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Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly attributable to inadequate glucagon secretion. Intra-islet somatostatin (SST) suppression of hypoglycemia-stimulated α-cell glucagon release plays an important role. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by SST receptor type 2 (SSTR2) antagonism of α-cells, which relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes-prone (BBDP) rats mimic insulin-dependent human autoimmune T1D, whereas nondiabetic BBDP rats mimic prediabetes. Diabetic and nondiabetic rats underwent a 3-h infusion of vehicle compared with SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycemia clamped at 3 ± 0.5 mmol/L. Diabetic rats treated with SSTR2a needed little or no glucose infusion compared with untreated rats. We attribute this effect to SSTR2a restoration of the attenuated glucagon response. Direct effects of SSTR2a on α-cells was assessed by resecting the pancreas, which was cut into fine slices and subjected to perifusion to monitor glucagon release. SSTR2a treatment enhanced low-glucose–stimulated glucagon and corticosterone secretion to normal levels in diabetic rats. SSTR2a had similar effects in vivo in nondiabetic rats and promoted glucagon secretion from nondiabetic rat and human pancreas slices. We conclude that SST contributes to impaired glucagon responsiveness to hypoglycemia in autoimmune T1D. SSTR2a treatment can fully restore hypoglycemia-stimulated glucagon release sufficient to attain normoglycemia in both diabetic and prediabetic stages.

Intense exercise (IE) (>80% O(2max)) causes a seven- to eightfold increase in glucose production (R(a)) and a fourfold increase in glucose uptake (R(d)), resulting in hyperglycemia, whereas moderate exercise (ME) causes both to double. If norepinephrine (NE) plus epinephrine (Epi) infusion during ME produces the plasma levels and R(a) of IE, this would prove them capable of mediating these responses. Male subjects underwent 40 min of 53% O(2max) exercise, eight each with saline (control [CON]), or with combined NE + Epi (combined catecholamine infusion [CCI]) infusion from min 26-40. In CON and CCI, NE levels reached 7.3 +/- 0.7 and 33.1 +/- 2.9 nmol/l, Epi 0.94 +/- 0.08 and 7.06 +/- 0.44 nmol/l, and R(a) 3.8 +/- 0.4 and 12.9 +/- 0.8 mg. kg(-1). min(-1) (P < 0.001), respectively, at 40 min. R(d) increased to 3.5 +/- 0.4 vs. 11.2 +/- 0.8 mg. kg(-1). min(-1) and glycemia 5.2 +/- 0.2 mmol/l in CON vs. 6.5 +/- 0.2 mmol/l in CCI (P < 0.001). The glucagon-to-insulin ratio did not differ. Comparing CCI data to those from 14-min IE (n = 16), peak NE (33.6 +/- 5.1 nmol/l), Epi (5.32 +/- 0.93 nmol/l), and R(a) (13.0 +/- 1.0 mg. kg(-1). min(-1)) were comparable. The induced increments in NE, Epi, and R(a), all of the same magnitude as in IE, strongly support that circulating catecholamines can be the prime regulators of R(a) in IE.

We recently established that in addition to plasma adrenocorticotrophic hormone (ACTH) and corticosterone, hypothalamic corticotrophin-releasing hormone (CRH) mRNA and hippocampal type 1 glucocorticoid receptor (GR1) mRNA were also upregulated in uncontrolled streptozotocin-induced diabetes. In the current study, control, diabetic, and insulin-treated diabetic rats underwent a hyperinsulinemic-hypoglycemic glucose clamp to evaluate central mechanisms of hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia. Increases in plasma ACTH, corticosterone, and epinephrine were significantly lower in diabetic rats versus controls. Insulin treatment restored ACTH and corticosterone but not epinephrine responses to hypoglycemia in diabetic rats. Glucagon and norepinephrine responses to hypoglycemia were not affected by diabetes or insulin treatment. In response to hypoglycemia, hypothalamic CRH mRNA and pituitary proopiomelanocortin mRNA expression increased in control and insulin-treated but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal GR1 mRNA expression in control and insulin-treated diabetic rats but not in diabetic rats. In contrast, type 2 glucocortoid receptor (GR2) mRNA was not altered by hypoglycemia. In conclusion, despite increased basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that the defect in CRH response could be related to the defective GR1 response. It is intriguing that insulin treatment restored the HPA response to hypoglycemia but, surprisingly, not the deficient epinephrine response. This is important because during severe hypoglycemia, epinephrine is an important counterregulatory hormone.

In intense exercise (>80% Vo2max), unlike at lesser intensities, glucose is the exclusive muscle fuel. It must be mobilized from muscle and liver glycogen in both the fed and fasted states. Therefore, regulation of glucose production (GP) and glucose utilization (GU) have to be different from exercise at

Combined kidney-pancreas transplantation (KPT) with anastomosis of the pancreatic vein to the systemic circulation (KPT-S) or to the portal circulation (KPT-P) provides a human model in which the chronic effects of portal versus systemic insulin delivery on glucose and VLDL metabolism can be examined. Despite similar plasma glucose and C-peptide levels, KPT-S (n = 9) had an approximate twofold elevation of fasting and intravenous glucose-stimulated plasma insulin levels compared with both KPT-P (n = 7) and healthy control subjects (n = 15). The plasma free fatty acid (FFA) levels were elevated in both transplant groups versus control subjects, but the plasma insulin elevation necessary to lower plasma FFA by 50% was approximately two times higher in KPT-S versus KPT-P and control subjects. Endogenous glucose production was similar in KPT-S and KPT-P, despite approximately 35% higher hepatic insulin levels in the latter, and was suppressed to a greater extent during a euglycemic-hyperinsulinemic clamp in KPT-S versus KPT-P. Total-body glucose utilization during the euglycemic-hyperinsulinemic clamp was approximately 40% lower in KPT-S versus KPT-P, indicating peripheral tissue but not hepatic insulin resistance in KPT-S versus KPT-P. Both transplant groups had an approximate twofold elevation of triglyceride (TG)-rich lipoprotein apolipoprotein B (apoB) and lipids versus control subjects. Elevation of VLDL-apoB and VLDL-TG in both transplant groups was entirely explained by an approximately 50% reduction in clearance of VLDL compared with healthy control subjects. In the presence of increased FFA load but in the absence of hepatic overinsulinization and marked hepatic insulin resistance, there was no elevation of VLDL secretion in KPT-S versus KPT-P and control subjects. These findings suggest that chronic systemic hyperinsulinemia and peripheral tissue insulin resistance with the consequent elevation of plasma FFA flux are insufficient per se to cause VLDL overproduction and that additional factors, such as hepatic hyperinsulinemia and/or gross insulin resistance, may be an essential prerequisite in the pathogenesis of VLDL overproduction in the common form of the insulin resistance syndrome.

This study was undertaken to characterize the effects of glycemia per se (glucose effectiveness) on muscle glucose transport. Isolated rat hindlimbs were perfused in situ for 2 h with perfusate containing either low (2 mmol/l, n = 7), normal (6.5 mmol/l, n = 6), or high (20 mmol/l, n = 6) concentrations of glucose, without insulin, to simulate hypo-, eu-, and hyperglycemic conditions. The effect of varying glucose concentrations on muscle glucose transport was assessed by an ensuing 30-min perfusion with 5.5 mmol/l glucose perfusate without insulin. The 2-h of low glucose perfusion induced significant increases in both muscle glucose clearance (approximately 2.3-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05) relative to normal. In contrast, high glucose perfusion decreased glucose clearance (approximately 1.7-fold, P < 0.01) and plasma membrane GLUT4 content (approximately 20%, P < 0.05). Glucose extraction during the following 30-min perfusion was 2.5-fold greater (P < 0.0001) in the low group and threefold less (P < 0.0001) in the high group, relative to normal. 2-[3H]deoxyglucose-6-phosphate content in both red (soleus) and white (extensor digitorum longus) muscles increased approximately twofold after 2 h of low glucose perfusion (P < 0.0001) and decreased > or =2-fold after high glucose perfusion (P < 0.0001), relative to normal. It is concluded that glycemia regulates glucose transport in skeletal muscle independently of insulin, achieved at least partially via changes in plasma membrane GLUT4. We propose that high glucose levels can acutely downregulate GLUT4 and glucose clearance, thus limiting excessive glucose uptake in muscle. Conversely, low glucose-induced upregulation of muscle glucose clearance and GLUT4 can compensate for reduced glucose availability in the circulation.

At rest and during exercise, chronic hyperglycemia, high free fatty acid (FFA) oxidation, and insulin deficiency in diabetes are well known to impair glucose clearance (metabolic clearance rate [MCR]). The effect of acute restoration of glycemia per se on MCR has been less well characterized. We therefore studied normal and alloxan-diabetic dogs both at rest and during exercise, as diabetic hyperglycemic or after acutely induced euglycemia (

We have shown previously in humans that insulin partly suppresses hepatic glucose production (HGP) by an extrahepatic (indirect) mechanism. In the present study, we investigated the role of free fatty acids (FFAs) in mediating the extrahepatic effects of insulin in humans and determined the extent to which insulin can regulate HGP by a non-FFA-mediated effect. Sixteen healthy men received an intravenous tolbutamide infusion for 3 h, and pancreatic insulin secretion was calculated by deconvolution of peripheral C-peptide levels. On a subsequent occasion, equimolar exogenous insulin was infused by peripheral vein. In both studies, glucose was clamped at euglycemia. We have previously validated this method and shown no independent insulin-like activity of tolbutamide. During the clamp, 9 of the 16 subjects received a low dose of heparin and Intralipid to prevent the insulin-induced suppression of FFAs, while 7 subjects received a high dose of heparin and Intralipid to raise FFAs approximately 2.5-fold. In both the high- and low-dose groups, peripheral insulin was higher and calculated portal insulin lower with peripheral versus portal insulin delivery. In the low-dose group, HGP decreased by 68.3 +/- 2.1% with portal insulin delivery and 64.7 +/- 3.7% with peripheral insulin delivery (NS). In the high-dose group, HGP decreased by 58.0 +/- 4.5% with portal insulin and 48.3 +/- 5.0% with peripheral insulin (P < 0.05). Four individuals who participated in the high-dose group underwent an additional peripheral insulin study in which the same dose of exogenous insulin was infused as in the high-dose group but in the absence of heparin and Intralipid. During this latter study, FFA levels declined by approximately 90% during hyperinsulinemia, and HGP was suppressed by 71.8 +/- 5.6%, which was a much greater suppression (P < 0.01) than when FFA levels were raised in these subjects during the equivalent rate insulin infusion. In summary, the previously observed greater suppression of HGP with equimolar peripheral versus portal insulin is eliminated or reversed, depending on plasma FFA levels, if FFAs are prevented from decreasing, suggesting an important role of FFAs in mediating the extrahepatic effects of insulin on HGP. However, the effect of FFA clamping is relatively small with a significant degree of suppression of HGP (by approximately 50%), which remains even when FFAs are elevated above basal levels, suggesting that in the physiological range FFAs only partially influence the suppression of HGP in humans. This suggests that other mechanisms, most likely hepatic, dominate the acute insulin-induced suppression of glucose production.

Pioglitazone, a thiazolidinedione derivative, ameliorates hyperglycemia by augmenting peripheral glucose disposal and suppressing hepatic glucose production in diabetic animals. However, the effect of this agent on hepatic glucose uptake has not been explored. To determine this, experiments were conducted in alloxan-induced diabetic dogs with (pioglitazone group, n = 7) or without (control group, n = 5) a 10-day oral treatment with pioglitazone (1 mg x kg(-1) x day(-1)). A euglycemic-hyperinsulinemic (insulin infusion rate 25.2 pmol x kg(-1) x min(-1)) clamp was maintained by adjusting the peripheral glucose infusion rate (GIR). After a 60-min basal period (period I), portal glucose infusion (Pinf, 33.3 micromol x kg(-1) x min(-1)) was administered for 120 min (period II). This was followed by a 60-min recovery period (period III). Arterial insulin levels were kept stable in the supraphysiological range throughout the experiment (1,623 +/- 52, pioglitazone group; 1,712 +/- 52 pmol/l, C group). There was no significant difference in whole-body glucose utilization determined by [3-3H]glucose between the pioglitazone and C groups in period I (68.4 +/- 2.8 vs. 70.1 +/- 2.8 micromol x kg(-1) x min(-1), respectively) and period III (81.2 +/- 5.0 vs. 74.5 +/- 3.3 micromol x kg(-1) x min(-1), respectively). Net hepatic glucose uptake (NHGU) determined by arteriovenous difference method was approximately zero in the basal period (-0.7 +/- 1.1, pioglitazone group; 0.1 +/- 1.2 micromol x kg(-1) x min(-1), C group). In period II, hepatic glucose uptake, determined by the changes in GIR, was significantly higher in the pioglitazone group (6.5 +/- 0.6 micromol x kg(-1) x min(-1)) than in the C group (-0.4 +/- 0.6 micromol x kg(-1) x min(-1), P < 0.001). This observation was also confirmed by NHGU during portal glucose infusion (6.9 +/- 1.4 vs. 2.1 +/- 1.8 micromol x kg(-1) x min(-1), pioglitazone vs. C, respectively; P < 0.025). We conclude that pioglitazone treatment enhances hepatic glucose uptake during portal glucose loading in alloxan-induced diabetic dogs. However, in hyperinsulinemic conditions, pioglitazone does not enhance the already high peripheral glucose uptake.

The present study examines the effect of the route of insulin delivery on glucose turnover in humans. By using a new noninvasive in vivo method, the acute effect of insulin secreted by the pancreas can be compared with that of insulin delivered by a peripheral vein. Three euglycemic-hyperinsulinemic studies were performed in lean healthy men. In the first study (n = 10), constant portal hyperinsulinemia was produced using a programmed intravenous tolbutamide infusion algorithm, and the insulin secretion rate was mathematically derived by deconvolution from peripheral plasma C-peptide levels. In the second study (n = 10), exogenous insulin was infused by peripheral vein at the same rate as that determined in the first study. In the third study (n = 7), the peripheral insulin levels in the first study were matched by infusing exogenous insulin into a peripheral vein at half that rate. Peripheral insulin levels were higher (P < 0.001) with the full-rate peripheral insulin infusion (266.3 ± 28.1 pmol/l) than with the portal delivery of insulin (171.1 ± 30.4 pmol/l) or the half-rate peripheral insulin infusion (158.6 ± 7.4 pmol/l) (portal versus half-rate peripheral insulin infusion, NS). Calculated hepatic insulin levels were higher (P < 0.001) in the portal insulin study (443.1 ± 52.6 pmol/l) than in the full-rate peripheral insulin study (303.6 ± 30.9 pmol/l) or in the half-rate peripheral insulin study (204.5 ± 9.8 pmol/l). Hepatic glucose production (HGP) was suppressed to a greater extent with the full-rate peripheral insulin infusion (69.3 ± 7.8%, P < 0.001 vs. portal or half-rate peripheral insulin) than portal (50.3 ± 9.8%) or half-rate peripheral insulin infusion (36.8 ± 3.8%). In the portal insulin study, however, suppression was > in the half-rate peripheral insulin study (P < 0.01), in spite of equal peripheral insulin levels. The assumption that tolbutamide, when used in this fashion, has no independent effect on glucose turnover, glucagon, or gluconeogenic precursor and energy substrates for gluconeogenesis was validated in five C-peptide-negative patients with IDDM. We conclude that in nondiabetic humans, 1) peripheral effects of insulin are important in suppressing HGP, as evidenced by the greater suppression of HGP with equivalent rate peripheral versus portal insulin delivery, and 2) because HGP was suppressed to a greater extent with portal verus peripheral insulin delivery at half the rate when peripheral insulin levels were matched, insulin-induced suppression of HGP is also partly mediated by a direct hepatic effect.

Exercise at > 85% VO2max causes the greatest known physiological increases in glucose production rates (Ra). To define the relative roles of catecholamine versus glucagon/insulin responses in stimulating Ra, normal subjects in the postabsorptive state exercised at 87 ± 2% VO2max during an islet cell clamp (IC): intravenous octreotide (somatostatin analog), 30 ng s· kg−1 · min−1; glucagon, 0.8 ng · kg−1 · min−1; growth hormone, 10 ng · kg−1 · min−1; and insulin adjusted to achieve euglycemia, then constant 56 ± 7 min before exercise. Seven control subjects exercised without an IC. In four subjects (IC-1) with hormone infusions held constant during exercise, plasma insulin rose 76% and glucagon 35%, perhaps because of altered hemodynamics. In seven subjects (IC-2), hormone infusions were decreased stepwise during exercise and returned stepwise to initial rates during early recovery. Ra increased sixfold in control and both IC groups. Plasma norepinephrine and epinephrine likewise increased > 12-fold with no differences among groups; both catecholamines correlated closely with Ra. Because mixed venous blood plasma insulin declined and glucagon did not change in control subjects, the glucagon-to-insulin ratio increased from 0.20 to 0.26 (P = 0.02). In IC subjects, plasma insulin increased and glucagon was either constant (IC-2) or increased < insulin, resulting in nonsignificant declines in the immunoreactive glucose-to-immunoreactive insulin ratio. Although a rise in insulin would have been expected to attenuate the Ra increment, this effect was overridden. The strong correlations of Ra with catecholamines and the similar Ra responses despite divergent glucagon-to-insulin responses are consistent with the primacy of catecholamines in regulation of Ra in intense exercise.

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G⇆G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6–9 h at a starting dose of 45–150 pmol-kg−1-min−1 to normalize plasma glucose from 23.9 ± 1.4 to 5.0 ± 0.4 mmol/1 and gradually decreased to and maintained at a basal rate (1.7 ± 1.0 pmol · kg−1 · min−1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 ± 2.7 and normalized at 1.3 ± 0.6 μmol · kg−1 · min−1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 ± 3.1 to 11.6 ± 1.2) than did glucose production (from 25.9 ± 1.9 to 10.3 ± 1.0 μmol · kg−1 · min−1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 ± 24 vs. 71 ± 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 ± 0.8 to 19.6 ± 2.6 U/g) and glucokinase (from 1.1 ± 0.2 to 1.0 ± 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content. We therefore conclude that in the depancreatized dog, 1) acute insulin infusion with a concurrent correction of hyperglycemia can markedly reduce and normalize GC, and 2) the acute reduction in GC is primarily mediated by reduced substrate fluxes through glucokinase and G6Pase and does not require stable changes in maximal activities of the two enzymes. However, in vivo allosteric changes in enzyme activities (particularly G6Pase), immeasurable in vitro, cannot be excluded. In addition to enzyme measurements, the importance of tracer methods that allow for in vivo measurements of fluxes through glucokinase and G6Pase is emphasized.

Diminished responsiveness to hypoglycemia contributes to defective counterregulation in diabetes. Pancreatic and/or circulating somatostatin are elevated in diabetes, which may inhibit counterregulatory hormone release during hypoglycemia. Thus, a selective somatostatin receptor type 2 antagonist (SSTR2a) should improve hormone counterregulation to hypoglycemia. Nondiabetic (N) and streptozotocin-induced diabetic (D) rats underwent 4-h infusion of saline or SSTR2a with insulin-induced hypoglycemia clamped at 2.5 ± 0.5 mmol/L. To evaluate the effect of the SSTR2a in the absence of hypoglycemia, rats underwent a 4-h infusion of saline (Ctrl:N, Ctrl:D) or SSTR2a (Ctrl:D+SSTR2a) only. The attenuated glucagon response to hypoglycemia in D (P < 0.0002) was fully restored by SSTR2a (P < 0.0001). Furthermore, the attenuated corticosterone response in D (P < 0.002) was also enhanced by SSTR2a (P < 0.05). In the absence of hypoglycemia, SSTR2a did not alter basal blood glucose levels. D exhibited 62% more pancreatic somatostatin than N after hypoglycemia. In N rats, SSTR2a did not augment the glucagon or corticosterone response to hypoglycemia. Thus, somatostatin may contribute to impaired glucagon responsiveness to hypoglycemia in diabetes. We demonstrate that SSTR2 antagonism enhances hypoglycemia-stimulated glucagon and corticosterone release in D but not in N rats. SSTR2 antagonism does not affect basal glycemia in D rats.

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-OHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca2+-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the α1-subunit of the Na+-K+-ATPase, a surface membrane marker. Isolated IM components such as LSR, HSR, and triads (terminal cisternae plus junctional TT) contained low or barely detectable amounts of GLUT4; furthermore, insulin treatment did not change the distribution of the transporter protein within these fractions. In contrast, a unique IM fraction that was not associated with either SR or triad markers, contained significant amounts of GLUT4 and showed an insulin-dependent decrease (40%) in GLUT4 protein content. These results show that acute insulin treatment induces the translocation of GLUT4s to both the PM and TTs from a unique intracellular organelle not associated with the SR.

This article is divided into two parts. A retrospective overview summarizes some of the work that provided the framework and tools of the more recent studies. The five novel areas of research are related to the indirect effects of insulin. Regulation of plasma glucose is of central importance in health and diabetes. Understanding this precise regulation requires sensitive isotope dilution methods that can measure the rates at which glucose is produced by the liver and used by the tissues on a minute-to-minute basis. Validation studies indicated that the non-steady-state tracer method yields reasonable results when the specific activity of plasma glucose does not change abruptly. During hyperinsulinemic glucose clamps, the decrease in specific activity of glucose can be prevented by the MSTI. During exercise, the decrease of specific activity can be only in part ameliorated by step-tracer infusion. Depancreatized dogs are used extensively as a model of selective insulin deficiency, because dog stomach secretes physiological amounts of glucagon. This strategy can avoid injections of somatostatin, which can have other affects in addition to the suppression of insulin and glucagon. In human diabetes, in addition to an increase of glucose production, there is also an increase in glucose cycling in the liver. In animal models of diabetes, mild NIDDM, and in glucose intolerance, the percentage of increments of glucose cycling are much larger than those of glucose production. We hypothesize, therefore, that measurements of glucose cycling can be used as an early marker of glucose intolerance. Application of different tracer strategies and use of the depancreatized dog as a model of diabetes, we investigated the importance of the indirect effects of insulin in the pathogenesis of diabetes. 1) Because, in the treatment of IDDM, insulin is administered by the peripheral routes we compared the relative importance of hepatic and peripheral effects of insulin in regulating the rate of glucose production. Experiments were performed in depancreatized dogs that were initially maintained at moderate hyperglycemia (10 mM) with subbasal portal insulin infusion. During the experimental period, insulin was infused either peripherally or portally at 0.9 mU · kg−1 · min−1. In addition, peripheral infusions were also given at 0.45 mU · kg−1 · min−1 We concluded that when suprabasal insulin levels are provided to moderately hyperglycemic depancreatized dogs, the suppression of glucose production is more dependent on peripheral than portal insulin concentrations. This indirect effect of insulin may be mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and/or by suppressive effect of insulin on glucagon secretion. These results suggest that the absence of a portal-peripheral gradient in insulin-treated diabetic subjects may not be important for postprandial suppression of glucose production. 2) The glucagon-insulin ratio is an important regulator of glucose production by the liver during moderate exercise, whereas during intense exercise the catecholamines play a prominent role. Regulation of glucose uptake during exercise is very complex. In vivo, insulin can play an indirect role by inhibiting the FFA-glucose cycle and by maintaining normoglycemia; both of these factors influence glucose uptake by the muscle. 3) Streptozocin-induced diabetes in rats decreases the number of glucose transporters when measured both by cytochalasin B binding and by assessment of GLUT4 transporters. Normalization of glycemia in the diabetic rats by a 2-day phlorizin treatment, which does not affect the insulin concentration, normalizes glucose transporter number in the plasma membrane. We concluded that hyperglycemia, per se, plays an important role in regulating glucose transporter number in the muscle. 4) In alloxan-induced diabetic dogs, similarly to IDDM, glucagon's response and, therefore, the response of glucose production to declining glucose is impaired. We present a new hypothesis that an increased ratio between somatostatin and glucagon in the residual diabetic islets of diabetic dogs can, at least in part, explain the lack of glucagon response to hypoglycemia. 5) To investigate the effect of stress in diabetic animals, we used an intracerebroventricular injection of a small amount of carbachol. This compound increases the release of all counterregulatory hormones, but without affecting insulin secretion. Surprisingly, this release of counterregulatory hormones induces only a marginal change in plasma glucose, because increased glucose production is matched by a similar increase in glucose uptake. In contrast, in hyperglycemic diabetic dogs, the same carbachol injection induces a sevenfold larger increment in plasma glucose. This occurred because the metabolic clearance rate of glucose does not increase. We therefore postulated a neural mechanism that controls peripheral glucose uptake and does not require a regulatory effect of insulin. A permissive role of insulin could be to maintain normal insulin sensitivity in the muscle. From the point of view of the clinician, if diabetic neuropathy affects this pathway, this would contribute to insulin resistance. However, even more important is the possibility that this pathway may take part in the pathogenesis of diabetes.

Diabetes-induced oxidative stress can lead to protein misfolding and degradation by the ubiquitin-proteasome system. This study examined protein ubiquitination in pancreatic sections from Zucker diabetic fatty rats. We observed large aggregates of ubiquitinated proteins (Ub-proteins) in insulin-expressing β-cells and surrounding acinar cells. The formation of these aggregates was also observed in INS1 832/13 β-cells after exposure to high glucose (30 mmol/l) for 8–72 h, allowing us to further characterize this phenotype. Oxidative stress induced by aminotriazole (ATZ) was sufficient to stimulate Ub-protein aggregate formation. Furthermore, the addition of the antioxidants N-acetyl cysteine (NAC) and taurine resulted in a significant decrease in formation of Ub-protein aggregates in high glucose. Puromycin, which induces defective ribosomal product (DRiP) formation was sufficient to induce Ub-protein aggregates in INS1 832/13 cells. However, cycloheximide (which blocks translation) did not impair Ub-protein aggregate formation at high glucose levels, suggesting that long-lived proteins are targeted to these structures. Clearance of Ub-protein aggregates was observed during recovery in normal medium (11 mmol/l glucose). Despite the fact that 20S proteasome was localized to Ub-protein aggregates, epoxomicin treatment did not affect clearance, indicating that the proteasome does not degrade proteins localized to these structures. The autophagy inhibitor 3MA blocked aggregate clearance during recovery and was sufficient to induce their formation in normal medium. Together, these findings demonstrate that diabetes-induced oxidative stress induces ubiquitination and storage of proteins into cytoplasmic aggregates that do not colocalize with insulin. Autophagy, not the proteasome, plays a key role in regulating their formation and degradation. To our knowledge, this is the first demonstration that autophagy acts as a defense to cellular damage incurred during diabetes.

Stress-induced hyperglycemia can lead to significant deterioration in glycemic control in individuals with diabetes. Previously, we have shown in normal dogs that, after intracerebroventricular (ICV) administration of carbachol (a model of moderate stress), increases in both the metabolic clearance rate (MCR) of glucose and endogenous glucose production (GP) occur. However, in hyperglycemic diabetic dogs subjected to the same stress, the MCR of glucose does not increase and glycemia therefore markedly deteriorates because of stimulation of GP. Our aims were to determine the following: 1) whether insulin-induced acute normalization of glycemia, with or without beta-blockade, would correct glucose clearance and prevent the hyperglycemic effect of stress, and 2) whether hyperinsulinemia per se could correct these abnormalities. Stress was induced by ICV carbachol in 27 experiments in five alloxan-administered diabetic dogs subjected to the following protocols in random order: 1) basal insulin infusion (BI) to restore normoglycemia; 2) basal insulin infusion with beta-blockade (BI+block); 3) normoglycemic-hyperinsulinemic clamp with threefold elevation of insulin above basal (3x BI); and 4) normoglycemic-hyperinsulinemic clamp with fivefold elevation of insulin above basal (5 x BI). The BI+block protocol fully prevented stress-induced hyperglycemia, both by increasing MCR (deltaMCR at peak: 0.72 +/- 0.25 ml x kg(-1) x min(-1) vs. no change in BI, P < 0.05) and by diminishing the stress-induced increment in GP observed in BI (deltaGP at peak: 3.72 +/- 0.09 micromol x kg(-1) x min(-1) for BI+block vs. 14.10 +/- 0.31 micromol x kg(-1) x min(-1) for BI, P < 0.0001). In contrast, 3x BI and 5x BI treatments with normoglycemic-hyperinsulinemic clamps proportionately increased basal MCR at baseline, but paradoxically were not associated with an increase in MCR in response to stress, which induced a twofold increase in GP. Thus, in alloxan-administered diabetic dogs, stress increased GP but not MCR, despite normalization of glycemia with basal or high insulin. In contrast, beta-adrenergic blockade almost completely restored the metabolic response to stress to normal and prevented marked hyperglycemia, both by limiting the rise in GP and by increasing glucose MCR. We conclude that acute normalization of glycemia with basal insulin or hyperinsulinemia does not prevent hyperglycemic effects of stress unless accompanied by beta-blockade, and we speculate that short-term beta-blockade may be a useful treatment modality under some stress conditions in patients with diabetes.

To determine whether glucagon-like peptide (GLP)-1 increases insulin sensitivity in addition to stimulating insulin secretion, we studied totally depancreatized dogs to eliminate GLP-1's incretin effect. Somatostatin was infused (0.8 microg x kg(-1) x min(-1)) to inhibit extrapancreatic glucagon in dogs, and basal glucagon was restored by intraportal infusion (0.65 ng x kg(-1) x min(-1)). To simulate the residual intraportal insulin secretion in type 2 diabetes, basal intraportal insulin infusion was given to obtain plasma glucose concentrations of approximately 10 mmol/l. Glucose was clamped at this level for the remainder of the experiment, which included peripheral insulin infusion (high dose, 5.4 pmol x kg(-1) x min(-1), or low dose, 0.75 pmol x kg(-1) x min(-1)) with or without GLP-1(7-36) amide (1.5 pmol x kg(-1) x min(-1)). Glucose production and utilization were measured with 3-[3H]glucose, using radiolabeled glucose infusates. In 12 paired experiments with six dogs at the high insulin dose, GLP-1 infusion resulted in higher glucose requirements than saline (60.9+/-11.0 vs. 43.6+/-8.3 micromol x kg(-1) x min(-1), P< 0.001), because of greater glucose utilization (72.6+/-11.0 vs. 56.8+/-9.7 micromol x kg(-1) x min(-1), P

We and others have shown that insulin acutely suppresses glucose production in fasting nondiabetic humans and dogs, by both a direct hepatic effect and an indirect (extrahepatic) effect, and in diabetic dogs by an indirect effect alone. In type 2 diabetes, there is resistance to insulin's ability to suppress hepatic glucose production, but it has not previously been determined whether the resistance is primarily at the level of the hepatocyte or the peripheral tissues. To determine whether the diabetic state reduces the direct effect of insulin in humans, we studied nine patients with untreated type 2 diabetes who underwent three studies each, 4-6 weeks apart. 1) Portal study (POR): intravenous tolbutamide was infused for 3 h with calculation of pancreatic insulin secretion from peripheral plasma C-peptide. 2) Peripheral study (PER): equidose insulin was infused by peripheral vein. 3) Half-dose peripheral insulin study (1/2 PER): matched peripheral insulin levels with study 1. In all studies, glucose was clamped at euglycemia, glucose turnover was measured with the constant specific activity method, and 3-[3H]glucose was purified by high-performance liquid chromatography. Peripheral insulin was lower in POR versus PER but slightly higher in POR versus 1/2 PER, although most of the difference could be accounted for by higher proinsulin levels in POR (stimulated by tolbutamide). Calculated portal insulin was approximately 1.3-fold higher in POR versus PER and approximately 2.2-fold higher in POR versus 1/2 PER. In the final 30 min of the clamp, glucose production reached a lower steady-state level in PER than in POR (4.0 +/- 0.4 vs. 5.3 +/- 0.5 pmol(-1) x kg(-1) x min(-1), P < 0.05), despite the higher hepatic insulin level in POR. In contrast with our studies in nondiabetic individuals, glucose production was not more suppressed at steady state in POR versus 1/2 PER (5.3 +/- 0.4 micromol x kg(-1) x min(-1)), despite much higher hepatic insulin levels in POR. In conclusion, this is the first study in patients with type 2 diabetes to characterize insulin resistance to the acute direct suppressive effect of insulin on hepatic glucose production.