoids (anandamide and 2-arachidonoyl glycerol) and CB₁Rs are present in peripheral tissues involved in energy homeostasis, such as adipose tissue, liver, skeletal muscle, and pancreas (1,2). CB₁R activation promotes lipogenesis in the liver and adipose tissue (1,2,5), reduces insulin responsiveness in skeletal muscle (3), and impairs insulin action and secretion in pancreatic β-cells (4). Consistent with these findings, clinical interventional trials suggest that CB₁R antagonism reduces body weight, improves dyslipidemia, and attenuates insulin resistance in humans (6,7).Δ9-Tetrahydrocannabinol (Δ9-THC), the primary psychoactive component of Cannabis sativa, activates peripheral and central CB₁Rs (2). Acute treatment of healthy human volunteers with cannabis induces glucose intolerance (8,9). Similarly, short-term (13 days) marijuana smoking increases appetite, food intake, and body weight in healthy men (10). In individuals with chronic hepatitis C, daily marijuana abuse was associated with a higher risk for hepatic steatosis (11). These findings from animal and human studies suggest that chronic cannabis use and CB₁R activation may negatively affect metabolic actions of insulin and facilitate hepatic steatosis. Cannabis (marijuana) is the most commonly used illicit drug in the U.S. with ∼17 million current users (12). Despite such widespread use, relatively little is known about the metabolic effects associated with chronic cannabis use. To that end, in a cross-sectional, case-control study, we examined if chronic cannabis smoking is associated with hepatic steatosis, insulin resistance, reduced β-cell function, and dyslipidemia in healthy individuals.     

Arterial Stiffness Is Inversely Related to Plasma Adiponectin Levels in Young Normotensive Patients With Type 1 Diabetes

OBJECTIVE

This study investigated the association between arterial stiffness and plasma adiponectin in patients with type 1 diabetes.

RESEARCH DESIGN AND METHODS

Participants were normotensive patients with type 1 diabetes who were up to age 40 years. Subjects on statins with macrovascular disease or overt nephropathy were excluded. Large artery stiffness was assessed by measurement of carotid-femoral pulse wave velocity (PWV), whereas plasma adiponectin was measured by radioimmunoassay.

RESULTS

Data from 80 patients (age 27.1 ± 6.1 years, BMI 24.2 ± 3.1 kg/m², HbA_1c 7.5 ± 1.6%, 39 men, adiponectin 13.9 ± 6.7 μg/mL, and PWV 5.6 ± 0.9 m/s) were analyzed. Log adiponectin inversely correlated with age-adjusted PWV (r = −0.291, P = 0.009) and waist circumference (r = −0.427, P < 0.001). In a fully adjusted model, age, expiration/inspiration index, and log adiponectin were independently associated with PWV, explaining 39.6% of its variance.

CONCLUSIONS

Arterial stiffness is inversely related to adiponectin concentration in young patients with type 1 diabetes without major complications.Arterial stiffness, an independent predictor of total and cardiovascular mortality, can be assessed noninvasively by measurement of pulse wave velocity (PWV) (1), which is increased at early stages of type 1 diabetes (2,3). Plasma adiponectin, an adipocytokine with insulin-sensitizing, antiatherogenic, and anti-inflammatory properties (4), is high in patients with type 1 diabetes (5,6). Although adiponectin is inversely related to arterial stiffness in subjects with essential hypertension (7,8), no adiponectin-PWV relationship has been shown in children/adolescents with type 1 diabetes (9). This study investigated the association between adiponectin and PWV in young adults with type 1 diabetes.     

Mechanisms of Glucose Lowering of Dipeptidyl Peptidase-4 Inhibitor Sitagliptin When Used Alone or With Metformin in Type 2 Diabetes: A double-tracer study

OBJECTIVE

To assess glucose-lowering mechanisms of sitagliptin (S), metformin (M), and the two combined (M+S).

RESEARCH DESIGN AND METHODS

We randomized 16 patients with type 2 diabetes mellitus (T2DM) to four 6-week treatments with placebo (P), M, S, and M+S. After each period, subjects received a 6-h meal tolerance test (MTT) with [¹⁴C]glucose to calculate glucose kinetics. Fasting plasma glucose (FPG), fasting plasma insulin, C-peptide (insulin secretory rate [ISR]), fasting plasma glucagon, and bioactive glucagon-like peptide (GLP-1) and gastrointestinal insulinotropic peptide (GIP) were measured.

RESULTS

FPG decreased from P, 160 ± 4 to M, 150 ± 4; S, 154 ± 4; and M+S, 125 ± 3 mg/dL. Mean post-MTT plasma glucose decreased from P, 207 ± 5 to M, 191 ± 4; S, 195 ± 4; and M+S, 161 ± 3 mg/dL (P < 0.01). The increase in mean post-MTT plasma insulin and in ISR was similar in P, M, and S and slightly greater in M+S. Fasting plasma glucagon was equal (∼65–75 pg/mL) with all treatments, but there was a significant drop during the initial 120 min with S 24% and M+S 34% (both P < 0.05) vs. P 17% and M 16%. Fasting and mean post-MTT plasma bioactive GLP-1 were higher (P < 0.01) after S and M+S vs. M and P. Basal endogenous glucose production (EGP) fell from P 2.0 ± 0.1 to S 1.8 ± 0.1 mg/kg ⋅ min, M 1.8 ± 0.2 mg/kg ⋅ min (both P < 0.05 vs. P), and M+S 1.5 ± 0.1 mg/kg ⋅ min (P < 0.01 vs. P). Although the EGP slope of decline was faster in M and M+S vs. S, all had comparable greater post-MTT EGP inhibition vs. P (P < 0.05).

CONCLUSIONS

M+S combined produce additive effects to 1) reduce FPG and postmeal plasma glucose, 2) augment GLP-1 secretion and β-cell function, 3) decrease plasma glucagon, and 4) inhibit fasting and postmeal EGP compared with M or S monotherapy.In diet-treated patients with type 2 diabetes mellitus (T2DM) and HbA_1c of ∼8.0%, sitagliptin (S) reduces HbA_1c by 0.6–0.7% over a 6-month period (1). A slightly greater HbA_1c decline (∼0.8–0.9%) is observed when metformin (M) therapy is added to S (2). Dipeptidyl peptidase (DPP)-4 inhibitors predominantly affect the postprandial plasma glucose excursion, but a significant, albeit modest, reduction in fasting plasma glucose (FPG) also is observed (1–3). The mechanism of action of the DPP-4 inhibitors has been well studied and includes increased plasma glucagon-like peptide (GLP)-1 and gastrointestinal insulinotropic peptide (GIP) levels, resulting in increased insulin and reduced glucagon secretion (4–6). The increase in plasma insulin and the decline in glucagon inhibit basal endogenous glucose production (EGP) and enhance the suppression of EGP without affecting splanchnic (hepatic) glucose uptake or gastric emptying (6,7). Therapy with S and M combined (M+S) exerts an additive effect to reduce HgA_1c; the mechanism of action of this combination has yet to be examined. Several studies have demonstrated that M inhibits DPP-4 activity, thus increasing plasma active GLP-1 levels (8–10). There are also reports indicating that the decline in plasma glucose with M therapy can restore the β-cells’ sensitivity to the stimulatory effect of incretins on insulin secretion (11,12). Despite these reports, we still do not fully understand the reasons why the use of M in diabetic patients is not accompanied by changes in insulin release. In the current study, we used the double-tracer technique (7) to examine the mechanism(s) via which M+S and each agent alone reduce the fasting and postmeal plasma glucose concentration in T2DM.     

Right Ventricular Involvement in Diabetic Cardiomyopathy

OBJECTIVE

To compare magnetic resonance imaging-derived right ventricular (RV) dimensions and function between men with type 2 diabetes and healthy subjects, and to relate these parameters to left ventricular (LV) dimensions and function.

RESEARCH DESIGN AND METHODS

RV and LV volumes and functions were assessed in 78 men with uncomplicated type 2 diabetes and 28 healthy men within the same range of age using magnetic resonance imaging. Steady-state free precession sequences were used to assess ventricular dimensions. Flow velocity mapping across the pulmonary valve and tricuspid valve was used to assess RV outflow and diastolic filling patterns, respectively. Univariate general linear models were used for statistical analyses.

RESULTS

RV end-diastolic volume was significantly decreased in patients compared with healthy subjects after adjustment for BMI and pulse pressure (177 ± 28 mL vs. 197 ± 47 mL, P < 0.01). RV systolic function was impaired: peak ejection rate across the pulmonary valve was decreased (433 ± 54 mL/s vs. 463 ± 71 mL/s, P < 0.01) and pulmonary flow acceleration time was longer (124 ± 17 ms vs. 115 ± 25 ms, P < 0.05). Indexes of RV diastolic function were impaired: peak filling rate and peak deceleration gradient of the early filling phase were 315 ± 63 mL/s vs. 356 ± 90 mL/s (P < 0.01) and 2.3 ± 0.8 mL/s² × 10⁻³ vs. 2.8 ± 0.8 mL/s² × 10⁻³ (P < 0.01), respectively. All RV parameters were strongly associated with its corresponding LV parameter (P < 0.001).

CONCLUSIONS

Diabetic cardiomyopathy affects the right ventricle, as demonstrated by RV remodeling and impaired systolic and diastolic functions in men with type 2 diabetes, in a similar manner as changes in LV dimensions and functions. These observations suggest that RV impairment might be a component of the diabetic cardiomyopathy phenotype.Cardiovascular disease is one of the major adverse consequences of type 2 diabetes. Patients with type 2 diabetes have an increased cardiovascular mortality rate (1). Even in the absence of significant coronary artery disease and hypertension, subclinical left ventricular (LV) dysfunction presents in type 2 diabetes (2). This so-called diabetic cardiomyopathy has a complex and multifactorial pathogenesis. Atherosclerosis, subclinical microinfarctions, mitochondrial dysfunction, and lipotoxicity all have been proposed as contributors to diabetic cardiomyopathy. Furthermore, it has been recognized that deposition of advanced glycation end products, caused by long-standing hyperglycemia, affects ventricular stiffness (3). The formation of advanced glycation end products yields fibrosis by cross-linking collagen (4), thus increasing myocardial stiffness. This may lead to a decreased LV end-diastolic volume and impaired subclinical LV function (5–7).All proposed mechanisms leading to LV impairment in type 2 diabetes are systemic changes and therefore also might hamper right ventricular (RV) function. RV involvement in diabetic cardiomyopathy might be of importance because the right ventricle has a substantial contribution to overall myocardial contractility. RV function has proven to be of importance for patient risk stratification in heart failure (8) and for prediction of development of atrial fibrillation (9). In general, RV dysfunction and fibrosis are associated with lethal ventricular arrhythmias, sudden death, exercise limitation, and impaired RV cardiac output (10). In addition, the prevalence of cardiac conduction abnormalities is increased in diabetic patients (11).However, only limited data exist on RV involvement in type 2 diabetes. Whereas animal studies have shown that dysfunction of the right ventricle might play a role in diabetic cardiomyopathy (12), the right ventricle is largely overlooked in human studies. Only a few echocardiographic studies discuss the right ventricle in diabetes (13–17). These studies were limited by inclusion of patients with cardiovascular diabetes-related complications (13–15,17), and study populations consisted partially (13,14) or entirely of type 1 diabetic patients (16). Moreover, none of these studies reported RV volumes.The right ventricle is a difficult structure from which to obtain reproducible echocardiographic signals because of the irregular geometrical shape and the anterior position within the thorax. Without mathematical modeling, conventional two-dimensional echo techniques commonly underestimate or overestimate the true size of the adult right ventricle (18). Cardiovascular magnetic resonance imaging (MRI) has become the reference standard for the assessment of RV function and volumes because good reproducibility has been shown (19,20).To our knowledge, no studies to date have evaluated volumetric as well as systolic and diastolic functional involvement of the right ventricle in uncomplicated type 2 diabetes compared with healthy subjects assessed by MRI. Accordingly, the purpose of the current study was to compare MRI-derived RV dimensions and systolic and diastolic function between well-controlled uncomplicated type 2 diabetic patients and healthy subjects, in relation to LV dimensions and function.     

Large Pre- and Postexercise Rapid-Acting Insulin Reductions Preserve Glycemia and Prevent Early- but Not Late-Onset Hypoglycemia in Patients With Type 1 Diabetes

OBJECTIVE

To examine the acute and 24-h glycemic responses to reductions in postexercise rapid-acting insulin dose in type 1 diabetic patients.

RESEARCH DESIGN AND METHODS

After preliminary testing, 11 male patients (24 ± 2 years, HbA_1c 7.7 ± 0.3%; 61 ± 3.4 mmol/mol) attended the laboratory on three mornings. Patients consumed a standardized breakfast (1 g carbohydrate ⋅ kg⁻¹ BM; 380 ± 10 kcal) and self-administered a 25% rapid-acting insulin dose 60 min prior to performing 45 min of treadmill running at 72.5 ± 0.9% VO_2peak. At 60 min postexercise, patients ingested a meal (1 g carbohydrate ⋅ kg⁻¹ BM; 660 ± 21 kcal) and administered a Full, 75%, or 50% rapid-acting insulin dose. Blood glucose concentrations were measured for 3 h postmeal. Interstitial glucose was recorded for 20 h after leaving the laboratory using a continuous glucose monitoring system.

RESULTS

All glycemic responses were similar across conditions up to 60 min postexercise. After the postexercise meal, blood glucose was preserved under 50%, but declined under Full and 75%. Thence at 3 h, blood glucose was highest under 50% (50% [10.4 ± 1.2] vs. Full [6.2 ± 0.7] and 75% [7.6 ± 1.2 mmol ⋅ L⁻¹], P = 0.029); throughout this period, all patients were protected against hypoglycemia under 50% (blood glucose ≤3.9; Full, n = 5; 75%, n = 2; 50%, n = 0). Fifty percent continued to protect patients against hypoglycemia for a further 4 h under free-living conditions. However, late-evening and nocturnal glycemia were similar; as a consequence, late-onset hypoglycemia was experienced under all conditions.

CONCLUSIONS

A 25% pre-exercise and 50% postexercise rapid-acting insulin dose preserves glycemia and protects patients against early-onset hypoglycemia (≤8 h). However, this strategy does not protect against late-onset postexercise hypoglycemia.Patients with type 1 diabetes are encouraged to engage in regular exercise as part of a healthy lifestyle (1,2). However, engaging in exercise is not without its difficulties (1). Defective glucose regulation presents a significant challenge in preventing hypoglycemia during, and particularly after, exercise (3,4). Exercise-induced hypoglycemia is both a frequent (5) and dangerous occurrence (6) and remains a major obstacle to patients who wish to engage in exercise (7).Much of the literature has focused on providing strategies to help combat hypoglycemia during, and early after, exercise (8–17), with investigations focusing on altering exercise modality (14,18), carbohydrate consumption (12,16,17), and reductions to pre-exercise, rapid-acting insulin dose (10–12,17,19). Prior to moderate-intensity, continuous, aerobic exercise, it is recommended that patients should reduce their prandial rapid-acting insulin dose by ∼75% to prevent hypoglycemia during exercise (10–12). However, despite best preserving blood glucose, it has been shown that this strategy is not fully protective against postexercise hypoglycemia (11,12). This has, in part, been attributed to iatrogenic causes (11), whereby patients administer their usual doses of rapid-acting insulin in a heightened insulin-sensitive state, potentially leading to unexpected falls in blood glucose and, consequently, hypoglycemia (11).A potential strategy to help minimize the risk of developing hypoglycemia after exercise could be to reduce the dose of rapid-acting insulin administered with the postexercise meal (20). Exercise increases the sensitivity of the body to insulin for many hours after exercise (3) and patients could be faced with a window of particularly high sensitivity around the postexercise meal, whereby greater rates of glucose uptake could occur to supplement the high metabolic priority of replenishing muscle glycogen (21). Thus, the meal consumed after exercise is important. With this in mind, it would be intuitive to reduce the amount of insulin administered with the meal consumed at this time; this may preserve glycemia and prevent postexercise hypoglycemia. Conversely, severe reductions in rapid-acting insulin dose may incur prolonged postexercise hyperglycemia, even more so if the pre-exercise dose is also reduced. However, there is a lack of data to confirm or refute these hypotheses. In addition, it would be prudent to examine the extent to which rapid-acting insulin dose adjustments may help combat late falls in glycemia after exercise, considering type 1 diabetic patients are susceptible to late-onset, postexercise hypoglycemia (3), suggested to be due to a biphasic response in glucose uptake occurring early and also late after exercise (22). Therefore, the aim of this study was to examine the acute and 24-h postexercise glycemic responses to reducing the postexercise rapid-acting insulin dose, when using the recommended pre-exercise insulin reductions, in type 1 diabetic patients.     

Sucralose Affects Glycemic and Hormonal Responses to an Oral Glucose Load

OBJECTIVE

Nonnutritive sweeteners (NNS), such as sucralose, have been reported to have metabolic effects in animal models. However, the relevance of these findings to human subjects is not clear. We evaluated the acute effects of sucralose ingestion on the metabolic response to an oral glucose load in obese subjects.

RESEARCH DESIGN AND METHODS

Seventeen obese subjects (BMI 42.3 ± 1.6 kg/m²) who did not use NNS and were insulin sensitive (based on a homeostasis model assessment of insulin resistance score ≤2.6) underwent a 5-h modified oral glucose tolerance test on two separate occasions preceded by consuming either sucralose (experimental condition) or water (control condition) 10 min before the glucose load in a randomized crossover design. Indices of β-cell function, insulin sensitivity (S_I), and insulin clearance rates were estimated by using minimal models of glucose, insulin, and C-peptide kinetics.

RESULTS

Compared with the control condition, sucralose ingestion caused 1) a greater incremental increase in peak plasma glucose concentrations (4.2 ± 0.2 vs. 4.8 ± 0.3 mmol/L; P = 0.03), 2) a 20 ± 8% greater incremental increase in insulin area under the curve (AUC) (P < 0.03), 3) a 22 ± 7% greater peak insulin secretion rate (P < 0.02), 4) a 7 ± 4% decrease in insulin clearance (P = 0.04), and 5) a 23 ± 20% decrease in S_I (P = 0.01). There were no significant differences between conditions in active glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, glucagon incremental AUC, or indices of the sensitivity of the β-cell response to glucose.

CONCLUSIONS

These data demonstrate that sucralose affects the glycemic and insulin responses to an oral glucose load in obese people who do not normally consume NNS.Most people, like other mammals, are innately attracted to consuming sweet-tasting foods. Nonnutritive sweeteners (NNS) are food additives that provide a sweet taste to food but have few, if any, calories. Therefore, the use of NNS has become a popular approach to help reduce energy intake and glycemic load (1,2). Currently, seven NNS (sucralose, saccharin, aspartame, acesulfame potassium, neotame, stevia, and Luo han guo extract) are approved by the U.S. Food and Drug Administration and are widely used in thousands of food products.Although it has been proposed that NNS do not affect glycemia (3), data from several recent studies suggest that NNS are not physiologically inert. First, it has been demonstrated that the gastrointestinal tract (4,5) and the pancreas (6,7) can detect sugars through taste receptors and transduction mechanisms that are similar to those indentified in taste cells in the mouth. Second, NNS-induced activation of gut sweet taste receptors in isolated duodenal L cells and pancreatic β-cells triggers the secretion of glucagon-like peptide 1 (GLP-1) (4,5) and insulin (6–9), respectively. Third, data from studies conducted in animal models demonstrate that NNS interact with sweet taste receptors expressed in enteroendocrine cells to increase both active and passive intestinal glucose absorption by upregulating the expression of sodium-dependent glucose transporter isoform 1 (5,10,11) and increasing the translocation of GLUT2 to the apical membrane of intestinal epithelia (12).The relevance of the findings from studies conducted in cell systems and rodent models to human physiology is not clear because the NNS data obtained from studies conducted in people often fail to replicate the metabolic outcomes observed in vitro and in animal models (rev. in 13). The results from most (14–18), but not all (19,20), studies conducted in people have found that NNS do not affect plasma glucose, insulin, or GLP-1. However, these studies did not exclude people who regularly consumed NNS, which could have chronic effects on glucose metabolism (5,10,11) that would blunt any acute effects of sucralose intake.The primary purpose of this study was to test the hypothesis that sucralose ingestion alters the glycemic and hormonal responses to glucose ingestion in obese subjects who are not regular users of NNS. We specifically studied obese people because NNS are often promoted to help decrease calorie intake and facilitate weight management in this population.     

Recombinant Expression and Characterization of the Major β-Lactamase of Mycobacterium tuberculosis

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of M. tuberculosis is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.Tuberculosis causes 3 million deaths annually, more than any other single infectious agent (2, 19, 35). Multidrug resistance is a growing clinical problem, with strains of Mycobacterium tuberculosis exhibiting resistance to 11 or more antimicrobial agents having been described (25). Although it was shown in the 1940s that under certain culture conditions penicillin inhibits the growth of M. tuberculosis (9, 10, 18, 31), the availability of other effective antimicrobial agents limited efforts to determine whether tuberculosis might respond to treatment with β-lactams. However, the recent rise in infections caused by multidrug-resistant strains has made it necessary to identify alternative treatment regimens, including the determination of whether some older classes of antibiotics such as the β-lactams might be effective in the clinical setting.The cell wall structure of M. tuberculosis contains a thick peptidoglycan layer. Cycloserine, a second-line drug in the treatment of tuberculosis, is a d-alanine analog that interferes with peptidoglycan synthesis (37). Recently, it has been shown that M. tuberculosis makes at least four penicillin-binding proteins (PBPs) that bind ampicillin and other β-lactams at clinically relevant antibiotic concentrations (3). The affinities of these agents for their PBP targets are of the magnitude seen for β-lactams that can be effectively used for the treatment of infections caused by other microbes. Also, the outer cellular structures of tubercle bacilli do not represent a major permeability barrier for β-lactams (3, 22). Therefore, the production of β-lactamase by M. tuberculosis appears to be its major mechanism of resistance to β-lactams.Most and possibly all isolates of M. tuberculosis produce β-lactamase (12, 13, 15, 42); however, data regarding its nature are limited. Opinions differ as to whether it is secreted, cytoplasmic, or bound to the cell membrane and as to whether its production is inducible or constitutive (10, 14, 15, 32, 42). Zhang et al. (42) have reported that isoelectric focusing of Triton X-100 extracts of acetone-precipitated cell pellets of strains of M. tuberculosis reveals two bands exhibiting β-lactamase activity with pI values of 4.9 and 5.1.Most of the information on the kinetic properties of M. tuberculosis β-lactamase comes from studies with relatively impure preparations of enzyme or has been inferred indirectly via the results of susceptibility tests involving β-lactams and β-lactam–β-lactamase inhibitor combinations. However, greater penicillinase activity than cephalosporinase activity is consistently reported (15, 20, 22, 42). M. tuberculosis β-lactamase is inhibited competitively by antistaphylococcal penicillins (13–15, 21, 22, 32) and by conventional β-lactamase inhibitors including clavulanic acid, sulbactam, and tazobactam (5, 8, 33, 38, 41, 42). β-Lactamase inhibitors improve the activities of some penicillins against M. tuberculosis in vitro (5, 8, 14, 33) and in vivo (13). In addition, some cephalosporins including ceforanide and cephapirin as well as carbapenems such as imipenem exhibit potent in vitro activities (23, 30, 36).Because a better understanding of the mechanisms by which M. tuberculosis expresses resistance to β-lactams might ultimately lead to strategies in which these agents could be used in the treatment of tuberculosis, we have worked to characterize its β-lactamase(s). In this report, we describe the isolation of three enzymes with distinct pI values directly from M. tuberculosis and the recombinant expression and kinetic characterization of the major enzyme.(Results of this study were presented in part at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., 15 to 18 September 1996, and at the 32nd U.S.-Japan Conference of Tuberculosis/Leprosy, Cleveland, Ohio, 21 to 23 July 1997.)     

Dietary Fat Acutely Increases Glucose Concentrations and Insulin Requirements in Patients With Type 1 Diabetes: Implications for carbohydrate-based bolus dose calculation and intensive diabetes management

OBJECTIVE

Current guidelines for intensive treatment of type 1 diabetes base the mealtime insulin bolus calculation exclusively on carbohydrate counting. There is strong evidence that free fatty acids impair insulin sensitivity. We hypothesized that patients with type 1 diabetes would require more insulin coverage for higher-fat meals than lower-fat meals with identical carbohydrate content.

RESEARCH DESIGN AND METHODS

We used a crossover design comparing two 18-h periods of closed-loop glucose control after high-fat (HF) dinner compared with low-fat (LF) dinner. Each dinner had identical carbohydrate and protein content, but different fat content (60 vs. 10 g).

RESULTS

Seven patients with type 1 diabetes (age, 55 ± 12 years; A1C 7.2 ± 0.8%) successfully completed the protocol. HF dinner required more insulin than LF dinner (12.6 ± 1.9 units vs. 9.0 ± 1.3 units; P = 0.01) and, despite the additional insulin, caused more hyperglycemia (area under the curve >120 mg/dL = 16,967 ± 2,778 vs. 8,350 ± 1,907 mg/dL⋅min; P < 0001). Carbohydrate-to-insulin ratio for HF dinner was significantly lower (9 ± 2 vs. 13 ± 3 g/unit; P = 0.01). There were marked interindividual differences in the effect of dietary fat on insulin requirements (percent increase significantly correlated with daily insulin requirement; R² = 0.64; P = 0.03).

CONCLUSIONS

This evidence that dietary fat increases glucose levels and insulin requirements highlights the limitations of the current carbohydrate-based approach to bolus dose calculation. These findings point to the need for alternative insulin dosing algorithms for higher-fat meals and suggest that dietary fat intake is an important nutritional consideration for glycemic control in individuals with type 1 diabetes.Current guidelines for the intensive treatment of type 1 diabetes focus exclusively on carbohydrate counting for mealtime bolus calculation (1,2). This carbohydrate-based approach to insulin dose calculation assumes that carbohydrate is the only dietary macronutrient that affects glucose levels and insulin requirements.Dietary fat and free fatty acids (FFAs) are known to impair insulin sensitivity and to enhance hepatic glucose production (3,4). Furthermore, pharmacologic interventions that lower FFA levels in nondiabetic and type 2 diabetic individuals lead to both improved insulin sensitivity and glucose tolerance (5,6). Studies in patients with type 1 and type 2 diabetes have shown that dietary fat delays gastric emptying, leading to a lag in glucose absorption (7,8). Although there has been considerable interest in the role of dietary fat and circulating FFAs in the pathogenesis of type 2 diabetes (9,10), relatively little attention has been given to the possible implications of FFA-induced insulin resistance for the treatment of type 1 diabetes. Review of continuous glucose monitoring and food log data from our adult patients with type 1 diabetes led to the observation that, contrary to the current treatment recommendations, higher-fat meals usually require more insulin coverage than lower-fat meals with similar carbohydrate content.Pizza is widely recognized to cause marked late postprandial hyperglycemia in patients with type 1 diabetes (11). Some studies have shown that use of an extended bolus with (12) or without (13–15) an increase in total dose is needed to attenuate hyperglycemia after higher-fat pizza meals. To our knowledge, a controlled study to determine whether changes in dietary fat intake, independent of other macronutrients, leads to alterations in glucose control and insulin requirements in type 1 diabetes has not been undertaken. In this study, we carefully regulated the macronutrient intake of patients with type 1 diabetes undergoing closed-loop glucose control to test the hypothesis that high-fat (HF) meals require more insulin coverage than low-fat (LF) meals with identical carbohydrate content.     

A protocol guided by transpulmonary thermodilution and lactate levels for resuscitation of patients with severe burns

Over-resuscitation is deleterious in many critically ill conditions, including major burns. For more than 15 years, several strategies to reduce fluid administration in burns during the initial resuscitation phase have been proposed, but no single or simple parameter has shown superiority. Fluid administration guided by invasive hemodynamic parameters usually resulted in over-resuscitation. As reported in the previous issue of Critical Care, Sánchez-Sánchez and colleagues analyzed the performance of a ‘permissive hypovolemia’ protocol guided by invasive hemodynamic parameters (PiCCO, Pulsion Medical Systems, Munich, Germany) and vital signs in a prospective cohort over a 3-year period. The authors’ results confirm that resuscitation can be achieved with below-normal levels of preload but at the price of a fluid administration greater than predicted by the Parkland formula (2 to 4 mL/kg per% burn). The classic approach based on an adapted Parkland equation may still be the simplest until further studies identify the optimal bundle of resuscitation goals.The recently published paper by Sanchez-Sanchez and colleagues [1] is a very interesting and well documented study showing how challenging it is to prevent fluid creep during early resuscitation after major burn trauma. The aim of resuscitation is to restore adequate organ and tissue oxygenation. Under- and over-resuscitation have been associated with complications and poor outcome not only in patients with burns [2] but in other critically ill patients as well [3]. Volume creep in major burns contributes to worsening of burn edema, conversion of superficial to deep burns, and compartment syndromes. Although the use of vital signs – heart rate and mean arterial pressure (MAP) – and urine output may lead to under-resuscitation in burned patients who are critically ill [4], several teams have attempted to optimize fluid resuscitation after major burns [5-7]. Basing fluid delivery on invasive hemodynamic monitoring has repeatedly been shown to cause over-resuscitation [8]. Some years ago, Arlati and colleagues [9] introduced the concept of permissive hypovolemia. They showed that fluid administration guided by a hemodynamics-oriented approach throughout the first 24-hour period limited to a urine output of 0.5 to 1 mL/kg per hour and a cardiac index of at least 2.2 L/minute per m² was safe and resulted in less organ dysfunction [9]. This year, Sánchez-Sánchez and colleagues [1] present interesting results of a 3-year prospective cohort study of 132 consecutive critically ill patients (age of 48 ± 18 years) with a burned body surface (total body surface area, or TBSA) of 35 ± 22%.The resuscitation algorithm was guided by blood pressure (MAP of more than 65 mm Hg), urinary output (0.5 to 1 mL/kg), transpulmonary thermodilution, and lactate levels: the crude 4 mL/kg per TBSA formula was used as a starting value at the beginning of the resuscitation but was constantly adjusted to achieve (a) a cardiac index of more than 2.5 L/minute per m², (b) an intrathoracic blood volume index (ITBVI) of more than 600 mL/m², and (c) normalization of lactate levels by 32 hours. The mean fluid rate required to achieve the protocol targets in the first 8 hours was 4.05 mL/kg per TBSA burned, increasing during the next 16 hours. The authors conclude that the initial burn trauma-induced hypovolemia is not optimally reflected by MAP and hourly urine output but can be detected by transpulmonary thermodilution during the early resuscitation phase. The authors consider that, thanks to their protocol, unnecessary fluid overload was prevented.However, there are some concerns with the results presented by Sánchez-Sánchez and colleagues [1] and doubts about the achievement of what they presented as ‘dry resuscitation’, especially when strong endpoints such as intra-abdominal pressure and Sepsis-related Organ Failure Assessment (SOFA) are considered. First, given a mean TBSA of 35%, the mortality rate is elevated (23%). Second, 12% of patients have an abdominal compartment syndrome (intra-abdominal pressure of more than 20 mm Hg), of whom 50% die. Third, acute renal failure occurred in 31.1% of patients and 11.4% finally required renal replacement therapy. Fourth, the length of mechanical ventilation is high (21.5 days), and 24.2% of patients developed acute respiratory distress syndrome, despite a very low prevalence (9.1%) of proven inhalation. The persistence of relatively high SOFA scores on days 5 and 7 differs from the fast organ failure decay observed by Arlati and colleagues [9] with a stricter hypovolemia protocol. Finally, early progressive enteral nutrition (EN) – that is, started within 12 hours of injury – has been shown to improve outcome after major burns and should be integrated in any resuscitation protocol since it enhances gut perfusion in both animals and humans, reduces both pyloric dysfunction [10] and gut edema [11], and hence improves gut function: further early EN is associated with a significant reduction of the requirement for parenteral nutrition (PN). In the Spanish cohort, EN was introduced only ‘whenever possible’, forcing the use of PN in 61.3% of patients, which is unusual and is likely to reflect gut dysfunction.Despite showing the willingness to reduce fluid delivery, the above events might still reflect over-resuscitation. Indeed, the ITBVI-driven resuscitation protocol results in a fluid delivery above the Parkland-determined target, which by no means can be qualified as a ‘hypovolemic’ target. Remember that the Advanced Trauma Life Support (ATLS) guidelines now recommend considering the low range of 2 to 4 mL/kg per% TBSA of the Parkland formula for calculation of the first 24 hours’ fluid administration [12]! Furthermore, the resuscitation algorithm does mention the use of inotropes and vasopressors, but only after attempting changes in fluid load. This may be the missing link to explain higher-than-expected fluid administration. Unfortunately, the results do not give details on the use of the latter or track the gain of weight.In conclusion, though not achieving the degree of over-resuscitation described by Holm and colleagues [8], the strategy by Sánchez-Sánchez and colleagues [1] still suffers from some degree of fluid overload and did not achieve the expected reduction of organ failures (pulmonary, renal, and digestive). Some degree of hypovolemia during the first 24 hours after burn does not require complete correction when occurring under close supervision. The ATLS proposed guidance by the low range of the Parkland formula remains a good alternative to an invasive hemodynamic target to guide initial resuscitation after major burns.     

β-Lactamase Inhibitors Are Substrates for the Multidrug Efflux Pumps of Pseudomonas aeruginosa

The MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including β-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether β-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance β-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using β-lactams which were readily hydrolyzed by the Pseudomonas aeruginosa class C chromosomal β-lactamase, it was demonstrated that elimination of the mexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase β-lactam susceptibility. With β-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance β-lactam susceptibility in MexAB-OprM⁺ strains, although BRL42715 did enhance β-lactam susceptibility in MexAB-OprM⁻ strains, suggesting that even with poorly hydrolyzed β-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to β-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of β-lactam–β-lactamase inhibitor combinations in treating bacterial infections.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to a variety of antimicrobial agents. Previously attributed to a highly impermeable outer membrane (22), this resistance is now recognized to result from the synergy between broadly specific drug efflux pumps and low outer membrane permeability (16). One such efflux system, encoded by the mexAB-oprM operon (8, 28, 29), effluxes a range of antibiotics, including tetracycline, chloramphenicol, quinolones, β-lactams, novobiocin, macrolides, and trimethoprim (8, 9, 12, 29). Expressed constitutively in wild-type cells, where it contributes to intrinsic drug resistance (5, 12, 29), the operon is hyperexpressed in nalB mutants (30), producing elevated levels of resistance to substrate antibiotics (8, 9, 12, 29). Homologous efflux systems encoded by the mexC-mexD-oprJ (27) and mexE-mexF-oprN (10) operons have also been described. Apparently not expressed during growth under normal laboratory conditions, these systems are expressed in nfxB (27) and nfxC (10) multidrug-resistant mutants, respectively. nfxB strains are resistant to chloramphenicol, tetracycline, quinolones, macrolides, novobiocin, and newer cephalosporins such as cefepime and cefpirome but display hypersusceptibility to most β-lactam antibiotics (18). nfxC strains exhibit resistance to chloramphenicol, trimethoprim, quinolones, and carbapenems, including imipenem, although the resistance to imipenem results from the loss of the porin protein OprD in these mutants and not from the overexpression of MexEF-OprN (6, 10).The tripartite efflux pumps consist of an inner membrane component (MexB, MexD, and MexF) which functions as a resistance-nodulation-division family H⁺ antiport exporter (21, 31), an outer membrane, a presumed channel-forming component (OprM, OprJ, and OprN) (16, 23), and a so-called membrane fusion protein predicted to link the membrane-associated efflux components (MexA, MexC, and MexE) (16, 23). Recent data suggest that the operation of MexAB-OprM (and by analogy the remaining efflux systems) is at least partially dependent upon the TonB energy-coupling protein implicated in the opening of outer membrane gated channels responsible for iron-siderophore uptake across the P. aeruginosa outer membrane (36). Thus, the outer membrane components of these efflux pumps may be gated channels.In an effort to further define the range of antibiotic compounds which are accommodated by the known P. aeruginosa efflux systems, we examined β-lactamase inhibitors as possible pump substrates by assessing the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance the efficacies of β-lactam compounds.     

GLP-1 Action and Glucose Tolerance in Subjects With Remission of Type 2 Diabetes After Gastric Bypass Surgery

OBJECTIVE

Glucagon like peptide-1 (GLP-1) has been suggested as a major factor for the improved glucose tolerance ensuing after Roux-en-Y gastric bypass (RYGBP) surgery. We examined the effect of blocking endogenous GLP-1 action on glucose tolerance in subjects with sustained remission of type 2 diabetes mellitus (T2DM) present before RYGBP.

RESEARCH DESIGN AND METHODS

Blood glucose, insulin, C-peptide, glucagon, GLP-1, and glucose-dependent insulinotropic peptide levels were measured after a meal challenge with either exendin-(9–39) (a GLP-1r antagonist) or saline infusion in eight subjects with sustained remission of T2DM after RYGBP and seven healthy controls.

RESULTS

Infusion of exendin-(9–39) resulted in marginal deterioration of the 2-h plasma glucose after meal intake in RYGBP subjects [saline 78.4 ± 15.1 mg/dL compared with exendin-(9–39) 116.5 ± 22.3 mg/dL; P < 0.001]. Furthermore, glucose response to meal intake was similarly enlarged in the two study groups [percent change in the area under the curve of glucose exendin-(9–39) infusion versus saline infusion: controls 10.84 ± 8.8% versus RYGBP 9.94 ± 8.4%; P = 0.884]. In the RYGBP group, the blockade of the enlarged GLP-1 response to meal intake resulted in reduced insulin (P = 0.001) and C-peptide (P < 0.001), but no change in glucagon (P = 0.258) responses.

CONCLUSIONS

The limited deterioration of glucose tolerance on blockade of GLP-1 action in our study suggests the resolution of T2DM after RYGBP may be explained by mechanisms beyond enhancement of GLP-1 action.The beneficial effect of Roux-en-Y gastric bypass (RYGBP) surgery on glycemic control in morbidly obese subjects with type 2 diabetes mellitus (T2DM) is well established (1,2). However, the precise mechanisms mediating T2DM remission after RYGBP are not yet clear (3–5). Although it traditionally has been asserted that bariatric operations are associated with improvement of glucose tolerance merely by caloric restriction and weight loss, several lines of evidence support weight-independent mechanisms are involved (6–11). An enhanced postsurgical glucagon-like peptide-1 (GLP-1) secretion, inducing a normalized or exaggerated insulin secretion after meal intake, has been hypothesized to play a major role in the improved glucose tolerance after RYGBP (3). Association studies have demonstrated larger improvements of glucose tolerance early after RYGBP being associated with a larger GLP-1 response to nutrient intake as compared with other surgical or nonsurgical interventions resulting in equivalent weight loss (7–9). Likewise, an exaggerated GLP-1 response has been reported up to 10 years after RYGB in subjects with sustained T2DM remission, suggesting a key role of GLP-1 in maintaining normal glucose tolerance in the long term after this type of surgery (12). However, because association does not prove causation, these data do not definitely prove GLP-1 plays a critical role in T2DM remission after RYGBP.Understanding the role of endogenous GLP-1 in metabolic physiology has been greatly enhanced by the availability of a potent GLP-1 receptor antagonist, exendin-(9–39). Exendin-(9–39) blockade of GLP-1 action in healthy volunteers results in a significant enlargement of postprandial glucose excursions (13–17). Moreover, using hyperglycemic clamp technique in combination with a mixed meal test, Salehi et al. (18) demonstrated that blocking GLP-1 action results in a larger decrease in the insulin secretion rate in RYGBP-operated subjects (−33%) as compared with nonoperated controls (−16%). This study clearly supports GLP-1 as an important determinant of insulin secretion after RYGBP. However, the use of hyperglycemic clamp limited the ability of the study to investigate the relative importance of GLP-1 secretion on glucose tolerance. Furthermore, because only one-third of the study participants presented with T2DM before surgery, the study also was limited in establishing the role of GLP-1 secretion in the remission of T2DM. Of note, in Goto-Kakizaki rats (a nonobese rat model of T2DM) administration of exendin-(9–39) has been shown to totally reverse the improved glucose tolerance resulting from duodeno-jejunal exclusion surgery (an experimental metabolic surgery similar to RYGBP) (19).Against this background, the main aim of our study was to examine the effect of endogenous GLP-1 blockade by exendin-(9–39) on glucose tolerance in subjects who had undergone RYGBP and with T2DM antedating surgery that had remitted after the surgical procedure. As secondary aims, we evaluated the effect of exendin-(9–39) on the insulin, C-peptide, glucagon, GLP-1, and glucose-dependent insulinotropic peptide (GIP) responses to meal intake. We evaluated individuals during the long-term after surgery to avoid the potential confounding effect of intense caloric restriction or rapid weight loss or both on glucose tolerance.     

Evaluation of Rodent Cage Processing Using Reduced Water Temperatures

Studies published in 1994 and 2000 established a temperature range of 143–180 °F for effective cage sanitization in animal facilities. These 2 studies were, respectively, theoretical and based on experiments using hot water to sanitize bacteria-coated test tubes. However, such experimental methods may not capture the practical advantages of modern washing technology or account for the routine use of detergent in cage wash. Moreover, these methods may not translate to the challenges of removing adhered debris and animal waste from the surfaces being sanitized. A sample of highly soiled cage bottoms, half of which were autoclaved with bedding to create challenging cleaning conditions, were processed at 6 combinations of wash and rinse cycles with 125 °F, 140 °F, and 180 °F water with detergent. All cycles were equipped with a data logging device to independently verify temperatures. After washing, cages underwent visual inspection and microbial sampling consisting of organic material detection using ATP detection and Replicate Organism Detection and Counting (RODAC) plates. Cages with any amount of visible debris failed inspection, as did cages that exceeded institutional sanitization thresholds. Results indicate that wash and rinse temperatures of 140 °F for a programmed wash duration of 450 s and rinse of 50 s effectively clean and disinfect both highly soiled and autoclaved cages. Accounting for both steam and electrical energy, these parameters result in an annual savings of $21,867.08 per washer on an equivalent run basis using the current institutional standard of 180 °F.

The Guide for the Care and Use of Laboratory Animals states that “effective disinfection can be achieved with wash and rinse water at 143–180°F or more.”¹³ Disinfection is defined as the reduction or elimination of microorganisms, whereas sanitization is the combined effect of cleaning, or removal of gross debris, with disinfection.¹³ In pursuing regulatory compliance and biosecurity, institutions commonly operate at the higher end of this range for both the wash and rinse stages, resulting in high utility usage and high-cost operation. Early research underlying recommendations for disinfection with water alone used a theoretical approach to establish time-heat combinations, known as cumulative heat factors.²⁶ Disinfection combinations are 1800 s at 143 °F (61.7 °C), 15 s at 161 °F (71.7 °C), and 0.1 s at 180 °F (82.2 °C).²⁶ Subsequent research has shown that contact times ranging from 2 to 5 s at 168 °F to 180 °F (75.6 to 82.2 °C) are sufficient to kill Pseudomonas aeruginosa, Salmonella cholerasuis, and Staphylococcus aureus,²⁷ and contact times of 2 min or more at 140 °F (60 °C) will kill Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia rettgeri, and Staphylococcus epidermidis.²¹ When accounting for chemical and mechanical factors in tunnel washers, washing at the domestic hot water temperature (110 °F [43.3 °C]), followed by a rinse at 180 °F, can adequately prevent transmission of mouse parvovirus, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus.⁶ The current study used performance standards in a practical study design to build on these disinfection principles and inform sanitization parameters for modern cage washing. Modern washers not only operate longer than the times required in the described cumulative heat factors, but they also use sophisticated mechanical delivery of water via high-pressure jets, dispense detergent, and allow programming of different temperatures at the wash and rinse stages. Test tube models and organism-directed research previously used to determine cage wash efficacy may not capture the challenges of debris removal encountered in day-to-day operation in an animal facility. Assessment of visible debris is recommended by cage processing working groups,¹² and should be included as a practical metric when establishing performance-based standards.The purpose of this study was to compare sanitization performance of reduced-temperature wash conditions to the standard wash cycle temperature of 180 °F. Experimental conditions combined wash and rinse temperatures of 125 °F, 140 °F, and 180 °F. Water sourced to the wash room in our facility is 125 °F (51.7 °C) and was therefore the minimal testable parameter. 140 °F (60 °C) is both commonly used in European facilities¹² and falls in the midrange of temperatures recommend for use with cage wash detergent (120 to 160 °F [48.9 to 71.1 °C]).²³ Highly soiled cages, half of which were autoclaved with bedding to represent the most challenging subset of cages to clean, were evaluated for disinfection and cleanliness. We hypothesized that cages washed and rinsed at 125 °F for 120 s would pass institutional sanitization standards, as assessed with visual inspection, ATP monitoring, and replicate organism detection and counting (RODAC). Given the energy usage, time, and utility costs necessary to reach temperatures of 180 °F, study aims were to verify sanitization performance at reduced temperatures, estimate time savings of operating at these various temperature parameters, and quantify cost and utility savings factoring in both electrical and steam-boosted heating.