Hyperglycemic Hyperinsulinemic Clamp Research Articles

Improved Afternoon Hepatic Glucose Disposal and Storage Requires Morning Engagement of Hepatic Insulin Receptors.

Glucose tolerance improves significantly upon consuming a second, identical meal later in the day (second meal phenomenon). We previously established that morning hyperinsulinemia primes the liver for increased afternoon hepatic glucose uptake (HGU). Although the route of insulin delivery is an important determinant of the mechanisms by which insulin regulates liver glucose metabolism (direct hepatic vs indirect insulin action), it is not known if insulin's delivery route affects the second meal response. To determine whether morning peripheral insulin delivery (as occurs clinically, i.e. subcutaneous) can enhance afternoon HGU, conscious dogs were treated in the morning with insulin delivered either via the portal vein or peripherally (leg vein), while glucose was infused to maintain euglycemia. Consequently, arterial insulin levels increased similarly in both groups, but relative hepatic insulin deficiency occurred with peripheral insulin delivery. In the afternoon, all animals were challenged with the same hyperinsulinemichyperglycemic clamp to simulate identical postprandial-like conditions. The substantial enhancement of HGU in the afternoon caused by morning portal vein insulin delivery was lost when insulin was delivered peripherally. This indicates that morning insulin does not cause the second meal phenomenon via its indirect actions on the liver, but rather through direct activation of hepatic insulin signaling.

Duration of morning hyperinsulinemia determines hepatic glucose uptake and glycogen storage later in the day.

The second-meal phenomenon refers to the improvement in glucose tolerance seen following a second identical meal. We previously showed that 4 h of morning hyperinsulinemia, but not hyperglycemia, enhanced hepatic glucose uptake (HGU) and glycogen storage during an afternoon hyperinsulinemic-hyperglycemic (HIHG) clamp. Our current aim was to determine if the duration or pattern of morning hyperinsulinemia is important for the afternoon response to a HIHG clamp. To determine this, the same total amount of insulin was administered either over 2 h in the first (Ins2h-A) or second (Ins2h-B) half of the morning or over the entire 4 h (Ins4h) of the morning. In the 4-h afternoon period, all three groups had 4x-basal insulin, 2x-basal glycemia, and portal glucose infusion to expose the liver to the primary postprandial regulators of hepatic glucose metabolism. During the afternoon clamp, there was a marked increase in HGU and hepatic glycogen synthesis in the Ins4h group compared with the Ins2h-A and Ins2h-B groups, despite matched hepatic glucose loads and total insulin infusion rates. Thus, the longer duration (Ins4h) of lower hyperinsulinemia in the morning seems to be the key to much greater liver glucose uptake during the afternoon clamp.NEW & NOTEWORTHY Morning insulin exposure primes the liver for increased hepatic glucose uptake and glycogen storage during a subsequent hyperinsulinemic-hyperglycemic clamp. This study addressed whether the pattern and/or duration of insulin delivery in the morning influences insulin's ensuing priming effect. We found that despite receiving equal total doses of insulin in the morning, a prolonged, lower rate of morning insulin delivery improved afternoon liver glucose metabolism more effectively than a shorter, higher rate of delivery.

1600-P: Duration of Morning Hyperinsulinemia Is Key to the Enhancement of Hepatic Glucose Uptake Later in the Day

The second meal phenomenon, which occurs in normal and diabetic individuals, refers to the improvement in glucose tolerance seen following a second identical meal. We previously showed that 4h of morning (AM) hyperinsulinemia, but not hyperglycemia, enhanced hepatic glucose uptake (HGU) and glycogen storage during a PM hyperinsulinemic hyperglycemic clamp (HIHG) later that day. Our aim was to determine if the duration of morning hyperinsulinemia is important for the PM response to a HIHG clamp. To determine this, we administered the same amount of insulin over 2h in the first half of the morning (Ins2h-A), over 2h in the 2nd half of the morning (Ins2h-B), or over the entire 4h (Ins4h) of the morning. We then assessed the impact of the AM insulin duration on HGU during a PM HIHG clamp in conscious dogs. The dogs underwent an AM hyperinsulinemic euglycemic clamp from 0-120 min, 120-240 min, or 0-240 min (Ins2h-A, Ins2h-B, or Ins4h; n=6/group). The insulin infusion rates used in the Ins4h were selected to mimic the rise in endogenous insulin secretion previously observed during a 4h AM duodenal glucose infusion (2.1 mU/kg/min (0-30 min), 2.4 mU/kg/min (30-60 min), and 1.5 mU/kg/min (60-240 min)). These rates were doubled in both 2h groups to match the total amount of insulin being infused in the 4h AM clamp. In the PM, all groups had 4x basal insulin, 2x basal glycemia, and portal glucose infusion to simulate a 2nd meal. During the 4h PM clamp, there were no significant differences between the two 2h groups. However, there was a marked effect on the mean HGU in the Ins4h group compared to the Ins2h-A and Ins2h-B groups (HGU of 6.3±0.9 vs 3.8±0.3 and 4.4±0.4 mg/kg/min; AUC of 1393±199 vs 822±68 and 971±93 mg/kg/4hr, P<0.05), respectively, despite matched hepatic glucose loads. The longer duration (Ins4h) of AM hyperinsulinemia allowed for a 41% and 66% greater uptake of glucose in the PM clamp (vs Ins2hA + B). Thus, the duration of AM hyperinsulinemia is an important determinant of hepatic metabolism later in the day. Disclosure H.L.Waterman: None. M.S.Smith: None. B.Farmer: None. G.Kraft: None. M.Scott: None. D.S.Edgerton: None. A.D.Cherrington: Advisory Panel; Metavention, vTv Therapeutics, Diakard, Sekkei Bio, Sensulin Labs, LLC, Other Relationship; Fractyl Health, Inc., Novo Nordisk, Abvance Therapeutics, Research Support; Cellular Longevity, Inc, dba Loyal, Senda Biosciences. M.C.Moore: None. Funding National Institutes of Health (5T32DK007563-34, 5R01DK131082-02)

205-OR: Hepatic Protein Kinase C-e Is Necessary and Sufficient in Mediating Lipid-Induced Hepatic Insulin Resistance

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with hepatic insulin resistance (HIR), however the key lipid species and molecular mechanisms linking these conditions are widely debated. In order to address these questions, we examined the metabolic impact of a liver-specific antisense oligonucleotide against PKCε and found that liver-specific PKCε knockdown ameliorated high-fat diet-induced HIR in rats, reflected by ∼2x increase in insulin-stimulated hepatic glycogen synthesis rate and improved insulin-mediated suppression of endogenous glucose production during a hyperinsulinemic-hyperglycemic (HH) clamp. This was associated with increased IRK Y1162 and Akt S473 phosphorylation and could be attributed to decreased PKCε-mediated IRK T1160 phosphorylation. Overexpressing a constitutively active PKCε (A159E) in the liver induced HIR in regular chow-fed rats, reflected by reduced insulin-stimulated glycogen synthesis rate during an HH clamp. This was associated with impaired IRK Y1162 and Akt S473 phosphorylation and could be attributed to increased PKCε-mediated IRK T1160 phosphorylation. We also developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum, mitochondria, plasma membrane (PM), lipid droplet and cytosol and applied this method to a cohort of obese human subjects with and without NAFLD and HIR. Liver PM sn-1,2 DAG content was ∼5x higher in HIR individuals with NAFLD compared to insulin-sensitive individuals without NAFLD, associated with ∼3x higher IRK T1160 phosphorylation, supporting the importance of PM sn-1,2 DAG-PKCε-IRK pT1160 pathway to mediate HIR in humans with NAFLD. In contrast there were no significant differences in ceramide content in any of the subcellular compartments. Conclusion: These data identify PM sn-1,2 DAGs as the key lipids that activate PKCε, and that hepatic PKCε is both necessary and sufficient in mediating HIR. Disclosure K. Lyu: None. D. Zhang: None. M. Kahn: None. M.R.S. Rodrigues: None. S. Hirabara: None. P. Luukkonen: None. S. Lee: None. S. Bhanot: None. J. Rinehart: None. N. Blume: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M. Rasch: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M.J. Serlie: None. J. Bogan: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. Funding U.S. Public Health Service (R01DK116774, R01DK119968, R01DK113984, P30DK045735, R01 DK092661); U.S. Department of Veternas Affairs (I01BX000901)

MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

GIP-induced vasodilation in human adipose tissue involves capillary recruitment.

Glucose-dependent insulinotropic polypeptide (GIP) in combination with hyperinsulinemia increase blood flow and triglyceride clearance in subcutaneous abdominal adipose tissue in lean humans. The present experiments were performed to determine whether the increase involves capillary recruitment. Eight lean healthy volunteers were studied before and after 1 h infusion of GIP or saline during a hyperglycemic–hyperinsulinemic clamp, raising plasma glucose and insulin to postprandial levels. Subcutaneous abdominal adipose tissue blood flow (ATBF) was measured by the 133Xenon clearance technique, and microvascular blood volume was determined by contrast-enhanced ultrasound imaging. During infusion of saline and the clamp, both ATBF (2.7 ± 0.5 mL/min 100 g/tissue) and microvascular blood volume remained unchanged throughout the experiments. During GIP infusion and the clamp, ATBF increased ~fourfold to 11.4 ± 1.9 mL/min 100 g/tissue, P < 0.001. Likewise, the contrast-enhanced ultrasound signal intensity, a measure of the microvascular blood volume, increased significantly 1 h after infusion of GIP and the clamp (P = 0.003), but not in the control experiments. In conclusion, the increase in ATBF during GIP infusion involves recruitment of capillaries in healthy lean subjects, which probably increases the interaction of circulating lipoproteins with lipoprotein lipase, thus promoting adipose tissue lipid uptake.

Sympathetic Denervation of the Common Hepatic Artery Lessens Glucose Intolerance in the Fat- and Fructose-Fed Dog.

This study assessed the effectiveness of surgical sympathetic denervation of the common hepatic artery (CHADN) in improving glucose tolerance. CHADN eliminated norepinephrine content in the liver and partially decreased it in the pancreas and the upper gut. We assessed oral glucose tolerance at baseline and after 4 weeks of high-fat high-fructose (HFHF) feeding. Dogs were then randomized to sham surgery (SHAM) (n = 9) or CHADN surgery (n = 11) and retested 2.5 or 3.5 weeks later while still on the HFHF diet. CHADN improved glucose tolerance by ∼60% in part because of enhanced insulin secretion, as indicated by an increase in the insulinogenic index. In a subset of dogs (SHAM, n = 5; CHADN, n = 6), a hyperinsulinemic-hyperglycemic clamp was used to assess whether CHADN could improve hepatic glucose metabolism independent of a change in insulin release. CHADN reduced the diet-induced defect in net hepatic glucose balance by 37%. In another subset of dogs (SHAM, n = 4; CHADN, n = 5) the HFHF diet was continued for 3 months postsurgery and the improvement in glucose tolerance caused by CHADN continued. In conclusion, CHADN has the potential to enhance postprandial glucose clearance in states of diet-induced glucose intolerance.

Regulation of Lipid Regulator ANGPTL8 Gene Transcript by Insulin Is Mediated by C/EBPß, and ANGPTL8 Deficiency Improves Glucose Homeostasis in Mice

Angiopoietin-like protein 8 (ANGPTL8) is a novel circulating protein synthesized by the liver and adipose tissue and plays a key role in regulating the plasma lipid profiles. Its expression is regulated acutely by fasting and refeeding in humans and animals. However, the molecular and physiological mechanisms, which regulate its expression, remain largely unknown. In this study, we investigated the regulation of ANPTL8 by insulin and glucose in multiple cellular and animal systems. Interestingly, insulin alone rapidly and dose-dependently increased ANGPTL8 gene expression in both cultured hepatocytes and mature adipocytes, while high glucose combined with insulin presented further stimulation on ANGPTL8 only in adipocytes. In mice, 2-hour euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic clamps markedly stimulated ANGPTL8 mRNA levels in liver to a similar extent. However, only combined hyperglycemia-/hyperinsulinemia caused a significant elevation of ANGPTL8 gene transcript in mouse adipose tissue. These findings imply that distinct pathways are responsible for the effects of insulin and glucose on ANGPTL8 expression in liver and adipose tissue. By cloning the ANGPTL8 gene promoter fragment into a luciferase reporter vector, we discovered that insulin-induced ANGPTL8 gene transcript was mediated by the CCAAT/enhancer-binding protein beta (C/EBPβ) transcription factor, which is likely through Janus Kinase 2 (JAK2) pathway. We also observed that AMPK activation had an inhibitory effect on insulin-induced ANGPTL8 gene expression; this could explain, in part, the low ANGPTL8 levels during fasting. Finally, in mice ANGPTL8 knockout did not alter body weight or food intake compared to wild type littermates, but significantly improved glucose tolerance in diet induced obese mice. This beneficial effect on glucose tolerance most likely is explained by the reduction in serum FFA and triglyceride levels. Disclosure L. Zhang: None. M. Abdul-Ghani: None. R.A. DeFronzo: Speaker's Bureau; Self; AstraZeneca, Novo Nordisk Inc.. Advisory Panel; Self; AstraZeneca, Novo Nordisk Inc., Janssen Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Elcelyx Therapeutics, Inc., Intarcia Therapeutics, Inc.. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Takeda Pharmaceuticals U.S.A., Inc.. L. Norton: None.

Morning Hyperinsulinemia Primes the Liver for Glucose Uptake and Glycogen Storage Later in the Day.

We observed that a 4-h morning (AM) duodenal infusion of glucose versus saline doubled hepatic glucose uptake (HGU) and storage during a hyperinsulinemic-hyperglycemic (HIHG) clamp that afternoon (PM). To separate the effects of AM hyperglycemia versus AM hyperinsulinemia on the PM response, we used hepatic balance and tracer ([3-3H]glucose) techniques in conscious dogs. From 0 to 240 min, dogs underwent a euinsulinemic-hyperglycemic (GLC; n = 7) or hyperinsulinemic-euglycemic (INS; n = 8) clamp. Tracer equilibration and basal sampling occurred from 240 to 360 min, followed by an HIHG clamp (360-600 min; four times basal insulin, two times basal glycemia) with portal glucose infusion (4 mg ⋅ kg-1 ⋅ min-1). In the HIHG clamp, HGU (5.8 ± 0.9 vs. 3.3 ± 0.3 mg ⋅ kg-1 ⋅ min-1) and net glycogen storage (6.0 ± 0.8 vs. 2.9 ± 0.5 mg ⋅ kg-1 ⋅ min-1) were approximately twofold greater in INS than in GLC. PM hepatic glycogen content (1.9 ± 0.2 vs. 1.3 ± 0.2 g/kg body weight) and glycogen synthase (GS) activity were also greater in INS versus GLC, whereas glycogen phosphorylase (GP) activity was reduced. Thus AM hyperinsulinemia, but not AM hyperglycemia, enhanced the HGU response to a PM HIHG clamp by augmenting GS and reducing GP activity. AM hyperinsulinemia can prime the liver to extract and store glucose more effectively during subsequent same-day meals, potentially providing a tool to improve glucose control.

The Gluco- and Liporegulatory and Vasodilatory Effects of Glucose-Dependent Insulinotropic Polypeptide (GIP) Are Abolished by an Antagonist of the Human GIP Receptor.

A truncated form of human glucose-dependent insulinotropic polypeptide (GIP), GIP(3-30)NH2, was recently identified as an antagonist of the human GIP receptor. This study examined the ability of GIP(3-30)NH2 to antagonize the physiological actions of GIP in glucose metabolism, subcutaneous abdominal adipose tissue blood flow (ATBF), and lipid metabolism in humans. Eight lean subjects were studied by measuring arteriovenous concentrations of metabolites and ATBF on three different occasions during hyperglycemic-hyperinsulinemic clamps with concomitant infusions of GIP, GIP(3-30)NH2, or both GIP and GIP(3-30)NH2 During infusion of GIP(3-30)NH2 alone and in combination with GIP, insulin levels and the total glucose amount infused to maintain the clamp were lower than during GIP alone. In addition, ATBF remained constant during the antagonist and increased only slightly in combination with GIP, whereas it increased fivefold during GIP alone. Adipose tissue triacylglyceride (TAG) and glucose uptake decreased, and the free fatty acid/glycerol ratio increased during the antagonist alone and in combination with GIP. The changes in glucose infusion rates and plasma insulin levels demonstrate an inhibitory effect of the antagonist on the incretin effect of GIP. In addition, the antagonist inhibited GIP-induced increase in ATBF and decreased the adipose tissue TAG uptake, indicating that GIP also plays a crucial role in lipid metabolism.

Correcting Postprandial Hyperglycemia in Zucker Diabetic Fatty Rats With an SGLT2 Inhibitor Restores Glucose Effectiveness in the Liver and Reduces Insulin Resistance in Skeletal Muscle.

Ten-week-old Zucker diabetic fatty (ZDF) rats at an early stage of diabetes embody metabolic characteristics of obese human patients with type 2 diabetes, such as severe insulin and glucose intolerance in muscle and the liver, excessive postprandial excursion of plasma glucose and insulin, and a loss of metabolic flexibility with decreased lipid oxidation. Metabolic flexibility and glucose flux were examined in ZDF rats during fasting and near-normal postprandial insulinemia and glycemia after correcting excessive postprandial hyperglycemia using treatment with a sodium–glucose cotransporter 2 inhibitor (SGLT2-I) for 7 days. Preprandial lipid oxidation was normalized, and with fasting, endogenous glucose production (EGP) increased by 30% and endogenous glucose disposal (E-Rd) decreased by 40%. During a postprandial hyperglycemic-hyperinsulinemic clamp after SGLT2-I treatment, E-Rd increased by normalizing glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake; activation of glucokinase was restored and insulin action was improved, stimulating muscle glucose uptake in association with decreased intracellular triglyceride content. In conclusion, SGLT2-I treatment improves impaired glucose effectiveness in the liver and insulin sensitivity in muscle by eliminating glucotoxicity, which reinstates metabolic flexibility with restored preprandial lipid oxidation and postprandial glucose flux in ZDF rats.

Insulin Plays a Permissive Role for the Vasoactive Effect of GIP Regulating Adipose Tissue Metabolism in Humans.

Glucose-dependent insulinotropic polypeptide (GIP) in combination with hyperinsulinemia increases blood flow and triglyceride (TAG) clearance in subcutaneous (sc) abdominal adipose tissue in lean humans. The present experiments were performed to further investigate the role of insulin for the vasoactive effect of GIP in adipose tissue metabolism and whether the vasodilatory effect of GIP is dependent on C-peptide. Six lean healthy subjects were studied. The sc abdominal adipose tissue metabolism was assessed by Fick's principle during GIP infusion (1.5 pmol/kg/min) in combination with 1) euglycemic-high insulinemic clamp (Eugluc-Hiinsu), raising plasma insulin concentrations to postprandial levels, 2) hyperglycemic-euinsulinemic clamp (Hygluc-Euinsu), and 3) hyperglycemic-hyperinsulinemic clamp, raising plasma insulin concentrations to supraphysiological levels. During the hyperglycemic clamps, endogenous insulin and C-peptide secretion were inhibited by infusion of the somatostatin analogue octreotide. During GIP infusion, Eugluc-Hiinsu, and hyperglycemic-hyperinsulinemic clamps, sc abdominal adipose tissue blood flow (ATBF) was similar and increased from 2.1 ± 0.2 and 2.2 ± 0.4 ml min(-1) (100 g tissue)(-1) to 7.1 ± 0.6 and 7.6 ± 0.1 ml min(-1) (100 g tissue)(-1), respectively (P < .01). ATBF remained virtually constant (2.7 ± 0.4 ml min(-1) [100 g tissue](-1)) during Hygluc-Euinsu and GIP infusion. In addition, adipose tissue TAG clearance increased significantly (P = .03), whereas free fatty acid output (P = .01), glycerol output (P = .02) and free fatty acid/glycerol release ratio (P = .04) decreased during the Eugluc-Hiinsu clamp compared to Hygluc-Euinsu clamp with GIP. In healthy lean humans, insulin is permissive for GIP to induce an increase in blood flow and TAG clearance in sc abdominal adipose tissue. This effect is independent of C-peptide.

The blunted effect of glucose-dependent insulinotropic polypeptide in subcutaneous abdominal adipose tissue in obese subjects is partly reversed by weight loss.

Background:Glucose-dependent insulinotropic polypeptide (GIP) appears to have impaired effect on subcutaneous abdominal adipose tissue metabolism in obese subjects. The aim of the present study was to examine whether weight loss may reverse the impaired effect of GIP on subcutaneous abdominal adipose tissue in obese subjects.Methods:Five obese males participated in a 12-week weight loss program, which consisted of caloric restriction (800 Cal day−1) followed by 4 weeks of weight-maintenance diet. Before and after weight loss, subcutaneous adipose tissue lipid metabolism was studied by conducting regional measurements of arterio-venous plasma concentrations of metabolites and blood flow (adipose tissue blood flow, ATBF) across a segment of the abdominal adipose tissue in the fasting state and during GIP infusion (1.5 pmol kg−1 min−1) in combination with a hyperinsulinemic–hyperglycemic clamp.Results:After weight loss (7.5±0.8 kg), glucose tolerance and insulin sensitivity increased significantly as expected. No significant differences were seen in basal ATBF before (1.3±0.4 ml min−1 100 g tissue−1) and after weight loss (2.1±0.4 ml min−1 100 g tissue)−1; however, a tendency to increase was seen. After weight loss, GIP infusion increased ATBF significantly (3.2±0.1 ml min−1 100 g tissue−1) whereas there was no increase before weight loss. Triacylglycerol (TAG) uptake did not change after weight loss. Baseline free fatty acid (FFA) and glycerol output increased significantly after weight loss, P<0.001. During the clamp period, FFA and glycerol output declined significantly, P<0.05, with no differences before and after weight loss. Weight loss increased glucose uptake and decreased FFA/glycerol ratio during the clamp period, P<0.05.Conclusions:In obese subjects, weight loss, induced by calorie restriction, improves the blunted effect of GIP on subcutaneous abdominal adipose tissue metabolism.

Regulation of the circadian clock gene expression in human adipose tissue by glucose-dependent insulinotropic polypeptide (GIP)

Aims: Recently was shown that Insulin has a direct impact on the circadian clock gene regulation. GIP is an incretin hormone released after food intake. Therefore GIP and Insulin are synergistically increased in the postprandial state. We hypothesised that GIP modulates the circadian gene expression in human adipose tissue. Methods: Moderately obese male subjects (n = 14) underwent one to three of the following procedures: 1) isotonic saline- or GIP-infusion (11); 2) hyperinsulinemic-euglycemic clamp (EC, n = 11) (+/-GIP); 3) hyperinsulinemic-hyperglycemic clamp (HC, n = 8) (+/-GIP). Subjects with Type 2 diabetes (n = 11) underwent HC (+/-GIP) under the same conditions. All clamps were performed for 4h. Subcutaneous adipose tissue biopsies were obtained before and after infusion. The expression of Clock genes PER2, PER3, RORα, REV-ERBα, TEF was measured by RT-PCR. Results: In healthy subjects, PER2 and TEF gene expression were increased by insulin and glucose after HC. We observed a trend up regulation of PER2, PER3 genes and a significant down regulation of REV-ERBα gene by insulin after EC+NaCl. Furthermore, we found a trend reduction of insulin-glucose impact on PER2, TEF and REV-ERBα genes by GIP in HC. GIP infusion alone alters the expression of the core clock genes PER3 and RORα. Otherwise in type 2 diabetes subjects we couldn't find any differences in the gene expression of PER2, TEF, REV-ERBα by the HC+GIP infusion. Conclusion: Our results demonstrate that GIP alters the clock-gene expression in human adipose tissue. Although GIP stimulates the insulin secretion in a glucose-dependent manner we could find that GIP suppresses and alters the insulin regulated circadian rhythms. In type 2 diabetes patients the inhibitory effect of GIP under the same conditions is missing.

Simultaneous GLP-1 and Insulin Administration Acutely Enhances Their Vasodilatory, Antiinflammatory, and Antioxidant Action in Type 2 Diabetes

To test the hypothesis that the simultaneous administration of GLP-1 and insulin may increase their vasodilatory, antiinflammatory, and antioxidant action in type 2 diabetes. In two groups of persons with type 2 diabetes, two sets of experiments were performed. The first group had two normoglycemic-normoinsulinemic clamps with or without GLP-1 and two normoglycemic-hyperinsulinemic clamps with or without GLP-1. The second group had two hyperglycemic-normoinsulinemic clamps and two hyperglycemic-hyperinsulinemic clamps with or without GLP-1. During the normoglycemic-hyperinsulinemic clamp, flow-mediated dilatation (FMD) increased, while soluble intercellular adhesion molecule (sICAM-1), plasma 8-iso-prostaglandin F2α (8-iso-PGF2α), nitrotyrosine, and interleukin (IL)-6 decreased compared with normoglycemic-normoinsulinemic clamp. Similar results were obtained with the infusion of GLP-1 during the normoglycemic-normoinsulinemic clamp. The combination of hyperinsulinemia and GLP-1 in normoglycemia was accompanied by a further FMD increase and sICAM-1, 8-iso-PGF2α, nitrotyrosine, and IL-6 decrease. During the hyperglycemic-normoinsulinemic clamp, FMD significantly decreased, while sICAM-1, 8-iso-PGF2α, nitrotyrosine, and IL-6 significantly increased. When hyperglycemia was accompanied by hyperinsulinemia or by the simultaneous infusion of GLP-1, these phenomena were attenuated. The simultaneous presence of hyperinsulinemia and GLP-1 had an increased beneficial effect. Our results show that the combination of insulin and GLP-1 is more effective than insulin or GLP-1 alone in improving endothelial dysfunction, inflammation, and oxidative stress in type 2 diabetes.

Glucotoxicity targets hepatic glucokinase in Zucker diabetic fatty rats, a model of type 2 diabetes associated with obesity

A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty (ZDF) rats. We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF rats. We measured expression and localization of GK and GK regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic hyperinsulinemic clamp after 1 or 6 wk of treatment with the sodium-glucose transporter 2 inhibitor (canaglifrozin) that was used to correct the persistent hyperglycemia of ZDF rats. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF rats was associated with the coresidency of GKRP with GK in the cytoplasm in the midstage of diabetes, which was followed by a decrease in GK protein levels due to impaired posttranscriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal conditions and normalizing translocation of GK from the nucleus to the cytoplasm, with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver's metabolic ability to respond to hyperglycemic hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness and is associated with altered GK regulation in the ZDF rat.

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

Effect of Avocado Soybean Unsaponifiables on Insulin Secretion and Insulin Sensitivity in Patients with Obesity

Aim: To evaluate the effect of avocado soybean unsaponifiables (ASU) on insulin secretion and insulin sensitivity in patients with obesity. Methods: A randomized, double-blind, placebo-controlled, clinical trial was carried out in 14 obese adult volunteers. After random allocation of the intervention, 7 patients received 300 mg of ASU or placebo during a fasting state for 3 months. A metabolic profile including IL-6 and high-sensitivity C-reactive protein (hs-CRP) levels was carried out prior to the intervention. A hyperglycemic-hyperinsulinemic clamp technique was used to assess insulin secretion and insulin sensitivity phases. Mann-Whitney U test and Wilcoxon test were performed for statistical analyses. The study was approved by the local ethics committee of our institution. Results: At baseline, both groups were similar according to clinical and laboratory characteristics. There was no significant difference in insulin secretion and insulin sensitivity with ASU. Conclusions: ASU administration for 3 months did not modify insulin secretion and insulin sensitivity in patients with obesity.

Hepatic glucose metabolism in late pregnancy: normal versus high-fat and -fructose diet.

Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol⋅100 g liver−1⋅min−1. The P and P-HFF dogs reached maximal NHGU in 90–120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol⋅100 g liver−1⋅min−1, respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.

Regulation of adiponectin secretion and expression by insulin and β‐agonists in rat

AbstractThe adipocytokines, including adiponectin, are important factors in the regulation of insulin sensitivity and carbohydrate and lipid metabolism. It is proved that concentrations of adiponectin are decreased in obesity, an insulin resistant state. The current study is to address potential mechanisms regulating adiponectin secretion and expression in vivo. To observe the regulation of adiponectin by fasting‐refeeding and β‐agonists, male Wistar rats were fasted for 18 h and allowed to refeed with/without a β3‐adrenergic receptor agonist infused into refeeding rats. We also investigated the effects of insulin clamp on adiponectin secretion and expression, including euglycemic–hyperinsulinemic clamp and hyperglycemic–hyperinsulinemic clamp. Plasma adiponectin levels were determined by radioimmunoassay. Using real‐time PCR assays, we analyzed the expression of adiponectin genes in rat primary adipocytes. Refeeding of 18‐h fasted rats increased plasma adiponectin concentration (∼2‐fold) and adipose tissue adiponectin expression (∼3‐fold), and these effects were mimicked by hyperinsulinemia in the absence of refeeding and completely blocked by administration of β‐agonists during refeeding. We conclude that (i) adiponectin secretion and expression are acutely regulated in vivo by nutritional status; (ii) in vivo, insulin and β‐agonists act directly at the adipocyte to regulate adiponectin secretion and expression.