Karen Kresge

Differential effects of sham feeding and meal ingestion on ghrelin and pancreatic polypeptide levels: evidence for vagal efferent stimulation mediating ghrelin release1

Abstract  Ghrelin has been suggested to function as an appetite‐stimulating signal from the gastrointestinal tract to the brain acting through a vagal afferent pathway. Ghrelin levels rise before meals and fall after meal ingestion. The purpose of this study was to investigate factors which regulate ghrelin release into the circulation by determining changes in systemic ghrelin concentrations after sham feeding and meal ingestion. Methods: Fifteen normal subjects underwent sham feeding of a bacon and cheese toasted sandwich. Serial blood samples were obtained before and every 5 min for another 30 min during sham feeding and for 30 min after actual meal ingestion. Radioimmunoassay was used to measure plasma ghrelin and pancreatic polypeptide concentrations. Results: During sham feeding, plasma ghrelin concentration increased from 1730 ± 237 to 1917 ± 269 pg/mL (P < 0.05) and plasma pancreatic polypeptide increased from 417 ± 50 to 841 ± 97 pg/mL (P < 0.01). Subsequent meal ingestion was characterized by an increase in pancreatic polypeptide from 782 ± 88 to 1710 ± 119 pg/mL (P < 0.01), but no significant change in ghrelin levels. Conclusions: Plasma ghrelin and pancreatic polypeptide concentrations increase with sham feeding. This suggests a vagal efferent pathway mediating ghrelin release. In contrast to pancreatic polypeptide which rises with actual meal ingestion, ghrelin levels did not change.

In Vivo Effects of Insulin and Free Fatty Acids on Matrix Metalloproteinases in Rat Aorta

OBJECTIVE—Obesity is associated with insulin resistance, hyperinsulinemia, elevated plasma free fatty acid (FFA), and increased risk for atherosclerotic vascular disease (ASVD). A part of this increased risk may be due to enhanced activation of matrix metalloproteinases (MMPs). Here, we have examined the effects of physiologically elevated levels of insulin and FFA on three MMPs and their inhibitors (tissue inhibitors of MMP [TIMPs]) in aortic tissue of male rats during euglycemic-hyperinsulinemic clamping. RESEARCH DESIGN AND METHODS—Four-hour euglycemic-hyperinsulinemic clamps with infusion of saline/glycerol, lipid/heparin, or insulin with or without lipid/heparin were performed in alert unrestrained male rats. RESULTS—Hyperinsulinemia increased MMP-2 (∼6-fold), MMP-9 (∼13-fold), membrane type 1-MMP (MT1-MMP; ∼8-fold) (all Western blots), and gelatinolytic activity (zymography) of MMP-2 (2-fold), while not affecting TIMP-1 and TIMP-2. Insulin increased IRS-1–associated PI 3-kinase (PI3K), extracellular signal–regulated kinases 1/2 (ERK1/2), and c-jun NH2-terminal kinase (JNK) (by Western blots with phospho-specific antibodies). FFA augmented the insulin-mediated increases in MMP-2 (from ∼6- to ∼11-fold), MMP-9 (from ∼3- to ∼23-fold), MT1-MMP (from ∼8- to ∼20-fold), MMP-2 gelatinolytic activity (from 2- to 3-fold), and JNK and p38 mitogen-activated protein kinase (MAPK) activities but decreased insulin-mediated activation of PI3K and ERK1/2. Raising FFA without raising insulin affected neither MMPs nor TIMPs. CONCLUSIONS—FFA augmented insulin stimulation of the MMP/TIMP balance of three proatherogenic MMPs and increased activities of two MAPKs (JNK and p38 MAPK), both of which are known to stimulate the production of proinflammatory cytokines. This may, over time, increase degradation of extracellular matrix and together with inflammatory changes promote development of ASVD.

Combined Use of Rosiglitazone and Fenofibrate in Patients With Type 2 Diabetes Prevention of Fluid Retention

Elevated plasma free fatty acid (FFA) levels are responsible for much of the insulin resistance in obese patients with type 2 diabetes. To lower plasma FFA levels effectively and long term, we have treated eight obese patients with type 2 diabetes for 2 months with placebo followed by 2 months of treatment with a combination of rosiglitazone (RGZ) (8 mg/day) and fenofibrate (FFB) (160 mg/day) in a single-blind placebo-controlled study design. Compared with placebo, RGZ/FFB lowered mean 24-h plasma FFA levels 30% (P < 0.03) and mean 24-h glucose levels 23% (P < 0.03) and increased insulin-stimulated glucose uptake (glucose rate of disappearance [GRd], determined using euglycemic-hyperinsulinemic clamp) 442% (P < 0.01), oral glucose tolerance (area under the curve for 3-h oral glucose tolerance test) 28% (P < 0.05), and plasma adiponectin levels 218% (P < 0.01). These RGZ/FFB results were compared with results obtained in five patients treated with RGZ alone. RGZ/FFB prevented the fluid retention usually associated with RGZ (−1.6 vs. 5.6%, P < 0.05), lowered fasting plasma FFA more effectively than RGZ alone (−22 vs. 5%, P < 0.05), and tended to be more effective than RGZ alone in lowering A1C (−0.9 vs. −0.4%) and triglyceride levels (−38 vs. −5%) and increasing GRd (442 vs. 330%). We conclude that RGZ/FFB is a promising new therapy for type 2 diabetes that lowers plasma FFA more than RGZ alone and in contrast to RGZ does not cause water retention and weight gain.

Abnormal Ghrelin and Pancreatic Polypeptide Responses in Gastroparesis

Vagal nerve dysfunction has been implicated in the pathogenesis of diabetic gastroparesis, but its role in idiopathic gastroparesis remains uncertain. The incre-ase in pancreatic polypeptide with sham feeding is often used as a measure of vagal integrity. Ghrelin has been suggested to function as an appetite-stimulating hormone from the gut to the brain acting through vagal afferent pathways. Systemic ghrelin also rises in part due to vagal efferent pathways. Alterations in ghrelin and its effects on appetite could play a role in gastroparesis. In this study we aimed [1] to investigate the presence of vagal nerve dysfunction in patients with idiopathic and diabetic gastroparesis and [2] to determine if alterations in ghrelin concentrations occur in gastroparesis. Normal subjects and patients with diabetic, idiopathic, or postsurgical gastroparesis underwent a sham feeding protocol. Serial blood samples were obtained for plasma ghrelin and pancreatic polypeptide. Sham feeding was characterized by an increase in pancreatic polypeptide and ghrelin in normal controls and patients with idiopathic gastroparesis. The changes in pancreatic polypeptide and ghrelin levels in diabetic and postsurgical gastroparesis were significantly less than those in normal subjects. Vagal nerve dysfunction, as evidenced by an impaired pancreatic polypeptide response with sham feeding, is present in diabetic gastroparesis but not idiopathic gastroparesis. Systemic ghrelin concentrations increased with sham feeding in normal subjects and patients with idiopathic gastroparesis but not in diabetic or postsurgical gastroparesis. Vagal function and regulation of ghrelin levels are impaired in diabetic gastroparesis.

Infusion of glucose and lipids at physiological rates causes acute endoplasmic reticulum stress in rat liver.

Endoplasmic reticulum (ER) stress has recently been implicated as a cause for obesity‐related insulin resistance; however, what causes ER stress in obesity has remained uncertain. Here, we have tested the hypothesis that macronutrients can cause acute (ER) stress in rat liver. Examined were the effects of intravenously infused glucose and/or lipids on proximal ER stress sensor activation (PERK, eIF2‐α, ATF4, Xbox protein 1 (XBP1s)), unfolded protein response (UPR) proteins (GRP78, calnexin, calreticulin, protein disulphide isomerase (PDI), stress kinases (JNK, p38 MAPK) and insulin signaling (insulin/receptor substrate (IRS) 1/2 associated phosphoinositol‐3‐kinase (PI3K)) in rat liver. Glucose and/or lipid infusions, ranging from 23.8 to 69.5 kJ/4 h (equivalent to between ∼17% and ∼50% of normal daily energy intake), activated the proximal ER stress sensor PERK and ATF6 increased the protein abundance of calnexin, calreticulin and PDI and increased two GRP78 isoforms. Glucose and glucose plus lipid infusions induced comparable degrees of ER stress, but only infusions containing lipid activated stress kinases (JNK and p38 MAPK) and inhibited insulin signaling (PI3K). In summary, physiologic amounts of both glucose and lipids acutely increased ER stress in livers 12‐h fasted rats and dependent on the presence of fat, caused insulin resistance. We conclude that this type of acute ER stress is likely to occur during normal daily nutrient intake.

Effects of hyperinsulinemia on hepatic metalloproteinases and their tissue inhibitors

To gain insight into the pathogenesis of hepatic fibrosis related to insulin resistance, we have examined the effects of euglycemic hyperinsulinemia on three matrix metalloproteinases (MMP-2, MMP-9, and MT1-MMP) and on two major tissue inhibitors of MMPs (TIMP-1 and TIMP-2) in liver of insulin-sensitive and insulin-resistant rats. Four hours of insulin infusion (4.8 mU.kg(-1).min(-1)) without or with lipid-heparin infusion (to produce insulin resistance) decreased hepatic MMP-2 mRNA (by RT-PCR), pro-MMP-2, MMP-2, MMP-9, and MT1-MMP (all by Western blots) and the gelatinolytic activity of MMP-2 (by gelatin zymography) by approximately 60-80%. Hyperinsulinemia ( approximately 1.6 mmol/l) increased TIMP-1 and TIMP-2 concentrations (by ELISA) in insulin-sensitive and insulin-resistant rats. Phosphoinositide 3-kinase was activated by insulin in insulin-sensitive rats and inhibited in insulin-resistant rats. Extracellular signal-regulated kinases 1/2 (ERK1/2) were activated by insulin in insulin-sensitive rats and partially inhibited in insulin-resistant rats; c-jun NH(2)-terminal kinase-1 (JNK1), JNK2/3, or p38 MAPK were only activated by lipid but not by insulin. We conclude that hyperinsulinemia, whether or not associated with insulin resistance, shifts the MMP/TIMP balance toward reduction of extracellular matrix degradation and thus may promote the development of hepatic fibrosis.

Insulin Resistance is Associated with Diminished Endoplasmic Reticulum Stress Responses in Adipose Tissue of Healthy and Diabetic Subjects

We recently showed that insulin increased ER stress in human adipose tissue. The effect of insulin resistance on ER stress is not known. It could be decreased, unchanged, or increased, depending on whether insulin regulates ER stress via the metabolic/phosphoinositide 3-kinase (PI3K) or alternate signaling pathways. To address this question, we examined effects of lipid-induced insulin resistance on insulin stimulation of ER stress. mRNAs of several ER stress markers were determined in fat biopsies obtained before and after 8-h hyperglycemic-hyperinsulinemic clamping in 13 normal subjects and in 6 chronically insulin-resistant patients with type 2 diabetes mellitus (T2DM). In normal subjects, hyperglycemia-hyperinsulinemia increased after/before mRNA ratios of several ER stress markers (determined by ER stress pathway array and by individual RT-PCR). Lipid infusion was associated with inhibition of the PI3K insulin-signaling pathway and with a decrease of hyperinsulinemia-induced ER stress responses. In chronically insulin-resistant patients with T2DM, hyperglycemic-hyperinsulinemia did not increase ER stress response marker mRNAs. In summary, insulin resistance, either produced by lipid infusions in normal subjects or chronically present in T2DM patients, was associated with decreased hyperinsulinemia-induced ER stress responses. This suggests, but does not prove, that these two phenomena were causally related.

A Single A1C >= 6.5% Accurately Identifies Type 2 Diabetes/Impaired Glucose Tolerance in African Americans.

Objective: In 2010, the American Diabetes Association revised its criteria for the diagnosis of diabetes to include A1C ≥ 6.5%; however, this has remained controversial, particularly for African Americans. The objective of this pilot study was to examine the usefulness of a single A1C determination in comparison with a same day 2-hour oral glucose tolerance test to diagnose type 2 diabetes in African Americans. Methods: In sum, 195 oral glucose tolerance tests and A1Cs were obtained on the same day from 77 overweight and obese African American women and 6 men over a period of 15 months. Results: A1C ≥ 6.5% was present in 31 of 195 patients, with 15 of these having type 2 diabetes by oral glucose tolerance test, another 12 having impaired glucose tolerance, and 4 having normal glucose tolerance. This gives a sensitivity of 50% and a specificity of 90%, with a positive predictive value of 48% and a negative predictive value of 91%. A1C ≤ 5.6%, proposed by the American Diabetes Association to indicate normal glucose tolerance, was present in only 28 patients, 10 (35.7%) of whom had normal glucose tolerance, whereas 18 (64.3%) had either impaired glucose tolerance (15 patients) or type 2 diabetes (3 patients). Fasting plasma glucose ≥ 126 mg/dL was present in 5 of 29 patients with type 2 diabetes (sensitivity, 17.2%; specificity, 100%). Conclusions: First, A1C ≥ 6.5% was a good “rule in” value to identify impaired glucose tolerance and type 2 diabetes (ie, patients at high risk for micro- and macrovascular complications). Second, A1C ≤ 5.6% did not rule out impaired glucose tolerance or type 2 diabetes. Last, fasting plasma glucose ≥ 126 mg/dL detected less than 1 in 5 cases with type 2 diabetes.