Preliminary evidence of effects of potassium chloride on a metabolomic path to diabetes and cardiovascular disease.

INTRODUCTION: Low potassium intake can affect cardiovascular disease (CVD) risk and cardiometabolic risk factors. OBJECTIVE: We hypothesize that potassium chloride (KCl) supplementation can improve cardiovascular risk metabolomic profile. METHODS: In this secondary analysis of a pilot randomized clinical trial (RCT) of 26 participants with prediabetes randomized to KCl or placebo, we performed targeted mass-spectrometry-based metabolomic profiling on baseline and 12-week (end-of-study) plasma samples. Principal component analysis (PCA) was used to reduce the many correlated metabolites into fewer, independent factors that retain most of the information in the original data. RESULTS: Those taking KCl had significant reductions (corresponding to lower cardiovascular risk) in the branched-chain amino acids (BCAA) factor (P = 0.004) and in valine levels (P = 0.02); and non-significant reductions in short-chain acylcarnitines (SCA) factor (P = 0.11). CONCLUSIONS: KCl supplementation may improve circulating BCAA levels, which may reflect improvements in overall cardiometabolic risk profile. CLINICAL TRIALS REGISTRY: Clinicaltrials.gov identifier: NCT02236598; https://clinicaltrials.gov/ct2/show/NCT02236598.

Effect of vitamin D supplementation on cardiovascular risk in type 2 diabetes.

BACKGROUND & AIMS: Whether vitamin D affects lipid profile and cardiovascular disease (CVD) risk is controversial. We evaluated the effect of oral daily vitamin D supplementation on lipid profile and CVD risk in patients with well-controlled type 2 diabetes. METHODS: Secondary analysis in the vitamin D for established type 2 diabetes (DDM2) study, a double-blind, randomized, placebo-controlled clinical trial. 127 patients (mean age 60 years) with stable (HbA1c ≤ 7.5%) diabetes managed with lifestyle only or lifestyle plus metformin were randomized to receive 4000 IU/day of vitamin D3 (n = 66) or placebo (n = 61) for 48 weeks. Changes in lipid profile (total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides [TG] and TG/HDL ratio), C-reactive protein and CVD risk (calculated according the American College of Cardiology/American Heart Association [ACC/AHA] guidelines) were assessed at week 24 and 48. RESULTS: The mean [±SEM] plasma 25-hydroxyvitamin D [25(OH)D] level was higher in the vitamin D vs. the placebo group (20.5 ± 1.18 vs. -1.6 ± 1.2 ng/mL respectively; p < 0.001). There was no statistically significant change in lipid profile, C-reactive protein or CVD risk. Among patients who were not on cholesterol medication (n = 32), vitamin D supplementation reduced TG compared to placebo at week 48 (-18.74 ± 8.91 vs. 9.69 ± 8.60 mg/dL respectively; p = 0.032). CONCLUSION: One year supplementation with vitamin D3 at 4000 IU/day did not affect lipid profile, C-reactive protein and CVD risk in patients with stable type 2 diabetes not selected for vitamin D deficiency, with the exception of improvement of TG among patients not on cholesterol medication. REGISTRATION: ClinicalTrials.gov Identifier NCT01736865.

Vitamin D Supplementation in Patients With Type 2 Diabetes: The Vitamin D for Established Type 2 Diabetes (DDM2) Study.

CONTEXT: Observational data support a role for vitamin D in type 2 diabetes, but evidence from trials is inconclusive. OBJECTIVE: To evaluate the effect of vitamin D supplementation on ß-cell function and hemoglobin A1c (HbA1c) in patients with well-controlled type 2 diabetes. DESIGN: Double-blind, randomized, placebo-controlled clinical trial. SETTING: Tufts Medical Center, Boston, MA; VA Medical Center, Cincinnati, OH. PARTICIPANTS: A total of 127 patients (mean age, 60 years) with stable (HbA1c ≤7.5%) diabetes managed with lifestyle only or lifestyle plus metformin. INTERVENTION: Subjects were given 4000 units of vitamin D3 (cholecalciferol) daily or placebo for 48 weeks. MAIN OUTCOME MEASURE: Insulin secretion rate (ISR) was estimated from peripheral plasma C-peptide levels after a 3-hour 75-g oral glucose tolerance test done at baseline and week 24. Changes in HbA1c were assessed at 16, 24, 36, and 48 weeks. RESULTS: Baseline mean plasma 25-hydroxyvitamin D [25(OH)D] concentration was 26.6 ng/mL, mean HbA1c was 6.6%, and 78% of patients were on metformin. At week 24, mean 25(OH)D changed by 20.5 and -1.6 ng/mL in the vitamin D and placebo groups, respectively (P < 0.001). The vitamin D and placebo groups did not differ in change in ISR or HbA1c. Among patients treated with lifestyle only (n = 28), vitamin D supplementation reduced HbA1c compared with placebo (-0.1% vs 0.3%, respectively; P = 0.034) at week 24. This result was not observed at the other time points and could be due to chance. CONCLUSION: Vitamin D3 at 4000 IU/d did not change ISR or HbA1c in patients with well-controlled type 2 diabetes on metformin not selected for vitamin D deficiency.

Effects of potassium supplements on glucose metabolism in African Americans with prediabetes: a pilot trial.

Background: Low potassium has been identified both as a risk factor for type 2 diabetes and as a mediator of the racial disparity in diabetes risk. Low potassium could be a potentially modifiable risk factor, particularly for African Americans.Objective: We sought to determine the effects of potassium chloride (KCl) supplements, at a commonly prescribed dose, on measures of potassium and glucose metabolism.Design: Among African-American adults with prediabetes, we conducted a double-blinded pilot randomized controlled trial that compared the effects of 40 mEq K/d as KCl supplements with a matching placebo, taken for 3 mo, on measures of potassium and glucose metabolism, with measures collected from frequently sampled oral-glucose-tolerance tests (OGTTs).Results: Twenty-seven of 29 recruited participants completed the trial. Participants had high adherence to the study medication (92% by pill count). Participants in both groups gained weight, with an overall mean ± SD weight gain of 1.24 ± 2.03 kg. In comparison with participants who received placebo, urine potassium but not serum potassium increased significantly among participants randomly assigned to receive KCl (P = 0.005 and 0.258, respectively). At the end of the study, participants taking KCl had stable or improved fasting glucose, with a mean ± SD change in fasting glucose of -1.1 ± 8.4 mg/dL compared with an increase of 6.1 ± 7.6 mg/dL in those who received placebo (P = 0.03 for comparison between arms). There were no significant differences in glucose or insulin measures during the OGTT between the 2 groups, but there was a trend for improved insulin sensitivity in potassium-treated participants.Conclusions: In this pilot trial, KCl at a dose of 40 mEq/d did not increase serum potassium significantly. However, despite weight gain, KCl prevented worsening of fasting glucose. Further studies in larger sample sizes, as well as with interventions to increase serum potassium more than was achieved with our intervention, are indicated to definitively test this potentially safe and inexpensive approach to reducing diabetes risk. This trial was registered at clinicaltrials.gov as NCT02236598.

Vitamin D status of black and white Americans and changes in vitamin D metabolites after varied doses of vitamin D supplementation.

BACKGROUND: Controversy exists over the disparate circulating 25-hydroxyvitamin D [25(OH)D] concentrations between black and white Americans. OBJECTIVE: We sought to determine whether there are differences in total and directly measured free 25(OH)D concentrations between black and white American adults and how daily supplementation with cholecalciferol changes these concentrations. DESIGN: Cross-sectional and longitudinal analyses were conducted with the use of data from 2 placebo-controlled, randomized trials at 2 academic medical centers in the United States: CaDDM (Calcium and Vitamin D in Type 2 Diabetes) and DDM2 (Vitamin D for Established Type 2 Diabetes). A total of 208 subjects with pre- or well-controlled diabetes with a mean age of 59.1 y and mean body mass index (BMI; in kg/m(2)) of 31.6 were randomly assigned to receive daily cholecalciferol supplementation at 1 of 2 doses (2000 or 4000 IU) or a matching placebo for 16 wk. We measured serum total 25(OH)D, vitamin D-binding protein (DBP) by 2 different immunoassays (with the use of monoclonal or polyclonal antibodies), parathyroid hormone, and albumin. Free 25(OH)D concentration was directly measured and calculated. RESULTS: Blacks had lower total 25(OH)D concentrations than whites [adjusted median: 20.3 ng/mL (95% CI: 16.2, 24.5 ng/mL) compared with 26.7 ng/mL (95% CI: 25.2, 28.1 ng/mL), respectively; P = 0.026)], and a higher proportion of blacks had total 25(OH)D concentrations <20 ng/mL (46% compared with 19%, respectively; P < 0.001). Directly measured free 25(OH)D concentrations were lower in blacks than in whites [adjusted median: 4.5 ng/mL (95% CI: 3.7, 5.4 ng/mL) compared with 5.7 ng/mL (95% CI: 5.4, 5.9 ng/mL), respectively; P = 0.044] and were strongly correlated with total 25(OH)D without an effect of race. DBP was lower in blacks when measured by the monoclonal but not the polyclonal antibody immunoassay. Cholecalciferol supplementation increased total and measured free 25(OH)D concentrations proportionally to the dose and without a difference between races. CONCLUSIONS: The relation between free and total 25(OH)D did not vary systematically by race in this multiracial population with pre- or well-controlled diabetes. The results need to be replicated in additional cohorts before concluding that the clinical assessment of vitamin D status in blacks and whites should follow a single standard. The CaDDM and DDM2 trials were registered at clinicaltrials.gov as NCT00436475 and NCT01736865, respectively.

Psyllium fiber improves glycemic control proportional to loss of glycemic control: a meta-analysis of data in euglycemic subjects, patients at risk of type 2 diabetes mellitus, and patients being treated for type 2 diabetes mellitus.

BACKGROUND: A number of health benefits are associated with intake of soluble, viscous, gel-forming fibers, including reduced serum cholesterol and the attenuation of postprandial glucose excursions. OBJECTIVE: We assess the effects of psyllium, which is a soluble, gel-forming, nonfermented fiber supplement, on glycemic control in patients who were being treated for type 2 diabetes mellitus (T2DM) and in patients who were at risk of developing T2DM. DESIGN: A comprehensive search was performed of available published literature (Scopus scientific database) and clinical records stored by Procter & Gamble with the use of key search terms to identify clinical studies that assessed the glycemic effects of psyllium in nondiabetic, pre-T2DM, and T2DM patients. RESULTS: We identified 35 randomized, controlled, clinical studies that spanned 3 decades and 3 continents. These data were assessed in 8 meta-analyses. In patients with T2DM, multiweek studies (psyllium dosed before meals) showed significant improvement in both the fasting blood glucose (FBG) concentration (-37.0 mg/dL; P < 0.001) and glycated hemoglobin (HbA1c) [-0.97% (-10.6 mmol/mol); P = 0.048]. Glycemic effects were proportional to baseline FBG; no significant glucose lowering was observed in euglycemic subjects, a modest improvement was observed in subjects with pre-T2DM, and the greatest improvement was observed in subjects who were being treated for T2DM. CONCLUSIONS: These data indicate that psyllium would be an effective addition to a lifestyle-intervention program. The degree of psyllium's glycemic benefit was commensurate with the loss of glycemic control. Because the greatest effect was seen in patients who were being treated for T2DM, additional studies are needed to determine how best to incorporate psyllium into existing prevention and treatment algorithms with concomitant hypoglycemic medications.

Mouse handling limits the impact of stress on metabolic endpoints.

Studies focused on end-points that are confounded by stress are best performed under minimally stressful conditions. The objective of this study was to demonstrate the impact of handling designed to reduce animal stress on measurements of glucose tolerance. A cohort of mice (CD1.C57BL/6) naïve to any specific handling was subjected to either a previously described "cup" handling method, or a "tail-picked" method in which the animals were picked up by the tail (as is common for metabolic studies). Following training, an elevated plus maze (EPM) test was performed followed by measurement of blood glucose and plasma corticosterone. A second cohort (CD1.C57BL/6) was rendered obese by exposure to a high fat diet, handled with either the tail-picked or cup method and subjected to an intraperitoneal glucose tolerance test. A third cohort of C57BL/6 mice was exposed to a cup regimen that included a component of massage and was subjected to tests of anxiety-like behavior, glucose homeostasis, and corticosterone secretion. We found that the cup mice showed reduced anxiety-like behaviors in the EPM coupled with a reduction in blood glucose levels compared to mice handled by the tail-picked method. Additionally, cup mice on the high fat diet exhibited improved glucose tolerance compared to tail-picked controls. Finally, we found that the cup/massage group showed lower glucose levels following an overnight fast, and decreased anxiety-like behaviors associated with lower stress-induced plasma corticosterone concentration compared to tail-picked controls. These data demonstrate that application of handling methods that reduce anxiety-like behaviors in mice mitigates the confounding contribution of stress to interpretation of metabolic endpoints (such as glucose tolerance).

Effect of vertical sleeve gastrectomy on food selection and satiation in rats.

Vertical sleeve gastrectomy (VSG) is a restrictive procedure that reduces food intake to produce weight loss. Here we assess volume and nutrient effects on the ingestive behavior of VSG and sham surgery animals. Rats given access to Ensure or pelleted chow were used to determine if liquid foods would adversely affect weight loss after surgery. Volume effects were studied by altering the caloric density of Ensure, and dietary preferences for fat and carbohydrate (sucrose) were assessed using a two-bottle test. c-Fos was used to measure neuronal activation in the nucleus of the solitary tract and area postrema in response to intragastric infusions of water, sucrose, or Intralipid. The degree of colocalization with catecholaminergic neurons was also assessed. VSG rats did not show the expected preference for a liquid diet over chow and lacked dietary preferences for fat seen in shams. Preferences for carbohydrate/sucrose solutions were unaffected by surgery. Meal size was reduced by VSG; however, VSG rats were able to alter their volume of intake to compensate for changes in caloric density, and intragastric infusions of water produced similar levels of neuronal activation among VSG, sham, and pair-fed rats. In comparison, nutrient-induced c-Fos activation was substantially increased by VSG. Colocalization between c-Fos and catecholaminergic-expressing neurons was similar among rats treated with water, sucrose, or Intralipid. VSG alters nutrient sensing in a manner that lowers the threshold for satiety and reduces fat preference to induce and maintain weight loss.

Chylomicron formation and secretion is required for lipid-stimulated release of incretins GLP-1 and GIP.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins produced in the intestine that play a central role in glucose metabolism and insulin secretion. Circulating concentrations of GLP-1 and GIP are low and can be difficult to assay in rodents. These studies utilized the novel intestinal lymph fistula model we have established to investigate the mechanism of lipid-stimulated incretin secretion. Peak concentrations of GLP-1 and GIP following an enteral lipid stimulus (Liposyn) were significantly higher in intestinal lymph than portal venous plasma. To determine whether lipid-stimulated incretin secretion was related to chylomicron formation Pluronic L-81 (L-81), a surfactant inhibiting chylomicron synthesis, was given concurrently with Liposyn. The presence of L-81 almost completely abolished the increase in lymph triglyceride seen with Liposyn alone (P < 0.001). Inhibition of chylomicron formation with L-81 reduced GLP-1 secretion into lymph compared to Liposyn stimulation alone (P = 0.034). The effect of L-81 relative to Liposyn alone had an even greater effect on GIP secretion, which was completely abolished (P = 0.004). These findings of a dramatic effect of L-81 on lymph levels of GLP-1 and GIP support a strong link between intestinal lipid absorption and incretin secretion. The relative difference in the effect of L-81 on the two incretins provides further support that nutrient-stimulation of GIP and GLP-1 is via distinct mechanisms.

Consumption of a high-fat diet induces central insulin resistance independent of adiposity.

Plasma insulin enters the CNS where it interacts with insulin receptors in areas that are related to energy homeostasis and elicits a decrease of food intake and body weight. Here, we demonstrate that consumption of a high-fat (HF) diet impairs the central actions of insulin. Male Long-Evans rats were given chronic (70-day) or acute (3-day) ad libitum access to HF, low-fat (LF), or chow diets. Insulin administered into the 3rd-cerebral ventricle (i3vt) decreased food intake and body weight of LF and chow rats but had no effect on HF rats in either the chronic or the acute experiment. Rats chronically pair-fed the HF diet to match the caloric intake of LF rats, and with body weights and adiposity levels comparable to those of LF rats, were also unresponsive to i3vt insulin when returned to ad libitum food whereas rats pair-fed the LF diet had reduced food intake and body weight when administered i3vt insulin. Insulin's inability to reduce food intake in the presence of the high-fat diet was associated with a reduced ability of insulin to activate its signaling cascade, as measured by pAKT. Finally, i3vt administration of insulin increased hypothalamic expression of POMC mRNA in the LF- but not the HF-fed rats. We conclude that consumption of a HF diet leads to central insulin resistance following short exposure to the diet, and as demonstrated by reductions in insulin signaling and insulin-induced hypothalamic expression of POMC mRNA.

The intestinal lymph fistula model--a novel approach to study ghrelin secretion.

The orexigenic hormone ghrelin is secreted from the stomach and has been implicated in the regulation of energy and glucose homeostasis. We hypothesized that ghrelin, like other gastrointestinal (GI) hormones, is present in intestinal lymph, and sampling this compartment would provide advantages for studying ghrelin secretion in rodents. Blood and lymph were sampled from catheters in the jugular vein and mesenteric lymph duct before and after intraduodenal (ID) administration of isocaloric Ensure, dextrin, or Liposyn meals or an equal volume of saline in conscious Sprague-Dawley rats. Total ghrelin levels were measured using an established radioimmunoassay. Acyl and des-acyl ghrelin were measured using two-site ELISA. Fasting ghrelin levels in lymph were significantly higher than in plasma (means +/- SE: 3,307.9 +/- 272.9 vs. 2,127.1 +/- 115.0 pg/ml, P = 0.004). Postingestive acyl and des-acyl ghrelin levels were also significantly higher, whereas the ratio of acyl:des-acyl ghrelin was similar in lymph and plasma (0.91 +/- 0.28 vs. 1.20 +/- 0.36, P = 0.76). The principle enzymes responsible for deacylation of ghrelin were lower in lymph than in plasma. Following ID Ensure, maximum ghrelin suppression occurred at 2 h in lymph compared with at 1 h in plasma. The return of suppressed ghrelin levels to baseline was also delayed in lymph. Similarly, dextrin also induced significant suppression of ghrelin (two-way ANOVA: P = 0.02), whereas Liposyn did not (P = 0.32). On the basis of these findings, it appears that intestinal lymph, which includes drainage from the interstitium of the GI mucosa, is enriched in ghrelin. Despite reduced deacylating activity in lymph, there is not a disproportionate amount of acyl ghrelin in this pool. The postprandial dynamics of ghrelin are slower in lymph than plasma, but the magnitude of change is greater. Assessing ghrelin levels in the lymph may be advantageous for studying its secretion and concentrations in the gastric mucosa.

Using the lymph fistula rat model to study the potentiation of GIP secretion by the ingestion of fat and glucose.

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin produced in the K cells of the intestine and secreted into the circulating blood following ingestion of carbohydrate- and fat-containing meals. GIP contributes to the regulation of postprandial insulin secretion and is essential for normal glucose tolerance. We have established a method of assaying GIP in response to nutrients using the intestinal lymph fistula model. Administration of Ensure, a mixed-nutrient liquid meal, stimulated a significant increase in intestinal lymphatic GIP levels that were approximately threefold those of portal plasma. Following the meal, lymph GIP peaked at 60 min (P < 0.001) and remained elevated for 4 h. Intraduodenal infusions of isocaloric and isovolumetric lipid emulsions or glucose polymer induced lymph GIP concentrations that were four and seven times the basal levels, respectively. The combination of glucose plus lipid caused an even greater increase of lymph GIP than either nutrient alone. In summary, these findings demonstrated that intestinal lymph contains high concentrations of GIP that respond to both enteral carbohydrate and fat absorption. The change in lymphatic GIP concentration is greater than the change observed in the portal blood. These studies allow the detection of GIP levels at which they exert their local physiological actions. The combination of glucose and lipid has a potentiating effect in the stimulation of GIP secretion. We conclude from these studies that the lymph fistula rat is a novel approach to study in vivo GIP secretion in response to nutrient feeding in conscious rats.

Apolipoprotein A-IV interacts synergistically with melanocortins to reduce food intake.

Apolipoprotein (apo) A-IV is an anorexigenic gastrointestinal peptide that is also synthesized in the hypothalamus. The goal of these experiments was to determine whether apo A-IV interacts with the central melanocortin (MC) system in the control of feeding. The third ventricular (i3vt) administration of a subthreshold dose of apo A-IV (0.5 microg) potentiated i3vt MC-induced (metallothionein-II, 0.03 nmol) suppression of 30-min feeding in Long-Evans rats. A subthreshold dose of the MC antagonist (SHU9119, 0.1 nmol, i3vt) completely attenuated the anorectic effect of i3vt apo A-IV (1.5 microg). The i3vt apo A-IV significantly elevated the expression of c-Fos in neurons of the paraventricular nucleus of the hypothalamus, but not in the arcuate nucleus or median eminence. In addition, c-Fos expression was not colocalized with proopiomelanocortin-positive neurons. These data support a synergistic interaction between apo A-IV and melanocortins that reduces food intake by acting downstream of the arcuate.

Obesity induced by a high-fat diet downregulates apolipoprotein A-IV gene expression in rat hypothalamus.

Apolipoprotein A-IV (apo A-IV) is an anorectic protein produced in the intestine and brain that has been proposed as a satiety signal. To determine whether diet-induced obesity alters apo A-IV gene expression in the intestine and hypothalamus, rats were fed a high-fat (HF), low-fat (LF), or standard chow (CHOW) diet for 2, 4, 6, 8, or 10 wk. Rats fed the HF diet had significantly greater body weights than rats given the LF and CHOW diets. Intestinal and plasma apo A-IV levels were comparable across dietary groups and time. LF and CHOW rats had comparable hypothalamic apo A-IV mRNA across the course of the experiment. However, HF rats had a slow and progressive diminution in hypothalamic apo A-IV mRNA over time that became significantly lower than that of LF or CHOW rats by 10 wk. Intragastric infusion of lipid emulsion to animals that were fasted overnight significantly stimulated hypothalamic apo A-IV mRNA in LF and CHOW rats but had no effect in HF rats. These results demonstrate that chronic consumption of a HF diet significantly reduces apo A-IV mRNA levels and the response of apo A-IV gene expression to dietary lipids in the hypothalamus. This raises the possibility that dysregulation of hypothalamic apo A-IV could contribute to diet-induced obesity.