IntroductionThe Staub-Traugott effect, or second-meal phenomenon, describes improved glucose disposal after a second identical meal. We previously showed that morning hyperinsulinemia primes the liver to enhance afternoon net hepatic glucose uptake and glycogen storage. However, mixed meals trigger co-secretion of insulin and glucagon, and glucagon is traditionally viewed as opposing insulin’s hepatic actions. Whether glucagon modifies the persistence of insulin’s priming effects across sequential metabolic challenges is unknown. Therefore, we investigated whether morning hyperglucagonemia alters the ability of morning hyperinsulinemia to prime subsequent hepatic glucose metabolism.MethodsConscious dogs underwent two pancreatic clamp periods separated by a 1.5h rest period. Endogenous insulin and glucagon were suppressed with somatostatin and replaced intraportally at defined rates. During a 4h morning hyperinsulinemic-euglycemic clamp, dogs received matched insulin prime infusions with either basal glucagon (AM INS; n=8) or elevated glucagon (AM INS+GCG; n=8). After the rest period, both groups underwent a 2.5h afternoon hyperinsulinemic-hyperglycemic clamp under identical hormonal conditions. Afternoon net hepatic glucose uptake, glycogen, glycolytic, and gluconeogenic flux rates were quantified using arteriovenous difference methods and [3-3H]-glucose tracer kinetics. Liver biopsies were collected before and after the afternoon clamp to assess gene transcription and protein regulators of hepatic glucose metabolism.ResultsDuring the afternoon clamp, despite matched insulin, glucagon, and glucose levels, net hepatic glucose uptake was 41% lower in AM INS+GCG (3.6±0.4 mg/kg/min) than in AM INS (6.1±0.6 mg/kg/min; p<0.003). This was accompanied by a trend toward incomplete suppression of hepatic glucose production in AM INS+GCG (1.4±0.4 mg/kg/min), whereas it was fully suppressed in the AM INS group (p=0.06). Direct glycogen synthesis was also 44% lower in AM INS+GCG (1.8±0.2 vs 3.2±0.7 mg/kg/min; p<0.015), along with reductions in net glycogen synthesis and glycolytic flux. Morning insulin with basal glucagon increased hepatic glucokinase mRNA and protein before the afternoon clamp, whereas concurrent glucagon prevented this induction.DiscussionIn summary, antecedent morning hyperglucagonemia attenuates insulin-mediated hepatic priming, reducing hepatic glucose flux during a later hyperinsulinemic-hyperglycemic challenge. These findings identify glucagon as a regulator of hepatic metabolic memory alongside insulin and demonstrate that early-day insulin-glucagon dynamics shape the liver’s response to subsequent challenges, providing mechanistic insight into postprandial glucose regulation and implications for metabolic health and diabetes risk.

This study investigated the efficacy of thiazolidinediones in mitigating diet-induced obesity and associated glucose intolerance in canines in vivo. We used a multitechnique approach and compared the results related to 1) fasting metabolites, hormones, and lipides; 2) oral glucose tolerance test (OGTT), and 3) hyperinsulinemic euglycemic (HIEG) clamps in 24 healthy dogs that were then fed a high-fat diet and assigned to a placebo group (n = 8) or received a daily dose of pioglitazone group (n = 16) for 56 days. The animals were studied before and after treatment, acting as their own controls, and we used a principal component analysis to combine the results obtained for the three different techniques. Both groups experienced weight gain, with 12% and 14% increases in the placebo and pioglitazone groups, respectively. The fasting level of free fatty acids was increased in both placebo (+17.5%) and pioglitazone groups (+7.7%), as were the insulin levels (+27% and 35%, respectively, in placebo and pioglitazone), but fasting glucose levels were reduced slightly in both (-2.7 and -2.9 mg/dL, respectively). Pioglitazone increased fasting adiponectin levels by 54% between day -1 and day 56. OGTTs revealed a significantly better glucose tolerance in the pioglitazone treatment group compared with the placebo group, as the level of insulin secreted during the OGTT was normalized. HIEG clamps demonstrated that, in the placebo group, glucose infusion rate and glucose utilization decreased in response to the diet, but this effect was prevented by pioglitazone. In conclusion, these findings show that pioglitazone is beneficial in preventing the consequences of dietary metabolic stress and overfeeding in canines. NEW & NOTEWORTHY This study validates the efficacy of the thiazolidinediones (TZD) drug pioglitazone in a fat-fed canine model. Using a novel principal component analysis integrating multimodal data (fasting metabolites, OGTT, and clamp results), we demonstrate that pioglitazone effectively reverses diet-induced glucose intolerance and significantly improves insulin sensitivity. Mechanistically, this improvement is driven by adiponectin action. These findings provide strong evidence supporting TZD use as a prophylactic tool for mitigating metabolic syndrome.

Introduction and Objective: Clinical (subcutaneous) insulin delivery abolishes the endogenous portal vein (PV) to arterial insulin gradient, leading to relative hepatic under-insulinization as well as excess insulin exposure of muscle. Our aim was to determine how route of insulin delivery affects liver vs muscle glucose disposal in dogs with high fat and fructose (FF) induced metabolic dysfunction. Methods: Dogs (n=6/group) were fed a chow (C) or FF diet for 4 weeks. Somatostatin was used to disable the pancreas and basal glucagon was infused. Postprandial hyperglycemia was created with PV (4 mg/kg/min) and leg vein (LV) glucose (infused as needed), while insulin was infused (1.2 mU/kg/min) either into the PV or a LV. Results: PV insulin was ~2x greater with PV infusion whereas arterial insulin was ~2x greater with LV infusion. Net hepatic glucose uptake was markedly reduced in FF compared to C-fed dogs. While the diet’s effect on NHGU was not altered by route of insulin delivery (6.2±0.5, 2.4±0.2 and 2.2±2.2 mg/kg/min in the C+PV ins, FF+PV ins and FF+LV ins groups, respectively), MGU was ~2.5x greater in FF+LV ins vs FF+PV ins. Conclusion: While PV nor LV insulin were able to overcome the severe reduction in liver glucose uptake caused by the FF diet, LV insulin delivery, in addition, caused much greater MGU, an effect that increases hypoglycemic risk. Therefore, diet induced impairment of postprandial hepatic glucose uptake cannot be normalized by increasing the dose of peripherally delivered insulin. Disclosure G. Kraft: None. K. Yankey: None. B. Farmer: None. H. Waterman: None. M. Lee: None. J. Hastings: None. D. Edgerton: None. Funding NIH RO1DK018243

The second-meal phenomenon refers to the improved glycemic response to a subsequent identical meal. We previously showed that morning (AM) hyperinsulinemia is a key mediator, priming the liver for enhanced net hepatic glucose uptake (NHGU) and glycogen storage during an afternoon (PM) hyperinsulinemic-hyperglycemic clamp. Postprandial NHGU is regulated by three primary mechanisms: insulin action (IA), initiated by hyperinsulinemia; glucose effectiveness (GE), driven by hyperglycemia; and the portal glucose signal (PGS), a neurally-mediated signal activated by glucose delivery into the hepatoportal circulation. It remained unclear, however, which of these mechanisms govern the increase in PM NHGU following AM insulin exposure. To address this, dogs underwent an AM clamp with either a 4-hour hyperinsulinemic prime (Prime, n=8) or basal insulin delivery (No Prime, n=8). After a 1.5-hour rest, both groups underwent a PM hyperglycemic clamp with portal glucose delivery under basal insulin conditions to isolate the effects of an AM insulin prime on PM glucose-mediated hepatic signals (GE/the PGS). Mean PM NHGU was significantly greater in the Prime group (2.2 ± 0.3 mg/kg/min) compared to the No Prime group (0.1 ± 0.3 mg/kg/min, p=0.005), accompanied by augmented net glycolytic and glycogen flux. These findings indicate that morning insulin can enhance glucose-mediated PM NHGU independently of a rise in PM insulin. However, maximal second-meal NHGU also requires elevated PM insulin. Together, this suggests that strategically timed early-day insulin or insulinotropic interventions could potentially improve hepatic responsiveness in settings of impaired postprandial glycemic control, such as insulin resistance or diabetes.

The second meal effect describes an improved glycemic response observed after consuming a second identical meal. We previously showed that morning (AM) exposure to hyperinsulinemia primes the liver for enhanced hepatic glucose uptake and glycogen storage in the afternoon (PM), with no significant effect on PM non-hepatic glucose uptake. Given that meals often trigger both insulin and glucagon secretion, we aimed to determine if AM hyperglucagonemia alters the priming effect of AM hyperinsulinemia on PM hepatic glucose metabolism. To test this, dogs were exposed to a 4h AM hyperinsulinemic-euglycemic clamp, with insulin delivered in a pattern mimicking the insulin profile observed earlier during a 4h AM duodenal glucose infusion. This period of hyperinsulinemia was paired with either basal (Prime, n=8) or elevated (Prime + ↑GGN, n=8) glucagon, maintaining a consistent insulin-to-glucagon molar ratio throughout the AM clamp. After a 1.5h rest period, the dogs underwent a 2.5h PM hyperinsulinemic-hyperglycemic clamp, during which glucose, insulin, and glucagon levels, along with the artery-to-portal vein glucose gradient, were carefully controlled to replicate postprandial conditions. During the PM clamp, the mean net hepatic glucose uptake (NHGU) in the Prime + ↑GGN group was only 59% of that in the Prime group (3.6±0.4 vs. 6.1±0.6 mg/kg/min, P<0.0027, respectively). Additionally, PM direct glycogen synthesis was two-fold greater in the Prime group compared to the Prime + ↑GGN group (3.2±0.7 vs. 1.5±0.2 mg/kg/min, P<0.0014, respectively). The observed difference in PM NHGU between the groups was not due to enhanced PM hepatic glucose uptake (HGU), which was similar in both groups (5.7±0.5 mg/kg/min in the Prime group vs. 5.2±0.3 mg/kg/min in the Prime + ↑GGN group), but rather a prolonged effect of AM hyperglucagonemia on PM hepatic glucose production (HGP) (-0.3±0.3 mg/kg/min in the Prime group vs. 1.7±0.4 mg/kg/min in the Prime + ↑GGN group, P<0.0072). This increase in PM HGP in the Prime + ↑GGN group was not driven by differences in PM gluconeogenic flux but by futile glucose cycling between glucose and glucose-6-phosphate, as well as hepatic glycogen storage and breakdown. In summary, these findings suggest that morning exposure to elevated glucagon shifts the insulin-driven priming effect on afternoon hepatic glucose metabolism by promoting sustained glucose cycling at the expense of glycogen synthesis and glycolysis, leading to persistent HGP despite identical PM insulin, glucose, and glucagon levels.

Glucose tolerance improves significantly upon consuming a second, identical meal later in the day (second-meal phenomenon). We previously established that morning hyperinsulinemia primes the liver for increased afternoon hepatic glucose uptake (HGU). Although the route of insulin delivery is an important determinant of the mechanisms by which insulin regulates liver glucose metabolism (direct hepatic vs. indirect insulin action), it is not known whether insulin’s delivery route affects the second-meal response. To determine whether morning peripheral insulin delivery (as occurs clinically, i.e., subcutaneously) can enhance afternoon HGU, conscious dogs were treated in the morning with insulin delivered either via the portal vein or peripherally (leg vein), while glucose was infused to maintain euglycemia. Consequently, arterial insulin levels increased similarly in both groups, but relative hepatic insulin deficiency occurred with peripheral insulin delivery. In the afternoon, all animals were challenged with the same hyperinsulinemic-hyperglycemic clamp to simulate identical postprandial-like conditions. The substantial enhancement of HGU in the afternoon caused by morning portal vein insulin delivery was lost when insulin was delivered peripherally. This indicates that morning insulin does not cause the second-meal phenomenon via its indirect actions on the liver but, rather, through direct activation of hepatic insulin signaling. Article Highlights Morning insulin delivery primes the liver for increased hepatic glucose uptake (HGU) later in the day, but until now, the mechanism (direct hepatic and/or indirect insulin action) remained unclear. This study compared insulin infusion via endogenous (hepatic portal vein) and clinical (peripheral) routes to assess their impact on afternoon hepatic glucose disposal. Arterial hyperinsulinemia in the morning, without a concomitant increase in insulin at the liver, failed to induce a significant enhancing effect on afternoon HGU and glycogen storage, unlike morning hepatic portal vein insulin delivery, which did. These findings highlight the importance of achieving appropriate hepatic insulin exposure in the morning to effectively prime the liver for enhanced glucose disposal later in the day.

The second meal phenomenon refers to the improved glycemic response observed after consuming a second identical meal. It is well known that postprandial hepatic glucose uptake (HGU) is determined by the combined effects of three regulatory factors: insulin action (hyperinsulinemia; HI), glucose effectiveness (hyperglycemia; GE), and glucose delivery into the hepatoportal circulation (portal glucose signal; PGS). While our previous studies demonstrated the importance of morning (AM) hyperinsulinemia in priming the liver for increased HGU later in the day, the question remains as to which aspect of the afternoon (PM) response is enhanced by morning insulin. Thus, to provide insight into the underlying mechanism of the 2nd meal effect, we sought to determine the extent to which PM HI, GE, and the PGS are impacted by AM hyperinsulinemia. Dogs underwent an AM clamp with either a 4h hyperinsulinemic prime (Prime, n=8) or basal insulin delivery (No Prime, n=8). After a 1.5h non-clamp period, both groups were challenged with a PM hyperglycemic clamp (with the PGS) in the presence of basal insulin. During the PM clamp, net HGU was significantly elevated in Prime vs. No Prime group (mean of 2.2±0.3 vs. 0.1±0.3 mg/kg/min, respectively, P<0.005), indicating an enhancement of GE and/or the PGS. There was no difference in PM non-HGU (4.5±0.3 vs. 4.0±0.3 mg/kg/min). In previous experiments, when all three factors (HI, GE, PGS) were present in the PM, net HGU was 6.3±1.7 vs. 2.4±1.1 mg/kg/min in groups receiving an AM insulin prime vs. basal insulin, respectively. Therefore, the AM insulin prime enhanced the effects of HI, GE, and the PGS on PM HGU by 3.9 mg/kg/min, whereas it enhanced the effects of glucose (GE and the PGS), in the absence of a rise in insulin, by 2.1 mg/kg/min. Taken together, the data show that AM HI enhanced PM insulin action and GE (including the PGS) to an equal extent. This suggests that AM insulin priming enhances cellular targets common to both insulin and glucose signaling in the PM.

The second meal effect describes an improvement in the glycemic response seen following the consumption of a 2nd identical meal. We previously showed that morning (AM) exposure of the liver to hyperinsulinemia can prime the liver so that there is increased hepatic glucose uptake and glycogen storage later in the day, with no effect on non-hepatic glucose uptake (non-HGU). Considering that most individuals consume meals that prompt both insulin and glucagon secretion, we sought to determine if AM hyperglucagonemia can alter the priming effect of AM hyperinsulinemia on PM hepatic glucose metabolism. To explore this aim, canines were exposed to 4h of hyperinsulinemia in the AM in a delivery pattern that mimicked the insulin secretory profile previously observed during a 4h AM duodenal glucose infusion. This 4h period of hyperinsulinemia was accompanied by either basal (INS, n=6) or elevated (I + G, n=8) glucagon. After a 1.5h non-clamp period, the dogs were challenged using a 2.5h afternoon (PM) hyperinsulinemic-hyperglycemic (HIHG) clamp during which the glucose load, insulin and glucagon levels, and the artery to portal vein glucose gradient were identical between the two groups. During the PM clamp, the mean NHGB in I+G was only 54% of that in INS (mean NHGB of -3.3±0.5 vs. -6.2±0.5 mg/kg/min; net AUC of 415±70 vs. 785±73 mg/kg/2.5h, P<0.003), respectively. Additionally, glycogen synthesized during the PM clamp was twice as high in INS vs. I+G (mean PM glycogen storage of 23.6±1.3 vs. 12.0±1.9 mg/g liver, P<0.001), respectively. There was no significant difference in mean PM non-HGU between the two groups. The mean PM NHGB for a group that received basal insulin and basal glucagon in the AM (No Prime) was -2.7±0.5 mg/kg/min, which is not statistically different from PM NHGB for I+G. In conclusion, exposing the liver to hyperglucagonemia in the morning has the potential to block the priming action of meal-induced AM hyperinsulinemia, preventing enhanced glucose uptake and glycogen storage by the liver later in the day. Disclosure H.L. Waterman: None. M.C. Moore: None. M.S. Smith: None. B. Farmer: None. K. Yankey: None. D.S. Edgerton: None. A.D. Cherrington: Consultant; Abvance Therapeutics. Research Support; Abvance Therapeutics. Advisory Panel; AdipoPharma. Research Support; Cellular Longevity, Inc. dba Loyal. Advisory Panel; Fractyl Health, Inc. Consultant; Fractyl Health, Inc. Research Support; Fractyl Health, Inc. Consultant; Novo Nordisk. Research Support; Novo Nordisk. Consultant; Paratus, Portal Insulin. Advisory Panel; Sekkei Bio, Sensulin Labs, LLC. Consultant; Thetis Pharmaceuticals, LLC. Funding National Institutes of Health (5R01DK131082); National Institutes of Health (5T32DK007563)

The second meal phenomenon refers to the improvement in glucose tolerance seen following a second identical meal. We previously showed that 4 hours of morning hyperinsulinemia, but not hyperglycemia, enhanced hepatic glucose uptake (HGU) and glycogen storage during an afternoon hyperinsulinemic-hyperglycemic (HIHG) clamp. Our current aim was to determine if the duration or pattern of morning hyperinsulinemia is important for the afternoon response to a HIHG clamp. To determine this, we administered the same total amount of insulin either over 2h in the first (Ins2h-A) or second (Ins2h-B) half of the morning, or over the entire 4h (Ins4h) of the morning. In the 4h afternoon period, all three groups had 4x-basal insulin, 2x-basal glycemia, and portal glucose infusion to expose the liver to the primary postprandial regulators of hepatic glucose metabolism. During the afternoon clamp, there was a marked increase in HGU and hepatic glycogen synthesis in the Ins4h group compared to the Ins2h-A and Ins2h-B groups, despite matched hepatic glucose loads and total insulin infusion rates. Thus, the longer duration (Ins4h) of lower hyperinsulinemia in the morning seems to be the key to much greater liver glucose uptake during the afternoon clamp.

Glucose tolerance is significantly improved in response to consuming a second, identical meal later in the day. It is known that physiologic (portal vein) exposure to insulin in the morning primes the liver for increased hepatic glucose uptake and glycogen storage in the afternoon. Although this phenomenon has been observed in both healthy and diabetic individuals, it has not yet been established whether or not morning peripheral insulin delivery could bring about the same response. Thus, the aim of this study was to determine how the route of morning insulin delivery impacts the liver’s ability to extract and store glucose later in the day. To explore this aim, we delivered insulin into the portal vein (Po Ins) or a leg vein (Pe Ins) in the morning (AM) and subsequently challenged conscious dogs to a hyperinsulinemic-hyperglycemic (HIHG) clamp in the afternoon (PM). We then assessed the impact that each route of AM insulin delivery had on hepatic glucose uptake (HGU), non-HGU, and glycogen storage in the PM. The insulin infusion rates used in the Po Ins group were selected to mimic the rise in endogenous insulin secretion previously observed during a 4hr AM duodenal glucose infusion (2.1 mU/kg/min [0-30 min], 2.4 mU/kg/min [30-60 min], and 1.5 mU/kg/min [60-240 min]). In efforts to expose both groups to the same amount of insulin in the periphery while creating a difference at the liver, these rates were halved in the Pe Ins group. In the PM clamp, all groups received 4x basal insulin, 2x basal glycemia, and portal glucose infusion to simulate a second meal. During the 2.5hr PM clamp, the mean HGU was 1.8-fold higher in the Po Ins group vs. the Pe Ins group (HGU of 6.28±0.62 vs. 3.49±0.31 mg/kg/min; net AUC of 827±82 vs. 474±40 mg/kg/2.5hr, p<0.002), respectively, with no significant difference in non-HGU (net AUC of 819±118 mg/kg/2.5hr in Po Ins vs. 684±83 mg/kg/2.5hr in Pe Ins). Additionally, mean PM hepatic glycogen content was 47% larger in Po Ins vs. Pe Ins (23.6±1.1 vs. 12.5±1.5 mg/g liver, p<0.001), respectively. When compared to a group from an earlier study that received morning saline and a HIHG clamp in the afternoon (Sal Ctrl), there was no significant difference between Pe Ins and Sal Ctrl for any parameter measured. Therefore, physiologic hepatic insulin exposure in the morning is critical for increased hepatic glucose disposition and glycogen storage later in the day. 5T32DK007563 5R01DK131082-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diets high in fat and sugar are associated with the development of metabolic disease, but pathogenic changes in the gut and downstream metabolic organs are not well understood. In the dog, we sought to assess 9 weeks of chow (n=5) versus high-fat, high-fructose diet (HFFD) (n=5) on the gut response and the impact on liver, pancreas fat, and muscle following a mixed meal (26g protein, 67g carbohydrates, 22g lipids). To measure the 6-hour postprandial rate of nutrient absorption and secretion of pancreatic and gut hormones, dogs underwent surgical placement of catheters in the portal vein, hepatic vein, and femoral artery, and flow probes around the portal vein and hepatic artery. Duodenal morphology and L and K cell distribution were evaluated. The meal-induced glucose absorption occurred rapidly with HFFD (peak 13.5±1.1 mg/kg/min at 90 min post-meal v 10.9±1.3 mg/kg/min at 120 min), resulting in a greater rise in glucose (231±25 v. 162±7 mg/dL, p=0.03) compared to chow. Insulin secretion rates for HFFD and chow groups were similar at the 90 min peak (1.6±0.3 v. 1.9±0.6 µU/kg/min) and 6h AUC (237±46 v 243±67 mU/kg). Meal-induced GIP secretion was markedly increased (542±70 v 295±74 pg/kg/min, p=0.05) while GLP-1 was reduced (100±40 v 219±64 pg/kg/min, p=0.16) in HFFD compared to chow. These changes were associated with increased duodenal total mucosal height (1817±133 v 1500±159 µm, p=0.04) but no change in L or K cell distribution. HFFD-induced liver response was markedly altered with a failure to switch from net hepatic glucose output pre-meal to glucose uptake and storage post-meal (output of 1.7±0.2 to output of 3.0±1.4 mg/kg/min) compared to chow (output of 1.6±0.3 to uptake of 3.4±1.1). Given the lack of hepatic uptake, meal glucose was likely taken up by muscle. In conclusion, HFFD-induced changes in duodenal morphology, gut function, and gut hormone secretion that were associated with abnormal hepatic and pancreatic responses, suggesting a role for the gut in metabolic pathogenesis. Disclosure G. Kraft: None. M. Scott: None. B. Farmer: None. K. Yankey: None. D.S. Edgerton: None. H. Rajagopalan: Board Member; Fractyl Health, Inc. Employee; Fractyl Health, Inc. Stock/Shareholder; Fractyl Health, Inc. E. Cozzi: Employee; Fractyl Health, Inc. Stock/Shareholder; Fractyl Health, Inc. K. Gibson-Corley: None. C.R. Flynn: None. A.D. Cherrington: Consultant; Abvance Therapeutics. Research Support; Abvance Therapeutics. Advisory Panel; AdipoPharma. Research Support; Cellular Longevity, Inc. dba Loyal. Advisory Panel; Fractyl Health, Inc. Consultant; Fractyl Health, Inc. Research Support; Fractyl Health, Inc. Consultant; Novo Nordisk. Research Support; Novo Nordisk. Consultant; Paratus, Portal Insulin. Advisory Panel; Sekkei Bio, Sensulin Labs, LLC. Consultant; Thetis Pharmaceuticals, LLC. Funding Fractyl Health

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

Diets high in fat and fructose (HFHF) disrupt glucose metabolism. This study compared the effects of a HFHF diet on liver insulin action vs glucose effectiveness. Dogs were fed either a chow or HFHF diet for 1 month. At the start of each study, 3-3H-glucose was infused and after a basal sampling period, somatostatin and basal glucagon were given. During the experimental period (4h) either insulin was infused into the hepatic portal vein at 4 fold basal, while glucose was delivered to maintain euglycemia (CHOW-INS and HFHF-INS), or glucose was infused to increase its level 2.5 fold, while insulin was maintained at basal (CHOW-GLC and HFHF-GLC) (n=6/grp). Plasma insulin, glucagon, glucose, and hepatic glucose load were matched within the respective groups. In response to selective hyperinsulinemia, net hepatic glucose balance (NHGB) was suppressed from output to uptake (basal period to last h; 1.2±0.1 to -1.0±0.2 vs 1.7±0.2 to 0.0±0.1; deltas of 2.2±0.2 vs 1.6±0.3 mg/kg/min in CHOW-INS vs HFHF-INS, respectively; p=0.2). Selective hyperglycemia, on the other hand, caused changes in NHGB of 1.1±0.2 to -2.5±0.4 vs 1.4±0.1 to 1.7±0.6; deltas of 3.6±0.4 vs -0.3±0.6 mg/kg/min in CHOW-GLC vs HFHF-GLC, respectively (p<0.001). Glucose turnover (Ra) was suppressed (basal period to last h) by 1.7±0.3 vs 1.3±0.1 mg/kg/min (p=0.4) in CHOW-INS vs HFHF-INS, and by 1.9±0.2 vs 0.5±0.1 mg/kg/min (p<0.001) in CHOW-GLC vs HFHF-GLC, respectively. Thus, the HFHF diet had a small, non-significant effect on liver insulin action under euglycemic conditions, whereas it severely impaired the effect of hyperglycemia in the presence of basal insulin (i.e. hepatic glucose effectiveness). These data demonstrate that the “gold standard” hyperinsulinemic euglycemic clamp may overlook substantial diet induced liver dysfunction. Furthermore, Ra accounts for glucose production but not liver uptake, thus has the potential to miss critical and substantial changes in liver glucose metabolism. Disclosure D.S. Edgerton: None. G. Kraft: None. H.L. Waterman: None. B. Farmer: None. K. Yankey: None. M.S. Smith: None. J.R. Hastings: None. M. Scott: None. A.D. Cherrington: Consultant; Abvance Therapeutics. Research Support; Abvance Therapeutics. Advisory Panel; AdipoPharma. Research Support; Cellular Longevity, Inc. dba Loyal. Advisory Panel; Fractyl Health, Inc. Consultant; Fractyl Health, Inc. Research Support; Fractyl Health, Inc. Consultant; Novo Nordisk. Research Support; Novo Nordisk. Consultant; Paratus, Portal Insulin. Advisory Panel; Sekkei Bio, Sensulin Labs, LLC. Consultant; Thetis Pharmaceuticals, LLC. Funding R01DK18243

Endogenous insulin secretion is a key regulator of postprandial hepatic glucose metabolism, but this process is dysregulated in diabetes. Subcutaneous insulin delivery alters normal insulin distribution, causing relative hepatic insulin deficiency and peripheral hyperinsulinemia, a major risk factor for metabolic disease. Our aim was to determine whether insulin’s direct effect on the liver is preeminent even when insulin is given into a peripheral vein. Postprandial-like conditions were created (hyperinsulinemia, hyperglycemia, and a positive portal vein to arterial glucose gradient) in healthy dogs. Peripheral (leg vein) insulin infusion elevated arterial and hepatic levels 8 and 2.8-fold, respectively. In one group, insulin’s full effects were allowed. In another, insulin’s indirect hepatic effects were blocked with the infusion of triglyceride, glucagon, and inhibitors of brain insulin action (intracerebroventricular) to prevent decreases in plasma free fatty acids and glucagon, while blocking increased hypothalamic insulin signaling. Despite peripheral insulin delivery the liver retained its full ability to store glucose, even when insulin’s peripheral effects were blocked, whereas muscle glucose uptake markedly increased, creating an aberrant distribution of glucose disposal between liver and muscle. Thus, the healthy liver’s striking sensitivity to direct insulin action can overcome the effect of relative hepatic insulin deficiency, whereas excess insulin in the periphery produces metabolic abnormalities in non-hepatic tissues.

Glucagon stimulates hepatic glucose production through its direct effects on the liver but may indirectly inhibit this process by acting on the brain. This was tested by delivering glucagon via the cerebral circulatory system. Central nervous system glucagon action reduced liver gluconeogenic flux, but glycogenolysis increased, resulting in no net change in hepatic glucose production. Surprisingly, brain glucagon also appeared to suppress lipolysis (plasma free fatty acid and glycerol levels were reduced).

We showed previously in people with T1D that by infusing glucagon along with insulin can buffer insulin’s hypoglycemic effect without worsening its ability to limit hyperglycemia. Our aim was to determine in the conscious dog the mechanisms by which glucagon limits the insulin driven fall in glucose. Somatostatin was infused to disable the endocrine pancreas and insulin and glucagon were infused into a leg vein at basal rates. After a 140 min control period, the insulin infusion rate was increased 4-7-fold for 3h to induce hypoglycemia. Glucagon was also infused into a leg vein, then was either increased proportionately to insulin (constant I/G molar ratio, HiGGN), or in a second study on the same dog, it was kept at its basal rate (Ba GGN). Hepatic sinusoidal insulin levels rose from 11±2 to 65±17 μU/ml regardless of the glucagon level, which remained basal in BaGGN (50±10pg/mL) but rose from 52±8 pg/ml to 238±50pg/ml in HiGGN. Plasma glucose fell over the first hour, reaching a nadir of 45±2 mg/dl when glucagon was basal, and fell more slowly when glucagon was elevated (nadir of 50±2 mg/dl). This difference was due to a greater increase in net hepatic glucose output in the HiGGN group (1.4 ± 0.2 vs 2.4 ± 0.2 mg/kg/min in BaGGN and HiGGN respectively). Interestingly the rise in plasma cortisol and epinephrine were reduced by 33 and 39%, respectively, in the group with elevated glucagon. Further, this lessening in the counterregulatory response was associated with reductions in lipolysis and lactate production by muscle. Thus, by maintaining a fixed I/G molar ratio the hypoglycemic potential of insulin is buffered. This could allow more aggressive insulin treatment which would in turn increase time in range for glucose. Disclosure G.Kraft: None. M.Scott: None. M.S.Smith: None. B.Farmer: None. D.S.Edgerton: None. A.D.Cherrington: Advisory Panel; Metavention, vTv Therapeutics, Diakard, Sekkei Bio, Sensulin Labs, LLC, Other Relationship; Fractyl Health, Inc., Novo Nordisk, Abvance Therapeutics, Research Support; Cellular Longevity, Inc, dba Loyal, Senda Biosciences.

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

High fat and fructose diets (HFFD) disrupt glucose metabolism. The aim of this study was to determine the effect of HFFD on glucagon (GGN) effectiveness. Dogs were fed either chow or HFFD for one month. After a basal sampling period, somatostatin and basal portal vein infusions of insulin and GGN were given for 4h, with the exception of GGN, which was either maintained at basal or increased 6-fold. Glucose was infused in the basal GGN groups (CHOW+GLC & HFFD+GLC) to match the 3-fold increase in plasma glucose that occurred with 6x-GGN (CHOW+GGN & HFFD+GGN) . There were no differences in plasma insulin or glucose and GGN levels were proportional to GGN infusion rates in all groups. 6x-GGN caused an identical increase in net hepatic glucose balance (NHGB) in the chow and HFFD groups (Fig 1A) after which NHGB declined. During the last hour NHGB was 1.8-fold greater in HFHF+GGN compared to Chow+GGN (A) . However, hyperglycemia per se caused a switch from net hepatic glucose output to uptake in Chow+GLC but not HFHF+GLC (there was a 3.7±0.4 vs. 0.3±0.4 mg/kg/min decrease in NHGB over time in the two groups, respectively) (B) . Thus, when expressed relative to each group’s respective glucose control group, the AUC for GGN stimulated NHGB was 1.9-fold greater in the Chow (C) compared to HFHF (D) groups (P<0.05) . In summary, HFHF feeding reduced hepatic glucagon action indirectly by impairing hepatic glucose effectiveness. Disclosure D.S.Edgerton: None. G.Kraft: None. K.C.Coate: None. B.Farmer: None. M.S.Smith: None. M.Scott: None. J.R.Hastings: None. M.C.Moore: None. A.D.Cherrington: Advisory Panel; Biocon, Diakard, Metavention, Sekkei Bio, Sensulin Labs, LLC, vTv Therapeutics, Consultant; Abvance, Fractyl Health, Inc., Novo Nordisk, Research Support; Bristol-Myers Squibb Company. Funding NIH R01DK18243

Iatrogenic hypoglycemia is a prominent barrier to achieving optimal glycemic control in patients with diabetes, in part due to dampened counterregulatory hormone responses. It has been demonstrated that elevated liver glycogen content can enhance these hormonal responses through signaling to the brain via afferent nerves, but the role that hypoglycemia in the brain plays in this liver glycogen effect remains unclear. During the first 4 h of each study, the liver glycogen content of dogs was increased by using an intraportal infusion of fructose to stimulate hepatic glucose uptake (HG; n = 13), or glycogen was maintained near fasting levels with a saline infusion (NG; n = 6). After a 2-h control period, during which the fructose/saline infusion was discontinued, insulin was infused intravenously for an additional 2 h to bring about systemic hypoglycemia in all animals, whereas brain euglycemia was maintained in a subset of the HG group by infusing glucose bilaterally into the carotid and vertebral arteries (HG-HeadEu; n = 7). Liver glycogen content was markedly elevated in the two HG groups (43 ± 4, 73 ± 3, and 75 ± 7 mg/g in NG, HG, and HG-HeadEu, respectively). During the hypoglycemic period, arterial plasma glucose levels were indistinguishable between groups (53 ± 2, 52 ± 1, and 51 ± 1 mg/dL, respectively), but jugular vein glucose levels were kept euglycemic (88 ± 5 mg/dL) only in the HG-HeadEu group. Glucagon and epinephrine responses to hypoglycemia were higher in HG compared with NG, whereas despite the increase in liver glycogen, neither increased above basal in HG-HeadEu. These data demonstrate that the enhanced counterregulatory hormone secretion that accompanies increased liver glycogen content requires hypoglycemia in the brain.NEW & NOTEWORTHY It is well known that iatrogenic hypoglycemia is a barrier to optimal glycemic regulation in patients with diabetes. Our data confirm that increasing liver glycogen content 75% above fasting levels enhances hormonal responses to insulin-induced hypoglycemia and demonstrate that this enhanced hormonal response does not occur in the absence of hypoglycemia in the brain. These data demonstrate that information from the liver regarding glycogen availability is integrated in the brain to optimize the counterregulatory response.

Hepatic glucose uptake (HGU) is critical for maintaining normal postprandial glucose metabolism. Insulin is clearly a key regulator of HGU, but the physiologic mechanisms by which it acts have yet to be established. This study sought to determine the mechanisms by which insulin regulates liver glucose uptake under postprandial-like conditions (hyperinsulinemia, hyperglycemia, and a positive portal vein-to-arterial glucose gradient). Portal vein insulin infusion increased hepatic insulin levels fivefold in healthy dogs. In one group (n = 7), the physiologic response was allowed to fully occur, while in another (n = 7), insulin's indirect hepatic effects, occurring secondary to its actions on adipose tissue, pancreas, and brain, were blocked. This was accomplished by infusing triglyceride (intravenous), glucagon (portal vein), and inhibitors of brain insulin action (intracerebroventricular) to prevent decreases in plasma free fatty acids or glucagon, while blocking increased hypothalamic insulin signaling for 4 h. In contrast to the indirect hepatic effects of insulin, which were previously shown capable of independently generating a half-maximal stimulation of HGU, direct hepatic insulin action was by itself able to fully stimulate HGU. This suggests that under hyperinsulinemic/hyperglycemic conditions insulin's indirect effects are redundant to direct engagement of hepatocyte insulin receptors.