Increased beta -oxidation but no insulin resistance or glucose intolerance in mice lacking adiponectin

Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generated Adipo(-/-) mice by gene targeting. Adipo(-/-) mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels of Adipo(-/-) and Adipo(+/+) mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed that Adipo(-/-) and Adipo(+/+) mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured beta-oxidation and found it to be significantly increased in muscle and liver of Adipo(-/-) mice. In conclusion, our study indicates that absence of adipo causes increased beta-oxidation but does not cause glucose intolerance or insulin resistance in mice.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) influences substrate utilization for hepatic glucose production

The hypoglycemia seen in the fasting PPARalpha null mouse is thought to be due to impaired liver fatty acid beta-oxidation. The etiology of hypoglycemia in the PPARalpha null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPARalpha null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-(13)C(6)]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-(13)C(3)]lactate or [2-(13)C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPARalpha null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPARalpha null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-(13)C(3)]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPARalpha null mice. In contrast, when [2-(13)C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPARalpha null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPARalpha null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPARalpha null mice than in controls. In conclusion, PPARalpha receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.     

Long-term administration of PACAP receptor antagonist, PACAP(6-27), impairs glucose tolerance and insulin sensitivity in obese diabetic ob/ob mice

Pituitary adenylate cyclase-activating peptide (PACAP) is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the PACAP receptor antagonist, PACAP(6-27) to evaluate the role of endogenous PACAP in genetic obesity-related diabetes and related metabolic abnormalities using ob/ob mice. Acute in vivo antagonistic potency of PACAP(6-27) was confirmed in ob/ob mice by blockade of the insulin-releasing action but not hyperglycaemia. In longer-term studies, ob/ob mice were given once daily injections of PACAP(6-27) or vehicle for 14 days. Feeding activity, body weight, basal plasma glucose and plasma insulin concentrations were not significantly affected by chronic PACAP(6-27) treatment. However, PACAP(6-27) treatment impaired glucose tolerance, insulin sensitivity and the glycaemic response to feeding. Plasma glucagon and lipids were unchanged. These observations indicate a role of endogenous PACAP for normal glucose homeostasis, but indicate a minor involvement in the regulation of insulin secretion in ob/ob mice.     

Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice

Growth hormone, acting through its receptor (GHR), plays an important role in carbohydrate metabolism and in promoting postnatal growth. GHR gene-deficient (GHR(-/-)) mice exhibit severe growth retardation and proportionate dwarfism. To assess the physiological relevance of growth hormone actions, GHR(-/-) mice were used to investigate their phenotype in glucose metabolism and pancreatic islet function. Adult GHR(-/-) mice exhibited significant reductions in the levels of blood glucose and insulin, as well as insulin mRNA accumulation. Immunohistochemical analysis of pancreatic sections revealed normal distribution of the islets despite a significantly smaller size. The average size of the islets found in GHR(-/-) mice was only one-third of that in wild-type littermates. Total beta-cell mass was reduced 4.5-fold in GHR(-/-) mice, significantly more than their body size reduction. This reduction in pancreatic islet mass appears to be related to decreases in proliferation and cell growth. GHR(-/-) mice were different from the human Laron syndrome in serum insulin level, insulin responsiveness, and obesity. We conclude that growth hormone signaling is essential for maintaining pancreatic islet size, stimulating islet hormone production, and maintaining normal insulin sensitivity and glucose homeostasis.     

Fetuin-null mice are protected against obesity and insulin resistance associated with aging

alpha2-HS glycoprotein (AHSG), also known as fetuin-A, inhibits insulin receptor autophosphorylation and tyrosine kinase activity in vitro and in vivo. Earlier we have shown that fetuin-null (KO) mice demonstrate improved insulin sensitivity and resistance to diet-induced obesity. Since aging is associated with insulin resistance and impaired glucose handling, we tested the hypothesis that fetuin-null (KO) mice are resilient to changes in insulin sensitivity associated with aging. Aged (80-week-old) fetuin-null mice were leaner and demonstrated significantly lower body weights compared to age- and sex-matched wild-type (WT) littermates. Leanness in aged fetuin KO mice was accompanied by a significant increase in dark-onset energy expenditure, without marked alteration of respiratory quotient. In comparison to WT mice, fetuin KO mice demonstrated a lower fasting insulin resistance index, and significantly lower blood glucose and insulin levels, following a 4h fast. Interestingly, despite significantly decreased insulin levels during a glucose tolerance test, aged fetuin-null mice demonstrated a similar glucose excursion as WT mice, indicative of improved insulin sensitivity. Analysis of aldehyde-fuchsin stained pancreas from aged fetuin KO mice indicated no difference in islet beta-cell size or number. An insulin tolerance test confirmed the increased insulin sensitivity of aged fetuin KO mice. Further, compared to WT mice, aged fetuin-null mice demonstrated increased skeletal muscle and liver IR autophosphorylation and TK activity. Taken together, this study suggests that the absence of fetuin may contribute to the improvement of insulin sensitivity associated with aging.     

A minor role for lipocalin 2 in high-fat diet-induced glucose intolerance

Adipose tissue controls energy homeostasis and systemic insulin sensitivity through the elaboration of a series of cytokines and hormones, collectively termed "adipokines." We and others have identified Lcn2 as a novel adipokine, but its exact role in obesity-induced insulin resistance remains controversial. The aim of this study was to examine the metabolic phenotype of Lcn2(-/-) mice to clarify the role of Lcn2 in metabolism. Male and female Lcn2(-/-) and wild-type (WT) littermates were placed on either chow or high-fat diet (HFD) to characterize their metabolic phenotype. Studies included body weight and body composition, glucose and insulin tolerance tests, and adipokine expression studies in serum and in white adipose tissue (WAT). Neither chow nor HFD cohorts showed any differences in body weight or body composition. Chow-fed Lcn2(-/-) mice did not exhibit any difference in glucose homeostasis compared with WT mice. Fasting serum glucose levels were lower in the chow-fed Lcn2(-/-) mice, but this finding was not seen in the HFD cohort. Serum adiponectin, leptin, resistin, and RBP4 levels were not different between WT and Lcn2(-/-) on chow diet. HFD-fed male Lcn2(-/-) mice did display a small improvement in glucose tolerance, but no difference in insulin sensitivity was seen in either male or female Lcn2(-/-) mice on HFD. We conclude that the global ablation of Lcn2 has a minimal effect on obesity-associated glucose intolerance but does not appear to affect either age- or obesity-mediated insulin resistance in vivo.     

Cathepsin L activity controls adipogenesis and glucose tolerance

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R beta-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR beta-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR beta-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders.     

Functional roles of the neuropeptide PACAP in brain and pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide implicated in a broad variety of physiological processes. To assess PACAP's function in vivo, we recently generated PACAP knockout mice (PACAP(-/-)) and transgenic mice overexpressing PACAP specifically in the pancreas (PACAP-Tg). In PACAP(-/-) mice, we have demonstrated a marked phenotypic changes including a high early mortality rate, increased novelty-seeking behavior and abnormal explosive jumping in a novel environment, as well as reduced female fertility. In this paper, we reevaluated these phenotypes in terms of the genetic background of the mice. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype. In PACAP-Tg mice, we have recently demonstrated that enhanced glucose-induced insulin secretion with normal glucose tolerance, amelioration of streptozotocin-induced diabetes with increased beta-cell proliferation, and a trend towards an increase in total islet mass with age. Here we show that PACAP(-/-) mice exhibit significantly impaired glucose-induced insulin secretion but still have normal glucose tolerance. These observations suggest that PACAP may play important roles in and beyond the regulation of insulin release. Taken together, the mutant phenotypes revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.     

Pituitary adenylate cyclase-activating peptide (PACAP): assessment of dipeptidyl peptidase IV degradation, insulin-releasing activity and antidiabetic potential

Pituitary adenylate cyclase-activating peptide (PACAP) is a member of the glucagon family of peptides. Like other members, most notably glucagon-like peptide-1 (GLP-1), PACAP is rapidly degraded by dipeptidylpeptidase IV (DPP IV). This study investigated how degradation by DPP IV affected the insulinotropic activity of PACAP, and whether PACAP exerted acute antihyperglycemic properties in normal or ob/ob mice. DPP IV degradation of PACAP(1-27) over 18 h led to the formation of PACAP(3-27), PACAP(5-27) and ultimately PACAP(6-27). In contrast to 1.4-1.8-fold concentration-dependent stimulation of insulin secretion by PACAP(1-27), these peptide fragments lacked insulinotropic activity. While PACAP(1-27) and PACAP(1-38) generated significant insulin responses when given alone or together with glucose in ob/ob and normal mice, they also elevated plasma glucose. These actions were eliminated following degradation of the peptide by incubation with DPP IV. The hyperglycemic effects may be explained at least partly by a potent glucagon-releasing action in ob/ob and normal mice. In conclusion, PACAP is inactivated by DPP IV and despite insulin-releasing effects, its actions on glucagon secretion and glucose homeostasis do not make it a good therapeutic tool for the treatment of type 2 diabetes.     

Overexpression of PACAP in the pancreas failed to rescue early postnatal mortality in PACAP-null mice

PACAP exerts multiple activities as a hormone and neurotransmitter, and has been proposed to play vital roles in a variety of neuronal functions. PACAP is also involved in insulin secretion from pancreatic beta-cells. Recently, we and other groups demonstrated that PACAP-deficient mice (PACAP(-/-)) are viable, but suffer from increased postnatal mortality. To ascertain whether this high mortality is rescued by overexpression of PACAP in peripheral tissue (such as pancreas), we performed a genetic cross between PACAP(-/-) and our recently developed transgenic mice overexpressing PACAP in pancreatic beta-cells; and then examined the survival rate of their F2 progeny. PACAP(-/-) mice were segregated into two groups based on mortality as well as body weight gain: PACAP(-/-) that survived >20 days of age with normal weight gain and PACAP(-/-) that died before 20 days with a marked weight loss. Kaplan-Meier survival analysis demonstrated that PACAP(-/-) mice and those carrying the PACAP transgene have similarly lower survival probability compared with their heterozygous littermates that served as positive controls. Further study using additional tissue-specific transgenic or knockout mouse models will be required to determine the causative defects underlying the high mortality of PACAP(-/-) mice.     

PACAP contributes to insulin secretion after gastric glucose gavage in mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic ganglia governing the parasympathetic nerves, which contribute to prandial insulin secretion. We hypothesized that this contribution involves PACAP and show here that the PACAP receptor antagonist PACAP-(6---27) (1.5 nmol/kg iv) reduces the 15-min insulin response to gastric glucose (150 mg/mouse) by 18% in anesthetized mice (P = 0.041). The reduced insulinemia was not due to inhibited release of the incretin factor glucagon-like peptide 1 (GLP-1) because PACAP-(6---27) enhanced the GLP-1 response to gastric glucose. Furthermore, the GLP-1 antagonist exendin-3-(9---39) (30 nmol/kg) exerted additive inhibitory effect on the insulin response when combined with PACAP-(6---27). The PACAP antagonism was specific because intravenous PACAP-(6---27) inhibited the insulin response to intravenous PACAP-27 plus glucose without affecting the insulin response to intravenous glucose alone (1 g/kg) or glucose together with other insulin secretagogues of potential incretin relevance of intestinal (GLP-1, gastric inhibitory polypeptide, cholecystokinin) and neural (vasoactive intestinal peptide, gastrin-releasing peptide, cholinergic agonism) origin. We conclude that PACAP contributes to the insulin response to gastric glucose in mice and suggest that PACAP is involved in the regulation of prandial insulin secretion.     

Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality.

Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/- mice were normal as shown by glucose tolerance tests. Normal Insr-/- pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post-natal growth retardation (up to 30-40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6-fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr-/- mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed.     

Suppressor of cytokine signaling-2 deficiency induces molecular and metabolic changes that partially overlap with growth hormone-dependent effects

Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2-/-) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2-/- phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2-/- mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2-/- mice. Moreover, increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.     

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis

The protein tyrosine phosphatase SHP-1 is a well-known inhibitor of activation-promoting signaling cascades in hematopoietic cells but its potential role in insulin target tissues is unknown. Here we show that Ptpn6(me-v/me-v) (also known as viable motheaten) mice bearing a functionally deficient SHP-1 protein are markedly glucose tolerant and insulin sensitive as compared to wild-type littermates, as a result of enhanced insulin receptor signaling to IRS-PI3K-Akt in liver and muscle. Downregulation of SHP-1 activity in liver of normal mice by adenoviral expression of a catalytically inert mutant of SHP-1, or after small hairpin RNA-mediated SHP-1 silencing, further confirmed this phenotype. Tyrosine phosphorylation of CEACAM1, a modulator of hepatic insulin clearance, and clearance of serum [125I]-insulin were markedly increased in SHP-1-deficient mice or SHP-1-deficient hepatic cells in vitro. These findings show a novel role for SHP-1 in the regulation of glucose homeostasis through modulation of insulin signaling in liver and muscle as well as hepatic insulin clearance.     

Role of PYK2 in the development of obesity and insulin resistance

Non-receptor proline-rich tyrosine kinase-2 (PYK2), which is activated by phosphorylation of one or more of its tyrosine residues, has been implicated in the regulation of GLUT4 glucose transporter translocation and glucose transport. Some data favor a positive role of PYK2 in stimulating glucose transport, whereas other studies suggest that PYK2 may participate in the induction of insulin resistance. To ascertain the importance of PYK2 in the setting of obesity and insulin resistance, we (1) evaluated the regulation of PYK2 in mice fed a high-fat diet and (2) characterized body and glucose homeostasis in wild type (WT) and PYK2(-/-) mice on different diets. We found that both PYK2 expression and phosphorylation were significantly increased in liver and adipose tissues harvested from high-fat diet fed mice. Wild type and PYK2(-/-) mice were fed a high-fat diet for 8 weeks to induce insulin resistance/obesity. Surprisingly, in response to this diet PYK2(-/-) mice gained significantly more weight than WT mice (18.7+/-1.2g vs. 9.5+/-0.6g). Fasting serum leptin and insulin and blood glucose levels were significantly increased in high-fat diet fed mice irrespective of the presence of PYK2 protein. There was a close correlation between serum leptin and body weight. Intraperitoneal glucose tolerance tests revealed that as expected, the high-fat diet resulted in increased blood glucose levels following glucose administration in wild type mice compared to those fed normal chow. An even greater increase in blood glucose levels was observed in PYK2(-/-) mice compared to wild type mice. These results demonstrate that a lack of PYK2 exacerbates weight gain and development of glucose intolerance/insulin resistance induced by a high-fat diet, suggesting that PYK2 may play a role in slowing the development of obesity, insulin resistance, and/or frank diabetes.