Empagliflozin and linagliptin combination therapy for treatment of patients with type 2 diabetes mellitus.

INTRODUCTION: Many patients with type 2 diabetes mellitus (T2DM) fail to achieve the desired A1c goal because the antidiabetic medications used do not correct the underlying pathophysiologic abnormalities and monotherapy is not sufficiently potent to reduce the A1c to the 6.5 - 7.0% range. Insulin resistance and islet (beta and alpha) cell dysfunction are major pathophysiologic abnormalities in T2DM. We examine combination therapy with linagliptin plus empagliflozin as a therapeutic approach for the treatment of inadequately controlled T2DM patients. AREAS COVERED: A literature search of all human diabetes, metabolism and general medicine journals from year 2000 to the present was conducted. Glucagon like peptide-1 (GLP-1) deficiency/resistance contributes to islet cell dysfunction by impairing insulin secretion and increasing glucagon secretion. DPP-4 inhibitors (DPP4i) improve pancreatic islet function by augmenting glucose-dependent insulin secretion and decreasing elevated plasma glucagon levels. Linagliptin, a DPP-4 inhibitor, reduces HbA1c, is weight neutral, has an excellent safety profile and a low risk of hypoglycemia. The expression of sodium-glucose cotransporter-2 (SGLT2) in the proximal renal tubule is upregulated in T2DM, causing excess reabsorption of filtered glucose. The SGLT2 inhibitor (SGLT2i), empagliflozin, improves HbA1c by causing glucosuria and ameliorating glucotoxicity. It also decreases weight and blood pressure, and has a low risk of hypoglycemia. EXPERT OPINION: The once daily oral combination of linagliptin plus empagliflozin does not increase the risk of hypoglycemia and tolerability and discontinuation rates are similar to those with each as monotherapy. At HbA1c values below 8.5% linagliptin/empagliflozin treatment produces an additive effect, whereas above 8.5%, there is a less than additive reduction with combination therapy compared with the effect of each agent alone. Linagliptin/empagliflozin addition is a logical combination in patients with T2DM, especially those with an HbA1c < 8.5%.

Initial combination therapy with metformin, pioglitazone and exenatide is more effective than sequential add-on therapy in subjects with new-onset diabetes. Results from the Efficacy and Durability of Initial Combination Therapy for Type 2 Diabetes (EDICT): a randomized trial.

AIM: To test our hypothesis that initiating therapy with a combination of agents known to improve insulin secretion and insulin sensitivity in subjects with new-onset diabetes would produce greater, more durable reduction in glycated haemoglobin (HbA1c) levels, while avoiding hypoglycaemia and weight gain, compared with sequential addition of agents that lower plasma glucose but do not correct established pathophysiological abnormalities. METHODS: Drug-naïve, recently diagnosed subjects with type 2 diabetes mellitus (T2DM) were randomized in an open-fashion design in a single-centre study to metformin/pioglitazone/exenatide (triple therapy; n = 106) or an escalating dose of metformin followed by sequential addition of sulfonylurea and glargine insulin (conventional therapy; n = 115) to maintain HbA1c levels at <6.5% for 2 years. RESULTS: Participants receiving triple therapy experienced a significantly greater reduction in HbA1c level than those receiving conventional therapy (5.95 vs. 6.50%; p < 0.001). Despite lower HbA1c values, participants receiving triple therapy experienced a 7.5-fold lower rate of hypoglycaemia compared with participants receiving conventional therapy. Participants receiving triple therapy experienced a mean weight loss of 1.2 kg versus a mean weight gain of 4.1 kg (p < 0.01) in those receiving conventional therapy. CONCLUSION: The results of this exploratory study show that combination therapy with metformin/pioglitazone/exenatide in patients with newly diagnosed T2DM is more effective and results in fewer hypoglycaemic events than sequential add-on therapy with metformin, sulfonylurea and then basal insulin.

The effect of muraglitazar on adiponectin signalling, mitochondrial function and fat oxidation genes in human skeletal muscle in vivo.

AIMS: The molecular mechanisms by which muraglitazar (peroxisome proliferator-activated receptor γ/α agonist) improves insulin sensitivity in Type 2 diabetes mellitus are not fully understood. We hypothesized that muraglitazar would increase expression of 5'-monophosphate-activated protein kinase and genes involved in adiponectin signalling, free fatty acid oxidation and mitochondrial function in skeletal muscle. METHODS: Sixteen participants with Type 2 diabetes received muraglitazar, 5 mg/day (n = 12) or placebo (n = 4). Before and after 16 weeks, participants had vastus lateralis muscle biopsy followed by 180 min euglycaemic hyperinsulinaemic clamp. RESULTS: Muraglitazar increased plasma adiponectin (9.0 ± 1.1 to 17.8 ± 1.5 µg/ml, P < 0.05), while no significant change was observed with placebo. After 16 weeks with muraglitazar, fasting plasma glucose declined by 31%, fasting plasma insulin decreased by 44%, insulin-stimulated glucose disposal increased by 81%, HbA1c decreased by 21% and plasma triglyceride decreased by 39% (all P < 0.05). Muraglitazar increased mRNA levels of 5'-monophosphate-activated protein kinase, adiponectin receptor 1, adiponectin receptor 2, peroxisome proliferator-activated receptor gamma coactivator-1 alpha and multiple genes involved in mitochondrial function and fat oxidation. In the placebo group, there were no significant changes in expression of these genes. CONCLUSIONS: Muraglitazar increases plasma adiponectin, stimulates muscle 5'-monophosphate-activated protein kinase expression and increases expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation. These changes represent important cellular mechanisms by which dual peroxisome proliferator-activated receptor agonists improve skeletal muscle insulin sensitivity.

The inflammatory status score including IL-6, TNF-α, osteopontin, fractalkine, MCP-1 and adiponectin underlies whole-body insulin resistance and hyperglycemia in type 2 diabetes mellitus.

A state of subclinical systemic inflammation is characteristically present in obesity/insulin resistance and type 2 diabetes mellitus (T2DM). The aim of the study was to develop an integrated measure of the circulating cytokines involved in the subclinical systemic inflammation and evaluate its relation with whole-body insulin sensitivity and glucose metabolism in T2DM. T2DM patients (n = 17, M/F 13/4, age = 55.0 ± 1.7 years, BMI = 33.5 ± 1.5 kg/m(2), HbA(1c) = 7.7 ± 0.3%) and normal glucose-tolerant (NGT) subjects (n = 15, M/F 7/8, age = 49.1 ± 2.5 years, BMI = 31.8 ± 1.2 kg/m(2), HbA(1c) = 5.6 ± 0.1%) were studied in a cross-sectional design. Whole-body insulin sensitivity was quantified by the euglycemic clamp. Beta-cell function [disposition index (DI)] was calculated using insulin and glucose values derived from an oral glucose tolerance test and the euglycemic clamp. Body fat mass was evaluated by dual-energy X-ray absorptiometry. Plasma cytokine [TNF-α, IL-6, MCP-1, osteopontin, fractalkine and adiponectin] values were divided into quintiles. A score ranging from 0 (lowest quintile) to 4 (highest quintile) was assigned. The inflammatory score (IS) was the sum of each cytokine score from which adiponectin score was subtracted in each study subject. Inflammatory cytokine levels were all higher in T2DM. IS was higher in T2DM as compared to NGT (10.0 ± 1.1 vs. 4.8 ± 0.8; p < 0.001). IS positively correlated with fasting plasma glucose (r = 0.638, p < 0.001), 1-h plasma glucose (r = 0.483, p = 0.005), 2-h plasma glucose (r = 0.611, p < 0.001) and HbA1c (r = 0.469, p = 0.007). IS was inversely correlated with insulin sensitivity (r = -0.478, p = 0.006) and DI (r = -0.523, p = 0.002). IS did not correlate with BMI and body fat mass. IS was an independent predictor of fasting plasma glucose and had a high sensibility and sensitivity to predict insulin resistance (M/I < 4). A state of subclinical inflammation defined and quantifiable by inflammatory score including TNF-α, IL-6, MCP-1, osteopontin, fractalkine and adiponectin is associated with both hyperglycemia and whole-body insulin resistance in T2DM.

Metabolic effects of muraglitazar in type 2 diabetic subjects.

AIM: To assess the effect of muraglitazar, a dual peroxisome proliferator-activated receptor (PPAR)γ-α agonist, versus placebo on metabolic parameters and body composition in subjects with type 2 diabetes mellitus (T2DM). METHODS: Twenty-seven T2DM subjects received oral glucose tolerance test (OGTT), euglycaemic insulin clamp with deuterated glucose, measurement of total body fat (DEXA), quantitation of muscle/liver (MRS) and abdominal subcutaneous and visceral (MRI) fat, and then were randomized to receive, in addition to diet, muraglitazar (MURA), 5 mg/day, or placebo (PLAC) for 4 months. RESULTS: HbA1c(c) decreased similarly (2.1%) during both MURA and PLAC treatments despite significant weight gain with MURA (+2.5 kg) and weight loss with PLAC (-0.7 kg). Plasma triglyceride, LDL cholesterol, free fatty acid (FFA), hsCRP levels all decreased with MURA while plasma adiponectin and HDL cholesterol increased (p < 0.05-0.001). Total body (muscle), hepatic and adipose tissue sensitivity to insulin and ß cell function all improved with MURA (p < 0.05-0.01). Intramyocellular, hepatic and abdominal visceral fat content decreased, while total body and subcutaneous abdominal fat increased with MURA (p < 0.05-0.01). CONCLUSIONS: Muraglitazar (i) improves glycaemic control by enhancing insulin sensitivity and ß cell function in T2DM subjects, (ii) improves multiple cardiovascular risk factors, (iii) reduces muscle, visceral and hepatic fat content in T2DM subjects. Despite similar reduction in A1c with PLAC/diet, insulin sensitivity and ß cell function did not improve significantly.

Effect of exenatide on splanchnic and peripheral glucose metabolism in type 2 diabetic subjects.

OBJECTIVE: Our objective was to examine the mechanisms via which exenatide attenuates postprandial hyperglycemia in type 2 diabetes mellitus (T2DM). STUDY DESIGN: Seventeen T2DM patients (44 yr; seven females, 10 males; body mass index = 33.6 kg/m(2); glycosylated hemoglobin = 7.9%) received a mixed meal followed for 6 h with double-tracer technique ([1-(14)C]glucose orally; [3-(3)H]glucose i.v.) before and after 2 wk of exenatide. In protocol II (n = 5), but not in protocol I (n = 12), exenatide was given in the morning of the repeat meal. Total and oral glucose appearance rates (RaT and RaO, respectively), endogenous glucose production (EGP), splanchnic glucose uptake (75 g - RaO), and hepatic insulin resistance (basal EGP × fasting plasma insulin) were determined. RESULTS: After 2 wk of exenatide (protocol I), fasting plasma glucose decreased (from 10.2 to 7.6 mm) and mean postmeal plasma glucose decreased (from 13.2 to 11.3 mm) (P < 0.05); fasting and meal-stimulated plasma insulin and glucagon did not change significantly. After exenatide, basal EGP decreased (from 13.9 to 10.8 µmol/kg · min, P < 0.05), and hepatic insulin resistance declined (both P < 0.05). RaO, gastric emptying (acetaminophen area under the curve), and splanchnic glucose uptake did not change. In protocol II (exenatide given before repeat meal), fasting plasma glucose decreased (from 11.1 to 8.9 mm) and mean postmeal plasma glucose decreased (from 14.2 to 10.1 mm) (P < 0.05); fasting and meal-stimulated plasma insulin and glucagon did not change significantly. After exenatide, basal EGP decreased (from 13.4 to 10.7 µmol/kg · min, P = 0.05). RaT and RaO decreased markedly from 0-180 min after meal ingestion, consistent with exenatide's action to delay gastric emptying. CONCLUSIONS: Exenatide improves 1) fasting hyperglycemia by reducing basal EGP and 2) postmeal hyperglycemia by reducing the appearance of oral glucose in the systemic circulation.

Effects of insulin and oral anti-diabetic agents on glucose metabolism, vascular dysfunction and skeletal muscle inflammation in type 2 diabetic subjects.

BACKGROUND: To test potential differences between the actions of anti-diabetic medications, we examined the effects of oral hypoglycaemic agents versus glargine-apidra insulin therapy in T2DM. METHODS: T2DM subjects were randomized to either oral hypoglycaemic agents (pioglitazone, metformin and glipizide, n = 9) or insulin therapy (n = 12) for 6 months. Carotid intimal media thickness, vascular reactivity (flow-mediated vasodilatation; percent change in brachial artery basal diameter post-ischaemia) and sublingual nitrate were measured with ultrasonography. Euglycemic hyperinsulinemic (80 mU/m(2) ) clamp with [3]-3H-glucose and muscle biopsies were performed. RESULTS: Fasting plasma glucose (~257 to ~124 mg/dL, oral hypoglycaemic agents and ~256 to ~142 mg/dL, IT) and HbA(1c) (~10.3 to ~6.4%, OHA and ~10.7 to ~7.1%, IT) improved comparably. Endogenous glucose production (~2.1 to ~1.7 mg/kg/min, oral hypoglycaemic agents and ~2.3 to ~2.0 mg/kg/min, insulin therapy) and endogenous glucose production suppression by insulin (~0.4 to ~0.3 mg/kg min, oral hypoglycaemic agents and ~0.5 to ~0.7 mg/kg min, insulin therapy) were different. Total glucose disposal × 100 increased in the oral hypoglycaemic agents group (~5.2 to ~8.1; p = 0.03), but not in insulin therapy (~6.0 to ~5.4 mg/kg/min/µU/mL × 100). OHA reduced CIMT (~0.080 to ~0.068 cm; p < 0.05), whereas insulin therapy did not (~0.075 to ~0.072 cm). After sublingual nitrate, brachial artery basal diameter increased in the OHA group (~8.7 to ~18.2%), but not in insulin therapy (~11.2 to ~15.0%; p < 0.02). Except for plasma adiponectin (~7 to ~15, oral hypoglycaemic agents versus ~6 to ~10, IT), changes in inflammatory markers in the circulation and in muscle (IκBα, super-oxidase dismutase 2, monocyte-chemo-attractant protein 1, p-ERK and JNK) were equivalent. CONCLUSIONS: Oral hypoglycaemic agents and insulin therapy treated patients achieved adequate glycemic control and the effects on circulating and muscle inflammatory biomarkers were similar, but only oral hypoglycaemic agents improved insulin sensitivity, vascular function and carotid intimal media thickness. These findings in a small sample suggest that the use of oral hypoglycaemic agents provides additional benefits to patients with T2DM.

Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial.

AIMS/HYPOTHESIS: The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle. METHODS: A randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n = 14) or (2) aggressive nutritional therapy (n = 12) to reduce HbA(1c) to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic-hyperinsulinaemic (80 mU m(-2) min(-1)) clamp. RESULTS: All patients in the pioglitazone (n = 14) or nutritional therapy (n = 12) group were included in the analysis. Pioglitazone significantly increased plasma adiponectin concentration by 79% and reduced fasting plasma NEFA by 35% (both p < 0.01). Following pioglitazone, insulin-stimulated glucose disposal increased by 30% (p < 0.01), and muscle AMPK and acetyl-CoA carboxylase (ACC) phosphorylation increased by 38% and 53%, respectively (p < 0.05). Pioglitazone increased mRNA levels for adiponectin receptor 1 and 2 genes (ADIPOR1, ADIPOR2), peroxisome proliferator-activated receptor gamma, coactivator 1 gene (PPARGC1) and multiple genes involved in mitochondrial function and fat oxidation. Despite a similar reduction in HbA(1c) and similar improvement in insulin sensitivity with nutritional therapy, there were no significant changes in muscle AMPK and ACC phosphorylation, or the expression of ADIPOR1, ADIPOR2, PPARGC1 and genes involved in mitochondrial function and fat oxidation. No adverse (or unexpected) effects or side effects were reported from the study. CONCLUSIONS/INTERPRETATIONS: Pioglitazone increases plasma adiponectin levels, stimulates muscle AMPK signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation. These changes may represent an important cellular mechanism by which thiazolidinediones improve skeletal muscle insulin sensitivity. TRIAL REGISTRATION: NCT 00816218 FUNDING: This trial was funded by National Institutes of Health Grant DK24092, VA Merit Award, GCRC Grant RR01346, Executive Research Committee Research Award from the University of Texas Health Science Center at San Antonio, American Diabetes Association Junior Faculty Award, American Heart Association National Scientist Development Grant, Takeda Pharmaceuticals North America Grant and Canadian Institute of Health Research Grant.

Rosiglitazone decreases albuminuria in type 2 diabetic patients.

Thiazolidinediones are insulin-sensitizing compounds that reduce plasma glucose and improve the lipid profile of type 2 diabetic patients. We determined the effect of rosiglitazone in 15 type 2 diabetic patients and compared these results to 14 randomly assigned placebo patients. After 3 months, the urinary albumin to creatinine ratio was significantly decreased, while the glucose metabolic clearance rate, during insulin clamp, was significantly increased by rosiglitazone compared to the placebo group. Fasting free fatty acid and tumor necrosis factor-alpha (TNF-alpha) levels were significantly decreased, while the adiponectin concentration was significantly increased by rosiglitazone treatment. The percentage decrease in albuminuria correlated with the decrease in fasting plasma glucose, free fatty acids TNF-alpha and the increase in fat mass, plasma adiponectin, and glucose metabolic clearance rate. Stepwise linear regression analysis showed the decrease in TNF-alpha and the increase in adiponectin were independently associated with decreased albuminuria. Our study indicates that thiazolidinediones may be useful to prevent nephropathy in type 2 diabetic patients.

Reduction in hematocrit and hemoglobin following pioglitazone treatment is not hemodilutional in Type II diabetes mellitus.

Peripheral edema, mild weight gain, and anemia are often observed in type II diabetic patients treated with thiazolidinediones (TZDs). Small decreases in hemoglobin (Hb) and hematocrit (Hct) appear to be a class effect of TZDs and are generally attributed to fluid retention, although experimental data are lacking. We analyzed 50 patients with type II diabetes mellitus undergoing either placebo or pioglitazone (PIO, 45 mg/day) for 16 weeks. Before and after therapy, we measured Hb/Hct and used (3)H(2)O and bioimpedance to quantitate total body water (TBW), extracellular water, and fat-free mass. The majority (89%) of the increment in body weight was accounted for by increased body fat. Hb and Hct fell significantly in the PIO group (-0.9+/-0.2 g/dl, -2.4+/-0.5%, both P<0.0001), without change in TBW. A decline in white blood cell (-0.8+/-0.1 x 10(3)/mm(3), P<0.0001) and platelet (-15+/-6 x 10(3)/mm(3), P<0.02) counts was seen after PIO. In conclusion, the small decreases in Hb/Hct observed after 16 weeks of PIO treatment cannot be explained by an increase in TBW. Other causes, such a mild marrow suppressive effect, should be explored.

Abnormal glucose handling by the kidney in response to hypoglycemia in type 1 diabetes.

The frequent occurrence of hypoglycemia in people with type 1 diabetes is attributed to abnormalities in the blood glucose counterregulatory response. In view of recent findings indicating that the kidney contributes to prevent and correct hypoglycemia in healthy subjects, we decided to investigate the role of renal glucose handling in hypoglycemia in type 1 diabetes. Twelve type 1 diabetic patients and 14 age-matched normal individuals were randomized to hyperinsulinemic-euglycemic (n = 6 diabetic subjects and n = 8 control subjects) or hypoglycemic (n = 6 each) clamps with blood glucose maintained either stable near 100 mg/dl (5.6 mmol/l) or reduced to 54 mg/dl (3.0 mmol/l). All study subjects had their renal vein catheterized under fluoroscopy, and net renal glucose balance and renal glucose production and utilization rates were measured using a combination of arteriovenous concentration difference with stable isotope dilution technique. Blood glucose and insulin were comparable in both groups in all studies. In patients with diabetes, elevations in plasma glucagon, epinephrine, and norepinephrine were blunted, and both the compensatory rise in endogenous glucose production and in the net glucose output by the kidney seen in normal subjects with equivalent hypoglycemia were absent. Renal glucose balance switched from a mean +/- SE baseline net uptake of 0.6 +/- 0.4 to a net output of 4.5 +/- 1.3 micromol x kg(-1) x min(-1) in normal subjects, but in patients with diabetes there was no net renal contribution to blood glucose during similar hypoglycemia (mean +/- SE net glucose uptake [baseline 0.7 +/- 0.4] remained at 0.4 +/- 0.3 micromol x kg(-1) x min(-1) in the final 40 min of hypoglycemia; P < 0.01 between groups). We conclude that adrenergic stimulation of glucose output by the kidney, which represents an additional defense mechanism against hypoglycemia in normal subjects, is impaired in patients with type 1 diabetes and contributes to defective glucose counterregulation.

Renal substrate metabolism and gluconeogenesis during hypoglycemia in humans.

To examine the potential contribution of precursor substrates to renal gluconeogenesis during hypoglycemia, 14 healthy subjects had arterialized hand vein and renal vein (under fluoroscopy) catheterized after an overnight fast. Net renal balance of lactate, glycerol, alanine, and glutamine was determined simultaneously with systemic and renal glucose kinetics using arteriovenous concentration differences and 6-[2H2]glucose tracer dilution. Renal plasma flow was measured by para-aminohippurate clearance and was converted to blood flow using the mathematical value (1-hematocrit). Arterial and renal vein samples were obtained in the postabsorptive state and during a 180-min hyperinsulinemic period during either euglycemia or hypoglycemia. Insulin increased from 49 +/- 14 to 130 +/- 25 pmol/l (hypoglycemia) and to 102 +/- 10 pmol/l (euglycemia). Arterial blood glucose decreased from 4.5 +/-0.2 to 3.0 +/- 0.1 mmol/l during hypoglycemia but did not change during euglycemia (4.3 +/- 0.2 mmol/l). After 150 min, endogenous glucose production reached a plateau value that was higher during hypoglycemia (10.3 +/0.6 micromol x kg(-1) x min(-1)) than during euglycemia (5.73 +/-0.6 micromol x kg(-1) x min(-1), P < 0.001). Hypoglycemia was associated with a rise in renal glucose production (RGP) from 3.0 +/- 0.7 to 5.4 +/- 0.6 micromol x kg(-1) x min(-1) (P < 0.05), although glucose utilization remained the same (2.0 +/- 0.8 vs. 2.1 +/-0.6 micromol x kg(-1) x min(-1)). As a result, net renal glucose output increased from 1.0 +/- 0.3 to 3.3 +/- 0.40 micromol x kg(-1) x min(-1). Elevations in net renal uptake of lactate (2.4 +/- 0.5 to 3.5 +/- 0.7 vs. 2.8 +/- 0.4 micromol x kg(-1) x min(-1)), glycerol (0.6 +/- 0.3 to 1.3 +/- 0.5 vs. 0.4 +/- 0.2 micromol x kg(-1) x min(-1)), and glutamine (0.7 +/- 0.2 to 1.1 +/- 0.3 vs. 0.1 +/- 0.3 micromol x kg(-1) x min(-1)) during hypoglycemia versus euglycemia (P < 0.05) could account for nearly 60% of all glucose carbons released in the renal vein during hypoglycemia. Our data indicate that extraction of circulating gluconeogenic precursors by the kidney is enhanced and responsible for a substantial fraction of the compensatory rise in RGP during sustained hypoglycemia. Increased renal gluconeogenesis from circulating substrates represents an additional physiological mechanism by which the decrease in blood glucose concentration is attenuated in humans.

Regulation of splanchnic and renal substrate supply by insulin in humans.

To determine the effects of peripheral insulin infusion on total, hepatic, and renal glucose production and on the percent contribution to glucose production of gluconeogenesis versus glycogenolysis, 10 healthy subjects had arterialized hand and hepatic vein catheterization after an overnight fast and the results were compared with data from 12 age- and weight-matched subjects with renal vein catheterization during a 180-minute infusion of either insulin (0.25 mU/kg x min) with dextrose, or saline. Endogenous, hepatic, and renal glucose production was measured with [6,6(-2)H2]glucose, regional lactate, alanine, and glycerol balance by arteriovenous difference; hepatic blood flow by indocyanine green clearance; and renal blood flow by p-aminohippurate clearance, before and every 30 minutes during each infusion period. Insulin increased from about 42 to 98 pmol/L and blood glucose remained constant in all studies (3.8 +/- 0.2 v4.4 +/- 0.1 micromol/ml, hepatic vrenal vein). In response to insulin infusion, endogenous, hepatic, and renal glucose production decreased immediately (30 minutes) and reached a lower plateau value (10.8 +/- 0.8 v6.4 +/- 0.7, 10.4 +/- 1.1 v7.8 +/- 1.0, and 2.8 +/- 0.6 v 1.5 +/- 0.6 micromol/kg x min, respectively) between 120 and 180 minutes (all P < .05). Net renal uptake of lactate (2.4 +/- 0.4 v0.9 +/- 0.6) decreased earlier (30 minutes) and returned to baseline between 120 and 180 minutes (2.4 +/- 0.5 micromol/kg x min), whereas net splanchnic uptake of lactate (5.7 +/- 0.7 v 0.7 +/- 0.6) and alanine (1.8 +/- 0.1 v 1.0 +/- 0.5 micromol/kg x min) decreased later (120 to 180 minutes). Net renal (0.3 +/- 0.1 v 0.1 +/- 0.1) and splanchnic (0.7 +/- 0.3 v 0.4 +/- 0.2 micromol/kg x min) glycerol uptake decreased 90 to 180 minutes after insulin and increased (P < .05) with saline infusion (0.4 +/- 0.1 v0.6 +/- 0.3 and 1.0 +/- 0.5 v1.8 +/- 0.4 micromol/kg x min, respectively). These data indicate that the rapid suppression of endogenous glucose production by insulin reflects primarily a decrease in hepatic glucose release, most likely due to inhibition of net glycogenolysis, combined with suppression of renal gluconeogenesis. Inhibition of hepatic gluconeogenesis presumably occurs later during hyperinsulinemia. We conclude that peripheral insulin, in addition to its inhibition of glycogen degradation, regulates endogenous glucose production, in part, by modifying the splanchnic and renal substrate supply.

Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo.

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0-100 nmol per injection of nine fatty acids was achieved within 60 min using [2H31]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0+/-0.3 to 1.0+/-0.1 micromol/kg min (p<0.01), whereas net renal balance remained neutral (-0.04+/-0.04 vs. -0.06+/-0.03 micromol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012+/-0.005 (myristic acid) to a high of 0.95+/-0.08 (oleic acid) micromol/kg min across the splanchnic tissues and from 0.005+/-0.002 (stearic acid) to 0.21+/-0.1 (oleic acid) micromol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.

Renal glucose production during insulin-induced hypoglycemia in humans.

We investigated the effects of hypoglycemia on renal glucose production (RGP) and renal glucose uptake (RGU) using arteriovenous balance combined with tracer technique in humans. Our 14 healthy subjects had arterialized hand veins (artery) and renal veins (under fluoroscopy) catheterized after an overnight fast. Systemic and renal glucose kinetics were measured with infusion of [6-(2)H2]glucose, and renal plasma flow was measured by para-aminohippurate clearance. After a 150-min equilibration period, artery and renal vein samples were obtained between -30 and 0 min, and subjects received a 180-min peripheral insulin infusion (0.250 mU kg(-1) x min(-1)) with a variable infusion of [6-(2)H2]dextrose adjusted to maintain plasma glucose at either approximately 60 mg/dl (hypoglycemic clamp) or approximately 90 mg/dl (euglycemic clamp). Blood samples were obtained between 150 and 180 min during the study period. Insulin increased from 49 +/- 14 to 130 +/- 25 (hypoglycemia) and to 102 +/- 10 (euglycemia) pmol/l. Glucose decreased from 5.32 +/- 0.11 to 3.58 +/- 0.07 micromol/ml during hypoglycemia, but it did not change during euglycemia (5.20 +/- 0.19 vs. 5.05 +/- 0.15 micromol/ml). Endogenous glucose production decreased (9.30 +/- 0.70 vs. 5.65 +/- 0.50) during euglycemia but not during hypoglycemia (9.80 +/- 0.50 vs. 10.25 +/- 0.60 micromol x kg(-1) x min(-1)). During hypoglycemia, net renal glucose output increased from 0.54 +/- 0.30 to 2.31 +/- 0.40, RGP increased from 1.88 +/- 0.70 to 3.65 +/- 0.50 (P < 0.05), and RGU did not change (1.34 +/- 0.50 vs. 1.34 +/- 0.60 micromol x kg(-1) x min(-1)). During euglycemia, renal glucose balance switched from a net output of 0.72 +/- 0.20 to a net uptake of 1.70 +/- 0.92, RGP decreased from 2.31 +/- 0.50 to 1.20 +/- 0.58, and RGU increased from 1.59 +/- 0.50 to 2.90 +/- 0.70 micromol x kg(-1) x min(-1) (P < 0.05). During hypoglycemia, arterial glucagon increased from 105 +/- 6 to 129 +/- 8, epinephrine increased from 116 +/- 28 to 331 +/- 33, norepinephrine increased from 171 +/- 9 to 272 +/- 9 (all P < 0.05), and renal vein norepinephrine increased from 236 +/- 13 to 426 +/- 50 (P < 0.001). These data indicate that, in addition to counterregulatory hormones, activation of the autonomic nervous system during hypoglycemia stimulates glucose production by the kidney, which may represent an important additional component of the body's defense against hypoglycemia in humans.

Insulin regulation of renal glucose metabolism in humans.

Eighteen healthy subjects had arterialized hand and renal veins catheterized after an overnight fast. Systemic and renal glucose and glycerol kinetics were measured with [6,6-2H2]glucose and [2-13C]glycerol before and after 180-min peripheral infusions of insulin at 0.125 (LO) or 0.25 (HI) mU. kg-1. min-1 with variable [6, 6-2H2]dextrose or saline (control). Renal plasma flow was determined by plasma p-aminohippurate clearance. Arterial insulin increased from 37 +/- 8 to 53 +/- 5 (LO) and to 102 +/- 10 pM (HI, P < 0.01) but not in control (35 +/- 8 pM). Arterial glucose did not change and averaged 5.2 +/- 0.1 (control), 4.7 +/- 0.2 (LO), and 5.1 +/- 0. 2 (HI) micromol/ml; renal vein glucose decreased from 4.8 +/- 0.2 to 4.5 +/- 0.2 micromol/ml (LO) and from 5.3 +/- 0.2 to 4.9 +/- 0.1 micromol/ml (HI) with insulin but not saline infusion (5.3 +/- 0.1 micromol/ml). Endogenous glucose production decreased from 9.9 +/- 0. 7 to 6.9 +/- 0.5 (LO) and to 5.7 +/- 0.5 (HI) micromol. kg-1. min-1; renal glucose production decreased from 2.5 +/- 0.6 to 1.5 +/- 0.5 (LO) and to 1.2 +/- 0.6 (HI) micromol. kg-1. min-1, whereas renal glucose utilization increased from 1.5 +/- 0.6 to 2.6 +/- 0.7 (LO) and to 2.9 +/- 0.7 (HI) micromol. kg-1. min-1 after insulin infusion (all P < 0.05 vs. baseline). Neither endogenous glucose production (10.0 +/- 0.4), renal glucose production (1.1 +/- 0.4), nor renal glucose utilization (0.8 +/- 0.4) changed in the control group. During insulin infusion, systemic gluconeogenesis from glycerol decreased from 0.67 +/- 0.05 to 0.18 +/- 0.02 (LO) and from 0.60 +/- 0.04 to 0.20 +/- 0.02 (HI) micromol. kg-1. min-1 (P < 0.01), and renal gluconeogenesis from glycerol decreased from 0.10 +/- 0.02 to 0.02 +/- 0.02 (LO) and from 0.15 +/- 0.03 to 0.09 +/- 0.03 (HI) micromol. kg-1. min-1 (P < 0.05). In contrast, during saline infusion, systemic (0.66 +/- 0.03 vs. 0.82 +/- 0.05 micromol. kg-1. min-1) and renal gluconeogenesis from glycerol (0.11 +/- 0.02 vs. 0. 41 +/- 0.04 micromol. kg-1. min-1) increased (P < 0.05 vs. baseline). We conclude that glucose production and utilization by the kidney are important insulin-responsive components of glucose metabolism in humans.

Effects of beta-adrenergic blockade on hepatic and renal glucose production during hypoglycemia in conscious dogs.

To investigate the role of beta-adrenergic mechanisms in the counterregulatory response of the liver and kidney to hypoglycemia, we studied 10 dogs before and after a 2-h constant infusion of insulin (4 mU. kg-1. min-1) either without (n = 4) or with (8 micrograms/min, n = 6) propranolol and variable dextrose to maintain hypoglycemia, 7 days after surgical placement of sampling catheters in left renal and hepatic veins and femoral artery. Systemic glucose appearance (Ra) and endogenous (EGP), hepatic (HGP), and renal (RGP) glucose production were measured by a combination of arteriovenous difference and peripheral infusion of [6-3H]glucose, renal blood flow with a flow probe, and hepatic plasma flow by indocyanine green clearance. Without beta-adrenergic blockade, arterial glucose decreased from 5.12 +/- 0.02 to 2.53 +/- 0.07 mmol/l, glucose Ra increased from 17.8 +/- 0.7 to 30.5 +/- 2.5 (P < 0.01) when EGP was 22.2 +/- 0.5, HGP from 13.5 +/- 1.1 to 19.3 +/- 1.3, and RGP from 2. 4 +/- 1.0 to 8.6 +/- 0.9 micromol. kg-1. min-1 (all P < 0.05). When propranolol was infused, glucose decreased from 5.97 +/- 0.02 to 2. 71 +/- 0.03 mmol/l, glucose Ra increased from 16.3 +/- 1.0 to 25.1 +/- 1.6 when EGP was 9.9 +/- 0.4, HGP decreased from 14.4 +/- 0.7 to 10.4 +/- 0.6, and RGP decreased from 3.8 +/- 1.3 to 1.1 +/- 0.8 micromol. kg-1. min-1 (all P < 0.05). Our data indicate that beta-adrenergic blockade impairs glucose recovery during sustained hypoglycemia, in part, by preventing the simultaneous compensatory increase in HGP and RGP.

Renal lactate metabolism and gluconeogenesis during insulin-induced hypoglycemia.

The contribution of gluconeogenic precursors to renal glucose production (RGP) during insulin-induced hypoglycemia was assessed in conscious dogs. Ten days after surgical placement of sampling catheters in the right and left renal veins and femoral artery and an infusion catheter in the left renal artery, systemic and renal glucose and glycerol kinetics were measured with peripheral infusions of [6-3H]glucose and [2-13C]glycerol. Renal blood flow was determined with a flowprobe, and the renal balance of lactate, alanine, and glycerol was calculated by arteriovenous difference. After baseline, six dogs received 2-h simultaneous infusions of peripheral insulin (4 mU x kg(-1) x min(-1)) and left intrarenal [6,6-2H]dextrose (14 micromol x kg(-1) x min(-1)) to achieve and maintain left renal normoglycemia during systemic hypoglycemia. Arterial glucose decreased from 5.3 +/- 0.1 to 2.2 +/- 0.1 mmol/l; insulin increased from 46 +/- 5 to 1,050 +/- 50 pmol/l; epinephrine, from 130 +/- 8 to 1,825 +/- 50 pg/ml; norepinephrine, from 129 +/- 6 to 387 +/- 15 pg/ml; and glucagon, from 52 +/- 2 to 156 +/- 12 pg/ml (all P < 0.01). RGP increased from 1.7 +/- 0.4 to 3.0 +/- 0.5 (left) and from 0.6 +/- 0.2 to 3.2 +/- 0.2 (right) micromol x kg(-1) x min(-1) (P < 0.01). Whole-body glycerol appearance increased from 6.0 +/- 0.5 to 7.7 +/- 0.7 micromol x kg(-1) x min(-1)(P < 0.01); renal conversion of glycerol to glucose increased from 0.13 +/- 0.04 to 0.30 +/- 0.10 (left) and from 0.11 +/- 0.03 to 0.25 +/- 0.05 (right) micromol x kg(-1) x min(-1), (P < 0.05). Net renal gluconeogenic precursor uptake increased from 1.5 +/- 0.4 to 5.0 +/- 0.4 (left) and from 0.9 +/- 0.2 to 3.8 +/- 0.4 (right) micromol x kg(-1) x min(-1) (P < 0.01). Renal lactate uptake could account for approximately 40% of postabsorptive RGP and for 60% of RGP during hypoglycemia. These results indicate that gluconeogenic precursor extraction by the kidney, particularly lactate, is stimulated by counterregulatory hormones and accounts for a significant fraction of the enhanced gluconeogenesis induced by hypoglycemia.

Role of the kidney in plasma glucose regulation during hyperglycemia.

Little is known about the role of the kidney in plasma glucose regulation during hyperglycemia. We studied 12 overnight-fasted conscious dogs after either intrarenal (IR, n = 6) or peripheral (PH, n = 6) dextrose infusion to maintain hyperglycemia without glycosuria. Systemic and renal glucose kinetics were measured with [6-3H]glucose, lactate balance was measured by arteriovenous difference, and glycogen content was assayed in the kidneys. Plasma glucose (approximately 5.5 vs. approximately 6.3 mM), insulin (approximately 70 vs. approximately 110 pM), and glucose appearance (approximately 14 vs. approximately 16 mumol.kg-1.min-1 increased comparably in both groups (P < 0.05). In IR, fractional extraction of glucose (FEGlc) increased from 4.1 +/- 0.2 to 16.1 +/- 0.5% (P < 0.001) and lactate balance reversed to renal output (+1.3 +/- 0.2 vs. -0.9 +/- 0.2 mumol.kg-1.min-1, P < 0.01). Glycogen content was twofold higher in the left (127 +/- 33 micrograms/g tissue) than in the right kidney (56 +/- 11 micrograms/g tissue, P < 0.01). In PH, FEGlc decreased from 4.9 +/- 0.6 to 2.2 +/- 0.3% (P < 0.05), renal glucose utilization did not change (approximately 1.3 mumol.kg-1.min-1); and glycogen content was equal in both kidneys (approximately 45 micrograms/g tissue). We conclude that, although the kidney plays a minor role in plasma glucose disposal in physiological hyperglycemia, increased glucose uptake, glycogen storage, and lactate formation precede glycosuria and may represent important mechanisms by which the kidney contributes to normalization of plasma glucose in diabetes.

Renal glucose production during insulin-induced hypoglycemia.

Recent in vivo studies have rekindled interest in the role of the kidney in glucose metabolism. We therefore undertook the present study to evaluate the contribution of the kidney to systemic glucose production and utilization rates during insulin-induced hypoglycemia using arteriovenous balance combined with a tracer technique. Ten days after the surgical placement of sampling catheters in the right and left renal veins and femoral artery and of an infusion catheter in the left renal artery of dogs, systemic and renal glucose kinetics were measured with the peripheral infusion of [6-3H]glucose. Renal blood flow was determined with a flowprobe. After baseline, six dogs received 2-h simultaneous infusions of peripheral insulin (4 mU x kg(-1) x min(-1)) and left intrarenal [6,6-2H]dextrose (14 micromol x kg(-1) x min(-1)) to achieve and maintain left renal normoglycemia during systemic hypoglycemia. Arterial glucose decreased from 5.3 +/- 0.1 to 2.2 +/- 0.1 mmol/l; insulin increased from 46 +/- 5 to 1,050 +/- 50 pmol/l; epinephrine increased from 130 +/- 8 to 1,825 +/- 50 pg/ml; norepinephrine increased from 129 +/- 6 to 387 +/- 15 pg/ml; and glucagon increased from 52 +/- 2 to 156 +/- 12 pg/ml (all P < 0.01). Systemic glucose appearance increased from 16.6 +/- 0.4 micromol x kg(-1) x min(-1) in the baseline to 24.2 +/- 0.6 micromol x kg(-1) x min(-1) during hypoglycemia when endogenous glucose production was 10.2 +/- 1.0 micromol x kg(-1) x min(-10 (P < 0.01). In the baseline, the liver accounted for 80% (13.3 +/- 0.8 micromol x kg(-1) x min(-1)) and each kidney contributed 10% (1.6 +/- 0.2 micromol x kg(-1) x min(-1)) to endogenous glucose production. During hypoglycemia, however, hepatic glucose production decreased to 4.0 +/- 0.4 micromol x kg(-1) x min(-1), whereas right renal glucose production doubled to 3.2 +/- 0.2 micromol x kg(-1) x min(-1) (P < 0.01). Left renal glucose production was 17 +/- 2 micromol x kg(-1) x min(-1), 14 of which were derived from the exogenous infusion. These results indicate that glucose production by the kidney is stimulated by counterregulatory hormones and represents an important component of the body's defense against insulin-induced hypoglycemia.