The goal in this study was to systematically analyze glucose metabolism in four representative inbred mouse strains using three clamp techniques to study in vivo physiology under well-controlled conditions. In C57BL/6, 129X1/Sv, FVB/N, and DBA/2 mice, we show strain-dependent differences in insulin action, hypoglycemic counter-regulation, and insulin secretion. These four strains were selected for comparison based on recommendations of the National MMPC Steering Committee because these strains are frequently used in metabolic studies. In these experiments, glucoregulatory phenotype was assessed using in vivo euglycemic, hypoglycemic, and hyperglycemic clamps developed to study whole-body physiology in mice. This research emphasizes that genetic background is a critical factor to consider when designing and interpreting experiments. These results are important because in vivo clamp techniques are increasingly used to study physiology in mice, and this is the first published study to comprehensively address the contribution of genetic background to results obtained during in vivo clamp experiments.
While a number of previous studies have investigated phenotypic glucoregulatory differences between different inbred strains (3
), a key distinction in these studies is that techniques to study physiology in conscious, unstressed mice under well-controlled glycemic conditions were used. One previously published study used in vivo clamp techniques to examine differences between two different inbred mouse strains (4
). This study assessed insulin action using methods (i.e., cut-tail blood sampling, large insulin prime, and overnight fast) previously shown to induce acute hepatic insulin resistance and higher catecholamines (2
The majority of previous studies performed in vivo to study whole-body physiology have used insulin and glucose tolerance tests to study glucose metabolism (5
). These assessments can be difficult to interpret because results are generally expressed as percent change relative to basal blood glucose or insulin levels, which may vary by strain or with genetic manipulation. Clamp methods are considered the gold standard for assessing glucose metabolism because glycemia is controlled, thus alleviating interpretation problems related to changes in blood glucose.
Euglycemic clamp results indicate strain-dependent differences in insulin action and highlight complexities of this method. GIR levels were different between strains, suggesting differences in insulin action, but steady-state insulin varied between strains. Normalizing GIR to clamp insulin accounts for differences and permits a more complete interpretation of insulin action. GIR normalized to insulin indicates that DBA/2 mice are insulin resistant compared with 129X1/Sv, C57BL/6, and FVB/N mice. This is consistent with higher basal insulin in DBA/2 mice, which may reflect some degree of β-cell compensation. There were no strain-dependent differences in GIR normalized to ΔInsulinClamp-Basal
(). The larger ΔInsulinClamp-Basal
in 129X1/Sv mice compared with FVB/N mice suggests strain-dependent differences in insulin clearance. One might also speculate that differences in clamp insulin are due to insulin-mediated suppression of β-cell insulin secretion. It is common not to report insulin levels (24
). The present study demonstrates that insulin must be reported to fully interpret results from clamp studies.
Insulin clamp GIR and Rd were significantly correlated with QUICKI and HOMA-IR (P < 0.01). The correlation coefficients comparing GIR to QUICKI and HOMA-IR were 0.53 and −0.54, respectively. The correlation coefficients comparing Rd to QUICKI and HOMA-IR were 0.53 and −0.50, respectively. These correlations were equally significant using GIR and Rd normalized to insulin compared with QUICKI and HOMA-IR.
Euglycemic clamp studies also suggest tissue-specific differences in insulin action. Rd
was lower in DBA/2 mice compared with other strains. The lower Rd
in DBA/2 mice did not correspond with differences in GLUT4 or HKII protein content or Akt activation in skeletal muscle. Lower Rd
in DBA/2 mice did correspond to a higher fat mass, which is consistent with an inverse relationship between fat mass and peripheral insulin action. This relationship also exists in humans. It is likely that the higher fat mass in DBA/2 mice contributes to insulin resistance, but it is impossible to establish a causal relationship from these studies. Hyperinsulinemia did not fully suppress endoRa
in FVB/N mice, indicating relative hepatic resistance to insulin. This corresponded with a lower activation of hepatic Akt. The insulin infusion used here was not ideal for resolving liver phenotypes because the dose was beyond the most sensitive region of insulin to endoRa
dose-response curve (2
). This was evident by negative endoRa
values in all but FVB/N mice. An insulin dose <15 pmol · kg−1
would better isolate hepatic insulin action phenotypes.
The results also indicate that the response to hypoglycemia is strain dependent. The hypoglycemic clamp, to our knowledge, had only been done in C57BL/6 mice (12
). Our results indicate that the endocrine response is largely absent in 129X1/Sv mice compared with C57BL/6, FVB/N, and DBA/2 mice. In contrast, the endocrine response in FVB/N mice is far more potent compared with the other strains. This marked endocrine response in FVB/N mice did not correspond with lower GIR (compared with C57BL/6 and DBA/2) during the hypoglycemic clamp. This could be due to different sensitivities to glucagon and/or catecholamines, but it is more likely due to high insulin levels, which may mask the effects of counter-regulatory hormones. These findings are not only critical factors for hypoglycemic clamps but are also important issues in insulin tolerance tests used to estimate insulin action. The key metric in both is the insulin-induced fall in glucose. Insulin tolerance tests could be complicated by differences in basal glycemia or differences in the counter-regulatory response when comparing mixed-background mice or different strains.
The insulin secretory response to hyperglycemia further highlights phenotypic differences between strains and the complexity underlying the physiology of insulin secretion. Several groups have previously investigated differences in insulin secretion in vitro and in vivo using glucose tolerance tests in inbred mouse strains (15
). Our measurements made in vivo using hyperglycemic clamps extend these results to fixed glycemic conditions. The objective was to match blood glucose between strains and quickly achieve hyperglycemia. The GIR is therefore dictated by the physiological response (i.e., blood glucose). The initial GIR is lower in FVB/N and DBA/2 mice compared with 129X1/Sv and C57BL/6 mice based on these glycemic responses (). When an initial GIR similar to that in 129X1/Sv and C57BL/6 mice was used in FVB/N and DBA/2 mice, severe hyperglycemia occurred, often exceeding the upper limit of detection (27 mmol/l).
The failure of FVB/N mice to respond to glucose in vivo under clamp conditions is interesting in light of the intact in vitro response. Catecholamines were elevated in FVB/N mice compared with other strains and might explain the failure to increase insulin secretion in vivo (28
). The robust insulin response in vivo in C57BL/6 mice contrasts with blunted secretion in vitro and previous work showing that C57BL/6 mice secrete less insulin in vivo compared with other strains (15
). In previous studies, this has been associated with deletion of the nicotinamide nucleotide transhydrogenase gene in C57BL/6 mice (22
). It is possible that there is long-term compensation for impaired insulin secretion in C57BL/6 mice (30
). However, there were no profound differences in C57BL/6 pancreatic insulin content, islet insulin content, or β-cell mass.
The correlation coefficient between in vitro and in vivo insulin secretion in response to hyperglycemia was low (r = −0.20). Insulin content in the total pancreas and individual islets normalized for size (islet equivalent) was assessed to understand the mechanism for differences in insulin secretion in vivo and in isolated islets. There was no systematic relationship between insulin content in isolated islets and insulin response (insulin secretion AUC) to high glucose in isolated islets (r = 0.25) or the insulin response to hyperglycemia in vivo (r = 0.01). There was also no relationship between pancreatic insulin content and insulin response to high glucose in isolated islets (r = −0.39) or in vivo (r = −0.17). This suggests that differences in secretion in isolated islet versus in vivo studies are due to differences in glucose sensing or stimulus/response coupling. These results highlight that unknown factors both intrinsic and extrinsic to islets contribute to these strain-dependent differences in insulin secretion and may complicate the interpretation of insulin secretion in vivo and in isolated islets.
An important observation is the strain-dependent differences in the ratio of whole-blood glucose to plasma glucose. This is an important consideration because clamp blood glucose may differ depending on whether whole-blood or plasma glucose is used. Also, using whole-blood glucose and plasma [3-3H]glucose concentrations to calculate glucose specific activity will underestimate endoRa. It is therefore important to use plasma glucose when calculating glucose turnover regardless of which method was used to clamp the mouse.
In summary, these results expose differences in glucose homeostasis in four commonly used mouse strains. Previous studies have documented the need to consider the contribution of the inbred strain to results from genetic manipulation on phenotype. Our results provide an important empirical reference for this under carefully controlled glycemic conditions. Our observations indicate that these four inbred mouse strains have unique underlying phenotypes in the basal condition and in response to clamp conditions. Because C57BL/6 mice are a common background strain used to study glucose metabolism, it is important to note that this strain has an intermediate physiological response to each of the three clamp experiments in comparison with 129X1/Sv, FVB/N, and DBA/2 mice. This is reassuring and suggests that C57BL/6 mice are a suitable model for studies of glucose homeostasis. Overall, these results demonstrate that it is critical to recognize the underlying phenotype of the inbred strain when performing metabolic testing on genetically modified mice and when comparing results within and between laboratories. These data are informative for selection of background strain, experimental design, and data interpretation.