α-Pinene, a Main Component of Pinus Essential Oils, Enhances the Expression of Insulin-Sensitive Glucose Transporter Type 4 in Murine Skeletal Muscle Cells.

Glucose transporter-4 (GLUT4) represents the major glucose transporter isoform responsible for glucose uptake into insulin-sensitive cells, primarily in skeletal muscle and adipose tissues. In insulin-resistant conditions, such as type 2 diabetes mellitus, GLUT4 expression and/or translocation to the cell plasma membrane is reduced, compromising cell energy metabolism. Therefore, the use of synthetic or naturally occurring molecules able to stimulate GLUT4 expression represents a good tool for alternative treatments of insulin resistance. The present study aimed to investigate the effects of essential oils (EOs) derived from Pinus spp. (P. nigra and P. radiata) and of their main terpenoid constituents (α- and ß-pinene) on the expression/translocation of GLUT4 in myoblast C2C12 murine cells. For this purpose, the chemical profiles of the EOs were first analyzed through gas chromatography-mass spectrometry (GC-MS). Cell viability was assessed by MTT assay, and GLUT4 expression/translocation was evaluated through RT-qPCR and flow cytometry analyses. The results showed that only the P. nigra essential oil (PnEO) and α-pinene can increase the transcription of the Glut4/Scl2a4 gene, resulting in a subsequent increase in the amount of GLUT4 produced and its plasma membrane localization. Moreover, the PnEO or α-pinene can induce Glut4 expression both during myogenesis and in myotubes. In summary, the PnEO and α-pinene emulate insulin's effect on the GLUT4 transporter expression and its translocation to the muscle cell surface.

The Attenuation of Insulin/IGF-1 Signaling Pathway Plays a Crucial Role in the Myo-Inositol-Alleviated Aging in Caenorhabditis elegans.

Myo-Inositol (MI) has been shown to alleviate aging in Caenorhabditis (C). elegans. However, the mechanism by which MI alleviates aging remains unclear. In this study, we investigate whether MI can modulate the PI3K so as to attenuate the insulin/IGF-1 signaling (IIS) pathway and exert the longevity effect. The wild-type C. elegans and two mutants of AKT-1 and DAF-16 were used to explore the mechanism of MI so as to extend the lifespan, as well as to improve the health indexes of pharyngeal pumping and body bend, and an aging marker of autofluorescence in the C. elegans. We confirmed that MI could significantly extend the lifespan of C. elegans. MI also ameliorated the pharyngeal pumping and body bend and decreased autofluorescence. We further adopted the approach to reveal the loss-of-function mutants to find the signaling mechanism of MI. The functions of the lifespan-extending, health-improving, and autofluorescence-decreasing effects of MI disappeared in the AKT-1 and DAF-16 mutants. MI could also induce the nuclear localization of the DAF-16. Importantly, we found that MI could dramatically inhibit the phosphoinositide 3-kinase (PI3K) activity in a dose-dependent manner with an IC50 of 90.2 µM for the p110α isoform of the PI3K and 21.7 µM for the p110ß. In addition, the downregulation of the PI3K expression and the inhibition of the AKT phosphorylation by MI was also obtained. All these results demonstrate that MI can inhibit the PI3K activity and downregulate the PI3K expression, and the attenuation of the IIS pathway plays a crucial role for MI in alleviating aging in C. elegans.

High Glucose Promotes Inflammation and Weakens Placental Defenses against E. coli and S. agalactiae Infection: Protective Role of Insulin and Metformin.

Placentas from gestational diabetes mellitus (GDM) patients undergo significant metabolic and immunologic adaptations due to hyperglycemia, which results in an exacerbated synthesis of proinflammatory cytokines and an increased risk for infections. Insulin or metformin are clinically indicated for the treatment of GDM; however, there is limited information about the immunomodulatory activity of these drugs in the human placenta, especially in the context of maternal infections. Our objective was to study the role of insulin and metformin in the placental inflammatory response and innate defense against common etiopathological agents of pregnancy bacterial infections, such as E. coli and S. agalactiae, in a hyperglycemic environment. Term placental explants were cultivated with glucose (10 and 50 mM), insulin (50-500 nM) or metformin (125-500 µM) for 48 h, and then they were challenged with live bacteria (1 × 105 CFU/mL). We evaluated the inflammatory cytokine secretion, beta defensins production, bacterial count and bacterial tissue invasiveness after 4-8 h of infection. Our results showed that a GDM-associated hyperglycemic environment induced an inflammatory response and a decreased beta defensins synthesis unable to restrain bacterial infection. Notably, both insulin and metformin exerted anti-inflammatory effects under hyperglycemic infectious and non-infectious scenarios. Moreover, both drugs fortified placental barrier defenses, resulting in reduced E. coli counts, as well as decreased S. agalactiae and E. coli invasiveness of placental villous trees. Remarkably, the double challenge of high glucose and infection provoked a pathogen-specific attenuated placental inflammatory response in the hyperglycemic condition, mainly denoted by reduced TNF-α and IL-6 secretion after S. agalactiae infection and by IL-1ß after E. coli infection. Altogether, these results suggest that metabolically uncontrolled GDM mothers develop diverse immune placental alterations, which may help to explain their increased vulnerability to bacterial pathogens.

Portal Glucose Infusion, Afferent Nerve Fibers, and Glucose and Insulin Tolerance of Insulin-Resistant Rats.

BACKGROUND: The role of hepatoportal glucose sensors is poorly understood in the context of insulin resistance. OBJECTIVES: We assessed the effects of glucose infusion in the portal vein on insulin tolerance in 2 rat models of insulin resistance, and the role of capsaicin sensitive nerves in this signal. METHODS: Male Wistar rats, 8 weeks old, weighing 250-275 g, were used. Insulin and glucose tolerance were assessed following a 4-hour infusion of either glucose or saline through catheterization in the portal vein in 3 paradigms. In experiment 1, for diet-induced insulin resistance, rats were fed either a control diet (energy content: proteins = 22.5%, carbohydrates = 64.1%, and lipids = 13.4%) or a high-fat diet (energy content: proteins = 15.3%, carbohydrates = 40.3%, and lipids =44.4%) for 4 months. In experiment 2, for centrally induced peripheral insulin resistance, catheters were inserted in the carotid artery to deliver either an emulsion of triglycerides [intralipid (IL)] or saline towards the brain for 24 hours. In experiment 3, for testing the role of capsaicin-sensitive nerves, experiment 2 was repeated following a periportal treatment with capsaicin or vehicle. RESULTS: In experiment 1, when compared to rats fed the control diet, rats fed the high-fat diet exhibited decreased insulin and glucose tolerance (P ≤ 0.05) that was restored with a glucose infusion in the portal vein (P ≤ 0.05). In experiment 2, infusion of a triglyceride emulsion towards the brain (IL rats) decreased insulin and glucose tolerance and increased hepatic endogenous production when compared to saline-infused rats (P ≤ 0.05). Glucose infusion in the portal vein in IL rats restored insulin and glucose tolerance, as well as hepatic glucose production, to controls levels (P ≤ 0.05). In experiment 3, portal infusion of glucose did not increase insulin tolerance in IL rats that received a periportal pretreatment with capsaicin. CONCLUSIONS: Stimulation of hepatoportal glucose sensors increases insulin tolerance in rat models of insulin resistance and requires the presence of capsaicin-sensitive nerves.

Physiologic Insulin Resensitization as a Treatment Modality for Insulin Resistance Pathophysiology.

Prevalence of type 2 diabetes increased from 2.5% of the US population in 1990 to 10.5% in 2018. This creates a major public health problem, due to increases in long-term complications of diabetes, including neuropathy, retinopathy, nephropathy, skin ulcers, amputations, and atherosclerotic cardiovascular disease. In this review, we evaluated the scientific basis that supports the use of physiologic insulin resensitization. Insulin resistance is the primary cause of type 2 diabetes. Insulin resistance leads to increasing insulin secretion, leading to beta-cell exhaustion or burnout. This triggers a cascade leading to islet cell destruction and the long-term complications of type 2 diabetes. Concurrent with insulin resistance, the regular bursts of insulin from the pancreas become irregular. This has been treated by the precise administration of insulin more physiologically. There is consistent evidence that this treatment modality can reverse the diabetes-associated complications of neuropathy, diabetic ulcers, nephropathy, and retinopathy, and that it lowers HbA1c. In conclusion, physiologic insulin resensitization has a persuasive scientific basis, significant treatment potential, and likely cost benefits.

Dietary Fat and Sugar Differentially Affect ß-Adrenergic Stimulation of Cardiac ERK and AKT Pathways in C57BL/6 Male Mice Subjected to High-Calorie Feeding.

BACKGROUND: High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. OBJECTIVE: The goal of this study was to investigate the mechanisms by which high amounts of dietary fat and sugar affect cardiac growth regulatory pathways. METHODS: Male C57BL/6 mice (9 wk old; n = 20/group) were fed a standard rodent diet (STD; kcal% protein-fat-carbohydrate, 29-17-54), a high-fat diet (HFD; 20-60-20), a high-fat and high-sugar Western diet (WD; 20-45-35), a high-sugar diet with mixed carbohydrates (HCD; 20-10-70), or a high-sucrose diet (HSD; 20-10-70). Body composition was assessed weekly by EchoMRI. Whole-body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After 6 wk on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO), and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. Data were analyzed by ANOVA with data from the STD group included for references only. RESULTS: Compared with HCD and HSD, WD and HFD increased body fat mass 2.7- to 3.8-fold (P < 0.001), induced glucose intolerance (P < 0.001), and increased insulin concentrations >1.5-fold (P < 0.05), thereby enhancing basal and ISO-stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+25-63%; P < 0.05). Compared with HFD, the high-sugar diets potentiated ISO-mediated stimulation of the glucose-sensitive kinases PYK2 (>47%; P < 0.05 for HCD and HSD) and ERK (>34%; P < 0.05 for WD, HCD, and HSD), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (>64%; P < 0.05 for WD, HCD, and HSD). CONCLUSIONS: Dietary fat and sugar affect cardiac growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological cardiac remodeling.

Peripherally delivered hepatopreferential insulin analog insulin-406 mimics the hypoglycaemia-sparing effect of portal vein human insulin infusion in dogs.

AIMS: We previously quantified the hypoglycaemia-sparing effect of portal vs peripheral human insulin delivery. The current investigation aimed to determine whether a bioequivalent peripheral vein infusion of a hepatopreferential insulin analog, insulin-406, could similarly protect against hypoglycaemia. MATERIALS AND METHODS: Dogs received human insulin infusions into either the hepatic portal vein (PoHI, n = 7) or a peripheral vein (PeHI, n = 7) for 180 minutes at four-fold the basal secretion rate (6.6 pmol/kg/min) in a previous study. Insulin-406 (Pe406, n = 7) was peripherally infused at 6.0 pmol/kg/min, a rate determined to decrease plasma glucose by the same amount as with PoHI infusion during the first 60 minutes. Glucagon was fixed at basal concentrations, mimicking the diminished α-cell response seen in type 1 diabetes. RESULTS: Glucose dropped quickly with PeHI infusion, reaching 41 ± 3 mg/dL at 60 minutes, but more slowly with PoHI and Pe406 infusion (67 ± 2 and 72 ± 4 mg/dL, respectively; P < 0.01 vs PeHI for both). The hypoglycaemic nadir (c. 40 mg/dL) occurred at 60 minutes with PeHI infusion vs 120 minutes with PoHI and Pe406 infusion. ΔAUCepinephrine during the 180-minute insulin infusion period was two-fold higher with PeHI infusion compared with PoHI and Pe406 infusion. Glucose production (mg/kg/min) was least suppressed with PeHI infusion (Δ = 0.79 ± 0.33) and equally suppressed with PoHI and Pe406 infusion (Δ = 1.16 ± 0.21 and 1.18 ± 0.17, respectively; P = NS). Peak glucose utilization (mg/kg/min) was highest with PeHI infusion (4.94 ± 0.17) and less with PoHI and Pe406 infusion (3.58 ± 0.58 and 3.26 ± 0.08, respectively; P < 0.05 vs Pe for both). CONCLUSIONS: Peripheral infusion of hepatopreferential insulin can achieve a metabolic profile that closely mimics portal insulin delivery, which reduces the risk of hypoglycaemia compared with peripheral insulin infusion.

Exploration of the Key Factors for Optimizing the in Vivo Oral Delivery of Insulin by Using a Noncovalent Strategy with Cell-Penetrating Peptides.

This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100-500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.

daf-16/FOXO and glod-4/glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans.

AIMS/HYPOTHESIS: The aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms. METHODS: Nematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment. Additionally, the activity of glyoxalase-1 and superoxide dismutase (SOD) and the formation of reactive oxygen species (ROS) and AGEs were determined. RESULTS: Insulin and its analogues reversed the life-shortening effect of HG conditions and prevented the glucose-induced neuronal impairment. Insulin treatment under HG conditions was associated with reduced concentration of glucose, as well as a reduced formation of ROS and AGEs, and increased SOD activity. These effects were dependent on the Forkhead box O (FOXO) homologue abnormal dauer formation (DAF)-16. Furthermore, glyoxalase-1 activity, which was impaired under HG conditions, was restored by human insulin. This was essential for the insulin-induced lifespan extension under HG conditions, as no change in lifespan was observed following either suppression or overexpression of glyoxalase-1. CONCLUSIONS/INTERPRETATION: Human insulin and its analogues prevent the reduction in lifespan and neuronal damage caused by HG conditions. The effect of human insulin is mediated by a daf-2/insulin receptor and daf-16/FOXO-dependent pathway and is mediated by upregulation of detoxifying mechanisms.

Open flow microperfusion: pharmacokinetics of human insulin and insulin detemir in the interstitial fluid of subcutaneous adipose tissue.

AIMS: To compare the time profile of insulin detemir and human insulin concentrations in the interstitial fluid (ISF) of subcutaneous adipose tissue during constant i.v. infusion and to investigate the relationship between the pharmacokinetics of both insulin molecules in plasma and the ISF of subcutaneous adipose tissue. METHODS: During a 6-h hyperinsulinaemic-euglycaemic clamp (plasma glucose level 8 mmol/l) human insulin (21 and 42 pmol/min/kg) or insulin detemir (209 and 417 pmol/min/kg) were infused i.v. in eight rats per dose level. Open flow microperfusion (OFM) was used to continuously assess interstitial insulin concentrations in subcutaneous adipose tissue. RESULTS: At the lower infusion rate, insulin detemir appeared significantly later in the ISF than in the plasma (p < 0.05) and also appeared later in the ISF relative to human insulin (p < 0.005). CONCLUSIONS: By using OFM we were able to monitor albumin-bound insulin detemir directly in the ISF of subcutaneous tissue and confirm its delayed transendothelial passage to a peripheral site of action.

EFFECTIVENESS OF INPATIENT INSULIN ORDER SETS USING HUMAN INSULINS IN NONCRITICALLY ILL PATIENTS IN A RURAL HOSPITAL.

OBJECTIVE: Recent guidelines recommend a physiologic approach to non-intensive care unit (ICU) inpatient glucose management utilizing basal-bolus with correctional (BBC) insulin over traditional sliding-scale insulin monotherapy. Unfortunately, few studies exist using a BBC approach restricted to human insulins (regular and neutral protamine Hagedorn [NPH]). This study evaluated changes in provider prescribing patterns, effects on blood glucose, and safety with implementation of hospital order sets for BBC using human insulins. METHODS: Order sets were developed for non-ICU inpatients, consisting of basal, prandial, and correctional insulin using NPH and regular human insulins. Evaluation compared a 4-month period before (admissions, n = 274) with a 4-month period after order set availability (n = 302). Primary outcome was change in insulin prescribing patterns. Secondary outcomes included use of nonpreferred diabetes treatments, hemoglobin A1c testing, mean daily blood glucose, and incidence of hypoglycemia. RESULTS: Use of BBC insulin regimen increased from 10.6 to 27.5% after order set implementation (P<.001). Use of oral antihyperglycemic agents decreased from 24.1 to 14.9% after implementation (P = .006). Hemoglobin A1c testing rose from 50.0 to 62.3% after (P = .003). Mean daily blood glucose improved, with an estimated mean difference of 14.4 mg/dL (95% confidence interval, 2.2 to 26.5 mg/dL) over hospital days 3 through 9 (P = .02). There was no significant change in the incidence of moderate or severe hypoglycemia. CONCLUSION: Implementation of hospital-wide human insulin order sets led to improvements in prescribing practices and blood glucose control, without increasing the incidence of hypoglycemia. These order sets may be useful for facilities limited by formulary and cost considerations to the use of older human insulins.

BASAL-BOLUS REGIMEN WITH INSULIN ANALOGUES VERSUS HUMAN INSULIN IN MEDICAL PATIENTS WITH TYPE 2 DIABETES: A RANDOMIZED CONTROLLED TRIAL IN LATIN AMERICA.

OBJECTIVE: Few randomized studies have focused on the optimal management of non-intensive care unit patients with type 2 diabetes in Latin America. We compared the safety and efficacy of a basal-bolus regimen with analogues and human insulins in general medicine patients admitted to a University Hospital in Asunción, Paraguay. METHODS: In a prospective, open-label trial, we randomized 134 nonsurgical patients with blood glucose (BG) between 140 and 400 mg/dL to a basal-bolus regimen with glargine once daily and glulisine before meals (n = 66) or Neutral Protamine Hagedorn (NPH) twice daily and regular insulin before meals (n = 68). Major outcomes included differences in daily BG levels and frequency of hypoglycemic events between treatment groups. RESULTS: There were no differences in the mean daily BG (157 ± 37 mg/dL versus 158 ± 44 mg/dL; P = .90) or in the number of BG readings within target <140 mg/dL before meals (76% versus 74%) between the glargine/glulisine and NPH/regular regimens. The mean insulin dose in the glargine/glulisine group was 0.76 ± 0.3 units/kg/day (glargine, 22 ± 9 units/day; glulisine, 31 ± 12 units/day) and was not different compared with NPH/regular group (0.75 ± 0.3 units/kg/day [NPH, 28 ± 12 units/day; regular, 23 ± 9 units/day]). The overall prevalence of hypoglycemia (<70 mg/dL) was similar between patients treated with NPH/regular and glargine/glulisine (38% versus 35%; P = .68), but more patients treated with human insulin had severe (<40 mg/dL) hypoglycemia (7.6% versus 25%; P = .08). There were no differences in length of hospital stay or mortality between groups. CONCLUSION: The basal-bolus regimen with insulin analogues resulted in equivalent glycemic control and frequency of hypoglycemia compared to treatment with human insulin in hospitalized patients with diabetes.

TWO TREATMENT APPROACHES FOR HUMAN REGULAR U-500 INSULIN IN PATIENTS WITH TYPE 2 DIABETES NOT ACHIEVING ADEQUATE GLYCEMIC CONTROL ON HIGH-DOSE U-100 INSULIN THERAPY WITH OR WITHOUT ORAL AGENTS: A RANDOMIZED, TITRATION-TO-TARGET CLINICAL TRIAL.

OBJECTIVE: To compare the efficacy and safety of 2 dosing regimens for human regular U-500 insulin (U-500R, 500 units/mL) replacing high-dose U-100 insulins with or without oral antihyperglycemic drugs in patients with inadequately controlled type 2 diabetes (T2D). METHODS: We conducted a 24-week, open-label, parallel trial in 325 patients (demographics [means]: age, 55.4 years; diabetes duration, 15.2 years; body mass index, 41.9 kg/m(2); glycated hemoglobin [HbA1c], 8.7%; U-100 insulin dose, 287.5 units administered in 5 injections/day [median; range, 2 to 10]). Patients were randomized to thrice-daily (TID, n = 162) or twice-daily (BID, n = 163) U-500R after a 4-week lead-in period. The primary outcome was HbA1c change from baseline. RESULTS: After 24 weeks, both treatments demonstrated significant HbA1c reductions (TID, -1.12%; BID, -1.22%; both, P<.001) and clinical equivalence (difference, -0.10%; 95% confidence interval, -0.33 to 0.12%; noninferiority margin, 0.4%). Comparable increases in total daily U-500R dose (TID, 242.7 to 343.1 units; BID, 249.0 to 335.0 units) were observed. Incidence and rate of documented symptomatic hypoglycemia (≤70 mg/dL) were lower for TID versus BID (P = .003 and P = .02, respectively); severe hypoglycemia was similar between treatments. Weight gain was similar in both groups (TID, 5.4 kg; BID, 4.9 kg). CONCLUSION: Initiation and titration of U-500R using either algorithm (TID or BID) improves glycemic control effectively and safely with fewer injections in patients with T2D treated with high-dose/high-volume U-100 insulin. These results provide clinicians with a practical framework for using U-500R in severely insulin-resistant patients with suboptimally controlled T2D.

OPTIMIZED HUMAN REGULAR U-500 INSULIN TREATMENT IMPROVES ß-CELL FUNCTION IN SEVERELY INSULIN-RESISTANT PATIENTS WITH LONG-STANDING TYPE 2 DIABETES AND HIGH INSULIN REQUIREMENTS.

OBJECTIVE: To assess ß-cell function and insulin sensitivity following improvement in glycemic control in severely insulin-resistant patients with poorly controlled type 2 diabetes (T2D). METHODS: A subset of patients in a 24-week, open-label, randomized trial comparing thrice-daily (n = 14/162) versus twice-daily (n = 11/163) human regular U-500 insulin (U-500R) underwent mixed meal tolerance testing at baseline and endpoint. Baseline characteristics were similar between treatment groups (combined means: age, 54.0 years; diabetes duration, 13.6 years; body mass index, 38.8 kg/m(2); glycated hemoglobin [HbA1c], 8.3%; U-100 insulin dose, 287.6 units/day, 2.6 units/kg/day). Primary outcome measure was ratio of area under the curve (AUC) for C-peptide to glucose (AUCC-peptide/AUCglucose) at 24-week endpoint. RESULTS: Change from baseline HbA1c, daily U-500R dose, and weight were -1.17% (P = .0002), +80.8 units (P = .0003), and +5.9 kg (P = .33), respectively. ß-Cell function significantly improved after 24 weeks of U-500R therapy in combined treatment groups. The AUCC-peptide/AUCglucose increased 34.0% (ratio of least-squares geometric mean, 1.34; 95% confidence interval, 1.18 to 1.52; P = .0001). Integral of total insulin secretion rate increased from 27.0 to 33.7 nmol/m(2), and glucose sensitivity improved from 18.3 to 24.0 pmol/min/m(2)/mM (both, P = .02). Matsuda index improved from 0.8 to 1.3 (P = .008). CONCLUSION: Despite long-standing diabetes and poor glycemic control at baseline, functional recovery of ß-cells was observed with improved glycemic control in these severely insulin-resistant patients with T2D, possibly due to alleviation of glucotoxicity.

Enhanced insulin action following subcutaneous co-administration of insulin and C-peptide in rats.

BACKGROUND: This study was undertaken to examine if C-peptide (C) may interact with hexameric insulin and facilitate its disaggregation into the physiologically active monomeric form. METHODS: Regular insulin (I) or an insulin analogue (IA) were injected s.c. in rats together with C or its C-terminal pentapeptide (PP). I or IA and C or PP were administered either as a physical mixture or into two separate s.c. depots. Whole body glucose utilization was evaluated using the euglycemic clamp technique. Phosphorylation of Akt/PKB and GSK in liver and skeletal muscles and 86Rb⁺ uptake by L6 cells were measured. RESULTS: S.c. injection of a mixture of I and C or I and PP resulted in a 30-55% greater (P < 0.01-0.001) and 15-27% (P < 0.05-0.001) longer stimulation of whole body glucose utilization than after separate injections. Insulin-stimulated phosphorylation of Akt/PKB in liver increased 35% more after injection of I and C in mixture compared with after separate injections. Phosphorylation of GSK3 was augmented by 50% (P < 0.05) following the injection of I and C in mixture compared with separate injections. Stimulation of myotubes with premixed I and C (1 nM) elicited 20% additional increase in ouabain-sensitive 86Rb⁺ uptake (P < 0.05) in comparison with the effect when I and C were added separately. CONCLUSIONS: Subcutaneous co-administration of insulin and C results in augmented insulin bioactivity at the level of tissue glucose uptake, intracellular signalling, and enzyme activation. These effects may be attributed to augmented C mediated disaggregation of hexameric insulin into its physiologically active monomeric form.

Long-acting insulins alter milk composition and metabolism of lactating dairy cows.

This study investigated the effect of 2 different types of long-acting insulin on milk production, milk composition, and metabolism in lactating dairy cows. Multiparous cows (n=30) averaging 88 d in milk were assigned to one of 3 treatments in a completely randomized design. Treatments consisted of control (C), Humulin-N (H; Eli Lilly and Company, Indianapolis, IN), and insulin glargine (L). The H and L treatments were administered twice daily at 12-h intervals via subcutaneous injection for 10d. Cows were milked twice daily, and milk composition was determined every other day. Mammary biopsies were conducted on d 11, and mammary proteins extracted from the biopsies were analyzed by Western blot for components of insulin and mammalian target of rapamycin signaling pathways. Treatment had no effect on dry matter intake or milk yield. Treatment with both forms of long-acting insulin increased milk protein content and tended to increase milk protein yield over the 10-d treatment period. Analysis of milk N fractions from samples collected on d 10 of treatment suggested that cows administered L tended to have higher yields of milk protein fractions than cows administered H. Milk fat content and yield tended to be increased for cows administered long-acting insulins. Lactose content and yields were decreased by treatment with long-acting insulins. Administration of long-acting insulins, particularly L, tended to shift milk fatty acid composition toward increased short- and medium-chain fatty acids and decreased long-chain fatty acids. Plasma concentrations of glucose and urea N were lower for cows administered long-acting insulins; interactions of treatment and sampling time were indicative of more pronounced effects of L than H on these metabolites. Concentrations of nonesterified fatty acids and insulin were increased in cows administered long-acting insulins. Decreased concentrations of urea N in both plasma and milk suggested more efficient use of N in cows administered long-acting insulins. Western blot analysis of mammary tissue collected by biopsy indicated that the ratios of phosphorylated protein kinase b (Akt) to total Akt and phosphorylated ribosomal protein S6 (rpS6) to total rpS6 were not affected by long-acting insulins. Modestly elevating insulin activity in lactating dairy cows using long-acting insulins altered milk composition and metabolism. Future research should explore mechanisms by which either insulin concentrations or insulin signaling pathways in the mammary gland can be altered to enhance milk fat and protein production.

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Glucose-Activated Switch Regulating Insulin Analog Secretion Enables Long-term Precise Glucose Control in Mice With Type 1 Diabetes.

Genetic modification of non-ß-cells to produce insulin is a promising therapeutic strategy for type 1 diabetes; however, it is associated with issues, including biosafety and precise regulation of insulin supply. In this study, a glucose-activated single-strand insulin analog (SIA) switch (GAIS) was constructed to achieve repeatable pulse activation of SIA secretion in response to hyperglycemia. In the GAIS system, the conditional aggregation domain-furin cleavage sequence-SIA fusion protein was encoded by the intramuscularly delivered plasmid and temporarily kept in the endoplasmic reticulum (ER) because it binds to the GRP78 protein; then, upon hyperglycemia, the SIA was released and secreted into the blood. In vitro and in vivo experiments systematically demonstrated the effects of the GAIS system, including glucose-activated and repeatable SIA secretion, long-term precise blood glucose control, recovered HbA1c levels, improved glucose tolerance, and ameliorated oxidative stress. Additionally, this system offers sufficient biosafety, as evidenced by the assays of immunological and inflammatory safety, ER stress, and histological evaluation. Compared with the viral delivery/expression system, the ex vivo implantation of engineered cells, and the exogenous inducer system, the GAIS system combines the advantages of biosafety, effectiveness, persistence, precision, and convenience, providing therapeutic potential for the treatment of type 1 diabetes. ARTICLE HIGHLIGHTS: We undertook this study to establish a glucose-responsive single-strand insulin analog (SIA) self-supply system in vivo. We sought to determine whether the endoplasmic reticulum (ER) can serve as a safe and temporary repository to store designed fusion proteins and release SIAs under hyperglycemic conditions for efficient blood glucose regulation. The intramuscularly expressed plasmid-encoded conditional aggregation domain-furin cleavage sequence-SIA fusion protein can be temporarily stored in the ER, and the SIA can be released under the stimulation of hyperglycemia, resulting in efficient and long-term regulation of stable blood glucose in mice with type 1 diabetes (T1D). The glucose-activated SIA switch system provides applicable potential for T1D therapy, integrating regulation and monitoring of blood glucose levels.

Fatty acids acutely enhance insulin-induced oxidative stress and cause insulin resistance by increasing mitochondrial reactive oxygen species (ROS) generation and nuclear factor-κB inhibitor (IκB)-nuclear factor-κB (NFκB) activation in rat muscle, in the absence of mitochondrial dysfunction.

AIMS/HYPOTHESIS: Insulin effects reportedly involve reactive oxygen species (ROS) and oxidative stress in vitro, but skeletal muscle oxidative stress is an emerging negative regulator of insulin action following high-fat feeding. NEFA may enhance oxidative stress and insulin resistance. We investigated the acute impact of insulin with or without NEFA elevation on muscle ROS generation and insulin signalling, and the potential association with altered muscle mitochondrial function. METHODS: We used hyperinsulinaemic-euglycaemic clamping, 150 min, without or with lipid infusion to modulate plasma NEFA concentration in lean rats. RESULTS: Insulin and glucose (Ins) infusion selectively enhanced xanthine oxidase-dependent muscle ROS generation. Ins with lipid infusion (Ins+NEFA) lowered whole-body glucose disposal and muscle insulin signalling, and these effects were associated with high muscle mitochondrial ROS generation and activation of the proinflammatory nuclear factor-κB inhibitor (IκB)-nuclear factor-κB (NFκB) pathway. Antioxidant infusion prevented NEFA-induced systemic insulin resistance and changes in muscle mitochondrial ROS generation, IκB-NFκB pathway and insulin signalling. Changes in insulin sensitivity and signalling were independent of changes in mitochondrial enzyme activity and ATP production, which, in turn, were not impaired by changes in ROS generation under any condition. CONCLUSIONS/INTERPRETATION: Acute muscle insulin effects include enhanced ROS generation through xanthine oxidase. Additional NEFA elevation enhances mitochondrial ROS generation, activates IκB-NFκB and reduces insulin signalling. These alterations are not associated with acute reductions in mitochondrial enzyme activity and ATP production, and are reversed by antioxidant infusion. Thus, NEFA acutely cause systemic and muscle insulin resistance by enhancing muscle oxidative stress through mitochondrial ROS generation and IκB-NFκB activation.