Ketone bodies are produced by the liver and used peripherally as an energy source when glucose is not readily available. The two main ketone bodies are acetoacetate (AcAc) and 3-beta-hydroxybutyrate (3HB), while acetone is the third, and least abundant, ketone body. Ketones are always present in the blood and their levels increase during fasting and prolonged exercise. They are also found in the blood of neonates and pregnant women. Diabetes is the most common pathological cause of elevated blood ketones. In diabetic ketoacidosis (DKA), high levels of ketones are produced in response to low insulin levels and high levels of counterregulatory hormones. In acute DKA, the ketone body ratio (3HB:AcAc) rises from normal (1:1) to as high as 10:1. In response to insulin therapy, 3HB levels commonly decrease long before AcAc levels. The frequently employed nitroprusside test only detects AcAc in blood and urine. This test is inconvenient, does not assess the best indicator of ketone body levels (3HB), provides only a semiquantitative assessment of ketone levels and is associated with false-positive results. Recently, inexpensive quantitative tests of 3HB levels have become available for use with small blood samples (5-25 microl). These tests offer new options for monitoring and treating diabetes and other states characterized by the abnormal metabolism of ketone bodies.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291520-7560%28199911/12%2915%3A6%3C412%3A%3AAID-DMRR72%3E3.0.CO%3B2-8

Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes

Arguably, a minimally invasive system using subcutaneous (s.c.) continuous glucose monitoring (CGM) and s.c. insulin delivery via insulin pump would be a most feasible step to closed-loop control in type 1 diabetes mellitus (T1DM). Consequently, diabetes technology is focusing on developing an artificial pancreas using control algorithms to link CGM with s.c. insulin delivery. The future development of the artificial pancreas will be greatly accelerated by employing mathematical modeling and computer simulation. Realistic computer simulation is capable of providing invaluable information about the safety and the limitations of closed-loop control algorithms, guiding clinical studies, and out-ruling ineffective control scenarios in a cost-effective manner. Thus computer simulation testing of closed-loop control algorithms is regarded as a prerequisite to clinical trials of the artificial pancreas. In this paper, we present a system for in silico testing of control algorithms that has three principal components: (1) a large cohort of n = 300 simulated “subjects” (n = 100 adults, 100 adolescents, and 100 children) based on real individuals’ data and spanning the observed variability of key metabolic parameters in the general population of people with T1DM; (2) a simulator of CGM sensor errors representative of Freestyle Navigator™, Guardian RT, or Dexcom™ STS™, 7-day sensor; and (3) a simulator of discrete s.c. insulin delivery via OmniPod Insulin Management System or Deltec Cozmo ® insulin pump. The system has been shown to represent adequate glucose fluctuations in T1DM observed during meal challenges, and has been accepted by the Food and Drug Administration as a substitute to animal trials in the preclinical testing of closed-loop control strategies.

/pdf/in-silico-preclinical-trials-a-proof-of-concept-in-closed-3llbpfenm5.pdf

In Silico Preclinical Trials: A Proof of Concept in Closed-Loop Control of Type 1 Diabetes

Type I diabetes may be an autoimmune disorder, although the evidence is largely circumstantial. The natural history of the disease after diagnosis includes partial remission in most patients, but only about 3 percent achieve transient insulin independence. beta Cell function, as indicated by the plasma concentration of C-peptide, is lost over 6 to 30 months and islet cell antibodies disappeared over 1 to 2 years. This article describes a pilot study in which 41 patients were treated with the immunosuppressive agent cyclosporine for 2 to 12 months. Of 30 patients treated within 6 weeks of diagnosis, 16 became insulin independent with concentrations of plasma C-peptide in the normal range and decreasing titers of islet cell antibodies. Of 11 patients who entered the study 8 to 44 weeks after diagnosis, two achieved this state. These results indicate that a controlled trial of the effects of cyclosporine in type I diabetes should be conducted.

https://science.sciencemag.org/content/sci/223/4643/1362.full.pdf

Effects of cyclosporine immunosuppression in insulin-dependent diabetes mellitus of recent onset

It has been suggested that elevated levels of insulin or insulin-like growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta. We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10(-9) M-1 and 10(-8) M-1 in all four cell types studied. Specific binding of IGF-I was between 7.2 and 7.9% per milligram of protein in endothelial cells and 9.1 and 10.4% in the vascular supporting cells. For 125I-MSA, retinal endothelial cells bound only 1.7-2.5%, whereas the aortic endothelial cells and the vascular supporting cells bound between 5.6 and 8.5% per milligram of protein. The specificity of the receptors for IGF-I and MSA differed, as insulin and MSA was able to compete with 125I-IGF-I for binding to the IGF-I receptors with 0.01-0.1, the potency of unlabeled IGF-I, whereas even 1 X 10(-6) M, insulin did not significantly compete with 125I-MSA for binding to the receptors for MSA. For growth-promoting effects, as measured by the incorporation of [3H]thymidine into DNA, confluent retinal endothelial cells responded to IGF-I and MSA by up to threefold increase in the rate of DNA synthesis, whereas confluent aortic endothelial cells did not respond at all. A similar differential of response to insulin between micro- and macrovascular endothelial cells was reported by us previously. In the retinal endothelium, insulin was more potent than IGF-I and IGF-I was more potent that MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In addition, insulin and IGF-I at 1 X 10(-6) and 1 X 10(-8) M, respectively, stimulated these cells to grow by doubling the number of cells as well. In all responsive tissues, the combination of insulin and IGFs were added together, no further increase in effect was seen. These data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones. These differences in biological response between cells from retinal capillaries and large arteries could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.

/pdf/receptors-and-growth-promoting-effects-of-insulin-and-h4o85hhx3k.pdf

Receptors and growth-promoting effects of insulin and insulinlike growth factors on cells from bovine retinal capillaries and aorta.

Background The last two decades have witnessed unprecedented technological progress in the development of continuous glucose sensors, resulting in the first generation of commercial glucose monitors. This has fuelled the development of prototypes of a closed-loop system based on the combination of a continuous monitor, a control algorithm, and an insulin pump.

Method  A review of electromechanical closed-loop approaches is presented. This is followed by a review of existing prototypes and associated glucose sensors. A literature review was undertaken from 1960 to 2004.

Results  Two main approaches exist. The extracorporeal s.c.–s.c. approach employs subcutaneous glucose monitoring and subcutaneous insulin delivery. The implantable i.v.–i.p. approach adopts intravenous sampling and intraperitoneal insulin delivery. Feasibility of both solutions has been demonstrated in small-scale laboratory studies using either the classical proportional–integral–derivative controller or a model predictive controller. Performance in the home setting has yet to be demonstrated.

Conclusions  The glucose monitor remains the main limiting factor in the development of a commercially viable closed-loop system, as presently available monitors fail to demonstrate satisfactory characteristics in terms of reliability and/or accuracy. Regulatory issues are the second limiting factor. Closed-loop systems are likely to be used first by health-care professionals in controlled environments such as intensive care units.

Continuous glucose monitoring and closed-loop systems

The diabetes which occurs spontaneously in the ‘BB’ Wistar rat has many affinities with human Type 1 (insulin-dependent) diabetes. It occurs in a non-obese, standard, laboratory rat derived from a non-inbred Wistar line. Both sexes are affected, with onset corresponding approximately to the time of sexual maturation. Both genetic and immune factors are involved in the aetiology, but their precise nature remains to be defined. Evolution of the overt clinical syndrome occurs over a period of hours to a few days. An intense insulitis is found, accompanied by selective destruction of B cells. Although insulitis may precede diabetes by many weeks, within 7–21 days after glycosuria the B cells are completely destroyed and have disappeared and the islets are few, small and with little residual inflammation. If untreated, marked wasting of body tissues, including fat and muscle protein, dehydration, and ketosis supervene. Careful study of littermates reveals glucose intolerance in 10%–25%, accompanied always by insulitis and these rats may subsequently develop insulin-dependent diabetes. Marked lymphopenia, mainly of thymus-derived (T) lymphocytes, both precedes and is sustained during glucose intolerance and overt diabetes. This lymphopenia appears to be associated reliably with insulitis, and may be a simple marker of susceptibility thereto. Abnormalities of nerves, testicles, and a tendency towards increased frequency of lymphomas have been found. Further research in this animal could lead to insights into aetiology, pathophysiology and complications potentially applicable to man.

/pdf/the-diabetic-syndrome-of-the-bb-wistar-rat-possible-biuwji6lti.pdf

The diabetic syndrome of the 'BB' Wistar rat: possible relevance to type 1 (insulin-dependent) diabetes in man.

Plasma from 14 diabetic and 6 nondiabetic BB Wistar rats along with plasma from 6 non-BB Wistar rats was evaluated for the presence of islet cells surface antibodies (ICSA) and antibodies to spleen lymphocytes by the protein-A radioligand assay. Dispersed Wistar rat islet cells incubated with plasma from diabetic rats bound 4255 ± 2208 cpm 125I-protein A/5 × 104 islet cells (mean ± SD) compared with 984 ± 454 cpm/5 × 104 islet cells in islet cells incubated with plasma from nondiabetic BB rats (P < 0.005). Twelve of the 14 diabetic rats with a duration of diabetes for 3–11 days bound radioactivity above the mean and 2 × SD of controls. The binding of 125I-protein A did not differ between nondiabetic BB rats and non-BB Wistar rats. Wistar rat spleen lymphocytes incubated in diabetic plasma bound 20,249 ± 10,783 cpm/2 × 106 spleen lymphocytes compared with 3460 ± 1809 in the controls (P < 0.005). Animals positive for ICSA correlated with those positive for spleen lymphocyte antibodies. It is concluded that ICSA and splenic lymphocyte antibodies are present in diabetic BB rats.

https://diabetes.diabetesjournals.org/content/diabetes/31/3/278.full.pdf

Islet Cell Surface Antibodies and Lymphocyte Antibodies in the Spontaneously Diabetic BB Wistar Rat

Exogenous protein in the absence of other calories can cause protein-sparing, but the mechanisms involved are controversial. It has been postulated that low insulin and high fat-derived substrate levels are necessary and sufficient conditions for such protein-sparing. We therefore established such conditions with differing protocols of protein input to define the role of protein input in mediating the response. Three groups of obese, nondiabetic subjects received the following diets: (1) 82.5±1.0 g protein/day (400 cal/day) for 21 days, n = 7; (2) the same, but as a refeeding diet for 7 days after 21-28 days of total fasts, n = 7; and (3) commencing with the same input, but with daily stepwise decrements over 14 days to 19.4±2.2 g/day, then maintained an additional 7 days, n = 4. Diet 3 gave approximately the amount and pattern of protein lost during total fasting.

The circulating hormone and substrate responses of diets 1 and 3 were comparable and resembled those of total fasts, in that plasma glucose and insulin fell and free fatty acids rose. Blood levels of alanine, pyruvate, and other glucogenic amino acids fell and blood levels of branched-chain amino acids rose transiently. Blood 3-hydroxybutyrate levels and urinary excretion were greater in diet 3 than diet 1, but less than in total fasting. Nitrogen balance in diet 1 was transiently negative, but in equilibrium from 12 to 21 days. In diet 3, it was constantly negative at −6 g/day, the values also observed at 21 days of fasting. Mean 3-methylhistidine excretion decreased by 170 μmol/day in diet 1 and 107 μmol/day in diet 3, reflecting decreased muscle protein catabolism. The refed, protein-depleted subjects, diet 2, showed an increase in plasma glucose without alteration in insulin levels. Free fatty acid and ketone body levels decreased to those of the steady state observed in diet 1. Glucogenic and branched-chain amino acids decreased transiently. Nitrogen balance became positive, and the low 3-methylhistidine excretion increased by 152 μmol/day.

The differing responses of nitrogen balance could not be accounted for on the basis of levels of insulin or of fat-derived substrates. The primary determinants of the protein-sparing observed appeared to be the protein supply itself, and the magnitude of the decrease in endogenous protein catabolism. The positive balance on refeeding after prior depletion of protein stores was likely due to the exogenous supply, combined with decreased catabolism and considerably increased reutilization.

/pdf/the-metabolic-response-to-hypocaloric-protein-diets-in-obese-168jucc3b4.pdf

The Metabolic Response to Hypocaloric Protein Diets in Obese Man

An extracorporeal “closed-loop” system has been employed to maintain glycemia in the normal range during consumption of meals in nine insulin-treated diabetics. This artificial pancreas system incorporated continuous blopd glucose monitoring (0.05 ml. per minute, delay time 90 seconds), a computer programed to respond to glycemia, and a hormone delivery system. Intravenous insulin delivery rates were determined by control parameters responsive to both glucose concentration and its rate of change. Because insulin-dependent diabetics often defend themselves poorly against hypoglycemie (in some cases due to inadequate glucagon responses), the instrument was also programed for exogenous glucagon delivery. A priori selection of ideal parameters for insulin and glucagon delivery for each individual is not yet possible. Con sequently, when the parameters were used for the first time on each subject, they were varied over a reasonable range. This approach resulted in a corresponding variety of glycemie responses, the average of which characterized a set of initial parameters that is generally applicable. Appropriate control parameters are presented that successfully prevented hypoglycemie. Glucagon delivery directly related to glycemia appeared sufficient for this purpose, thus obviating the need for dextrose administration. This system provides a technique for complete normalization of blood glucose concentration in the types of diabetics tested, during both fed and interprandial periods. It has yielded insights essential to the development of more sophisticated future devices.

Normalization of Glycemia in Diabetics During Meals with Insulin and Glucagon Delivery by the Artificial Pancreas

The artificial endocrine pancreas (AEP) can normalize glycemia at rest and with meals. To determine whether insulin, glucagon, and amino acid profiles are also normalized, nine diabetics on subcutaneous insulin ( S C ) and AEP control were compared to ten normal controls (NC). Glycemia was monitored continuously over 10 hr during which meals were consumed. Insulin infusion rate, and the levels of immunoreactive insulin (IRI) (in NC), free insulin (in S C and AEP), C-peptide, glucagon, and amino acids are reported. Glycemia in AEP started at somewhat higher levels than in NC, but with breakfast and thereafter, it was identical. In S C , hyperglycemia prevailed throughout, with no systematic change in free IRI. In AEP, both basal and peak free insulin levels, measured in four patients, were significantly higher than in NC. C-peptide values were significantly lower in diabetics and did not change with meals. Basal glucagon values were not different in the three groups and changes with meals were of small magnitude. Branched chain amino acids were higher in S C and did not increase as in NC. In AEP, levels were lower than NC after the first two meals. Similarly, lysine and threonine were lower in AEP than in NC at the same times. Alanine, though similar at the onset, was lower 2 hr postbreakfast and higher 2 hrs postsupper in AEP and S C compared to NC. These studies demonstrate that glycemic control with AEP is accompanied by hyperinsulinemia, which could account for the amino acid responses and the small alterations in immunoreactive glucagon (IRG) patterns. Further refinement is needed to obtain full normalization of metabolic profiles.

Azima F. Nakhooda

Papers

The diabetic syndrome of the 'BB' Wistar rat: possible relevance to type 1 (insulin-dependent) diabetes in man.

Islet Cell Surface Antibodies and Lymphocyte Antibodies in the Spontaneously Diabetic BB Wistar Rat

The Metabolic Response to Hypocaloric Protein Diets in Obese Man

Normalization of Glycemia in Diabetics During Meals with Insulin and Glucagon Delivery by the Artificial Pancreas

Insulin, glucagon, and amino acids during glycemic control by the artificial pancreas in diabetic man.